WO2010002940A2

WO2010002940A2 - Heterogeneous synthesis of multivalent chimeric immunomodulatory compounds using platform based molecules

Info

Publication number: WO2010002940A2
Application number: PCT/US2009/049325
Authority: WO
Inventors: Tracy Matray; Karen L. Fearon; Hesham A. Sherif
Original assignee: Dynavax Technologies Corp
Current assignee: Dynavax Technologies Corp
Priority date: 2008-06-30
Filing date: 2009-06-30
Publication date: 2010-01-07
Anticipated expiration: 2010-12-30
Also published as: WO2010002940A3

Abstract

The invention provides chimeric immunomodulatory compounds, methods for making such compounds and methods for immunomodulation of individuals using the immunomodulatory compounds.

Description

HETEROGENEOUS SYNTHESIS OF MULTIVALENT CHIMERIC IMMUNOMODULATORY COMPOUNDS

USING PLATFORM BASED MOLECULES

FIELD OF THE INVENTION

[0001] The present invention relates to improved chimeric immunomodulatory compounds ('CICs') containing nucleic acid and non-nucleic acid moieties, improved methods of synthesizing such CICs, and to the use of such compounds to modulate an immune response. The invention finds use in the fields of biomedicine and immunology.

BACKGROUND OF THE INVENTION

[0002] At a high level, immunity can generally be classified as innate immunity or as adaptive immunity. Innate immune responses typically occur immediately upon infection to provide an early barrier to infectious disease whereas adaptive immune responses occur later with the generation of antigen- specific effector cells and provides for long term protective immunity. Innate immune responses do not generate lasting protective immunity but appear to play a role in the generation of the later arising adaptive immune response.

[0003] Innate immunity uses germ-line encoded receptors to recognize features that are common to many pathogens and to activate signaling events that result in the expression of effector molecules. Some of these effector molecules may eventually induce an adaptive immune response. The family of Toll-like receptors (TLRs) has been associated with innate immune response signaling and microbial ligands have been identified for several mammalian TLRs. For example, TLR2 interacts with peptidoglycan, bacterial lipopeptides and certain types of lipopolysaccharide (LPS), TLR3 interacts with double- stranded RNA, TLR4 interacts with LPS and TLR-5 interacts with bacterial flagellin. See, for example, Poltorak et al. (1998) Science 282:2085-2088; Akira et al. (2003) Immunol. Lett. 85:85-95; Alexopoulou et al. (2001) Nature 413:732-738; Hayashi et al. (2001) Nature 410:1099- 1103. TLR-7 is activated by guanosine analogs, by small antiviral compounds such as imidazoquinolines, imiquimod and R-848, and by single- stranded viral RNA, and TLR-8 is also activated by R-848 and single- stranded viral RNA. See, for example, Lee et al. (2003) Proc. Natl. Acad. ScL USA 100:6646-6651; Hemmi et al. (2002) Nat. Immunol. 3:196-200; Jurk et al. (2002) Nat. Immunol. 3:499; Heil et al. (2004) Science 303:1526-1529; Diebold et al. (2004) Science 303:1529-1531. TLR-9 has been shown to recognize immuno stimulatory nucleic acid molecules such as bacterial DNA and immunostimulatory DNA containing a 5'- CG-3' sequence. See, for example, Hemmi et al. (2000) Nature 408:740-745; Bauer et al. (2001) Proc. Natl. Acad. ScL USA 98:9237-9242; Takeshita et al. (2001) J. Immunol. 167:3555-3558. In addition, certain TLRs (for example, TLR-I, TLR-2 and TLR-6) can heterodimerize, interact with their microbial ligands and lead to cell activation, thus expanding the ligand repertoire of the TLR family. Ozinsky et al. (2000) J. Endotoxin Res. 6:393-396; Ozinsky et al. (2000) Proc. Natl. Acad. ScL USA 97:13766-13771.

[0004] Immunostimulatory nucleic acid (ISNA) molecules, such as bacterial DNA or a polynucleotide containing unmethylated 5'-CG-3' sequences, can stimulate innate immune responses, such as cytokine production, and dendritic cell and macrophage activation, and then lead to a Thl-type immune response. Immunostimulatory nucleic acid molecules stimulate the immune response through interaction with and signaling through the mammalian TLR9 receptor. See Hemmi et al. (2000), Supra. Mammalian DNA does not generally possess immunostimulatory activity due apparently to a low frequency of CG sequences and to most of the CG sequences having a methylated cytosine. Mammalian immune system cells thus appear to distinguish bacterial DNA from self DNA through the TLR9 receptor.

[0005] With respect to adaptive immune response, the type of adaptive immune response generated by infection or other antigenic challenge can generally be distinguished by the subset of T helper (Th) cells involved in the response. The ThI subset is responsible for classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs), whereas the Th2 subset functions more effectively as a helper for B-cell activation. The type of immune response to an antigen is generally influenced by the cytokines produced by the cells responding to the antigen. Differences in the cytokines secreted by ThI and Th2 cells are believed to reflect different biological functions of these two subsets. See, for example, Romagnani (2000) Ann. Allergy Asthma Immunol. 85:9-18.

[0006] The ThI subset may be particularly suited to respond to viral infections, intracellular pathogens, and tumor cells because it secretes IL-2 and IFN-gamma, which activate CTLs. The Th2 subset may be more suited to respond to free-living bacteria and helminthic parasites and may mediate allergic reactions, since cytokines produced by Th2 cells such as IL-4, IL-5 and IL- 13 are known to induce IgE production and eosinophil activation, respectively. In general, ThI and Th2 cells secrete distinct patterns of cytokines and so one type of response can moderate the activity of the other type of response. A shift in the Thl/Th2 balance can result in an allergic response, for example, or, alternatively, in an increased CTL response.

[0007] It has been recognized for some time that a ThI -type immune response can be induced in mammals by administration of certain immunomodulatory polynucleotides. The immunomodulatory polynucleotides include sequences referred to as immunostimulatory sequences ('ISS'), often including a CG. See, e.g., PCT Publications WO 98/55495, WO 97/28259, U.S. Pat. Nos. 6,194,388 and 6,207,646; and Krieg et al. (1995) Nature 374:546- 49. For many infectious diseases, such as tuberculosis and malaria, Th2-type responses are of little protective value against infection. Protein-based vaccines typically induce Th2-type immune responses, characterized by high titers of neutralizing antibodies but without significant cell-mediated immunity. Moreover, some types of antibody responses are inappropriate in certain indications, most notably in allergy where an IgE antibody response can result in anaphylactic shock.

[0008] Chimeric immunomodulatory compounds (CICs) have been disclosed in the art. See, for example, U.S. Patent App. Pub. Nos. 2007/0049550 and 2009/055076. However, these previous CICs had different generic structures and different methods of synthesis. In addition, these previous methods were not conducive to scaling up to manufacture large quantities of such CICs without having to undergo tedious purification processes and which were concomitant with significant loss of product. In particular, multimeric CICs manufactured on a large scale may be more difficult to purify to state-of-the-art levels for drug development when synthesized by the previous methods.

[0009] Accordingly, there is a need for an improved method of making CICs that is relatively straightforward, high yielding and scaleable for manufacturing large quantities of such CICs without the need for difficult purification steps. In addition, methods for increasing the purity of multimeric CICs manufactured on a large scale are required to meet state-of-the-art or greater purity requirements, such as those required for drug development. The compositions and methods provided herein fulfill these needs and also further provide CICs, as well as platform compounds for the synthesis of CICs, for use in the modulation or regulation of an immune response.

BRIEF SUMMARY OF THE INVENTION

[0010] The invention provides for novel platform molecules, novel methods of making platform molecules, novel methods of making chimeric immunomodulatory compounds (CICs) from the platform molecules, and novel CICs. CICs, as referenced herein with respect to the present invention, contain one or more immunomodulatory sequences, such as immuno stimulatory sequences (ISS) or immunoregulatory sequences (IRS). For example, CICs of the present invention include chimeric immuno stimulatory compounds (CISCs) that include one or more immuno stimulatory sequences and chimeric immunoregulatory comopounds (CIRCs) that include one or more immunoregulatory sequences.

[0011] In another aspect, the present invention provides novel platform molecules, novel methods of making platform molecules, novel methods of making CICs from the platform molecules, and novel CICs, wherein preparations of said compounds, or preparations of compounds made according to said methods, are substantially pure after purification by conventional means. As used herein, a preparation of a compound of the present invention is substantially pure if the compound is at least 80% by weight to at least 99% by weight on an anhydrous basis (e.g.., after correction of the total weight of the preparation for water). In some embodiments, the preparation is substantially pure if non-conforming compounds are less than 20% by weight to less than 1% by weight on an anhydrous basis Such non- conforming compounds may refer to compounds that resulted from incomplete synthesis of the given compound or an intermediate thereof, or other side products that arise during the synthesis of the given compound, as described herein.

[0012] In one aspect the invention provides a novel method for synthesizing a platform molecule comprising the steps:

(a) obtaining a modified solid support having the structure (1)

SS FGG R₁ O APG₁

(b) removing the APGi group to obtain modified solid support (2) SS FFG — R₁ OH

(c) reacting the modified solid support with a phosphoramidite having the structure (3)

\

-o- -Rv -o- -APG_V

FL -N

Ry -

to obtain intermediate (4):

SS — FGG — R₁ O P O Ry O APGy

ZPPGy

(d) oxidizing or sulfurizing intermediate (4) to obtain intermediate (5):

Y

SS — FGG — R₁ O P O Ry O APGy

ZPPGy

(e) removing the APG_y group from intermediate (5) to obtain intermediate (6):

Y

SS — FGG — R₁ O P O Ry OH

ZPPGy

steps (c) to (e) may be performed m times, wherein m is an integer from 0 to 30, such as m : 0, 1, 2 or 3, with each APG_y, PPG_y, R_y, R_y' and R_y" chosen independently in each step, to obtain intermediate (7):

(f) reacting intermediate (7) with a phosphoramidite having the structure (8)

o obtain intermediate (9):

(g) oxidizing or sulfurizing intermediate (9) and removing the APG groups as in steps (d) and (e), respectively, to obtain intermediate (10):

(h) reacting intermediate (10) with a phosphoramidite having the structure (10-A) PPGz Z

P O Rz O APGz

Rz N

Rz'

to obtain intermediate (11):

(i) oxidizing or sulfurizing intermediate (11) and removing the APG_Z groups as in steps (d) and (e), respectively, to obtain intermediate (12):

steps (h) and (i) may be performed m times, wherein m is an integer from 0 to 30, such as m = 0, 1, 2 or 3, with each APG_Z, PPG_Z, R_z, R_z' and R_z" chosen independently in each step to obtain intermediate (13):

(]) reacting intermediate (13) with a phosphoramidite having the structure

PPG₂ Z

P O R₅ PMRG(PJ_n

Rc N

Rd

to obtain intermediate (14):

SS FGG R₁-J-O P O R_y 1-0 P O R₂ B

ZPPG_V / ZPPG₁

(k) oxidizing or sulfurizing intermediate (14) as in steps (d) to obtain intermediate (15):

and (1) deprotecting intermediate (15) and releasing it from the solid support to obtain the tri- arm platform formula (16):

wherein SS is a solid support; FGG is a functional group generator attached at one end to the solid support; FG is a functional group; R₁, R₂, R₃, R₄, Rs, R7, Ra, Rb, Rc, Rd, Rz, Rz', Rz-, Ry, R_y' and R_y >> are independently selected substituent groups; APGi, APG₂, APG₃, APG_Z and APG_y are acid-labile protecting groups; PPGi_, PPG₂, PPG_y and PPG_Z are phosphate protecting groups; BP is a branch point having three bonds, consisting of CR₇ or N; each PMRG is independently a platform molecule reactive group; Pr is a PMRG protecting group; n is 0 or 1; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S. In some aspects, suitable protecting groups PPGi PPG₂ PPG_y, PPG_Z and P_r and suitable functional group generator FGG may be selected to allow releasing from the solid support and deprotection of formula (15) to be performed as separated steps, instead of concurrently as in step (1). For example, the protecting groups may be selected to be orthogonal to the FGG. In some embodiments in which the protecting groups and the FGG have been selected to be mutually orthogonal, the deprotection of intermediate (15) is performed prior to release of the deprotected intermediate from the solid support.

[0013] In another aspect, the invention provides a novel tri-arm platform molecule having the structure (16):

wherein FG is a functional group; BP is a branch point having three bonds, consisting of CR₇ or N; R₁, R₂, R3, R₄, R5, R7, R_z and R_y, are independently selected substituent groups; each PMRG is independently a platform molecule reactive group; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S. In one embodiment, the platform molecule comprises at least one spacer. In another embodiment the platform molecule is symmetrical, wherein FG is the same as PMRG. In another embodiment, the platform molecule has one unique arm, wherein FG and PRMG are different.

[0014] In still another aspect, the invention provides a method for synthesizing a branched CIC comprising the steps:

(a) reacting a tri-arm platform molecule of formula (16):

with an oligonucleotide of formula (17):

ORG R₆ — N₂ "OH

to obtain formula (18):

wherein BP is a branch point having three bonds, consisting of CR7 or N; R₁, R₂, R3, R₄, R5, R_O, R₇, R_z and R_y are independently selected substituent groups; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; each Sp is independently a reaction product of PMRG and ORG or a reaction product of FG and ORG, wherein each

PMRG is independently a platform molecule reactive group, FG is a functional group, and each ORG is independently an oligonucleotide reactive group that can react with PMRG and FG; and each N_z is independently a linear oligonucleotide of z mer units, wherein each N is an independently selected nucleotide and each z is independently an integer from 1 to 30. In one embodiment, at least one branch is capable of immunomodulatory activity. In certain embodiments, the branched CIC optionally comprises at least one spacer. In other embodiments, the branched CIC comprises nucleic acid moieties wherein the nucleic acid moieties are each independently between 5- to 30-mers, between 6- to 12-mers or between 6- to 20-mers. In another embodiment, the branched CIC comprises nucleic acid moieties wherein at least one of the nucleic acid moieties is 6-mer or greater, 7- mer or greater, 8- mer or greater, 9- mer or greater, 10- mer or greater, 11- mer or greater, 12- mer or greater, 15- mer or greater, 20- mer or greater, 25- mer or greater or 30- mer or greater. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are each independently 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14- mers,15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers, 25-mers or 30-mers. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are 10-mers. In some embodiments, the branched CIC comprises both 7-mer and 10-mer nucleic acid moieties. In some embodiments, the branched CIC comprises two 7-mer nucleic acid moieties and one 10-mer nucleic acid moiety. In some embodiments, the branched CIC comprises two 10-mer nucleic acid moieties and one 7-mer nucleic acid moiety. In some embodiments, the branched CIC comprises only 7-mer nucleic acid moieties. In some embodiments, the branched CIC comprises only 10-mer nucleic acid moieties. In another embodiment, the branched CIC is symmetrical, wherein all the nucleic acid moieties are the same. In another embodiment, the branched CIC is asymmetrical, wherein all of the nucleic acid moieties are different from each other. In another embodiment, the branched CIC is asymmetrical, wherein two of the nucleic acid moieties are the same as each other, these two moieties being different from the third nucleic acid moiety.

[0015] In yet another aspect, the invention provides a branched tri-arm CIC wherein each branch comprises a nucleic acid moiety having the structure (18)

wherein BP is a branch point having three bonds, consisting of CR₇ or N; R₁, R₂, R3, R₄, R5, R_O, R₇, R_z and R_y are independently selected substituent groups; each PMRG is independently a platform molecule reactive group and PMRG = FG; each m is independently an integer from 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; each ORG is independently an oligonucleotide reactive group that can react with PMRG and FG; each Sp is independently the reaction product of PMRG and ORG or the reaction product of FG and ORG; and each N_z is a linear oligonucleotide of z mer units, wherein each N is an independently selected nucleotide and each z is independently an integer from 1 to 30. In one embodiment, at least one branch is capable of immunomodulatory activity. In one embodiment, the branched CIC optionally comprises at least one spacer. In another embodiment, the branched CIC comprises nucleic acid moieties wherein the nucleic acid moieties are each independently between 5- to 30-mers, between 6- to 12-mers or between 6- to 20-mers. In another embodiment, the branched CIC comprises nucleic acid moieties wherein at least one of the nucleic acid moieties is 6-mer or greater, 7- mer or greater, 8- mer or greater, 9- mer or greater, 10- mer or greater, 11- mer or greater, 12- mer or greater, 15- mer or greater, 20- mer or greater, 25- mer or greater or 30- mer or greater. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are each independently 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14- mers,15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers, 25-mers or 30-mers. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are 7-mers. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are 10-mers. In some embodiments, the branched CIC comprises both 7-mer and 10-mer nucleic acid moieties. In some embodiments, the branched CIC comprises two 10-mer nucleic acid moieties and one 7-mer nucleic acid moiety. In some embodiments, the branched CIC comprises only 7-mer nucleic acid moieties. In some embodiments, the branched CIC comprises only 10-mer nucleic acid moieties. In another embodiment, the branched CIC is symmetrical, wherein all the nucleic acid moieties are the same. In another embodiment, the branched CIC is asymmetrical, wherein all of the nucleic acid moieties are different from each other. In another embodiment, the branched CIC is asymmetrical, wherein two of the nucleic acid moieties are the same as each other, these two moieties being different from the third nucleic acid moiety.

[0016] In yet another aspect, a method is provided for synthesizing a tri-arm branched

CIC with one unique arm, comprising the steps of:

(a) activating a tri-arm platform molecule with one unique arm of formula (19):

R, — Oi — P O — Rz O- -o- -FU PMRG

HO — R₀-N_z— O-J— P O R_y O— j-P O R₂- BP_V

R_Λ — O — P O — Rz O- -o- -FU PMRG

by reacting (19), wherein each PMRG is independently a primary amine (-NH₂) or a secondary amine, with a heterobifunctional activator ALG-C(O)-Rx-W to obtain an activated platform formula (20):

, R",3 — O' -O — Rz- ^■°-π -R₅- -N R_x — W

H

HO-R_n-N₇-O-I-P O— R_y O-j-P O R₂-BP.

R₄ — Oj-P O — Rz O— J— P O R₅ N R_x — W

Z^"

(b) reacting the activated platform formula (20) with an oligonucleotide of formula (48):

ORG R₆ N'_z. — OH to obtain formula (20- A):

wherein ORG is an oligonucleotide reactive group; each Sp is independently the reaction product of W and ORG; R₀, R₁, R₂, R3, R₄, R5, R_O, R7, R_x, Ry and R_z are independently selected substitutent groups; BP is a branch point having three bonds, consisting of CR₇ or N; each PMRG is independently a platform molecule reactive group; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; each Nz and NV are independently linear oligonucleotides of z mer or z'-mer units, respectively, each N and N' is an independently selected nucleotide and each z and z' is independently selected integer from 1 to 30; and ALG-C(O)-Rx-W is a heterobifunctional activator, wherein ALG is the leaving group of an activated carboxylic acid, and W is an electrophilic group.

[0017] In certain aspects of the method, platform formula (19) is activated with a heterobifunctional activator, ALG-C(O)-Rx-W, wherein Rx is CH₂ and W is a halogen. In certain embodiments, W is chlorine.

[0018] In certain aspects of the method, ORG of oligonucleotide (48) is a thiol. For such aspects, an oligonucleotide having the thiol reactive group may be generated from the reduction of a disulfide precursor, e.g., HO - N'z - R₆ - S - S - R₆ - N'z - OH, or any other suitable precursor that generates the desired reactive oligonucleotide (48). In aspects where ORG is a thiol and W is a halogen, such as chlorine, Sp is a thioether (-S-).

[0019] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (20- A) include branched CIC formula (21 -A): 3^'

and exemplary embodiments of platform formula (19) include platform formula (21- B):

wherein in certain embodiments of CISCs of the present invention, each Ni is a first oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise or more rodent (e.g., rat or mouse) motifs, N₂ is independently a second oligonucleotide comprising one or more human and/or rodent (e.g., rat or mouse) motifs, HEG is hexaethylene glycol, and R₁, R₂, R₃, R₄ and R₅ are independently selected substituent groups. In certain embodiments of branched CIC formula (21 -A) and platform formula (21-B), Ri if present is poly₍i_i₂₎ethyleneglycol-OPSO₂ or (CH₂)i_₈-OPSO₂; R₂ is poly₍i-i₂₎ethyleneglycol or (CH₂)i_g; R₃ if present is poly₍i_i₂₎ethyleneglycol or (CH₂)i_g; R₄ if present is poly(i_i₂)ethyleneglycol-OPSO₂ or (CH₂)i_g-OPSO₂; R5 is poly(_1-12)ethyleneglycol- OPSO₂ or (CH₂) i_g-OPSO₂ In some embodiments, each oligonucleotide comprises phosphorothioate linkage. In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences.

[0020] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formulae (20- A) and (21 -A) include branched CIC formula (21):

and exemplary embodiments of platform formulae (19) and (21 -B) include platform formula (22):

wherein in certain embodiments of CISCs of the present invention, each Ni is a first oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, N₂ is independently a second oligonucleotide comprising one or more human and/or rodent (e.g., rat or mouse) motifs, HEG is hexaethylene glycol, and R₁, R₂, R₃ and R₄ are independently selected substituent groups. In certain embodiments of branched CIC formula (21) and platform formula (22), Ri if present is hexaethyleneglycol - OPSO₂, R₂ is (CH₂)₆ or (CH₂)₃, R₃ is CH₂CH₂OCH₂CH₂ or (CH₂)₃, R₄ if present is hexaethyleneglycol - OPSO₂ In some embodiments, each oligonucleotide comprises phosphorotioate linkages. In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences.

[0021] In certain embodiments of CISCs of the present invention, an oligonucleotide having a 'human motif generally includes one or more TCG trinucleotides. In certain preferred embodiments, the oligonucleotide having a human motif contains at least one additional CG dinucleotide. In certain preferred embodiments, the oligonucleotide having a human motif includes a TCG trinucleotide at the oligonucleotide's 5 '-end. [0022] In certain embodiments of CISCs of the present invention, a 'rodent motif,' such as a 'mouse motif generally includes the hexanucleotide sequence 5'- purine-purine-CG- pyrimidine-pyrimidine-3', where each purine is independently A or G (or other modified purines) and each pyrimidine is independently C and T (or other modified pyrimidines). Preferred embodiments of the rodent motif include, for example, AACGTT and GACGTT.

[0023] In certain aspects of the method and compounds, platform formula (22) for the synthesis of CICs of the present invention is activated with a heterobifunctional activator, ALG-C(O)-Rx-W, wherein Rx is CH₂ and W is a halogen, such as fluorine, chlorine, bromine or iodine. In certain embodiments, W is chlorine. The activated derivative of platform formula (22) is a example of activated platform formula (20), such as formula (22- A):

wherein in certain embodiments of platform molecules for the synthesis of CISCs of the present invention, N₂ is an oligonucleotide comprising one or more human and/or rodent (e.g., rat or mouse) motifs, HEG is hexaethylene glycol, and R₃, R₄ and R_x are independently selected substituent groups. In certain embodiments of activated branched CIC formula (22- A), R₃ is CH₂CH₂OCH₂CH₂ or (CH₂)₃, R₄ if present is hexaethyleneglycol - OPSO₂. In some embodiments, each oligonucleotide comprises phosphorotioate linkages, Rx is methylene (CH₂) and W is a halogen. In certain preferred embodiments, W is chlorine. In certain embodiments of platform molecules for the synthesis of CIRCs of the present invention, N₂ is an oligonucleotide comprising one or more immunoregulatory sequences.

[0024] In certain aspects of the method and compounds, activated platform formula (22-

A) can be reacted with an embodiment of oligonucleotide of formula (48), such as formula

(22-B):

5' N₁ OPSO₂-R₁ R₂ ORG to obtain embodiments of branched CIC formula (21), including formula (22-C):

wherein in certain embodiments of CISCs of the present invention, each Ni is a first oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e g , rat or mouse) motifs, N₂ is a second oligonucleotide comprising one or more human and/or rodent (e g rat or mouse) motifs, HEG is hexaethylene glycol, and R₁, R₂ R3, R₄ and R_x are independently selected substituent groups In certain embodiments of oligonucleotide (22-B) and branched CIC formula (22-C), Ri if present is hexaethyleneglycol - OPSO₂, R₂ is (CH₂)₆ or (CH₂)₃, R₃ is CH₂CH₂OCH₂CH₂ or (CH₂)3, R₄ if present is hexaethyleneglycol - OPSO₂ In some embodiments, each oligonucleotide comprises phosphorotioate linkages, ORG is a thiol, Rx is CH₂, W is a halogen, such as fluorine, chlorine, bromine or iodine, and Sp is thioether In certain embodiments, W is chlorine In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide compπsing one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences

[0025] In certain aspects of the described methods and compounds, exemplary embodiments of branched CISC (21), (21 -A) and (22-C), platform molecules having formulae (22), (21 -B) and (22-A) and oligonucleotide (22-B) include those in which the first oligonucleotide Ni is independently any one of the oligonucleotides listed in column 1 of Table A First oligonucleotide Ni may be selected independently of any other group such as second oligonucleotide N₂ and substituent groups ORG, W, R₁, R₂, R₃, R₄, R₅ and R_x

TABLE A (all sequences are listed 5' to 3')

Ni ID Ni Oligonucleotide Sequences N₂JD N? Oliεonucleotide Sequences

No No

Nl-I TCGTCGACTT (SEQ ID NO: 1) N2-1 TCGTCGACTT (SEQ ID NO 1)

Nl-2 TCGTCGAGAT (SEQ ID NO: 2) N2-2 TCGTCGAGAT (SEQ ID NO: 2)

[0026] In the present invention, embodiments of branched CISC molecule having formulae (21), (21 -A) and (22-C) and platform molecule having formulae (22), (21-B) and (22- A) may include those in which the second oligonucleotide N₂ is independently any one of the oligonucleotides listed in column 2 of Table A. Second oligonucleotide N₂ may be selected independently of any other group, such as first oligonucleotide Ni and substituent groups W, R_x, R₁, R₂, R₃, R₄ and R₅. Exemplary combinations of first oligonucleotide Ni and second nuceleotide N₂ in branched CIC molecules of the present invention are identified herein as 'C-N1-N2,' where Nl is the indentifying number from Table A of the first oligonucleotide Ni and N₂ is the identifying number from Table A of second nuceleotide N₂. Thus, for example, a branched CIC identified with 'C- 1-22' includes the first oligonucleotide Nl-I ( 5'- TCGTCGACTT -3') and second oligonucleotide N2-22 (5'- TAACGTTCGT -3').

[0027] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC molecules having formulae (21), (21-A) and (22-C) include formula (23):

3'

and exemplary embodiments of platform molecules having formulae (22), (21-B) and (22- A) include forumula (24):

3'

wherein in certain embodiments of CISCs of the present invention, each Ni is independently a first oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as those listed in column 1 of Table A, N₂ is independently a second oligonucleotide comprising one or more human and/or rodent (e.g., rat or mouse) motifs, such as those listed in column 2 of Table A, and HEG is hexaethylene glycol. In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences.

[0028] In certain aspects of the method and compounds, platform formula (24) for the synthesis of CICs of the present invention is activated with a heterobifunctional activator, ALG-C(O)-Rx-W, wherein Rx is CH₂ and W is a halogen, such as fluorine, chlorine, bromine or iodine. In certain embodiments, W is chlorine. The activated derivative of platform formula (24) is a example of activated platform molecule having formulae (20) and (22- A), such as formulae (24- A):

3'

wherein in certain embodiments of platform molecules for the synthesis of CISCs of the present invention, N₂ is an oligonucleotide comprising one or more humand and/or rodent (e.g., rat or mouse) motifs, such as those listed in column 2 of Table A, HEG is hexaethylene glycol, and R_x is an independently selected substituent group. In certain embodiments of activated branched platform formula (24- A), each oligonucleotide comprises phosphorotioate linkages, Rx is methylene (CH₂) and W is a halogen. In certain preferred embodiments, W is chlorine. In certain embodiments of platform molecules for the synthesis of CIRCs of the present invention, N₂ is an oligonucleotide comprising one or more immunoregulatory sequences.

[0029] In certain aspects of the method and compounds, activated platform molecule having formula (24- A) can be reacted with an example of oligonucleotide of formula (48) and (22-B), such as formula (24-B):

5' N ₁ O PSO₂ (CH₂)₆ ORG to obtain embodiments of branched CIC (21), including formula (22-D):

wherein in certain embodiments of CICs of the present invention, each Ni is independently a first oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as those listed in column 1 of Table A, N₂ is a second oligonucleotide comprising one or more human and/or rodent (e.g., rat or mouse) motifs, such as those listed in column 2 of Table A, HEG is hexaethylene glycol and each oligonucleotide comprises phosphorotioate linkages. In certain embodiments, ORG is a thiol, Rx is CH₂, W is a halogen, such as fluorine, chlorine, bromine or iodine, and Sp is thioether. In certain embodiments, W is chlorine. In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences.

[0030] In certain aspects of the described methods and compounds, exemplary embodiments of branched CIC (23) and (22-D), platform (24) and (24- A) and oligonucleotide (24-B) include those in which the first oligonucleotide Ni is independently any one of the oligonucleotides listed in column 1 of Table A. First oligonucleotide Ni may be selected independently of any other group, such as second oligonucleotide N₂ and substituent groups ORG, W, Ri, R₂, R₃, R₄ and R_x.

[0031] In the present invention, embodiments of branched CIC (21) and platform (22) may include those in which the second oligonucleotide N₂ is independently any one of the oligonucleotides listed in column 2 of Table A. Second oligonucleotide N₂ may be selected independently of any other group, such as first oligonucleotide Ni and substituent groups W, R_x, R₁, R₂, R₃ and R₄.

[0032] In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the first oligonucleotide Ni is Nl-I (5'- TCGTCGACTT -3') and the second oligonucleotide N₂ is any other suitable oligonucleotide, preferably a second oligonucleotide N₂ selected from Table A. In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the first oligonucleotide Ni is N 1-2 (5'- TCGTCG AG AT -3') and the second oligonucleotide N₂ is any other suitable oligonucleotide, preferably a second oligonucleotide N₂ selected from Table A. In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the first oligonucleotide Ni is N 1-3 (5'- TCGTGATCGT -3') and the second oligonucleotide N₂ is any other suitable oligonucleotide, preferably a second oligonucleotide N₂ selected from Table A. In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the first oligonucleotide Ni is N 1-4 (5'- TCGTTCGAAT -3') and the second oligonucleotide N₂ is any other suitable oligonucleotide, preferably a second oligonucleotide N₂ selected from Table A. In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the first oligonucleotide Ni is N 1-5 (5'- TCGTCGA -3') and the second oligonucleotide N₂ is any other suitable oligonucleotide, preferably a second oligonucleotide N₂ selected from Table A. In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the first oligonucleotide Ni is Nl- 19 (5'- TCGAACGTTT -3') and the second oligonucleotide N₂ is any other suitable oligonucleotide, preferably a second oligonucleotide N₂ selected from Table A. In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the first oligonucleotide Ni is N 1-20 (5'- TCGGACGTTT -3') and the second oligonucleotide N₂ is any other suitable oligonucleotide, preferably a second oligonucleotide N₂ selected from Table A.

[0033] In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the second oligonucleotide N₂ is N2-21 (5'- TGACGTTCGT -3') and the first oligonucleotide Ni is any other suitable oligonucleotide, preferably a first oligonucleotide Ni selected from Table A. In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the second oligonucleotide N₂ is N2-22 (5'- TAACGTTCGT -3') and the first oligonucleotide Ni is any other suitable oligonucleotide, preferably a first oligonucleotide Ni selected from Table A. In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the second oligonucleotide N₂ is N2- 23 (5'- AACGTTC -3') and the first oligonucleotide Ni is any other suitable oligonucleotide, preferably a first oligonucleotide Ni selected from Table A. In certain embodiments of the present invention, embodiments of branched CIC formula (21) may include those in which the second oligonucleotide N₂ is N2-24 (5'- GACGTTC -3') and the first oligonucleotide Ni is any other suitable oligonucleotide, preferably a first oligonucleotide Ni selected from Table A.

[0034] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (21) inlcude:

(5' Ni 3' OPSO₂ Ri R₂ S CH₂C(O)NH R₃ OPSO₂ R₄ CH₂)₂ CH OPSO₂ HEG OPSO₂-5'-N₂-3' such as depicted in the following exemplary formula (21 -C):

3'

and exemplary embodiments of the platform formula (22) include: (H₂N - R₃ -OPSO₂- R₄ -CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-N₂ -3' such as depicted in the following exemplary formula (22-E):

3'

wherein

HEG is hexaethylene glycol,

Ri is absent,

R₂ is (CH₂)₆ ,

R₃ is CH₂CH₂OCH₂CH₂,

R₄ is hexaethylene glycol-OPSO2, in certain embodiments of CICs of the present invention, Ni is selected from the group consisting of the oligonucleotides listed in column 1 of Table A, and N₂ is selected from the group consisting of the oligonucleotides listed in column 2 of Table A. In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[0035] Exemplary embodiments of branched CIC formula (21) of the present invention include the following compounds:

D-I: (5'-TCGTCGACTT-S'- OPSO₂- R_I - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3',

D-13: (5'- TCGTCGAGAT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TGACGTTCGT -3',

D-2: (5'- TCGTGATCGT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3',

D-14: (5'- TCGTGATCGT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TGACGTTCGT -3',

D-3: (5'- TCGTCGA-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3',

D-4: (5'- TCGTCGA-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3',

D-12: (5'- TCGTTCG-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3' and

D-I l: (5'- TCGTTCGAAT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3'; and exemplary embodiments of the platform (22) include the following compounds:

P-I: (H₂N- R₃ -OPSO₂- R₄ -CH₂)₂-CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3',

P-I l: (H₂N - R₃ -OPSO₂- R₄ -CH₂)^CH-OPSO₂-HEG-OPSO₂- 5'-TGACGTTCGT - 3'

P-3: (H₂N - R₃ -OPSO₂- R₄ -CH₂)^CH-OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3' and

P- 12: (H₂N - R₃ -OPSO₂- R₄ -CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-GACGTTC -3' wherein

HEG is hexaethylene glycol,

Ri is absent,

R₂ is (CH₂)₆ ,

R₃ is CH₂CH₂OCH₂CH₂, and

R₄ is hexaethylene glycol-OPSO₂. In some embodiments, each oligonucleotide comprises phosphorothioate linkages. [0036] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (21) include:

(5'-Ni-3'- OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R4 -CH2)₂-CH-OPSθ2-HEG- OPSO₂-5'-N₂-3' such as depicted in the following formula (21-D):

-3'

and exemplary embodiments of platform (22) include:

(H₂N - R₃ -OPSO₂- R₄ -CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-N₂ -3' such as depicted in the following formula (22-F):

-3'

wherein

HEG is hexaethylene glycol, Ri is absent, R₂ is (CH₂)₆ , R₃ is CH₂CH₂OCH₂CH₂ , R₄ is absent, and

in certain embodiments of CISCs of the present invention, Ni is selected from the group consisting of the oligonucleotides listed in column 1 of Table A, and N₂ is selected from the group consisting of the oligonucleotides listed in column 2 of Table A. In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[0037] Exemplary embodiments of branched CIC formula (21) of the present invention include the following compounds:

D-5: (5'-TCGTCGACTT-S'- OPSO₂- R_I - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3',

D-8: (5'-TCGTCGA-S'- OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)^CH- OPSO₂-HEG-OPSO₂-5'-AACGTTC-3' and

D-15: (5'- TCGTGATCGT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3'; and exemplary embodiments of the platform formula (22) include the following exemplary compounds:

P-2: (H₂N - R₃ -OPSO₂- R₄ -CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3' and

P-4: (H₂N - R₃ -OPSO₂- R₄ -CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3'; wherein:

HEG is hexaethylene glycol,

Ri is absent,

R₂ is (CH₂)₆ ,

R₃ is CH₂CH₂OCH₂CH₂ , and R₄ is absent. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[0038] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (21) include:

(5'-Ni-3'- OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)^CH-OPSO₂-HEG- OPSO₂-5'-N₂-3' such as depicted in the following formula (21-E): N₂ -3'

and embodiments of platform formula (22) include:

(H₂N - R₃ -OPSO₂- R₄ -CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-N₂ -3' such as the following exemplary platform formula (22-G):

-3'

wherein

HEG is hexaethylene glycol, Ri is absent, R₂ is (CH₂)₃ , R₃ is CH₂CH₂OCH₂CH₂ , and R₄ is hexaethylene glycol -OPSO₂. and

[0039] Exemplary embodiments of branched CIC formula (21) of the present invention include the following compounds:

D-6: (5'-TCGTCGACTT-S'- OPSO₂- R_I - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3', D-9: (5'-TCGTCGA-S'- OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CHJ₂-CH- OPSO₂-HEG-OPSO₂-5'-AACGTTC-3' and

D-16: (5'- TCGTGATCGT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5' TAACGTTCGT -3'; and exemplary embodiments of the platform (22) include the following exemplary compounds:

P-I: (H₂N - R₃ -OPSO₂- R^CHJrCH-OPSOrHEG-OPSO^S'-TAACGTTCGT^' and

P-3- (H₂N - R₃ -OPSO₂- R₄ -CH₂)₂-CH-OPSO₂-HEG-OPSO₂-5'-AACGTTC-3' wherein:

HEG is hexaethylene glycol,

Ri is absent,

R₂ is (CH₂)₃ ,

R₃ is CH₂CH₂OCH₂CH₂ , and R₄ is hexaethylene glycol -OPSO₂ In some embodiments, each oligonucleotide comprises phosphorothioate linkages

[0040] In certain aspects of the described methods and compounds, exemplary embodiments of branched CIC formula (21) include:

(5'-Ni-3'- OPSO₂- Ri - R₂-S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CHJ₂-CH-OPSO₂-HEG- OPSO₂-5'-N₂-3' such as depicted in the following formula (21 -F):

— N₂ 3

and exemplary embodiments of the branched formula (22) include- (H₂N - R₃ -OPSO₂- R₄ -CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-N₂ -3' such as the following exemplary platform formula (22-H): CH₂CH₂OCH₂CH₂ H₂N OPSO₂ HEG — OPSO₂ CH₂

HC OPSO₂ HEG OPSO₂ N₂ -3'

.CH₂CH₂OCH₂CH₂ /

/ --. /

H₂N OPSO₂ HEG — OPSO₂ CH₂ wherein

HEG is hexaethylene glycol, Ri is hexaethylene glycol-OPSO2, R₂ is (CH₂)₃ , R₃ is CH₂CH₂OCH₂CH₂ , and R₄ is hexaethylene glycol -OPSO₂. and

in certain embodiments of CIRCs of the present invention, Ni is selected from the group consisting of the oligonucleotides listed in column 1 of Table A, and N₂ is selected from the group consisting of the oligonucleotides listed in column 2 of Table A. In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[0041] Exemplary embodiments of branched CIC formula (21) of the present invention include the following compounds:

D-7: (5'-TCGTCGACTT-S'- OPSO₂- RI - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)2-CH-OPSO₂-HEG-OPSθ2-5'-TAACGTTCGT-3',

D-IO: (5'-TCGTCGA-S'- OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂-5'-AACGTTC-3' and

D-17: (5'- TCGTGATCGT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂VCH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3'; and exemplary embodiments of the platform formula (22) include the following exemplary compounds:

P-I: (H₂N - R₃ -OPSO₂- R₄ -CH₂)2-CH-OPSO₂-HEG-OPSθ2-5'-TAACGTTCGT-3' and

P-3: (H₂N - R₃ -OPSO₂- R₄ -CH₂)2-CH-OPSO₂-HEG-OPSθ2-5'-AACGTTC-3' and wherein:

HEG is hexaethylene glycol,

Ri is hexaethylene glycol-OPSO2,

R₂ is (CH₂)₃ ,

R₃ is CH₂CH₂OCH₂CH₂ , and R₄ is hexaethylene glycol-OPSO₂. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[0042] In yet another aspect, the invention provides a novel method for synthesizing a symmetrical tri-arm branched oligonucleotide comprises the steps of:

(a) activating a platform molecule of formula (16):

by reacting (16), wherein each PMRG and FG are both a primary amine (-NH₂) or both a secondary amine, with a heterobifunctional activator, ALG-C(O)-Rx-W to obtain an activated platform formula (25):

(b) reacting the activated platform formula (25) with an oligonucleotide of formula (17):

ORG R₆ — N_z OH

to obtain formula (26):

wherein ORG is an oligonucleotide reactive group; each Sp is independently the reaction product of a W and an ORG; BP is a branch point having three bonds, consisting of CR₇ or N; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; Ri, R₂, R₃, R₄, R₅, Re, R₇, R_x, R_y and R_z are substitutent groups; Nz is a linear oligonucleotide of z mer units, and each N is an independently selected nucleotide and each z is an independently selected integer from 1 to 30; and ALG-C(O)-Rx-W is a heterobifunctional activator, wherein ALG is the leaving group of an activated carboxylic acid, and W is an electrophilic group.

[0043] In certain aspects of the method, platform formula (16) includes one or more substituents that each comprises a suitable chromophoric and/or fluorophoric moiety. For example, the chromophore- and/or fluorophore-containing substituent can be at one or more of Ri, R₂, R₃, R₄, R5, R₇, R_z and R_y in formula (16). Such moieties may allow improved detection and purification of formula (16) and its precursors, particularly when the platform molecule does not contain other significant chromophores or fluorophores, such as oligonucleotides. Examples of suitable chromophoric and/or fluorophoric substituents include natural and non-natural nucleosides, such as adenosine, thymidine, cytidine, guanosine and other suitable bases known in the art. Such nucleosides may be ribonucleosides, 2'-deoxyribonucleosides, or other suitable sugars or modified versions thereof known in the art. One or more of such nucleosides can be incorporated by use of suitable phosphoramidite precursors as shown herein, and as are known in the art.

[0044] In certain aspects of the method, the platform formula (16) is activated with a heterobifunctional activator, ALG-C(O)-Rx-W, to yield activated platform formula (25), wherein Rx is CH₂ and W is a halogen. In certain embodiments, W is chlorine.

[0045] In certain aspects of the method, ORG of oligonucleotide (17) is a thiol. For such aspects, an oligonucleotide having the thiol reactive group may be generated from the reduction of a disulfide precursor, e.g., HO - Nz - Re - S - S - Re - Nz - OH , or any other suitable precursor that generates the desired reactive oligonucleotide (17). In aspects where ORG is a thiol and W is a halogen, such as chlorine, Sp is a thioether (-S-).

[0046] In certain aspects of the method and compounds, exemplary embodiments of symmetrical tri-arm platform formula (16) are defined by formula (25-A):

wherein each R₃ if present is independently poly(i_i2)ethyleneglycol-OPSO2 or (CH₂)_1-8 - OPSO₂, each R₄ if present is poly(i_i2)ethyleneglycol-OPSO₂, each R₅ if present is independently 5'- ribo- or deoxyribonucleoside-3' or 3'- ribo- or deoxyribonucleoside-5' , which can be activated with a heterobifunctional activator, ALG-C(O)-CH₂-Cl, wherein ALG is the leaving group of an activated carboxylic acid, to obtain an exemplary embodiment of activated platform formula (25), as defined by formula (25-B):

H CIH₂C(O)C N R₃ R₄ R₅-OPSO₂

H

-OPSO₉ FU R_d R, -N- -C(O)CH₂CI

H CIH₂C(O)C N R₃ R₄ R₅-OPSO₂-

which can be reacted with an exemplary embodiment of activated oligonucleotide (17) , as defined by formula (25-C):

to obtain an exemplary embodiment of CIC formula (26), as defined by formula (25-D): 5'

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

wherein in certain embodiments of CISCs of the present invention, each Ni is independently an oligonucleotide comprising one or more of human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as the oligonucleotides listed in column 1 of Table A, each Ri if present is independently poly₍i_i₂₎ethyleneglycol - OPSO₂, each R₂ if present is independently (CH₂)i_g or poly₍i_i₂₎ethyleneglycol, each R₃ if present is independently poly₍i_i₂₎ethyleneglycol-OPSO₂ or (CH₂)i_g - OPSO₂, each R₄ if present is poly₍i_i₂₎ethyleneglycol-OPSO₂, and each R₅ if present is independently 5'- ribo- or deoxyribonucleoside-3' or 3'- ribo- or deoxyribonucleoside-5' . In certain embodiments of CIRCs of the present invention, each Ni is an oligonucleotide comprising one or more immunoregulatory sequences. In some preferred embodiments, all Ni are the same oligonucleotide moiety. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[0047] In certain aspects of the method and compounds, exemplary embodiments of symmetrical tri-arm platform formula (25-A) are defined by:

H₂N-CH₂CH₂OCH₂CH₂ — OPSO₂-H EG-OPSO₂ — T-OPSO₂-V y — OPSO₂ — T — OPSO₂-HEG-OPSO₂ — (CH₂)₃— NH₂

H₂N-CH₂CH₂OCH₂CH₂ — OPSO₂-H EG-OPSO₂ — T-OPSO₂-/

and branched CIC formula (25-D): 5'

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

wherein each T is independently 5'- thymidine-3' or 3'- thymidine -5' and HEG is hexaethyleneglycol, and wherein in certain embodiments of CISCs of the present invention, each Ni is independently an oligonucleotide comprising one or more of human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as such as the oligonucleotides listed in column 1 of Table A. In some preferred embodiments of CICs, all Ni are the same oligonucleotide moiety.

[0048] In another aspect, the invention provides a branched tetra arm CIC having the structure (27)

and branched tetra arm platform formula (28):

wherein BP is a branch point having four bonds, consisting ofC; R₁, R₂, R3, R₄, R5, Re, R_z and R_y are independently selected substituent groups; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; p is the reaction product of PMRG and ORG and FG and ORG, wherein each PMRG is independently a platform molecule reactive group, FG is a functional group, and each ORG is independently an oligonucleotide reactive group that can react with PMRG and FG; and each N_z is independently a linear oligonucleotide of z mer units, wherein each N is an independently selected nucleotide and each z is independently 1 to 30. In certain embodiments, at least one branch of formula (27) comprises a nucleic acid moiety that is capable of immunomodulatory activity. In another embodiment, the branched tetra arm CIC or branched tetra arm platform molecule optionally comprises at least one spacer.

[0049] In any embodiment of a CIC, CISC or CIRC of the present invention described herein, the oligonucleotides Ni and N₂ contained therein may be the same oligonucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 depicts a synthesis scheme for a symmetrical Tri Arm platform molecule.

[0051] Figure 2 depicts a synthesis scheme for a Tri Arm symmetrical conjugation.

[0052] Figure 3 depicts a synthesis scheme for a Tri Arm platform molecule with one unique branch of polynucleotides. [0053] Figure 4 depicts a synthesis scheme for a Tri Arm single unique branch conjugation.

[0054] Figure 5 depicts a synthesis scheme for a Tri Arm platform molecule with all unique branches of polynucleotides.

[0055] Figure 6 depicts a synthesis scheme for a Tri Arm conjugation with all unique branches of polynucleotides.

[0056] Figure 7 depicts a synthesis scheme for a symmetrical Tetra Arm platform molecule.

[0057] Figure 8 depicts a synthesis scheme for the conjugation of a symmetrical Tetra Arm platform molecule.

[0058] Figure 9 depicts a synthesis scheme for a Tetra Arm platform molecule with one unique branch of polynucleotides.

[0059] Figure 10 depicts a synthesis scheme for the conjugation of a Tetra Arm platform molecule with one unique arm.

[0060] Figure 11 depicts a synthesis scheme for a Tetra Arm platform molecule with two unique branches of polynucleotides.

[0061] Figure 12 depicts a synthesis scheme for the conjugation of a Tetra Arm platform molecule with two unique branches of polynucleotides.

[0062] Figure 13 depicts a synthesis scheme for a Tetra Arm platform molecule with all unique branches of polynucleotides.

[0063] Figure 14 depicts a synthesis scheme for the conjugation of a Tetra Arm with all unique branches of polynucleotides.

[0064] Figure 15 depicts a synthesis scheme for a Click platform molecule with one unique branch of polynucleotides.

[0065] Figure 16 depicts a synthesis scheme for the conjugation of a Click platform molecule. [0066] Figure 17 depicts a synthesis scheme for a Click hexavalent platform molecule.

[0067] Figure 18 depicts a synthesis scheme for the conjugation of a Click hexavalent platform molecule.

[0068] Figure 19 depicts an exemplary reverse -phase HPLC chromatogram of a purified branched CIC molecule synthesized in accordance with the present invention.

[0069] Figure 20 depicts an exemplary reverse -phase HPLC chromatogram of a purified branched CIC molecule synthesized by the previously described stepwise synthesis route.

[0070] Figures 21-24 depict exemplary anion exchange HPLC chromatograms of reactions performed in accordance with the present invention.

[0071] Figure 25 depicts the induction of IFN-alpha by human peripheral blood mononuclear cells (PBMCs) in the presence of immunostimulatory nucleic acids and a branched CIC of the present invention.

[0072] Figure 26 shows data of IFN-alpha induction (EC50 and IFN-alpha maximum) by human PBMC in the presence of immunostimulatory nucleic acid sequences and branched CIC of the present invention.

[0073] Figure 27 shows data of B cell proliferation (EC50 and proliferation maximum values) of immunostimulatory nucleic acid sequences and branched CIC of the present invention.

[0074] Figures 28-31 show data of IFN-alpha induction by human PBMCs in the presence of immunostimulatory nucleic acids and a branched CIC of the present invention.

[0075] Figure 32 shows data of expression of maturation markers by plasmacytoid dendritic cells in the presence of immunostimulatory nucleic acid sequences and branched CICs of the present invention.

[0076] Figure 33 shows data of IL-6 induction in mouse splenocytes in the presence of immunostimulatory nucleic acid sequences and branched CICs of the present invention.

[0077] Figure 34 shows data of IL-10 induction in PBMC in the presence of immunostimulatory nucleic acid sequences and branched CICs of the present invention. DETAILED DESCRIPTION OF THE INVENTION

/. General Methods

[0078] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly and individually referred to herein as 'Sambrook'). Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Handbook of Experimental Immunology (D. M. Weir & CC. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller & M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); The Immunoassay Handbook (D. Wild, ed., Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996); Methods of Immunological Analysis (R. Masseyeff, W.H. Albert, and N.A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993), Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, and Harlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (jointly and individually referred to herein as 'Harlow and Lane'), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000); and Agrawal, ed., Protocols for Oligonucleotides and Analogs, Synthesis and Properties Humana Press Inc., New Jersey, 1993).

//. Definitions

[0079] As used herein, the singular form 'a', 'an', and 'the' includes plural references unless otherwise indicated or clear from context. For example, as will be apparent from context, 'a' chimeric immunomodulatory compound ('CIC) can include one or more CICs. Likewise, 'a' chimeric immunoregulatory compound ('CIRC) or 'a' chimeric immuno stimulatory compound ('CISC') can include one or more CIRCs or CISCs, respectively. Similarly, reference in the singular form of a component element of a CIC (i.e., nucleic acid moiety or non-nucleic acid spacer moiety) can include multiple elements. For example, a description of 'a nucleic acid moiety' in a CIC can also describe two or more 'nucleic acid moieties' in the CIC.

[0080] As used interchangeably herein, the terms 'polynucleotide,' 'oligonucleotide',

'nucleic acid' and 'nucleic acid moiety' include single- stranded DNA (ssDNA), double- stranded DNA (dsDNA), single- stranded RNA (ssRNA) and double- stranded RNA (dsRNA), modified oligonucleotides and oligonucleosides, or combinations thereof. The nucleic acid can be linearly or circularly configured, or the oligonucleotide can contain both linear and circular segments. Nucleic acids are polymers of nucleosides joined, e.g., through phosphodiester linkages or alternate linkages, such as phosphorothioate esters. A nucleoside consists of a purine (adenine (A) or guanine (G) or derivative thereof) or pyrimidine (thymine (T), cytosine (C) or uracil (U), or derivative thereof) base bonded to a sugar. The four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxy guano sine, deoxythymidine, and deoxy cytidine. A nucleotide is a phosphate ester of a nucleoside.

[0081] The term '3" generally refers to a region or position in a polynucleotide or oligonucleotide 3' (downstream) from another region or position in the same polynucleotide or oligonucleotide.

[0082] The term '5" generally refers to a region or position in a polynucleotide or oligonucleotide 5' (upstream) from another region or position in the same polynucleotide or oligonucleotide.

[0083] An element, e.g., region, portion, non-nucleic acid spacer moiety, nucleic acid moiety, or sequence is 'adjacent' to another element, e.g., region, portion, non-nucleic acid spacer moiety, nucleic acid moiety, or sequence, when it directly abuts that region, portion, spacer or sequence.

[0084] The term "branch point" refers to a moiety in a CIC having 3 or 4 bonds to which other moieties can be or are attached. Suitable exemplary branch points include, for example, substituted or unsubstituted carbon, nitrogen, silicon, and phosphorous.

[0085] The term "optionally" as used herein, such as in "optionally includes X",

"optionally X", "optionally comprises X", wherein X is any element, is intended to refer to include embodiments in which X is optional, as well as embodiments in which X is expressly included.

[0086] The term 'CIC-antigen conjugate' refers to a complex in which a CIC and an antigen are linked. Such conjugate linkages include covalent and/or non-covalent linkages.

[0087] The term 'antigen' means a substance that is recognized and bound specifically by an antibody or by a T cell antigen receptor. Antigens can include peptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids; portions thereof and combinations thereof. The antigens can be those found in nature or can be synthetic. Antigens suitable for administration with a CIC includes any molecule capable of eliciting a B cell or T cell antigen- specific response. Preferably, antigens elicit an antibody response specific for the antigen. Haptens are included within the scope of 'antigen.' A hapten is a low molecular weight compound that is not immunogenic by itself but is rendered immunogenic when conjugated with an immunogenic molecule containing antigenic determinants. Small molecules may need to be haptenized in order to be rendered antigenic. Preferably, antigens of the present invention include peptides, lipids (e.g. sterols, fatty acids, and phospholipids), polysaccharides such as those used in Hemophilus influenza vaccines, gangliosides and glycoproteins.

[0088] 'Adjuvant' refers to a substance which, when added to an immunogenic agent such as antigen, nonspecifically enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture.

[0089] The term 'peptide' are polypeptides that are of sufficient length and composition to effect a biological response, e.g., antibody production or cytokine activity whether or not the peptide is a hapten. Typically, the peptides are at least six amino acid residues in length. The term 'peptide' further includes modified amino acids (whether or not naturally or non- naturally occurring), such modifications including, but not limited to, phosphorylation, glycosylation, pegylation, lipidization and methylation.

[0090] 'Antigenic peptides' can include purified native peptides, synthetic peptides, recombinant peptides, crude peptide extracts, or peptides in a partially purified or unpurified active state (such as peptides that are part of attenuated or inactivated viruses, cells, microorganisms), or fragments of such peptides. An 'antigenic peptide' or 'antigen polypeptide' accordingly means all or a portion of a polypeptide which exhibits one or more antigenic properties. Thus, for example, an 'Amb a 1 antigenic polypeptide' or 'Amb a 1 polypeptide antigen' is an amino acid sequence from Amb a 1, whether the entire sequence, a portion of the sequence, and/or a modification of the sequence, which exhibits an antigenic property (i.e., binds specifically to an antibody or a T cell receptor).

[0091] A 'delivery molecule' or 'delivery vehicle' is a chemical moiety which facilitates, permits, and/or enhances delivery of a CIC or CIC-antigen mixture, or CIC- antigen conjugate to a particular site and/or with respect to particular timing. A delivery vehicle may or may not additionally stimulate an immune response.

[0092] An 'allergic response to antigen' means an immune response generally characterized by the generation of eosinophils (usually in the lung) and/or antigen- specific IgE and their resultant effects. As is well-known in the art, IgE binds to IgE receptors on mast cells and basophils. Upon later exposure to the antigen recognized by the IgE, the antigen cross-links the IgE on the mast cells and basophils causing degranulation of these cells, including, but not limited, to histamine release. It is understood and intended that the terms 'allergic response to antigen', 'allergy', and 'allergic condition' are equally appropriate for application of some of the methods of the invention. Further, it is understood and intended that the methods of the invention include those that are equally appropriate for prevention of an allergic response as well as treating a pre-existing allergic condition.

[0093] As used herein, the term 'allergen' means an antigen or antigenic portion of a molecule, usually a protein, which elicits an allergic response upon exposure to a subject. Typically the subject is allergic to the allergen as indicated, for instance, by the wheal and flare test or any method known in the art. A molecule is said to be an allergen even if only a small subset of subjects exhibit an allergic (e.g., IgE) immune response upon exposure to the molecule. A number of isolated allergens are known in the art. These include, but are not limited to, those provided in Table 7 herein.

[0094] The term 'desensitization' refers to the process of the administration of increasing doses of an allergen to which the subject has demonstrated sensitivity. Examples of allergen doses used for desensitization are known in the art, see, for example, Fornadley (1998) Otolaryngol. Clin. North Am. 31:111-127. [0095] 'Antigen- specific immunotherapy' refers to any form of immunotherapy which involves antigen and generates an antigen-specific modulation of the immune response. In the allergy context, antigen- specific immunotherapy includes, but is not limited to, desensitization therapy.

[0096] The term 'microcarrier' refers to a particulate composition which is insoluble in water and which has a size of less than about 150, 120 or 100 μm, more commonly less than about 50-60 μm, and may be less than about 10 μm or even less than about 5 μm. Microcarriers include 'nanocarriers', which are microcarriers have a size of less than about 1 μm, preferably less than about 500 nm. Microcarriers include solid phase particles such a particles formed from biocompatible naturally occurring polymers, synthetic polymers or synthetic copolymers, although microcarriers formed from agarose or cross-linked agarose may be included or excluded from the definition of microcarriers herein as well as other biodegradable materials known in the art. Solid phase microcarriers are formed from polymers or other materials which are non-erodible and/or non-degradable under mammalian physiological conditions, such as polystyrene, polypropylene, silica, ceramic, polyacrylamide, gold, latex, hydroxyapatite, and ferromagnetic and paramagnetic materials. Biodegradable solid phase microcarriers may be formed from polymers which are degradable (e.g., poly(lactic acid), poly(glycolic acid) and copolymers thereof, such as poly(D, L-lactide- co-glycolide) or erodible (e.g., poly(ortho esters such as 3,9-diethylidene-2,4,8,10- tetraoxaspiro[5.5] undecane (DETOSU) or poly(anhydrides), such as poly(anhydrides) of sebacic acid) under mammalian physiological conditions. Microcarriers are typically spherical in shape, but microcarriers which deviate from spherical shape are also acceptable (e.g., ellipsoidal, rod-shaped, etc.). Due to their insoluble nature, solid phase microcarriers are filterable from water and water-based (aqueous) solutions (e.g., using a 0.2 micron filter). Microcarriers may also be liquid phase (e.g., oil or lipid based), such as liposomes, iscoms (immune- stimulating complexes, which are stable complexes of cholesterol, phospholipid and adjuvant-active saponin) without antigen, or droplets or micelles found in oil-in- water or water- in-oil emulsions. Biodegradable liquid phase microcarriers typically incorporate a biodegradable oil, a number of which are known in the art, including squalene and vegetable oils. The term 'nonbiodegradable', as used herein, refers to a microcarrier which is not degraded or eroded under normal mammalian physiological conditions. Generally, a microcarrier is considered nonbiodegradable if it not degraded (i.e., loses less than 5% of its mass or average polymer length) after a 72 hour incubation at 37° C in normal human serum.

[0097] A microcarrier is considered 'biodegradable' if it is degradable or erodable under normal mammalian physiological conditions. Generally, a microcarrier is considered biodegradable if it is degraded (i.e., loses at least 5% of its mass or average polymer length) after a 72 hour incubation at 37° C in normal human serum.

[0098] The term 'CIC/microcarrier complex' or 'CIC/MC complex' refers to a complex of a CIC and a microcarrier. The components of the complex may be covalently or non- covalently linked. Non-covalent linkages may be mediated by any non-covalent bonding force, including by hydrophobic interaction, ionic (electrostatic) bonding, hydrogen bonds and/or van der Waals attractions. In the case of hydrophobic linkages, the linkage is generally via a hydrophobic moiety (e.g., cholesterol) covalently linked to the CIC.

[0099] An 'individual' or 'subject' is a vertebrate, such as avian, preferably a mammal, such as a human. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, experimental animals, rodents (e.g., mice and rats) and pets.

[00100] An 'effective amount' or a 'sufficient amount' of a substance is that amount sufficient to effect a desired biological effect, such as beneficial results, including clinical results, and, as such, an 'effective amount' depends upon the context in which it is being applied. In the context of administering a composition that modulates an immune response to a co-administered antigen, an effective amount of a CIC and antigen is an amount sufficient to achieve such a modulation as compared to the immune response obtained when the antigen is administered alone. In the context of administering a composition that suppresses a TLR9 dependent immune response, an effective amount of a CIRC is an amount sufficient to inhibit or decrease a cellular response to stimulation through TLR9. In the context of administering a composition that suppresses a TLR7 dependent immune response, an effective amount of an CIRC is an amount sufficient to inhibit or decrease a cellular response to stimulation through TLR7. An effective amount can be administered in one or more administrations.

[00101] The term 'co-administration' as used herein refers to the administration of at least two different substances sufficiently close in time to modulate an immune response. Preferably, co-administration refers to simultaneous administration of at least two different substances.

[00102] 'Stimulation' of a response or parameter includes eliciting and/or enhancing that response or parameter. For example, "stimulation" of an immune response, such as innate immune response or ThI response, means an increase in the response, which can arise from eliciting and/or enhancement of a response. Similarly, "stimulation" of a cytokine or cell type (such as CTLs) means an increase in the amount or level of cytokine or cell type. B cell "stimulation" includes, for example, enhanced B cell proliferation, induced B cell activation and/or increased production of cytokines, such as IL-6 and/or TNF-α, from the stimulated B cell.

[00103] The term "immuno stimulatory nucleic acid" or "immuno stimulatory polynucleotide" as used herein refers to a nucleic acid molecule (e.g., polynucleotide) that effects and/or contributes to a measurable immune response as measured in vitro, in vivo and/or ex vivo. Examples of measurable immune responses include, but are not limited to, antigen- specific antibody production, secretion of cytokines, activation or expansion of lymphocyte populations such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, and the like. Immunostimulatory nucleic acid (ISNA) sequences are known to stimulate innate immune responses, in particular, those responses that occur through TLR-9 signaling in the cell. As known in the art, immunostimulatory nucleic acid (ISNA) molecules can be isolated from microbial sources, such as bacteria, can be present in nucleic acid vectors for use in gene therapy, or can be synthesized using techniques and equipment described herein and known in the art. Generally, an immunostimulatory nucleic acid sequence includes at least one CG dinucleotide, with the C of this dinucleotide being unmethylated. Accordingly, microbial infection and administered DNA can in some cases result in stimulation of innate immune responses.

[00104] The term "immunostimulatory" or "stimulating an immune response" as used herein includes stimulation of cell types that participate in immune reactions and enhancement of an immune response to a specific antigenic substance. An immune response that is stimulated by an immunostimulatory nucleic acid is generally a "ThI -type" immune response, as opposed to a "Th2-type" immune response. ThI -type immune responses are normally characterized by "delayed-type hypersensitivity" reactions to an antigen and activated macrophage function and can be detected at the biochemical level by increased levels of ThI -associated cytokines such as IFN-γ, IL-2, IL-12, and TNF-β. Th2-type immune responses are generally associated with high levels of antibody production, especially IgE antibody production and enhanced eosinophils numbers and activation, as well as expression of Th2-associated cytokines such as IL-4, IL-5 and IL- 13.

[00105] The term "innate immune response" or "innate immunity" as used herein includes a variety of innate resistance mechanisms by which a cell or individual recognizes and responds to the presence of a pathogen. As used herein, an "innate immune response" includes the intracellular and intercellular events and reactions that occur when the cell recognizes pathogen associated molecular patterns or signals. Cellular receptors active in an innate immune response include a family of Toll-like receptors (TLRs) and microbial ligands have been identified for several TLRs, as described herein.

[00106] The term "immunoregulatory sequence" or "IRS", as used herein, refers to a nucleic acid sequence that inhibits and/or suppresses a measurable innate immune response as measured in vitro, in vivo and/or ex vivo. The term "immunoregulatory sequence" or "IRS", as used herein, refers to both nucleic acid sequences that comprise a modification (i.e., modified IRS) as well as nucleic acids which do not comprise a modification (i.e., unmodified IRS).

[00107] The term "chimeric immunoregulatory compound" or "CIRC", as used herein, refers to a molecule which has immunoregulatory activity and which comprises one or more nucleic acid moieties and one or more non-nucleic acid moieties. The nucleic acid moieties in a CIRC with more than one nucleic acid moiety may be the same or different. The non- nucleic acid moieties in a CIRC with more than one non-nucleic acid moiety may be the same or different. Thus, in one embodiment the CIRC comprises two or more nucleic acid moieties and one or more non-nucleic acid spacer moieties, where at least one non-nucleic acid spacer moiety is covalently joined to two nucleic acid moieties refers to a molecule which has immunoregulatory activity and which comprises a nucleic acid moiety comprising an IRS. A CIRC of the present invention preferably inhibits and/or suppresses a measurable innate immune response as measured in vitro, in vivo and/or ex vivo. Inhibition of a TLR, e.g., TLR-7 or 9, includes without limitation inhibition at the receptor site, e.g., by blocking ligand - receptor binding, and inhibition of the downstream signal pathway after ligand - receptor binding. Examples of measurable innate immune responses include, but are not limited to, secretion of cytokines, activation or expansion of lymphocyte populations such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, maturation of cell populations such as plasmacytoid dendritic cells and the like.

[00108] The term "modified chimeric immunoregulatory compound" or "modified CIRC", as used herein, refers to a molecule which has immunoregulatory activity and which comprises a nucleic acid moiety comprising at least one modified IRS. The modified CIRC may consist of a nucleic acid moiety that comprises more than one modified IRS, comprises one or more modified IRS and one or more unmodified IRS, consists of a modified IRS, or has no immunostimulatory activity on its own. The modified CIRC may consist of a polynucleotide (a "modified polynucleotide CIRC") or it may comprise additional moieties. Accordingly, the term modified IRC includes compounds which incorporate one or more nucleic acid moieties, at least one of which comprises a modified CIRC, covalently linked to a non-nucleotide spacer moiety.

[00109] The term "unmodified immunoregulatory sequence" or "unmodified IRS" as used herein refers to a nucleic acid sequence consisting of no modifications (i.e. absent of modifications) of the nucleic acid sequence, that alone or contained in a CIRC inhibits and/or suppresses a measurable innate immune response as measured in vitro, in vivo and/or ex vivo. Inhibition of a TLR, e.g., TLR-7 or 9, includes without limitation inhibition at the receptor site, e.g., by blocking ligand - receptor binding, and inhibition of the downstream signal pathway after ligand - receptor binding. Examples of measurable innate immune responses include, but are not limited to, secretion of cytokines, activation or expansion of lymphocyte populations such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, maturation of cell populations such as plasmacytoid dendritic cells and the like.

[00110] An 'IgE associated disorder' is a physiological condition which is characterized, in part, by elevated IgE levels, which may or may not be persistent. IgE associated disorders include, but are not limited to, allergy and allergic reactions, allergy-related disorders (described below), asthma, rhinitis, atopic dermatitis, conjunctivitis, urticaria, shock, Hymenoptera sting allergies, food allergies, and drug allergies, and parasite infections. The term also includes related manifestations of these disorders. Generally, IgE in such disorders is antigen- specific. In some cases, multiple allergies can occur in an individual, and thus IgE can be specific for multiple antigens in such multi-allergy disorders.

[00111] An 'allergy-related disorder' means a disorder resulting from the effects of an antigen- specific IgE immune response. Such effects can include, but are not limited to, hypotension and shock. Anaphylaxis is an example of an allergy-related disorder during which histamine released into the circulation causes vasodilation as well as increased permeability of the capillaries with resultant marked loss of plasma from the circulation. Anaphylaxis can occur systemically, with the associated effects experienced over the entire body, and it can occur locally, with the reaction limited to a specific target tissue or organ.

[00112] The term 'viral disease', as used herein, refers to a disease which has a virus as its etiologic agent. Examples of viral diseases include hepatitis B, hepatitis C, influenza, acquired immunodeficiency syndrome (AIDS), and herpes zoster.

[00113] As used herein, and as well-understood in the art, 'treatment' is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. 'Treatment' can also mean prolonging survival as compared to expected survival if not receiving treatment.

[00114] 'Palliating' a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder. Especially in the allergy context, as is well understood by those skilled in the art, palliation may occur upon modulation of the immune response against an allergen(s). Further, palliation does not necessarily occur by administration of one dose, but often occurs upon administration of a series of doses. Thus, an amount sufficient to palliate a response or disorder may be administered in one or more administrations. [00115] An 'antibody titer', or 'amount of antibody', which is 'elicited' by a CIC and antigen refers to the amount of a given antibody measured at a time point after administration of the CIC and antigen.

[00116] A 'ThI -associated antibody' is an antibody whose production and/or increase is associated with a ThI immune response. For example, IgG2a is a Thl-associated antibody in the mouse. For purposes of this invention, measurement of a Thl-associated antibody can be measurement of one or more such antibodies. For example, in humans, measurement of a Thl-associated antibody could entail measurement of IgGl and/or IgG3.

[00117] A Th2-associated antibody' is an antibody whose production and/or increase is associated with a Th2 immune response. For example, IgGl is a Th2-associated antibody in the mouse. For purposes of this invention, measurement of a Th2-associated antibody can be measurement of one or more such antibodies. For example, in human, measurement of a Th2-associated antibody could entail measurement of IgG2 and/or IgG4.

[00118] To 'suppress' or 'inhibit' a function or activity, such as cytokine production, antibody production, or histamine release, is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, a composition comprising a CIC and antigen which suppresses histamine release reduces histamine release as compared to, for example, histamine release induced by antigen alone. As another example, a composition comprising a CIC and antigen which suppresses antibody production reduces extent and/or levels of antibody as compared to, for example, extent and/or levels of antibody produced by antigen alone. As another example, a composition comprising a CIC reduces Th2 cytokine productions, such as the production of one or more of IL-4, IL-5 and/or IL- 13. The reduction of such Th2 cytokines by CICs may be useful in the treatment of allergies and/or asthma. In another example, a composition comprising a CIRC which suppresses immuno stimulatory nucleic acid induced cytokine production reduces cytokine production as compared to, for example, cytokine production induced by the immuno stimulatory nucleic acid alone. As another example, a composition comprising a CIRC which suppresses cytokine production associated with an innate immune response reduces the extent and/or levels of cytokine production as compared to, for example, extent and/or levels of cytokine produced by the innate immune response alone. B cell "suppression" includes, for example, reduced B cell proliferation, reduced B cell activation and/or reduced production of cytokines, such as IL-6 and/or TNF-α, from the stimulated B cell. Inhibition of a TLR response, e.g., a TLR7 or 9 response, includes, but is not limited to, inhibition at the receptor site, e.g., by preventing or blocking effective ligand - receptor binding, and inhibition of the downstream signal pathway, e.g., after effective ligand - receptor binding.

[00119] As used herein manufactured or formulated 'under GMP standards,' when referring to a pharmaceutical composition means the composition is formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[00120] As used herein, the term 'immunogenic' has the normal meaning in the art and refers to an agent (e.g., polypeptide) that elicits an adaptive immune response upon injection into a person or animal. The immune response may be B cell (humoral) and/or T cell (cellular).

[00121] All ranges are intended to be inclusive of the terminal values. Thus, a polymer of 'from 2 to 7 nucleotides' or 'between 2 and 7 nucleotides' includes polymers of 2 nucleotides and polymers of 7 nucleotides. Where a lower limit and an independently selected upper limit are described, it is understood that the upper limit is higher than the lower limit. In addition, all numerical ranges of integers are intended to included every integer in the range, including the terminal values. For example, a range of integers from 0 to 30 includes the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29 and 30.

[00122] As used herein, the term 'substantially pure' in respect to a given compound of the present invention is intended to mean a preparation of the compound that includes at least 80% to at least 99% of the compound by weight on an anhydrous basis (e.g., after correction of the total weight for water content, as described below). As used herein, the term 'compound' refers to a structurally-defined product, in which the defined product includes, for example, particular oligonucleotide sequence(s), spacer(s) and backbone configuration. In some embodiments, a preparation of a compound of the present invention that is substantially pure is at least 85% pure by weight, at least 86% pure by weight, at least 87% pure by weight, at least 88% pure by weight, at least 89% pure by weight, at least 90% pure by weight, at least 91% pure by weight, at least 92% pure by weight, at least 93% pure by weight, at least 94% pure by weight, at least 95% pure by weight, at least 96% pure by weight, at least 97% pure by weight, at least 98% pure by weight or at least 99% pure by weight on an anhydrous basis (e.g., after correction of the total weight for water content). The total weight is preferably corrected for water content because CICs and oligonucleotides isolated by lyophilization often contain high, and variable, levels of water, e.g., 3-20%. The water content can be determined on a weight percent basis by known methods, such as Karl Fischer analysis (U.S. Pharmacopoeia, vol. 23, 1995, method 921, U. S. P. Pharmacopeial Convention, Inc., Rockville, MD, USA). For instance, if 100 mg (total weight) of material were weighed and the water content was determined to be 10%, then the total weight corrected for the water content would be 90 mg (100 mg x (100- 10)/ 100). In this example, a compound with a purity of 90% by weight would contain 81 mg (90 mg x 90/100) of the defined compound on an anhydrous basis (e.g., after correction of the total weight for the water content).

[00123] The purity of the compound on an area percent basis can be determined, for instance, by a HPLC method that resolves the compound from the compound-related impurities (e.g., non-conforming compounds) on a chromatography column and uses detection at a suitable characteristic wavelength where the compound absorbs light, e.g., at 260 nm. See Example 25 for an exemplary suitable HPLC method. As the response factor (area counts per weight) of the compound and compound-related impurities are highly similar, the area percent result can be taken as the weight percent result. For instance, if the area percent purity by HPLC is 90% and the total weight after correction for water content is 90 mg, then 81 mg of the defined compound would be present in the sample (90 mg x 90/100).

[00124] In another aspect, a given compound of the present invention that is 'substantially pure' is intended to mean that a preparation of the compound is substantially free of non-conforming compound. As used herein, a 'non-conforming compound' of a given compound differs from the given compound with respect to one or more of the following exemplary characteristics: one or more of the compound's oligonucleotide sequences, one or more of the compound's spacers, the compound's backbone configuration, or any other stable attribute of the compound. Such non-conforming compounds may result from incomplete synthesis of the given compound, or other side products that arise during the synthesis of the given compound. For example, for compounds of the present invention that include phosphorothioate oligonucleotides, typical non-conforming compounds include, for example, deletions in one or more of the oligonucleotides (e.g., n-1, n-2, etc.) in which the non-conforming compound is missing one or more nucleotide -phosphorothioate groups with respect to the defined compound; PO defects, in which the non-conforming compound contains one or more phosphodiester backbone linkages instead of a phosphorothioate linkage as in the compound; hydrophobic modifications, in which the non-conforming compound contains one or more hydrophobic modifications, such as cyanoethyl, acetyl, t- butyl, etc., which are not present in the compound; additions in one or more of the oligonucleotides (e.g., n+1, n+2, etc.), in which the non-conforming compound contains one or more extra nucleotide-phosphorothioate groups than the compound; depurination, in which the non-conforming compound is missing one or more bases in at least one of the defined oligonucleotide sequences of the compound; depurination cleavage products, in which the non-conforming compound comprises or is missing one or more fragments of the compound that have resulted from cleavage at a depurination site. Accordingly, in some embodiments, a preparation of a compound of the present invention that is substantially pure includes less than 20% non-conforming compounds by weight, less than 15% non-conforming compounds by weight, less than 14% non-conforming compounds by weight, less than 13% non- conforming compounds by weight, less than 12% non-conforming compounds by weight, less than 11% non-conforming compounds by weight, less than 10% non-conforming compounds by weight, less than 9% non-conforming compounds by weight, less than 8% non-conforming compounds by weight, less than 7% non-conforming compounds by weight, less than 6% non-conforming compounds by weight, less than 5% non-conforming compounds by weight, less than 4% non-conforming compounds by weight, less than 3% by weight non-conforming compounds by weight, less than 2% by weight non-conforming compounds by weight or less than 1% by weight non-conforming compounds by weight on an anhydrous basis (e.g., after correction of the total weight for the water content). The respective weights of the compound and non-conforming compounds can be determined as described herein.

///. Chimeric Immunomodulatory Compounds

[00125] The invention provides chimeric immunomodulatory compounds ('CICs') useful, inter alia, for modulating an immune response in individuals such as mammals, including humans. CICs of the present invention also provides chimeric immunoregulatory compounds ('CIRCs') useful, inter alia, for regulating an innate immune response in individuals such as mammals, including humans. The CICs of the present invention also provides chimeric immuno stimulatory compounds ('CISCs') useful, inter alia, for stimulating an immune response in individuals such as mammals, including humans. The invention provides novel methods of heterogeneous synthesis of multivalent CICs using platform based molecules. The invention also provides compositions comprising such CICs. Thus, the invention provides reagents and methods for modulating or regulating an immune response, including treatment and prophylaxis of disease in humans and other animals.

[00126] The following sections describe the structure and properties of the CICs of the invention, as well as the structure and properties of the component nucleic acid moieties and non-nucleic acid spacer moieties. In all cases where variables are used more than once, each instance of the variable is independently selected unless otherwise noted. Choices for variables may be found at the section beginning at paragraph [0185] unless otherwise noted.

A. Branched Tri-Arm Platform Molecules

[00127] In one aspect, the invention provides compositions comprising branched platform molecules and methods for synthesizing them. These platform molecules are useful for synthesizing branched or multivalent CICs, such as CICs with three (or more) arms or branches. The invention is, in part, the synthetic pathway that allows for one of skill in the art to synthesize these platform molecules. As shown in the Examples and the accompanying Figures, both Tri Arm (i.e., three arms) and Tetra Arm (i.e., four arms) platform molecules can be made in relatively few steps instead of many (e.g., 30) discreet coupling steps which can lead to impurities which are difficult to remove from the final product.

[00128] For these tri-arm platform molecules, one of skill in the art can make them either symmetrically or asymmetrically. The use of the term 'symmetrical' with respect to the Tri Arm platform molecule means that the termini or reactive groups at the end of the three arms are identical. It then follows that the use of: (a) the term 'one unique arm' with respect to the Tri-Arm platform means that the termini or reactive groups of two of the three arms are identical and the terminus or reactive group of the third arm is unique; and (b) the term 'asymmetrical' with respect to the Tri-Arm platform molecule means that the three termini or reactive groups all differ in structure. In some embodiments, a symmetrical Tri Arm platform molecule that has spacer groups has the same three termini or reactive groups, but the spacer groups between the branch point and the termini or reactive groups are not the same. In some embodiments, a symmetrical Tri Arm platform molecule has the same three termini or reactive groups and the same spacer groups between the branch point and the termini or reactive groups.

[00129] Tri Arm platform molecules may also be synthesized such that they have one unique arm or all unique arms comprising a first polynucleotide sequence attached to the unique terminus or reactive group and the remaining termini or reactive groups are available for additional arms to be grafted on to introduce a second or a third polynucleotide sequence on the second and third arms, respectively. See, for example, Figs. 3-6 and also Examples 3- 6.

[00130] One method for synthesizing a symmetrical Tri- Arm platform molecule comprises the following steps:

(a) obtaining a modified solid support having the structure (1)

SS FGG R₁ O APG₁

(b) removing the APGi group to obtain modified solid support (2)

SS FFG — R₁ OH

to obtain intermediate (4):

SS — FGG — R₁ O P O Ry O APGy

ZPPGy (d) oxidizing or sulfurizing intermediate (4) to obtain intermediate (5):

Y

SS — FGG — R -o- -o- -Ry O- -APGy

ZPPGy

(e) removing the APG_y group from intermediate (5) to obtain intermediate (6):

Y

SS — FGG — R₁ O P O Ry OH

ZPPGy

steps (c) to (e) may be performed m times, wherein m is an integer from 0 to 30, such as m = 0, 1, 2 or 3, with each APGY_y, PPG_y, R_y, R_y' and R_y" chosen independently in each step, to obtain intermediate (7):

(f) reacting intermediate (7) with a phosphoramidite having the structure (8)

to obtain intermediate (9):

(h) reacting intermediate (10) with a phosphoramidite having the structure (10- A)

PPGz-

-O Rz O APGz

/

Rz"" -N M

Rz' o obtain intermediate (11):

ZPPGz

steps (h) and (i) may be performed m times, wherein m is an integer from 0 to 30, such as m = 0, 1, 2 or 3, with each APG_Zi PPG_Z, R_z, R_z' and R_z' ' chosen independently in each step to obtain intermediate (13):

(]) reacting intermediate (13) with a phosphoramidite having the structure (13-A):

to obtain intermediate (14):

and

(1) deprotecting intermediate (15) and releasing it from the solid support to obtain the tri- arm platform formula (16):

wherein SS is a solid support; FGG is a functional group generator attached at one end to the solid support; FG is a functional group; BP is a branch point having three bonds, consisting of CR₇ or N; Ri, R₂, R₃, R₄, R₅, R₇, Ra, Rb, Rc, Rd, Rz, R_z>, Rz-, R_y, Ry and R_y ^» are independently selected substituent groups; APGi, APG₂, APG₃, APG_Z and APG_y are acid- labile protecting groups; PPGi_, PPG₂, PPG_y and PPG_y are phosphate protecting groups; PMRG is a platform molecule reactive group; Pr is a PMRG protecting group; n is 0 or 1; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S. These variables are defined further herein. In some aspects, suitable protecting groups PPGi PPG₂ PPG_y, PPG_z and P_r and suitable functional group generator FGG may be selected to allow releasing from the solid support and deprotection of formula (15) to be performed as separated steps, instead of concurrently as in step (1). For example, the protecting groups may be selected to be orthogonal to the FGG. In some embodiments in which the protecting groups and the FGG have been selected to be mutually orthogonal, the deprotection of intermediate (15) is performed prior to release of the deprotected intermediate from the solid support. [00131] Another method for synthesizing a tri-arm platform molecule with one unique arm comprising the steps:

(a) obtaining a solid support containing a first protected nucleoside (PN), having the structure (29)

SS R₀ (PN)₁ — O APG₁

(b) removing the APGi group to obtain modified solid support (30)

SS R₀ (PN)₁ — OH

(c) reacting the solid support containing (PN)₁ with a protected nucleoside phosphoramidite having the structure (31)

to obtain intermediate (32)

(d) oxidizing or sulfurizing intermediate (32) to obtain intermediate (33):

(e) removing the APG₂ group from intermediate (33) to obtain intermediate (34)

steps (c) to (e) may be performed z-1 times, wherein z is an integer from 1 to 30, with each B, PPG2, Ra and Rb chosen independently in each step, to obtain intermediate (35):

SS R₀ (PN)_Z — OH thereby (PN)₂ is a linear oligonucleotide of z-mer units, (f) reacting intermediate (35) with a phosphoramidite having the structure (3)

to obtain intermediate (36):

SS R₀ — (PN)_Z O P O R_y O APGy

ZPPGy

(g) oxidizing or sulfurizing intermediate (36) to obtain intermediate (37):

Y

SS R₀ — (PN)_Z O P O Ry O APGy

ZPPGy (h) removing the APG_y group from intermediate (37) to obtain intermediate (38): Y

SS R₀ — (PN)_Z O P O R_y OH

ZPPGy steps (f) to (h) may be performed m times, wherein m is an integer from 0 to 30, such as m = 0, 1, 2 or 3, with each PPG_y, R_y, R_y' and R_y" chosen independently in each step, to obtain intermediate (39)

(i) reacting intermediate (39) with a phosphoramidite having the structure (40)

PP

to obtain intermediate (41):

(j) oxidizing or sulfurizing intermediate (41) and removing the APG groups as in steps (g) and (h), respectively, to obtain intermediate (42):

(k) reacting intermediate (42) with a phosphoramidite having the structure (10- A)

to obtain intermediate (43)

(1) oxidizing or sulfurizing intermediate (43) and removing the APG_Z groups as in steps (g) and (h), respectively, to obtain intermediate (44):

steps (k) to (1) may be performed m times, wherein m is an integer from 0 to 30, such as m = 0, 1, 2 or 3, with each PPG_Z, R_z, R_z' and R_z" chosen independently in each step, to obtain intermediate (45)

(m) reacting intermediate (45) with a phosphoramidite having the structure (45-A) PPG

to obtain intermediate (46):

(n) oxidizing or sulfurizing intermediate (46) as in step (g) to obtain intermediate

(47):

and

(o) deprotecting intermediate (47) and releasing it from the solid support to obtain the tri-arm platform molecule with one unique arm that already includes a nucleic acid moiety (19):

wherein (PN) is a protected nucleotide or protected nucleoside analog; (PN)z is a protected linear oligonucleotide of z mer units, wherein each z is independently an integer from 1 to 30, and each (PN) is an independently selected protected nucleotide; Nz is a linear oligonucleotide of z mer units, wherein each z is independently an integer from 1 to 30, and each N is an independently selected nucleotide; B is a heterocyclic base, such as adenine, guanine, cytosine, thymine, uracil, or analogs thereofand may be in its protected form during synthesis; SS is a solid support; FGG is a functional group generator attached at one end to the solid support; FG is a functional group; BP is a branch point having three bonds, consisting of CR₇ or N; Ri, R₂, R₃, R₄, Rs, R₇, R_a, Rb, R_c, Rd, Re, Rf, Rz, Rz', Rz-, R_y, Ry' and R_y" are independently selected substituent groups; APGi, APG₂, APG₃, APG₄, APG_Z and APG_y are acid-labile protecting groups; PPGi₁ PPG₂, PPG_y and PPG_y are phosphate protecting groups; PMRG is a platform molecule reactive group; Pr is a PMRG protecting group; n is 0 or 1; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S. These variables are defined further herein. In some aspects, suitable protecting groups PPGi PPG₂ PPG_y, PPG_Z and P_r and suitable functional group generator FGG may be selected to allow releasing from the solid support and deprotection of formula (15) to be performed as separated steps, instead of concurrently as in step (1). For example, the protecting groups may be selected to be orthogonal to the FGG. In some embodiments in which the protecting groups and the FGG have been selected to be mutually orthogonal, the deprotection of intermediate (15) is performed prior to release of the deprotected intermediate from the solid support.

[00132] Another method for synthesizing a tri-arm platform molecule with all unique arms comprising the steps:

(a) obtaining a solid support containing a first protected nucleoside(PN), having the structure (29): SS R₀ (PN)₁ — O APG₁

(b) removing the APGi group to obtain modified solid support (30)

SS R₀ (PN)₁ — OH

to obtain intermediate (32)

(d) oxidizing or sulfurizing intermediate (32) to obtain intermediate (33):

steps (c) to (e) may be performed z-1 times, wherein z is an integer from 1 to 30, with each B, APG₂, PPG₂, Ra and Rbchosen independently in each step, to obtain intermediate

(35):

SS R₀ (PN)_Z — OH thereby (PN)₂ is a linear oligonucleotide of z-mer units, wherein z is an integer from 1 to 30,

(f) reacting intermediate (35) with a phosphoramidite having the structure (3)

to obtain intermediate (36):

SS R₀ — (PN)_Z O P O Ry O APGy

ZPPGy

(g) oxidizing or sulfurizing intermediate (36) to obtain intermediate (37):

Y

SS R₀ — (PN)_Z O P O Ry O APGy

SS R₀ — (PN)_Z O P O R_y OH

(i) reacting intermediate (39) with a phosphoramidite having the structure (49)

to obtain intermediate (50):

(j) oxidizing or sulfurizing intermediate (50) and selectively removing the APG₃ group as in steps (g) and (h), respectively, to obtain intermediate (51):

R₃ OH

SS R₀ — (PN)zH-O P O Ry — HO P O R₂ BP

ZPPGy ZPPG₃ R₄ O BPGi

(k) reacting intermediate (51) with a phosphoramidite having the structure (10- A) PPGz-

\

-O Rz O APGz

Rz"- -N \

Rz' to obtain intermediate (52)

R₀ — (PN)₇-Ho P O R_y — HO P O R₂ BP

ZPPGy ZPPG₃

R₄ O BPG₁

(1) oxidizing or sulfurizing intermediate (52) and selectively removing the APG_Z group as in steps (g) and (h), respectively, to obtain intermediate (53):

SS R₀ — (PN)z- -o- ^■o- -R₂ BP.

ZPPGy ZPPG₃

R₄ O BPG₁ steps (k) to (1) may be performed m times, wherein m is an integer from 0 to 30, such as m = 0, 1, 2 or 3, with each PPG_y, R_y, R_y' and R_y" chosen independently in each step, to obtain intermediate (54):

(m) reacting intermediate (54) with a phosphoramidite having the structure (54- A) PPG₄ Z

P O R₅ PMRG₁(Pr)_n

R, -N

\

Rd to obtain intermediate (55):

/S₃-J-O P O R_z O — J-P O R₅ PMRG₁(P₁

^ZPPGz / ZPPG₄

SS R₀- (PN)_Z{-O P O R_y O "J— P O R₂ — BP_N

ZPPGy / ZPPG₃

™ R₄ — O BPG₁

(n) oxidizing or sulfurizing intermediate (55) as in step (g) to obtain intermediate (56):

(o) selectively removing the BPGi group by treatment with a suitable base on intermediate (56) to obtain intermediate (57):

(p) reacting intermediate (57) with a phosphoramidite having the structure (10- A) PPGz-

^"\

P O Rz O APGz

Rz N

Rz' to obtain intermediate (58):

Y R₃ "ho P O — R_z — J-O P O R₅ PMRGi(Pr)_n

^ZPPQ; _m ZPPG₄ R₀- (PN)z-f-O P O — R Iyy--JJ--OC P O R₂-BP_N

ZPPG₃

R₄ — O — P O — R_z O APGz

ZPPGz

(q) oxidizing or sulfurizing intermediate (58) and removing the APG_y group as in steps (g) and (h), respectively, to obtain intermediate (59):

steps (p) to (q) may be performed m times, wherein m is an integer from 0 to 30, such as m = 0, 1, 2 or 3, with each PPG_y, R_y, Ry and R_y" chosen independently in each step, to obtain intermediate (60):

(r) reacting intermediate (61) with a phosphoramidite having the structure (61-A)

to obtain intermediate (62)

(s) oxidizing or sulfurizing intermediate (62) as in step (g) to obtain intermediate (63):

and

(t) deprotecting intermediate (63) and releasing from the solid support to obtain the tri- arm platform molecule with all unique arms (64):

wherein SS is a solid support; (PN) is a protected nucleoside or protected nucleoside analog; (PN)z is a protected linear oligonucleotide of z mer units, and each (PN) is an independently selected protected nucleotide; Nz is a linear oligonucleotide of z mer units, and each N is an independently selected nucleotide, and each z is independently an integer from 1 to 30; B is a heterocyclic base, such as adenine, guanine, cytosine, thymine, uracil and analogs thereof and may be in its protected form during synthesis; R₁, R₂, R₃, R₄, R5, Re, R7, R_z, Rz', R_Z", Ry, Ry', R_y", R_a, R_b, R_c and R_d are independently selected substituent groups; APGi, APG₂, APG₃, APGz and APG_y are acid-labile protecting groups; PPGi, PPG₂, PPG₃, PPG₄, PPG₅, PPG_y and PPG_z are phosphate protecting groups; BPGi is a base-labile protecting group; BP is a branch point having three bonds consisting of CR₇ or N groups; PMRGi and PMRG₂ are orthogonal platform molecule reactive groups; each Pr is an independently selected PMRG protecting group; n is 0 or 1; each m is independently an integer from 0 to 30, such as m = 0, 1, 2 or 3; and each Y and Z is independently O or S. In some aspects, suitable protecting groups P, PPGi PPG₂ PPG₃ PPG₄ PPG₅ PPG_y, PPG_Z and P_r and the attachment to the solid support SS may be selected to allow releasing from the solid support and deprotection of formula (63) to be performed as separated steps, instead of concurrently as in step (t). For example, the protecting groups may be selected to be orthogonal to conditions that release the intermediate from the solid support. In some embodiments in which the protecting groups and the solid support have been selected to be mutually orthogonal, the deprotection of intermediate (63) is performed prior to release of the deprotected intermediate from the solid support.

[00133] A Tri-Arm platform molecule has the structure (16):

wherein FG is a functional group; BP is a branch point having three bonds, consisting of CR₇ or N; R₁, R₂, R₃, R₄, R₅, R₇ and R_y, are independently selected substituent groups; PMRG is a platform molecule reactive group; each m is independently an integer from 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S. [00134] A Tri-Arm platform molecule with one unique arm has the structure (19):

wherein BP is a branch point having three bonds, consisting of CR₇ or N; Ro, Ri, R₂, R3, R₄, R₅, R₇, R_z and R_y, are independently selected substituent groups; Nz is a linear oligonucleotide of z mer units, z is an integer from 1 to 30, and each N is an independently selected nucleotide; each PMRG is independently a platform molecule reactive group; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S.

[00135] A Tri-Arm platform molecule with all unique arms has the structure (64):

wherein BP is a branch point having three bonds, consisting of CR₇ or N; Ro, Ri, R₂, R3, R₄, R₅, Re, R₇, R_z and R_y, are independently selected substituent groups; Nz is a linear oligonucleotide of z mer units, z is an integer froml to 30, and each N is an independently selected nucleotide; PMRGi and PMRG₂ are orthogonal platform molecule reactive groups; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S.

B. Tetra Arm Platform Molecules [00136] Platform molecules with four arms (i.e., Tetra-Arm platform molecules) are also provided herein. Tetra-Arm platform molecules allow for both symmetrical as well as asymmetrical synthesis as exemplified in Examples 7-14. Symmetrical Tetra Arm platform molecules can be synthesized in as few as two steps and can be used for conjugation without rigorous purification steps. The conjugation of a polynucleotide to the symmetrical Tetra Arm platform molecule results in a CIC that has four branches comprising the same polynucleotide sequence.

[00137] Tetra Arm platform molecules can be made where there is one, two, three or four unique termini. Alternatively, Tetra Arm platform molecules can be made with two distinct sets of two matching termini.

[00138] A tetra-arm platform molecule has the formula (28):

[00139] A tetra-arm platform molecule with one unique arm that already includes a nucleic acid moiety has the formula (66):

wherein, for formulae (28) and (66), Ro, R₁, R₂, R₃, R₄, Rs, RO, R7, Rz and R_y, are independently selected substituent groups; each Nz and Nz' is a linear oligonucleotide of z mer units, each z is an independently selected integer from 1 to 30, and each N is an independently selected nucleotide; PMRG is a platform molecule reactive group; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; and each Y and Z is independently O or S.

C. TriArm Branched CICs

[00140] In one aspect, the invention provides compositions and methods for synthesizing branched CICs, including CISCs and CIRCs with three arms or branches. The invention is, in part, the conjugation methodology that allows for one of skill in the art to synthesize branched CISCs and CIRCs, such as those with three arms or four arms, in a manner that allows for exact control over each sequence in a branch. As shown in the Examples and the accompanying Figures, both Tri-Arm and Tetra Arm CICs can be made from the corresponding platform molecules in relatively few steps instead of many discreet coupling steps which can lead to impurities which are difficult to remove from the final product. Use of smaller branch and platform compounds that can be purified prior to their conjugation to form the CIC leads to CICs of higher purity than those made by a stepwise procedure.

[00141] In one embodiment, at least one branch of a CIC of the present invention is capable of immunomodulatory activity. In one embodiment, at least one branch of a CIRC of the present invention is capable of immunomregulatory activity. In one embodiment, at least one branch of a CIsC of the present invention is capable of immuno stimulatory activity. In one embodiment, the branched CIC optionally comprises at least one spacer. In another embodiment, the branched CIC comprises nucleic acid moieties wherein the nucleic acid moieties are each independently between 5- to 30-mers, between 6- to 12-mers, or between 6- to 20-mers. In another embodiment, the branched CIC comprises nucleic acid moieties wherein at least one of the nucleic acid moieties is 6-mer or greater, 7- mer or greater, 8- mer or greater, 9- mer or greater, 10- mer or greater, 11- mer or greater, 12- mer or greater, 15- mer or greater, 20- mer or greater, 25- mer or greater or 30- mer or greater. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are each independently 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14- mers,15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers, 25-mers or 30-mers. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are 10-mers. In some embodiments, the branched CIC comprises both 7-mer and 10-mer nucleic acid moieties. In some embodiments, the branched CIC comprises two 7-mer nucleic acid moieties and one 10-mer nucleic acid moiety. In some embodiments, the branched CIC comprises two 10-mer nucleic acid moieties and one 7-mer nucleic acid moiety. In some embodiments, the branched CIC comprises only 7-mer nucleic acid moieties. In some embodiments, the branched CIC comprises only 10-mer nucleic acid moieties. Tri-Arm CICs can be made by one of skill in the art either symmetrically or asymmetrically from the appropriate Tri-Arm platform molecule. The use of the term 'symmetrical' with respect to the Tri-Arm CIC means that the Tri-Arm CIC is made from the symmetrical Tri-Arm platform molecule which contains the same three termini or reactive groups and comprises the same polynucleotide sequence, e,g,, 5'-XXXXXXX-3' on all three arms, as exemplified in Examples 1 and 2 and seen in Figs. 1 and 2. It then follows that the use of the terms 'with one unique arm' or 'asymmetrical' with respect to the Tri Arm CIC means that the CIC can be made from the corresponding asymmetrical Tri-Arm platform molecule. In some embodiments, a symmetrical Tri Arm platform molecule that includes spacer groups has the same three termini or reactive groups, but the spacer groups between the branch point and the termini are not the same. In some embodiments, a symmetrical Tri Arm platform molecule has the same three termini or reactive groups and the same spacer groups between the branch point and the termini or reactive groups.

[00142] Tri Arm CICs may also be synthesized such that the platform molecule precursor has one unique arm comprising a first polynucleotide sequence and then additional arms are grafted (conjugated) on to introduce a second or a third polynucleotide sequence on the second and third arms, respectively. See, for example, Figs. 3-6 and also Examples 3-6. 1. Methods of Making Tri Ann Branched CICs

[00143] A method of making a symmetrical tri-arm branched oligonucleotide comprises the steps of:

(a) reacting a tri-arm platform molecule of formula (16):

with an oligonucleotide of formula (17):

ORG R₆ — N₂ -OH

to obtain formula (18):

wherein FG is a functional group; BP is a branch point having three bonds, consisting of CR₇ or N; R₁, R₂, R₃, R₄, R₅, Re, R₇, R_z and R_y are independently selected substituent groups; each PMRG is independently a platform molecule reactive group and PMRG = FG; each m is independently 0 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; ORG is an oligonucleotide reactive group that can react with PMRG; each Sp is the reaction product of a PMRG and an ORG or a FG and an ORG; and N_z is a linear oligonucleotide of z mer units, wherein each N is an independently selected nucleotide and each z is independently an integer from 1 to 30.

[00144] In certain embodiments of the present invention, platform formula (16) includes one or more substituents that each comprises a suitable chromophoric and/or fluorophoric moiety. For example, the chromophore- and/or fluorophore-containing substituent can be at one or more of R₁, R₂, R₃, R₄, R5, R7, R_z and R_y in formula (16). Such moieties may allow improved detection and purification of formula (16) and its precursors, particularly when the platform molecule does not contain other significant chromophores or fluorophores, such as oligonucleotides. Examples of suitable chromophoric and/or fluorophoric substituents include natural and non-natural nucleosides, such as adenosine, thymidine, cytosine, guanosine and other suitable bases known in the art. Such nucleosides may be ribonucleosides, 2'-deoxyribonucleosides, or other suitable sugars or modified versions thereof known in the art. One or more of such nucleosides can be incorporated by use of suitable phosphoramidite precursors as shown herein, and as are known in the art.

[00145] Another method of making a symmetrical tri-arm branched oligonucleotide comprises the steps:

(a) activating an oligonucletotide of formula (17): ORG R₆ — N_z OH

by reacting (16), wherein ORG is an amine, with a heterobifunctional activator, ALG- C(O)-Rx-W, to obtain an activated oligonucleotide formula (67):

(b) reacting the activated oligonucleotide formula (67) with platform of formula (16):

to obtain formula (68):

wherein FG is a functional group; BP is a branch point having three bonds, consisting of CR₇ or N; R₁, R₂, R₃, R₄, Rs, Re, R7, R_z, R_x and R_y are independently selected substituent groups; each PMRG is independently a platform molecule reactive group and PMRG = FG; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; ALG is a leaving group of an activated carboxylic acid; W is an electrophilic group that can react with PMRG; ORG is an oligonucleotide reactive group, particularly amine, that can react with the heterobifunctional activator by displacing ALG; each Sp is independently the reaction product of a PMRG and a W or a FG and a W; and N_z is a linear oligonucleotide of z mer units, wherein each N is an independently selected nucleotide and each z is independently an integer from 1 to 30.

[00146] Still another method of making a symmetrical tri-arm branched oligonucleotide comprises the steps:

(a) activating a platform molecule of formula (16):

by reacting (16), wherein PMRG = FG = amine, with a heterobifunctional activator, ALG-C(O)-Rx-W to obtain an activated platform formula (69): W

(b) reacting the activated platform formula (69) with an oligonucleotide of formula

(17):

ORG R₆ — N_z OH

to obtain formula (70):

wherein FG is a functional group; BP is a branch point having three bonds, consisting of CR₇ or N; R₁, R₂, R₃, R₄, Rs, Re, R7, R_z, R_x and R_y are independently selected substituent groups; each PMRG is independently a platform molecule reactive group and PMRG = FG, particularly amine, that can react with the heterobifunctional activator by displacing ALG; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; ALG is a leaving group of an activated carboxylic acid; W is an electrophilic group that can react with ORG; each Sp is the reaction product of an ORG and a W; and N_z is a linear oligonucleotide of z mer units, wherein each N is an independently selected nucleotide and each z is independently an integer from 1 to 30.

[00147] In certain embodiments of the present invention, platform formula (16) includes one or more substituents that each comprises a suitable chromophoric and/or fluorophoric moiety. For example, the chromophore- and/or fluorophore-containing substituent can be at one or more of R₁, R₂, R₃, R₄, R₅, R₇, R_z and R_y in formula (16). Such moieties may allow improved detection and purification of formula (16) and its precursors, particularly when the platform molecule does not contain other significant chromophores or fluorophores, such as oligonucleotides. Examples of suitable chromophoric and/or fluorophoric substituents include natural and non-natural nucleosides, such as adenosine, thymidine, cytosine, guanosine and other suitable bases known in the art. Such nucleosides may be ribonucleosides, 2'-deoxyribonucleosides, or other suitable sugars or modified versions thereof known in the art. One or more of such nucleosides can be incorporated by use of suitable phosphoramidite precursors as shown herein, and as are known in the art.

[00148] In certain embodiments, platform formula (16) is activated with a heterobifunctional activator, ALG-C(O)-Rx-W, to yield activated platform formula (69), wherein Rx is CH₂ and W is a halogen. In certain embodiments, W is chlorine.

[00149] In certain embodiments, ORG of oligonucleotide (17) is a thiol. For such embodiments, an oligonucleotide having the thiol reactive group may be generated from the reduction of a disulfide precursor, e.g., HO - Nz - Re - S - S - Re - Nz - OH , or any other suitable precursor that generates the desired reactive oligonucleotide.

[00150] In certain aspects of the method and compounds, exemplary embodiments of symmetrical tri-arm platform formula (16) are defined by formula (25-A):

wherein each R₃ if present is independently poly(i_i2)ethyleneglycol-OPSO₂ or (CH₂)i-8- OPSO₂, each R₄ if present is poly(i_i₂)ethyleneglycol-OPSO₂, each R₅ if present is independently 5'- ribo- or deoxyribonucleoside-3' or 3'- ribo- or deoxyribonucleoside-5' , which can be activated with a heterobifunctional activator, ALG-C(O)-CH2-C1, wherein ALG is the leaving group of an activated carboxylic acid, to obtain an exemplary embodiment of activated platform formula (25) as defined by formula (25-B):

which can be reacted with an exemplary embodiment of activated oligonucleotide (17) as defined by formula (25-C):

HS R₂ R₁-OPSO₂-N₁ 5' ₅

to obtain an exemplary embodiment of CIC formula (26) as defined by formula (25-D):

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ N₁ 5'

-OPSO₂ R₅ R₄-R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

wherein in certain embodiments of CICs of the present invention, each Ni is independently an oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as those listed, for example, in column 1 of Table A, each Ri if present is independently poly₍i_i₂₎ethyleneglycol - OPSO₂, each R₂ if present is independently (CH₂) _1-8 or poly₍i_i₂₎ethyleneglycol. In certain embodiments of CIRCs of the present invention, each Ni is an oligonucleotide comprising one or more immunoregulatory sequences.

[00151] In certain aspects of the method and compounds, exemplary embodiments of symmetrical tri-arm platform formula (16) are defined by formula (25-A):

wherein each R₃ if present is independently poly(i_i2)ethyleneglycol-OPSO2 or (CH₂)i-8- OPSO₂, each R₄ if present is poly(i_i2)ethyleneglycol-OPSO₂, each R₅ if present is independently 5'- ribo- or deoxyribonucleoside-3' or 3'- ribo- or deoxyribonucleoside-5' , which can be activated with a heterobifunctional activator, ALG-C(O)-CH2-C1, wherein ALG is the leaving group of an activated carboxylic acid, to obtain an exemplary embodiment of activated platform formula (25) as defined by formula (25-B):

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ N₁ 5'

-OPSO₂ R₅ R₄-R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

wherein each Ni is oligonucleotide Nl- 19 (5'-TCGAACGTTT-S' ; (SEQ ID NO: 19)) or oligonucleotide N 1-20 (5'-TCGGACGTTT-S' ; (SEQ ID NO:20))

TCGGACGTTT (SEQ ID NO:20) each Ri if present is independently poly₍i_i₂₎ethyleneglycol - OPSO₂, each R₂ if present is independently (CH₂) _1-8 or poly₍i_i₂₎ethyleneglycol.

[00152] In certain aspects of the method and compounds, exemplary embodiments of symmetrical tri-arm platform formula (16) are defined by formula (25-A):

wherein each R₃ if present is independently poly(i_i2)ethyleneglycol-OPSO₂ or (CH₂)_1-8- OPSO₂, each R₄ if present is poly(i_i₂)ethyleneglycol-OPSO₂, each R₅ if present is independently 5'- ribo- or deoxyribonucleoside-3' or 3'- ribo- or deoxyribonucleoside-5' , which can be activated with a heterobifunctional activator, ALG-C(O)-CH₂-Cl, wherein ALG is the leaving group of an activated carboxylic acid, to obtain an exemplary embodiment of activated platform formula (25) as defined by formula (25-B):

to obtain an exemplary embodiment of CIC formula (26) as defined by formula (25-D): OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ N₁ 5'

-OPSO₂ R₅ R₄ — R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

wherein in certain embodiments of CISCs of the present invention, each Ni is independently an oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as those listed, for example, in column 1 of Table A, each Ri if present is independently poly₍i_i₂₎ethyleneglycol - OPSO₂, each R₂ if present is independently (CH₂)i_g or poly₍i_i₂₎ethyleneglycol. In certain embodiments of CIRCs of the present invention, each Ni is an oligonucleotide comprising one or more immunoregulatory sequences.

[00153] In certain aspects of the method and compounds, exemplary embodiments of symmetrical tri-arm platform formula (16) are defined by formula (25-A):

wherein each R₃ if present is independently poly₍i_i₂₎ethyleneglycol-OPSO₂ or (CH₂)i_₈- OPSO₂, each R₄ if present is poly(i_i₂)ethyleneglycol-OPSO₂, each R₅ if present is independently 5'- ribo- or deoxyribonucleoside-3' or 3'- ribo- or deoxyribonucleoside-5' , which can be activated with a heterobifunctional activator, ALG-C(O)-CH₂-Cl, wherein ALG is the leaving group of an activated carboxylic acid, to obtain an exemplary embodiment of activated platform formula (25) as defined by formula (25-B):

HS R₂ R₁-OPSO₂-N₁ 5' ₅

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ N₁ 5'

-OPSO₂ R₅ R₄-R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

wherein each Ni is oligonucleotide Nl-20 (5'- TCGGACGTTT -3'; (SEQ ID NO:20))

each Ri if present is independently poly₍i_i₂₎ethyleneglycol - OPSO₂, each R₂ if present is independently (CH₂) _1-8 or poly(_1-12)ethyleneglycol.

[00154] In certain aspects of the method and compounds, exemplary embodiments of symmetrical tri-arm platform formula (16) are defined by formula (25-A):

and branched CIC formula (25-D): 5'

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

wherein in certain embodiments of CISCs of the present invention,each Ni is independently an oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as those listed, for example, in column 1 of Table A, each Ri if present is independently ethylene glycol- OPSO₂, each R₂ if present is independently (CH₂)₆ or (CH₂)₃, each R₃ if present is independently CH₂CH₂OCH₂CH₂-OPSO₂ or (CH₂)₆-OPSO₂ or (CH₂)₃-OPSO₂, each R₄ if present is hexaethylene glycol-OPSO₂, each R₅ if present is independently 5'- ribo- or deoxyribonucleoside-3' or 3'- ribo- or deoxyribonucleoside-5' . In certain embodiments of CIRCs of the present invention, each Ni is an oligonucleotide comprising one or more immunoregulatory sequences.

[00155] In certain aspects of the method and compounds, exemplary embodiments of symmetrical tri-arm platform formula (16) are defined by formula (25-A):

and branched CIC formula (25-D): OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ N₁ 5'

-OPSO₂ R₅ R₄ — R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5'

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5' wherein in certain embodiments of CISCs of the present invention, each Ni is independently an oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as those listed, for example, in column 1 of Table A,

Ri is absent,

R₂is (CH₂)₆, each R₃ is independently CH₂CH₂OCH₂CH₂-OPSO₂ or (CH₂)₃-OPSO₂,

R₄ is hexaethylene glycol-OPSO₂, each R₅ is independently 5'- thymidine-3' or 3'- thymidine -5'. In certain embodiments of CIRCs of the present invention, each Ni is an oligonucleotide comprising one or more immunoregulatory sequences.

[00156] A method of making a tri-arm branched oligonucleotide with one unique arm comprises the steps of:

(a) reacting a tri-arm platform molecule with one unique arm of formula (19):

with an oligonucleotide of formula (48):

to obtain formula (71):

wherein BP is a branch point having three bonds, consisting of -CR₇- or -N-; Ro, R₁, R₂, R₃, R₄, R₅, Re, R₇, R_z and R_y are independently selected substituent groups; PMRG is a platform molecule reactive group; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; - Sp - is the reaction product of - ORG and PMRG-; and N_z and N'_z' each is a linear oligonucleotide of z mer and z' mer units, wherein each N and N' is an independently selected nucleotide and z and z' are independently selected integers from 1 to 30.

[00157] Another method of making a tri-arm branched oligonucleotide with one unique arm comprises the steps of:

(a) activating an oligonucletotide of formula (48):

ORG R₆ N'_z. — OH

by reacting (48), wherein ORG is an amine, with a heterobifunctional activator, ALG- C(O)-Rx-W, to obtain an activated oligonucleotide formula (72):

(b) reacting the activated oligonucleotide formula (72) with a tri-arm platform molecule with one unique arm of formula (19):

to obtain formula (73):

wherein BP is a branch point having three bonds, consisting Of -CR₇- or -N-; Ro, R₁, R₂, R3, R₄, R₅, Re, R_7, R_z, R_x and R_y are independently selected substituent groups; PMRG is a platform molecule reactive group; ALG is a leaving group of an activated carboxylic acid; W is an electrophilic group that can react with PMRG; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; - Sp - is the reaction product of - W and PMRG-; and N_z and NV are linear oligonucleotides of z mer and z' mer units, respectively, wherein each N and N' is an independently selected nucleotide and z and z' are independently selected integers from 1 to 30.

[00158] Still another method of making a tri-arm branched oligonucleotide with one unique arm comprises the steps of:

(a) activating a tri-arm platform molecule with one unique arm of formula (19):

by reacting (19), wherein PMRG is an amine, with a heterobifunctional activator, ALG-C(O)-Rx-W to obtain an activated platform formula (73- A):

(b) reacting the activated platform formula (73) with an oligonucleotide of formula (48):

to obtain formula (74):

wherein BP is a branch point having three bonds, consisting of CR₇ or N; Ro, R₁, R₂, R₃, R₄, R5, Re, R₇, Rz, R_x and R_y are independently selected substituent groups; PMRG is an amine; ALG is a leaving group of an activated carboxylic acid; W is an electrophilic group that can react with PMRG; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; Sp is the reaction product of W and ORG; and N_z and NV are linear oligonucleotides of z mer and z' mer units, respectively, wherein each N and N' is an independently selected nucleotide and z and z' are independently selected integers from 1 to 30.

[00159] In certain embodiments, platform formula (19) is activated with a heterobifunctional activator, ALG-C(O)-Rx-W, wherein Rx is CH₂ and W is a halogen. In certain embodiments, W is chlorine.

[00160] In certain embodiments, ORG of oligonucleotide (48) is a thiol. For such embodiments, an oligonucleotide having the thiol reactive group may be generated from the reduction of a disulfide precursor, e.g., HO - NV - R₆ - S - S - R₆ - NV - OH , or any other suitable precursor that generates the desired reactive oligonucleotide.

[00161] In certain aspects of the method and compounds, exemplary embodiments of formula (19) include platform formula (22):

3'

which can be activated with a heterobifunctional activator, ALG-C(O)-CH₂-Cl to obtain an exemplary embodiment of activated platform formulae (19) and (22- A), such as formula (74):

3'

which can be reacted with an exemplary embodiment of activated oligonucleotide (48) such as formula (22-B):

HS R₂ R₁-OPSO₂-N₁ 5' to obtain an exemplary embodiment of branched CIC formula (20- A) such as formula (75):

wherein in certain embodiments of CISCs of the present invention, each Ni is a first oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as those listed, for example, in column 1 of Table A,

N₂ is independently a second oligonucleotide comprising one or more of human and/or rodent (e.g., rat or mouse)motifs, such as those listed, for example, in column 2 of Table A,

Ri if present is poly(i_i2)ethyleneglycol-OPSO₂,

R₂ is (CH₂)i-8 or poly(i_i2)ethyleneglycol,

R₃ is poly₍i_i2)ethyleneglycol or

R₄ if present is poly(i_i₂)ethyleneglycol-OPSO₂, and

R₅ is poly₍i_i₂₎ethyleneglycol or

In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[00162] In certain aspects of the described methods and compounds, exemplary embodiments include the branched CIC formula (75):

3'

and the platform formula (76): 3'

wherein in certain embodiments of CICs of the present invention, each Ni is a first oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as those listed, for example, in column 1 of Table A,

Ri if present is hexaethylene glycol-OPSO₂,

R₃ is CH₂CH₂OCH₂CH₂ or (CH₂)₆ or (CH₂)₃,

R₄ if present is hexaethylene glycol-OPSO₂, and

R₅ is hexaethylene glycol. In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[00163] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (75) include:

3'

and the platform formula (76): 3'

wherein in certain embodiments of CISCs of the present invention, each Ni is a first oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent (e.g., rat or mouse) motifs, such as those listed, for example, in column 1 of Table A, N₂ is independently a second oligonucleotide comprising one or more of human and/or rodent (e.g., rat or mouse)motifs, such as those listed, for example, in column 2 of Table A,

Ri is absent,

R₂ is (CH₂)₆,

R₃ is CH₂CH₂OCH₂CH₂,

R₄ is hexaethylene glycol-OPSO₂ and

R₅ is hexaethylene glycol.

[00164] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CISC formula (75) include:

3'

and the platform formula (76):

in which Ni is TCGT(N3)CG(N4)(N5) and N₂ is (N6)(N7)ACGTTC(N8), wherein N3 if present is GAT or T, N4 if present is A or T, N5 if present is CTT or GAT or AT, N6 if present is T, N7 is G or A, N8 if present is GT, and

Ri if present is poly(i_i2)ethyleneglycol-OPSO₂, R₂ is (CH₂)i-₈ or poly₍i_i₂₎ethyleneglycol, R₃ is poly(i_i2)ethyleneglycol or (CH₂)i-8, R₄ if present is poly(i_i2)ethyleneglycol-OPSO₂, and

R₅ is poly₍i_i₂₎ethyleneglycol or (CH₂)i-8. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[00165] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CISC formula (75) include:

S'

and the platform formula (76):

in which Ni is TCGT(N3)CG(N4)(N5) and N₂ is (N6)(N7)ACGTTC(N8), wherein N3 if present is GAT or T, N4 if present is A or T, N5 if present is CTT or GAT or AT, N6 if present is T, N7 is G or A, N8 if present is GT, and Ri if present is hexaethylene glycol-OPSO2,

R₃ is CH₂CH₂OCH₂CH₂ or (CH₂)₆ or (CH₂)₃, R₄ if present is hexaethylene glycol-OPSO₂, and

R₅ is hexaethylene glycol₂. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[00166] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CISC formula (75) include:

3'

and the platform formula (76):

in which Ni is TCGT(N3)CG(N4)(N5) and N₂ is (N6)(N7)ACGTTC(N8), wherein

N3 if present is GAT or T,

N4 if present is A or T,

N5 if present is CTT or GAT or AT,

N6 if present is T,

N7 is G or A,

N8 if present is GT, and Ri is absent, R₂ is (CH₂)₆, R₃ is CH₂CH₂OCH₂CH₂, R₄ is hexaethylene glycol-OPSO₂ and

R₅ is hexaethylene glycol. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[00167] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (75) include:

3'

and the platform formula (76):

3'

wherein in certain embodiments of CISCs of the present invention, Ni is selected from the group consisting of the oligonucleotides listed in column 1 of Table A, N₂ is selected from the group consisting of the oligonucleotides listed in column 2 of Table A, and

Ri if present is poly(i_i₂)ethyleneglycol-OPSO₂,

R₂ is (CH₂)i_₈ or poly₍i_i₂₎ethyleneglycol,

R₃ is poly₍i_i₂₎ethyleneglycol or (CH₂)i_g,

R₄ if present is poly₍i_i₂₎ethyleneglycol-OPSO₂, and

R5 is poly₍i_i₂₎ethyleneglycol or (CH₂)i_g. In certain embodiments of CIRCs of the present invention, each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences. In some embodiments, each oligonucleotide comprises phosphorothioate linkages. [00168] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (75) include:

3'

and the platform formula (76):

3'

Ri if present is hexaethylene glycol-OPSO₂,

R₃ is CH₂CH₂OCH₂CH₂ or (CH₂)₆ or (CH₂)₃,

R₄ if present is hexaethylene glycol-OPSO₂, and

[00169] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (75) include: 3'

and the platform formula (76):

3'

Ri is absent,

R₂ is (CH₂)₆,

R₃ is CH₂CH₂OCH₂CH₂,

R₄ is hexaethylene glycol-OPSO₂ and

[00170] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CISC formula (21) include the following compounds:

D-2: (5'- TCGTGATCGT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3', D-14: (5'- TCGTGATCGT-S' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TGACGTTCGT -3',

D-4: (5'- TCGTCGA-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH2)₂- CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3'

D-I l: (5'- TCGTTCGAAT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3'; and exemplary embodiments of the platform formula (19) include the following embodiments:

HEG is hexaethylene glycol,

Ri is absent,

R₂ is (CH₂)₆ ,

R₃ is CH₂CH₂OCH₂CH₂, and and R₄ is hexaethylene glycol-OPSO₂. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[00171] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (75) include:

S^'

and the platform formula (76):

wherein in certain embodiments of CISCs of the present invention, Ni is selected from the group consisting of the oligonucleotides listed in column 1 of Table A, N₂ is selected from the group consisting of the oligonucleotides listed in column 2 of Table A,

Ri is absent,

R₂ is (CH₂)₆ ,

R₃ is CH₂CH₂OCH₂CH₂ ,

R₄ is absent, and

[00172] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CISC formula (74) include the following compounds:

D-5: (5'-TCGTCGACTT-S'- OPSO₂- R_I -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3' ,

D-15: (5'- TCGTGATCGT-3' - OPSO₂- Ri -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3' and

D-8: (5'- TCGTCGA-3' - OPSO₂- Ri -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3'; and exemplary embodiments of the platform formula (19) include the following embodiments:

P-2: (H₂N - R₃ -OPSO₂- R₄ -CHI)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3' and

P-3: (H₂N - R₃ -OPSO₂- R₄ -CHI)₂-CH-OPSO₂-HEG-OPSO₂- 5'- AACGTTC -3'; wherein:

HEG is hexaethylene glycol, Ri is absent,

R₂ is (CH₂)₆ ,

[00173] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC formula (75) include:

3'

and the platform formula (76):

3'

Ri is absent,

R₂ is (CH₂)₃ ,

R₃ is CH₂CH₂OCH₂CH₂ ,

R₄ is HEG-OPSO₂, and

[00174] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CISC formula (74) include the following compounds: D-6: (5'-TCGTCGACTT-S'- OPSO₂- R_I -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3',

D-9: (5'- TCGTCGA-3' - OPSO₂- Ri -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3' and

D-16: (5'-TCGTGATCGT-S'- OPSO₂- R_I -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3'; and exemplary embodiments of the platform formula (19) include the following embodiment:

P-I: (H₂N - R₃ -OPSO₂- R₄ -CH2)2-CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3' and

P-3: (H₂N - R₃ -OPSO₂- R₄ -CHI)₂-CH-OPSO₂-HEG-OPSO₂-S'- AACGTTC -3', wherein:

HEG is hexaethylene glycol,

Ri is absent,

R₂ is (CH₂)₃ ,

R₃ is CH₂CH₂OCH₂CH₂ , and R₄ is hexaethylene glycol. In some embodiments, each oligonucleotide comprises phosphorothioate linkages.

[00175] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC compound (75) include:

S^'

and the platform formula (76):

wherein in certain embodiments of CIRCs of the present invention, Ni is selected from the group consisting of the oligonucleotides listed in column 1 of Table A, N₂ is selected from the group consisting of the oligonucleotides listed in column 2 of Table A,

Ri is hexaethylene glycol-OPSO₂,

R₂ is (CH₂)₃ ,

R₃ is CH₂CH₂OCH₂CH₂ ,

R₄ is hexaethylene glycol-OPSO₂, and

[00176] In certain aspects of the described methods and compounds, exemplary embodiments of the compound (21) include the following compounds:

D-7: (5'-TCGTCGACTT-S'- OPSO₂- R_I -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3' ,

D-17: (5'- TCGTGATCGT-3' - OPSO₂- Ri -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ - CH₂)₂-CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3' and

D-IO: (5'- TCGTCGA-3' - OPSO₂- Ri -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO2-HEG-OPSO2- 5'-AACGTTC -3'; and exemplary embodiments of the platform formula (19) include the following embodiment:

P-I: (H₂N - R₃ -OPSO₂- R4 -CH2)₂-CH-OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3' and

P-3: (H₂N - R₃ -OPSO₂- R₄ -CH2)₂-CH-OPSO₂-HEG-OPSO₂-5'-AACGTTC-3' and wherein:

HEG is hexaethylene glycol,

Ri is hexaethylene glycol-OPSO₂,

R₂ is (CH₂)₃ ,

R₃ is CH₂CH₂OCH₂CH₂ , and R₄ is hexaethylene glycol. In some embodiments, each oligonucleotide comprises phosphorothioate linkages. [00177] In certain aspects of the described methods and compounds, exemplary embodiments of the branched CIC compound (74) include:

S^'

and the platform formula (19):

3'

Ri is hexaethylene glycol-OPSO₂,

R₂ is (CH₂)₃ ,

R₃ is (CH₂)₆ ,

R₄ is hexaethylene glycol-OPSO₂, and

2. Structure of Tri Arm Branched CICs [00178] A symmetrical tri-arm branched oligonucleotide has the structure (18):

wherein BP is a branch point having three bonds, consisting of CR₇ or N; R₁, R₂, R3, R₄, R5, R_O, R₇, R₅ and R_y are independently selected substituent groups; each PMRG is independently a platform molecule reactive group and PMRG = FG; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; each Y and Z is independently O or S; ORG is an oligonucleotide reactive group that can react with PMRG; each Sp is a spacer moiety (which is the reaction product of a PMRG and an ORG or a FG and an ORG); and N_z is a linear oligonucleotide of z mer units, wherein each N is an independently selected nucleotide and each z is independently an integer from 1 to 30.

[00179] Another symmetrical tri-arm branched oligonucleotide has the structure (68)

[00180] Still another symmetrical tri-arm branched oligonucleotide has the structure (26):

[00181] A tri-arm branched oligonucleotide with one unique arm has the structure (71):

[00182] Another tri-arm branched oligonucleotide with one unique arm has the structure

(73):

[00183] Another tri-arm branched oligonucleotide with one unique arm has the structure

(74)

[00184] As further detailed below, any number of polynucleotide sequences can be used as part of any of the CICs. The length of the polynucleotide sequence can be variable. In one embodiment, the polynucleotide sequences are each independently between 5- to 30-mers, between 6- to 12-mers or between 6- to 20-mers., as shown in the Figures and described in the Examples. In another embodiment, the branched CIC comprises nucleic acid moieties wherein at least one of the nucleic acid moieties is 6-mer or greater, 7- mer or greater, 8- mer or greater, 9- mer or greater, 10- mer or greater, 11- mer or greater, 12- mer or greater, 15- mer or greater, 20- mer or greater, 25- mer or greater or 30- mer or greater. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are each independently 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14- mers,15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers or 30-mers. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are 10-mers. In some embodiments, the branched CIC comprises both 7-mer and 10-mer nucleic acid moieties. In some embodiments, the branched CIC comprises two 7-mer nucleic acid moieties and one 10-mer nucleic acid moiety. In some embodiments, the branched CIC comprises two 10-mer nucleic acid moieties and one 7-mer nucleic acid moiety. In some embodiments, the branched CIC comprises only 7-mer nucleic acid moieties. In some embodiments, the branched CIC comprises only 10-mer nucleic acid moieties. However, other lengths of polynucleotide sequences are contemplated within the scope of the invention. The polynucleotide sequences, also referred to herein as nucleic acid moieties, are generally capable of immunomodulatory activities.

3. Components of the Syntheses, Platform Molecules and CICs

[00185] SJ>: The preferred solid supports of the invention include controlled pore glass (CPG) beads, and polystyrene. However, the term 'solid support,' is intended to include all forms of support known to one of ordinary skill in the art for the synthesis of oligomeric compounds and related compounds such as peptides. Some representative support medium that are amenable to the methods of the present invention include but are not limited to the following: controlled pore glass (CPG); oxalyl-controlled pore glass (see, e.g., AM, et al., Nucleic Acids Research 1991, 19, 1527); silica-containing particles, such as porous glass beads and silica gel such as that formed by the reaction of trichloro-[3-(4- chloromethyl)phenyl]propylsilane and porous glass beads (see Parr and Grohmann, Angew. Chem. Internal Ed. 1972, 11, 314, sold under the trademark 'PORASIL E' by Waters Associates, Framingham, Mass., USA); the mono ester of 1,4-dihydroxymethylbenzene and silica (see Bayer and Jung, Tetrahedron Lett., 1970, 4503, sold under the trademark 'BIOPAK' by Waters Associates); TENTAGEL (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373); cross-linked styrene/divinylbenzene copolymer beaded matrix or POROS, a copolymer of polystyrene/divinylbenzene (available from Perceptive Biosystems); soluble support medium, polyethylene glycol PEG's (see Bonora et al., Organic Process Research & Development, 2000, 4, 225-231). [00186] Further support medium amenable to the present invention include without limitation particles based upon copolymers of dimethylacrylamide cross-linked with N,N'- bisacryloylethylenediamine, including a known amount of N-tertbutoxycarbonyl-beta-alanyl- N'-acryloylhexamethylenediamine. Several spacer molecules are typically added via the beta alanyl group, followed thereafter by the amino acid residue subunits. Also, the beta alanyl- containing monomer can be replaced with an acryloyl saf cosine monomer during polymerization to form resin beads. The polymerization is followed by reaction of the beads with ethylenediamine to form resin particles that contain primary amines as the covalently linked functionality. The polyacrylamide-based supports are relatively more hydrophilic than are the polystyrene-based supports and are usually used with polar aprotic solvents including dimethylformamide, dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, et al, J. Am. Chem. Soc, 1975, 97, 6584, Bioorg Chem. 1979, 8, 351, and J. C. S. Perkin I 538 (1981)).

[00187] Further support medium amenable to the present invention include without limitation a composite of a resin and another material that is also substantially inert to the organic synthesis reaction conditions employed. One exemplary composite (see Scott, et al., J. Chrom. ScL, 1971, 9, 577) utilizes glass particles coated with a hydrophobic, cross-linked styrene polymer containing reactive chloromethyl groups, and is supplied by Northgate Laboratories, Inc., of Hamden, Conn., USA. Another exemplary composite contains a core of fluorinated ethylene polymer onto which has been grafted polystyrene (see Kent and Merrifield, Israel J. Chem. 1978, 17, 243 and van Rietschoten in Peptides 1974, Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp. 113-116).

[00188] FGG and FG represent the preferred 'functional group generators' and 'functional groups' of the invention. These preferred embodiments are shown in the context of their incorporation during the syntheses of the platform molecules in accordance with Table 1. Table 1

SS-FGG-R₁-OAPG₁ FG-R₁

O — R₁-S-S — R₁-O-APG₁ HS-R₁-

Generated in situ by removal of FG-R₁

Z, Y = O,S

[00189] As used herein 'substituent groups' refers to the R groups of the invention Ro_, R₁, R₂, R₃, R₄, R₅, R₆, R₇, Ry, R_x, Rz, Ra, Rb, Rc Rd, Re, Rf, Rz', Rz-, Ry and R_r which are independently selected from group consisting of methyl, C₂-Ci₂ unsubstituted or substituted, branched or linear alkyl, C₂-Ci₂ unsubstituted or substituted, branched or linear alkenyl, C₂- Ci₂ unsubstituted or substituted, branched or linear alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, C₂-Ci₂ unsubstituted or substituted cycloalkyl; unsubstituted or substituted cycloalkylmethyl; unsubstituted or substituted (CH₂)_m- cycloalkyl, unsubstituted or substituted (CH₂)_m-aryl, wherein m is 1, 2, 3, 4, or 5; (CH₂)_nA wherein n is a natural number being 0, 1, 2, 3, 4, or 5 and A is selected from alkenyl, alkynyl, aryloxy, heteroaryl, substituted alkenyl, substituted alkynyl, substituted aryloxy, and substituted heteroaryl; hydroxyalkyl, carboxyalkyl, — NHRo wherein Ro is selected from hydrogen, alkylsulfonyl, acyl, acyl substituted by acylamido and hydroxyalkyl; CO₂Y, B(OY)₂, CHO, CH₂OY, CH(CO₂Y)₂, PO(OY)₂ wherein Y is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, cycloalkyl, substituted aryl or substituted heteroaryl; tetrazolyl; and CONHZ wherein Z is selected from hydrogen, hydroxy, alkyl, alkylsulfonyl, arylsulfonyl, and cyanoalkyl; (CH₂CH₂O)_n where n is from 1-10; any group listed above with a heteroatom replacing one or more carbons; any group listed above with a hydroxyl or carboxyl or amine or amide group attached, any group listed above with an amide group replacing one or more carbons, a nucleoside or substituted nucleoside group, and a sugar linked to a heterocylic group. [00190] In some cases, R₁, R₂ and R₅ are bonds.

[00191] R_a and Rb, R_c and Rd, R_e and Rf, R_z> and R_Z", and R_y> and R_y" are preferably methyl, ethyl, isopropyl, or R_a and R_b or R_c and R_d or R_e and R_f or R_z> and R_z>> or R_y and R_y> together with N form N-pyrrolidino, N-morpholino, N-2,6-dimethylpiperidino, N-piperidino or N-2,2,6,6-tetramethylpiperidino, or other cycloalkylamine group.

[00192] ALG is a leaving group of an activated carboxylic acid, wherein the activated carboxylic acid is a carboxylic acid, carboxylic ester, carboxylic anhydride, carboxylic acid halide, amide, imidate ester or cyano.

[00193] APGi, APG₂, APG₃, APG₄, APG_Z and APG_y are acid-labile protecting groups. In preferred embodiments, each APG is independently selected from the group consisting of trityl, substituted trityl including monomethoxytrityl (MMT), 4,4'-dimethoxytrityl (DMT); pixyl (9-phenylxanthen-9-yl) (Px), substituted pixyl, moxyl (9-(p-methoxyphenyl)xanthen-9- yl) (Mox) and substituted moxyl. Other substituted trityl and pixyl groups are listed in reference: Beaucage, S.L. and Iyer, R.P., 1992 Tetrahedron, 48, 2223-2311.

[00194] PPGi, PPG₂, PPG₃, PPG₄, PPG₅, PPG_Z and PPG_y are phosphate protecting groups. In preferred embodiments, each PPG is independently selected from the group consisting of cyanoethyl, methyl, dichlorobenzyl, beta-thiobenzoylethyl, NCCH₂CH(Me)-, NCCH₂C(Me)₂-, Cl₃CCH₂-, Cl₃C(Me)₂-, allyl, (CF₃)₂CH-, methylsulfonylethyl, p- nitrophenylethyl, 4-N pyridinylethyl, o-methylbenzyl, o-chlorobenzyl, phenyl, p-nitrophenyl, hexachlorophenyl, and o-chlorophenyl.

[00195] BP is a branch point having three bonds selected from the group consisting of - CR₇ and N (wherein CH and N have three available bonds).

[00196] BPGi is a base-labile protecting group selected from the group consisting of levulinyl and 9H-fluoren-9-ylmethoxycarbonyl.

[00197] PMRG, PMRGi and PMRG₂ are platform molecule reactive groups. In preferred embodiments, each PMRG is independently selected from a primary amine (-NH₂), a secondary amine, thiol, carboxylate, aldehyde, ketone, phosphate, thiophosphate, phosphorodithioate, alkyl halide and haloacetyl; and Pr is a PMRG protecting group. ] Specific PMRG / PMRG(Pr)_n combinations are shown in table 2 below:

Table 2

PMRG-Pr Deprotection conditions PMRG

Strong Base

Oxidation

Reduction

PMRG-Pr Deprotection conditions PMRG

situ by

Z, Y = O,S

[00199] In some cases, PMRG, ORG and Sp are selected as shown in Table 3 below:

Table 3

[00200] In some cases, PMRG, W and Sp are selected as shown in Table 4 below:

Table 4

[00201] In some cases, PMRG, ORG and Sp are selected as shown in Table 5 below:

Table 5

[00202] Heterobifunctional activators: In preferred embodiments, the heterobifunctional activators of the invention are selected from the various leaving groups (ALG) and electrophilic groups (W) as shown in Table 6 below:

Table 6

Heterobif unctional Activator

Leaving Group (ALG) ALG

T Electrop o hiliG

D. Tetra Arm CICs

[00203] Multivalent CICs with four arms (i.e., Tetra Arm) are also provided herein. Tetra- Arm CICs can be made from Tetra- Arm platform molecules, which allow for both symmetrical as well as asymmetrical synthesis as exemplified in Examples 7-14. Symmetrical Tetra Arm platform molecules can be synthesized in as few as two steps and can be used for conjugation reactions without rigorous purification steps. The conjugation of a polynucleotide to the symmetrical Tetra Arm platform molecule results in a CIC that has four branches comprising the same polynucleotide sequence. In some embodiments, all branches can each contain a free 5' termini, thereby enhancing biological activity. [00204] In contrast, asymmetrical Tetra Arm CICs can be made where there is one unique branch of polynucleotides, e.g., 5'-XXXXXXX-3', already incorporated into the platform molecule. Other arms can be subsequently added onto the platform structure, each thereby introducing a second, third or a fourth polynucleotide sequence. In one aspect, the CIC has one arm with a polynucleotide sequence of 5'-XXXXXXX-3' and three arms with a polynucleotide sequence of 5'-YYYYYYY-3'. In another aspect, the CIC has two arms with a polynucleotide sequence of 5'-XXXXXXX-3' and two arms with a polynucleotide sequence of 5'-YYYYYYY-3' . In another aspect, the CIC has one arm with a polynucleotide sequence of 5'-XXXXXXX-3'; the second arm with a polynucleotide sequence of 5'-YYYYYYY-3'; the third arm with a polynucleotide sequence of 5'- QQQQQQQ-3'; and a fourth arm with a polynucleotide sequence of 5'-ZZZZZZZ-3'.

[00205] For both the Tri Arm and Tetra Arm CICs, one benefit of using the methodology described herein is the ability to control the sequence independently for each of the branches. Normal DNA synthesis methods are unable to synthesize CICs of this nature with comparable purity. The methodology described herein also allows for flexibility to make many unique CICs from a relatively small number of short oligonucleotides in parallel fashion. Accordingly, this allows for one of skill in the art to make multiple CICs using a parallel synthesis approach instead of making each individual CIC one at a time on a DNA synthesizer. One of skill in the art can make and/or use a library of branches of unique sequences or motifs which are stored in high purity on a large scale, and use this library to graft onto platform molecules in a variety of combinations.

[00206] Without being bound by theory, the size of each CIC may play a role in biological activity. For example, larger CICs may be taken up by early endosomes while smaller CICs are taken up by late endosomes. The early endosomes results in a different immune response than the late endosomes. The immune response for early vs. late endosomes are described in, for example, Guiducci, C , et al, J. Exp. Med., 203, 1999-2008 (2006), which is herein incorporated by reference for all purposes in its entirety. In addition, the polynucleotide sequence can affect the type of immune response elicited, which affects which disease is being targeted. Accordingly, the ability to control the size and/or the content of the polynucleotide sequence of the CICs is very valuable. [00207] The methodology described herein also provides the additional benefit of being carried out in aqueous environments. In addition, although the methodology described herein mainly refers to branched CICs, the methodology is applicable to making linear CICs as well. As such, compositions comprising linear CICs made according to the methodology described herein are contemplated by this invention as well.

[00208] A symmetrical tetra-arm branched CIC has the formula (76):

[00209] A tetra-arm branched CIC with one unique arm has the formula (77):

wherein, for formulae (76) and (77), R₀, R₁, R₂, R₃, R₄, R5, Re, R7, R_z and R_y, are independently selected substituent groups; each Nz and Nz' is a linear oligonucleotide of z mer and z' mer units, respectively, each z and z' are independently selected integers from 1 to 30, and each N of Nz and Nz' is an independently selected nucleotide; - Sp - is the reaction product of - PMRG and ORG -; each m is independently 0 to 30, such as m = 0, 1, 2 or 3; and each Y and Z is independently O or S.

E. CICs Having Specified Tertiary Structure, and CIC Multimers

[00210] The branched CICs described herein include variants having particular structural features. CICs and CIC multimers described in this section may be targeted to, or efficiently taken up by phagocytic cells or antigen-presenting cells, may present a high density of nucleic acid moiety 5'-ends, may change structure in vivo (e.g., due to nuclease or other degradative activity, acidification in the endosome, and/or dilution of the CIC or multimer thereof in vivo (thereby changing properties after administration to a subject or in a particular biological compartment).

i) CICs having specified tertiary structure

[00211] As noted elsewhere herein, linear CICs with at least two nucleic acid moieties having sequences complementary or partially complementary to each other can form hairpin duplexes (and/or CIC dimers or concatamers). As used herein, 'hairpin duplex' refers to the structure formed by hybridization of two nucleic acid moieties that are in the same orientation in the CIC (e.g., one nucleic acid moiety is bound at the 3' terminus to the spacer moiety and the other nucleic acid moiety is bound at the 5' terminus to the spacer moiety) in a CIC. In one embodiment, the two nucleic acid moieties are separated by no more than one additional nucleic acid moiety. In another embodiment, there is no intervening nucleic acid moiety between the base-paired nucleic acid moieties. In a hairpin duplex, the pair of nucleic acid moieties with complementary sequences can be reverse complements of each other (e.g., palindromic), or the pair can have one or more positions that deviate from such reverse complementarity. It will be appreciated that exact complementarity is not required so long as the nucleic acid moieties are of sufficient complementarity and length to form a duplex at 37°C in an aqueous solution at physiological pH (i.e., 7 ^'.0-7.4, e.g., 7.2) and ionic strength (e.g., 150 mM NaCl).

[00212] The presence of a duplex structure can be detected using well-known methods. These include detecting a change in CIC structure based on size exclusion chromatography, and detecting a change in A₂6o or A₂8o upon raising or lowering the temperature of the CIC- containing composition (indicative of melting or formation of the duplex). Absorbance increases as a double- stranded DNA separates into the single- stranded forms. [00213] As noted, certain CICs can form hairpin structures or can form dimers or concatamers. It is believed the latter structures are favored when the CICs are allowed to anneal at high concentration and/or when the spacer is of sufficient length and flexibility (e.g., [HEG]_ό) to favor the kinetics of dimer formation by providing increased degrees of freedom of movement of the nucleic acid moieties.

[00214] Like linear CICs, branched CICs can form a variety of types of structures, including the 'fork,' 'H,' 'comb,' 'central spacer,' and 'dendrimer' structures described below and in the Examples.

[00215] A 'fork' structure has only a single branching spacer (e.g. glycerol, glycerol- [HEG]₂, symmetrical doubler- [HEG]₂, and the like), which is bound to three nucleic acid moieties. The three nucleic acid moieties can all have the same sequence, or can have different sequences. In one embodiment, at least 2 of the nucleic acid moieties has the same sequence. In one embodiment, at least 1, at least 2, or at least 3 of the nucleic acid moieties is a 5-prime moiety. In an embodiment, at least 1, at least 2, or at least 3 of the nucleic acid moieties includes the sequence CG, optionally TCG, optionally 5'^F-TCG (i.e., TCG in the 5- prime position of a 5-prime moiety).

[00216] A 'trident' structure has only a single branching spacer (e.g., trebler, [HEG]- trebler-[HEG]₃, and the like), which is bound to four nucleic acid moieties. The four nucleic acid moieties can all have the same sequence, or can have different sequences. In one embodiment, at least 3 of the nucleic acid moieties have the same sequence. In one embodiment, at least 1, at least 2, at least 3, or at least 4 of the nucleic acid moieties is a 5- prime moiety. In an embodiment, at least 1, at least 2, at least 3, or at least 4 of the nucleic acid moieties includes the sequence CG, optionally TCG, optionally 5'^F-TCG (i.e., TCG in the 5-prime position of a 5-prime moiety). The reader will recognize that one or more of the nucleic acid moieties can have a sequence, motif or property described herein below.

[00217] A 'polydent' structure has at least 3 branched spacers (e.g., 3-15, usually 3-7) and at least 4 nucleic acid moieties, where all of the nucleic acid moieties in the structure have an unbound terminus (a free 5' end or a free 3' end). In one embodiment all of the nucleic acid moieties have a free 5 '-end. [00218] An 'H' structure is defined by having exactly two branching spacers, each of which is linked to the other via (a) a nucleic acid moiety or (b) a combination of nucleic acid moieties and nonbranching spacers (e.g., -ATTT-HEG- ATTT-) and each of which is linked to two additional nucleic acid moieties. In embodiments, at least 1, at least 2, at least 3 or at least 4 (i.e., all) of the 'two additional nucleic acid moieties' is a 5-prime moiety. In one embodiment, at least 1, at least 2, at least 3, or at least 4 of the two additional nucleic acid moieties is a 5-prime moiety. In embodiments, at least 1, at least 2, at least 3, or at least 4 of the nucleic acid moieties includes the sequence CG, optionally TCG, optionally 5'^F-TCG (i.e., TCG in the 5-prime position of a 5-prime moiety) . The reader will recognize that one or more of the nucleic acid moieties can have a sequence, motif or property described herein below. The nucleic acid moiety(s) linking the two branching spacers may also comprise a sequence CG or other sequence or motif described herein.

[00219] A 'central spacer' structure is defined by having spacer moiety bound to 4 or more nucleic acid moieties, where at least 3 of said 4 or more nucleic acid moieties is a 5- prime moiety, and wherein at least 3 of the 5-prime moieties include the sequence CG, optionally TCG, optionally 5'^F-TCG (i.e., TCG in the 5-prime position of a 5-prime moiety). The reader will recognize that one or more of the nucleic acid moieties can have a sequence, motif or property described herein. In various embodiments, the number of nucleic acid moieties bound to the spacer may be less than 500 (e.g., for CICs made by conjugation strategies, such as CICs with Ficoll-based central spacers) or less than about 10 (e.g., for compounds made using a DNA synthesizer).

[00220] A 'CIC dendrimer' is a discrete, highly branched polymer created by covalent linking of multiple (e.g., 3-15) branched CICs. Usually all or most of the component CICs has the same structural motif (e.g., all are fork structures or all are trident structures). The CIC dendrimer should not be confused with dendrimers that may serve as spacer moieties but which do not comprise nucleic acid moieties (e.g., the 'dense star or 'starburst' polymers).

ii) CIC Multimers

[00221] Certain CIC linear or branched CICs of the invention can form 'multimers' of 2 or more CICs that stably associate with each other due to Watson-Crick hybridization between pairs of at least partially complementary nucleic acid moieties. Examples of such CIC multimers are multimers comprising only linear CICs, and CIC multimers comprising at least one, and usually at least two, branched CICs.

[00222] In various alternative embodiments CIC multimers may comprise at least 2, at least 3, at least 4, at least 5, at least 10, and sometimes more than 10 individual CICs. The individual CIC subunits need not all be the same.

[00223] As noted, individual CICs in CIC multimers associate with each other. As used in this context, 'stably associate' means the CICs remain associated at 37°C in a buffered aqueous salt solution of near physiological ionic strength and pH, e.g., 150 mM NaCl, pH 7.2. It will be recognized, of course, that even 'stably associated' multimeric macromolecules may exist in a state of equilibrium such that an individual CICs may be unassociated with the multimer for relatively brief periods of time, or there may be exchange between CICs in the multimeric structure and unassociated monomers in solution. CIC multimers may be self assembling (i.e., the component CICs may spontaneously associate under physiological conditions). Usually, a CIC multimer will form when the component CICs are dissolved at a concentration of approximately 1.0 mg/ml in 50 mM sodium phosphate/150 mM sodium chloride/pH 7.2, heated to 95°C for 3 min., and allowed to slowly (e.g., over a period of approximately 2 hours) to 37°C or room temperature.

[00224] Because the association between CICs in a CIC multimer relies, at least in part, on hybrids formed between nucleic acid moieties that are at least partially complementary, and sometimes exactly complementary, the normal parameters for formation of nucleic acid hybrids apply. That is, the hybridizing regions of nucleic acid moieties are of sufficient length and/or sequence composition (e.g., GC content) to form stable CIC multimers. Generally the nucleic acid moieties of one CIC will comprise at least 8, more often at least 10, and usually at least 12 contiguous bases that are exactly complementary to nucleic acid moieties of a second CIC in the multimer. However, when there are a large number of hybridizing nucleic acid moieties, the region of complementarity or contiguity may be shorter.

[00225] Conditions under which two polynucleotides, or regions of a self-complementary polynucleotide, will form a duplex can be determined empirically or can be predicted using well known methods (taking into consideration base sequence, polynucleotide length, type of ester linkage [e.g., phosphorothioate or phosphodiester linkage], temperature, ionic strength, presence of modified bases or sugars, etc.). The annealing nucleic acid moieties in the associating CICs may be self-complementary or alternatively, a nucleic acid moiety(s) on one CIC may be complementary to a nucleic acid moiety(s) on a second CIC , but not to itself.

[00226] As noted above, examples of CIC multimers include multimers having a 'central axis' structure, a 'cage' structure, and a 'starfish' structure.' A 'central axis' structure refers to a dimer of two branched CICs, in which one nucleic acid moiety of each CIC forms a double- stranded region with a complementary nucleic acid moiety of the second CIC, and each spacer is bound to at least two other nucleic acid moieties.

[00227] A 'cage' structure refers to a CIC multimer in which at least two nucleic acid 5- prime moieties from each component CIC are hybridized to a nucleic acid moiety of another CIC in the multimer. In some embodiments, all of the 5 -prime moieties from one or all of the CICs are hybridized to a nucleic acid moiety of another CIC in the multimer. A 'cage' structure is characterized in that each of the nucleic acid 5-prime moieties in a duplex is linked to the spacer moiety with the same polarity (i.e., the spacer moitey-nucleic acid moiety linkage for each nucleic acid moiety in a particular duplex is either 3' or is 5'). In an embodiment, the cage structure CIC multimer contains no more than two CICs.

[00228] A 'starfish' structure has the same properties as the cage structure, supra, except (a) the starfish is always a dimer and (b) the two nucleic acid moieties in each duplex are linked to the spacer moieties with different polarities (i.e., one is linked at the 5' terminus and one is linked at the 3' terminus).

[00229] In each type of CIC multimer, it will be understood that nucleic acid moieties in the multimer may have any of the sequence, structural features or properties described herein for nucleic acid moieties, so long as the feature is consistent with the multimer structure. Thus, one or more nucleic acid moieties may be a 5-prime moiety, may include the sequence CG, TCG, or 5'^F-TCG (i.e., TCG in the 5-prime position of a 5-prime moiety), or have other sequence, motif or property described herein.

Spacer CICs

[00230] In a different aspect of the invention, the branched CIC comprises a structure in which one or more of the nucleic acid moieties in the CIC are covalently conjugated to the platform molecule through one or more spacer moieties S_n. Examples of suitable spacers used in the present invention are described herein.

[00231] For example, in one embodiment, S_n has the structure of a multimer comprising smaller units (e.g., oligoethylene glycols, [e.g., HO-(CH2CH2-O)_N-H, where N = 2-10; e.g., HEG and TEG], glycerol, l'2'-dideoxyribose, C2 alkyl - C12 alkyl subunits [preferably , C2 alkyl - ClO alkyl subunits], and the like), typically connected by an ester linkage (e.g., phosphodiester or phosphorothioate ester), e.g., as described infra. The multimer can be heteromeric or homomeric. In one embodiment, the spacer is a heteromer of monomeric units (e.g., HEG, TEG, glycerol, l'2'-dideoxyribose, C2 alkyl to C12 alkyl linkers, preferably C2 alkyl to ClO alkyl linkers, and the like) linked by an ester linkage (e.g., phosphodiester or phosphorothioate ester). Other examples of suitable spacers are described herein.

Immunomodulatory Activity of CICs

[00232] The CICs of the invention have immunomodulatory activity. The terms 'immunomodulatory,' 'immunomodulatory activity,' or 'modulating an immune response,' as used herein, include immunostimulatory as well as immunosuppressive effects. An immune response that is immuno stimulated according to the present invention is generally one that is shifted towards a 'Thl-type' immune response, as opposed to a 'Th2-type' immune response. An immunomodulated immune response according to the present invention may also be characterized by an inhibition of the Th2-type immune response in conjunction with a low or absent Thl-type response. Thl-type responses are typically considered cellular immune system (e.g., cytotoxic lymphocytes) responses, while Th2-type responses are generally 'humoral', or antibody-based. Thl-type immune responses are normally characterized by, for example, 'delayed-type hypersensitivity' reactions to an antigen. Thl-type responses can also be detected at a biochemical level by increased levels of one or more ThI -associated cytokines such as IFN-gamma, IFN-alpha, IL-2, IL- 12, and TNF-alpha, as well as IL-6, although IL-6 may also be associated with Th2-type responses as well. Th2-type immune responses are generally associated with higher levels of antibody production, including IgE production, an absence of or minimal CTL production, as well as expression of Th2- associated cytokines such as IL-4, IL-5 and IL- 13. IL-10 also plays a role in immunoregulation by down-regulating the expression of ThI cytokines and MHC class II antigens and enhancing B cell survival and antibody production, and inducing the development of regulatory T cells.

[00233] Immunomodulation in accordance with the invention may be recognized by measurements (assays) in vitro, in vivo and/or ex vivo. Examples of measurable immune responses indicative of immunomodulatory activity include, but are not limited to, antigen- specific antibody production, secretion of cytokines, activation or expansion of lymphocyte populations such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, presence or absence of eosinophils, changed patterns of co- stimulatory molecules on antigen- presenting cells, disease modification, and the like. See, e.g., WO 97/28259; WO 98/16247; WO 99/11275; Krieg et al. (1995) Nature 374:546-549; Yamamoto et al. (1992) J. Immunol. 148:4072-4076; Ballas et al. (1996) J. Immunol. 157:1840-1845; Klinman et al. (1997) J. Immunol. 158:3635-3639; Sato et al. (1996) Science 273:352-354; Pisetsky (1996) J. Immunol. 156:421-423; Shimada et al. (1986) Jpn. J. Cancer Res. 77:808-816; Cowdery et al.

(1996) J. Immunol. 156:4570-4575; Roman et al. (1997) Nat Med. 3:849-54; Lipford et al.

(1997) Eur. J. Immunol. 27:2340-2344; WO 98/55495, WO 00/61151, Pichyangkul et al. (2001) J. Imm. Methods 247:83-94, Hessel et al. (2005) J. Exp. Med. 202:1563-1573; Coffman et al. (2005) 201:1875-1879. Certain useful assays are described herein below for purposes of illustration and not for limitation.

[00234] Assays are generally carried out by administering or contacting a cell, tissue, animal or the like with a test sample (e.g., containing a CIC, polynucleotide, and/or other agent) and measuring a response. The test samples containing CICs or polynucleotides can be in a variety of forms or concentrations, which will be understood by the ordinarily skilled practitioner to be appropriate for the assay type. For example, for purposes of a cell-based assay, CICs or polynucleotides are often used at a concentration of 20 ug/ml or 10 ug/ml or 2 ug/ml. Alternatively, in cell-based assays, CICs or polynucleotides can be tested in concentration ranges based on micromolar concentration of CIC or polynucleotide. Typically, for the purposes of the assay, concentration is determined by measuring absorbance at 260 nm and using the conversion 0.5 OD₂₆o/ml = 20 ug/ml. This normalizes the amount of total nucleic acid in the test sample and may be used, for example, when the spacer moiety does not have a significant absorbance at 260 nm. Alternatively, concentration or weight can be measured by other methods known in the art. If desired, the amount of nucleic acid moiety can be determined by measuring absorbance at 260 nm, and the weight of the CIC calculated using the molecular formula of the CIC. This method is sometimes used when the ratio of weight contributed by the spacer moiety(s) to weight contributed by the nucleic acid moieties in a CIC is high (i.e., greater than 1).

[00235] It will similarly be understood that positive and negative controls are useful in assays for immunomodulatory activity. Suitable positive controls for immunomodulatory activity are the immunomodulatory phosphorothioate DNA having the sequences 5'- TGACTGTGA ACGTTCGAGATG A-3' (SEQ ID NO:25) and 5'-

TCGTCGAACGTTCGAGATGAT -3' (SEQ ID NO:26), although other suitable positive controls with immunomodulatory activity will be apparent to the ordinarily skilled practitioner. One suitable negative control is no test agent (i.e., excipient or media alone, also referred to as 'cells alone' for certain in vitro assays). Alternatively, a phosphorothioate DNA having the sequence 5'-TGACTGTGAACCTTAGAGATGA-S' is used as a negative control in some embodiments. Alternatively, a phosphorothioate DNA having the sequence 5'-TGCTTGCAAGCTTGCAAGCA-S' is used as a negative control in some embodiments. Other negative controls can be designed by the practitioner guided by the disclosure herein and ordinary assay design.

[00236] One useful class of assays is 'cytokine response assays.' An exemplary assay for immuno stimulatory activity measures the cytokine response of human peripheral blood mononuclear cells ('PBMCs') (e.g., as described in Bohle et al. [1999], Eur. J. Immunol. 29:2344-53; Verthelyi et al. [2001] J. Immunol. 166:2372-77). In one embodiment of this assay, peripheral blood is collected from one or more healthy human volunteers and PBMCs are isolated. Typically blood is collected by venipuncture using a heparinized syringe, layered onto a FICOLL^® (Amersham Pharmacia Biotech) cushion and centrifuged. Alternativley, blood is collected, a buffy coat prepared, and then layered onto a FICOLL® (Amersham Pharmacia Biotech) cushion and centrifuged. PBMCs are then collected from the FICOLL^® interface and washed twice with cold phosphate buffered saline (PBS). The cells are resuspended and cultured (e.g., in 48- or 96-well plates) at 2 x 10⁶ cells/mL in RPMI 1640 with 10% heat- inactivated human AB serum, 50 units/mL penicillin, 50 μg/mL streptomycin, 300 μg/mL glutamine, 1 mM sodium pyruvate, and 1 x MEM non-essential amino acids (NEAA) in the presence and absence of test samples or controls for 24 hours. Alternatively, the cells may be resuspended and cultured in RPMI 1640, 10% fetal bovine serum, 50 U/ml Penicillin, 50 ug/ml Streptomycin, 2 rnM L-glutamine, 10 rnM HEPES, 1 rnM Sodium Pyruvate.

[00237] Cell-free medium is collected from each well and assayed for IFN-gamma and/or IFN-alpha concentration. Immunomodulatory activity is detected when the amount of IFN- gamma secreted by PBMCs contacted with the test compound is significantly greater (e.g., at least about 2-fold greater, at least about 2.5-fold greater, at least about 3-fold greater, at least about 4-fold greater, at least about 5-fold greater) than the amount secreted by the PBMCs in the absence of the test compound or, in some embodiments, in the presence of an inactive control compound (e.g., 5'-TGACTGTGAACCTTAGAGATGA-S'). Conversely, a test compound does not have immunomodulatory activity if the amount of IFN-gamma secreted by PBMCs contacted with the test compound is not significantly greater (e.g., less than 2-fold greater) than in the absence of the test compound or, alternatively, in the presence of an inactive control compound (e.g., 5'-TGACTGTGAACCTTAGAGATGA-S').

[00238] When IFN-alpha concentration is assayed, the amount of IFN-alpha secreted by PBMCs contacted with the test compound is often significantly greater (e.g., in the case of IFN-alpha sometimes at least about 2-fold or at least about 3-fold greater) than the amount secreted by the PBMCs in the absence of the test compound or, in some embodiments, in the presence of an inactive control compound (e.g., 5'-TGACTGTGAACCTTAGAGATGA-S'). In some embodiments, the significantly increased IFN-alpha secretion level is at least about 5-fold, at least about 10-fold, or even at least about 20-fold greater than controls. Conversely, a test compound does not have immunomodulatory activity if the amount of IFN-alpha secreted by PBMCs contacted with the test compound is not significantly greater (e.g., less than 2-fold greater) than in the absence of the test compound or, alternatively, in the presence of an inactive control compound (e.g., 5 '-TG ACTGTG AACCTT AG AG ATGA- 3'). Alternatively, a phosphorothioate DNA having the sequence 5'- TGCTTGC A AGCTTGC A AGC A- 3' is used as a negative control in some embodiments.

[00239] Another useful class of assays are cell proliferation assays, e.g., B cell proliferation assays. The effect of an agent (e.g. a CIC) on B cell proliferation can be determined using any of a variety of assays known in the art. Another useful class of assays are cell cytokine production (assays, e.g., B cell cytokine production (assays. The effect of an agent (e.g. a CIC) on B cell cytokine production (e.g., IL-6, IL-IO) can be determined using any of a variety of assays known in the art.

[00240] To account for donor variation, e.g., in cell-based assays, such as cytokine and proliferation assays, preferably assays are carried out using cells (e.g., PBMCs) from multiple different donors. The number of donors is usually at least 2 (e.g. 2), preferably at least 4 (e.g. 4), sometimes at least 10 (e.g. 10). Immunomodulatory activity is detected when the amount of IFN-gamma secreted in the presence of the test compound (e.g. in at least half of the healthy donors tested, preferably in at least 75%, most preferably in at least 85%) is at least about 3-fold greater or at least about 5-fold greater than secreted in the absence of the test compound, or in some embodiments, than in the presence of an inactive control compound such as described supra.

[00241] Immunomodulatory activity may also be detected by measuring interferon- induced changes in expression of cytokines, chemokines and other genes in mammalian cells (e.g., PBMCs, bronchial alveolar lavage (BAL) cells, and other cells responsive to interferon). For example, expression of the chemokines interferon-induced-protein 1OkDa (IP-10), monokine induced by IFN-gamma (MIG) and monocyte chemotactic protein 1 (MCP-I) are increased in the presence of IFN-alpha and IFN-gamma. Expression of these proteins, or their corresponding mRNA, may be used as markers of immuno stimulatory activity in cultured cells or tissues or blood of animals to which a CIC has been administered. Expression of such markers can be monitored any of a variety of methods of assessing gene expression, including measurement of mRNAs (e.g., by quantitative PCR), immunoassay (e.g., ELISA), DNA microarrays, oligonucleotide microarrays, and the like.

[00242] Biological activity of CICs can also be measured by measuring the induction of gene products known to have antiviral activities, including 2'-5' Oligoadenylate synthetase (2'-5'OAS), Interferon-stimulated gene - 54kD (ISG-54kD), Guanylate binding protein-1 (GBP-I), MxA and MxB. Expression of these proteins, or their corresponding mRNA, may be used as markers of immunostimulatory activity in cultured cells or tissues or blood of animals to which a CIC has been administered. Expression of such markers can be monitored any of a variety of methods of assessing gene expression, including measurement of mRNAs (e.g., by quantitative PCR), immunoassay (e.g., ELISA), and the like. In vitro or ex vivo assays can also be carried out using mouse cells and in other mammalian cells. [00243] In some embodiments, immuno stimulatory CICs (i.e., CISCs) of the present invention are able to effect biological responses even at relatively high dose levels, thereby resulting in a extended dose curve. For example, CISCs of the present invention may induce production cytokines, such as IFN-alpha, IL-6 or IL-IO, by cells (such as by B-cells in PBMCs or splenocytes) even at relatively high levels of the CISC. This extended dose curve may allow the CISC to maintain a pharmacological effective amount of the produced cytokine over a broad concentration range of the CISC. This extended dose curve potentially may also result in a corresponding manner with the use of CIRCs of the present invention.

Nucleic Acid Moieties

[00244] The CICs of the invention comprise one or more polynucleotide sequences (also referred to herein as 'nucleic acid moieties'). The term 'nucleic acid moiety,' as used herein, refers to a nucleotide monomer (i.e., a mononucleotide) or polymer (i.e., comprising at least 2 contiguous nucleotides). As used herein, a nucleotide comprises (1) a purine or pyrimidine base linked to a sugar that is in an ester linkage to a phosphate group, or (2) an analog in which the base and/or sugar and/or phosphate ester are replaced by analogs, e.g., as described infra. In a CIC comprising more than one nucleic acid moiety, the nucleic acid moieties may be the same or different. The nucleic acid moieties may confer immunomodulatory activities. In one embodiment, the immunomodulation is immuno stimulation. In another embodiment, the immunomodulation is immunosuppression.

[00245] The next three sections describe characteristics of nucleic acid moieties such as length, the presence, and the position of sequences or sequence motifs in the moiety, as well as describing (without intending to limit the invention) the properties and structure of nucleic acid moieties and CICs containing the moieties.

Length

[00246] Usually, a nucleic acid moiety is from 1 to 100 nucleotides in length, although longer moieties are possible in some embodiments. In some embodiments, the length of one or more of the nucleic acid moieties in a CIC is less than 8 nucleotides (i.e., 1, 2, 3, 4, 5, 6 or 7 nucleotides). In various embodiments, a nucleic acid moiety (such as a nucleic acid moiety fewer than 8 nucleotides in length) is at least 2 nucleotides in length, often at least 3, at least 4, at least 5, at least 6, or at least 7 nucleotides in length. In other embodiments, the nucleic acid moiety is at least 10, at least 20, or at least 30 nucleotides in length. In one embodiment, the nucleic acid moiety is 7 nucleotides in length. In one embodiment, the nucleic acid moiety is 10 nucleotides in length. In other embodiments, the nucleic acid moiety is between 5- to 30-mers, between 6- to 12-mers or between 6- to 20-mers. In another embodiment, the nucleic acid moiety is 6-mer or greater, 7- mer or greater, 8- mer or greater, 9- mer or greater, 10- mer or greater, 11- mer or greater, 12- mer or greater, 15- mer or greater, 20- mer or greater, 25- mer or greater or 30- mer or greater. In some embodiments, the the nucleic acid moiety is a 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer,15-mer, 16- mer, 17-mer, 18-mer, 19-mer, 20-mer, 25-mer or 30-mer. In some embodiments, the branched CIC comprises one or more of the nucleic acid moieties that are 10-mers. In some embodiments, the branched CIC comprises both 7-mer and 10-mer nucleic acid moieties. In some embodiments, the branched CIC comprises two 7-mer nucleic acid moieties and one 10-mer nucleic acid moiety. In some embodiments, the branched CIC comprises two 10-mer nucleic acid moieties and one 7-mer nucleic acid moiety. In some embodiments, the branched CIC comprises only 7-mer nucleic acid moieties. In some embodiments, the branched CIC comprises only 10-mer nucleic acid moieties.

[00247] It is contemplated that, in some embodiments, a CIC will comprise at least one nucleic acid moiety shorter than 8 nucleotides. In some embodiments, all of the nucleic acid moieties in a CIC will be shorter than 8 nucleotides (e.g., having a length in a range defined by a lower limit of 2, 3, 4, 5, of 6 and an independently selected upper limit of 5, 6, or 7 nucleotides, where the upper limit is higher than the lower limit). For example, in one embodiment, specified nucleic acid moieties in a CIC (including all of the the nucleic acid moieties in the CIC) may be either 6 or 7 nucleotides in length. In one embodiment, the CIC comprises two spacer moieties and an intervening nucleic acid moiety that is less than 8 bases in length (e.g., 5, 6, or 7 bases in length). In some embodiments, a CIC will comprise at least one nucleic acid moiety that is at least 7 nucleotides. In some embodiments, a CIC will only include nucleic acid moieties that are at least 7 nucleotides. In some embodiments, a CIC will comprise at least one nucleic acid moiety that is at least 10 nucleotides. In some embodiments, a CIC will only include nucleic acid moieties that are at least 10 nucleotides. In some embodiments, the branched CIC comprises both 7-mer and 10-mer nucleic acid moieties. In some embodiments, the branched CIC comprises two 7-mer nucleic acid moieties and one 10-mer nucleic acid moiety. In some embodiments, the branched CIC comprises two 10-mer nucleic acid moieties and one 7-mer nucleic acid moiety. In some embodiments, the branched CIC comprises only 7-mer nucleic acid moieties. In some embodiments, the branched CIC comprises only 10-mer nucleic acid moieties.

[00248] It is contemplated that in a CIC comprising multiple nucleic acid moieties, the nucleic acid moieties can be the same or different lengths. In one embodiment, the length of one or more, or most (e.g., at least about 2, at least about 4, or at least about 25%, at least about 50%, at least about 75%) or all of the nucleic acid moieties in a CIC is fewer than 8 nucleotides, in some embodiments fewer than 7 nucleotides, in some embodiments fewer than 6 nucleotides, in some embodiments between 2 and 6 nucleotides, in some embodiments between 2 and 7 nucleotides, in some embodiments between 3 and 7 nucleotides, in some embodiments between 4 and 7 nucleotides, in some embodiments between 5 and 7 nucleotides, and in some embodiments between 6 and 7 nucleotides. In other embodiments, the CIC comprises nucleic acid moieties wherein the nucleic acid moieties are each independently between 5- to 30-mers, between 6- to 12-mers or between 6- to 20-mers.

[00249] As is discussed in greater detail infra, often at least one nucleic acid moiety of a CIC includes the sequence CG, e.g. TCG, or a CG-containing motif described herein. In one embodiment, at least one nucleic acid moiety comprises a CG-containing nucleic acid motif and is less than 8 nucleotides in length (e.g., has a specified length as described supra less than 8 nucleotides). In a related embodiment, none of the nucleic acid moieties in a CIC that are longer than 8 nucleotides comprise the sequence 'CG' or optionally the sequence 'TCG' or 'ACG' (i.e., all of the nucleic acid moieties in the CIC that comprise the sequence CG are less than 8 nucleotides in length). In an embodiment, at least one nucleic acid moiety in the CIC does not comprise a CG sequence.

Sequences and Motifs

[00250] As noted supra, a particular nucleic acid moiety can have a variety of lengths. In one embodiment, the nucleic acid moiety has a length shorter than 8 nucleotides. In one embodiment, the nucleic acid moiety has a length of 8 nucleotides or longer. In various embodiments at least one nucleic acid moiety of a CIC of the invention comprises a sequence as disclosed infra.

[00251] In the formulas provided below, all sequences are in the 5' -^3' direction and the following abbreviations are used: B = 5-bromocytosine; bU = 5-bromouracil; a-A = 2-amino- adenine; g = 6-thio-guanine; t = 4-thio-thymine; H = a modified cytosine comprising an electron- withdrawing group, such as halogen in the 5 position; and X = any nucleotide. In various embodiments, a cytosine (C) in a sequence referred to infra is replaced with N4- alkylcytosine, such as N4-ethylcytosine or N4-methylcytosine, or 5-hydroxycytosine. In various embodiments, a guanosine (G) in the formula is replaced with 7-deazaguanosine.

[00252] In CICs reported thus far, the presence of CG correlates with cytokine-inducing activity. Thus, in one embodiment, at least one nucleic acid moiety of a CIC comprises at least one 5'-cytosine, guanine-3' (5'-CG-3') sequence. The cytosine is not methylated at the C- 5 position and, preferably is not methylated at any position. In some embodiments, the C and/or the G of the CG motif may be replaced with a non-natural base, such as N4- alkylcytosine, such as N4-ethylcytosine or N4-methylcytosine, or 5-hydroxycytosine for cytosine (C), or 7-deazaguanosine for guanosine (G).

[00253] In one embodiment, one or more nucleic acid moieties comprises 3 to 7 bases. In one embodiment, the nucleic acid moiety comprises 3 to 7 bases and has the sequence 5'- [(X)o-2]TCG[(X)₂-₄]-3\ or 5'-TCG[(X)₂-₄]-3\ or 5'-TCG(A/T)[(X)i_₃]-3\ or 5'- TCG(A/T)CG(A/T)-3\ or 5'-TCGACGT-S' or 5'-TCGTCGA-3', wherein each X is an independently selected nucleotide. In some embodiments, the CIC contains at least 3, at least 10, at least 30 or at least 100 nucleic acid moieties having an aforementioned sequence.

[00254] In an embodiment, the nucleic acid moiety comprises the sequence 5 '-thymidine, cytosine, guanine-3' (5'-TCG-3'), for example (without limitation), the 3-mer TCG, the 4- mer TCGX (e.g., TCGA), the 5-mers TCGXX (e.g., TCGTC and TCGAT), the 6-mers TCGXXX, XTCGXX and TCGTCG, and the 7-mers TCGXXXX, XTCGXXX, XXTCGXX and TCGTCGX, where X is any base. Often, at least one nucleic acid moiety comprises the sequence 5'-thymidine, cytosine, guanine, adenosine-3' (5'-TCGA-3'), e.g., comprises a sequence 5'-TCGACGT-3'. In one embodiment, the nucleic acid moiety comprises a heptameric sequence 5'-TCGXCGX, 5'-TCGXTCG (e.g., 5'-TCGTTCG, 5'-TCGATCG, 5'- TCGCTCG, 5'-TCGGTCG), 5'-TCGXXCG, 5'-TCGCGXX, 5'-TCGTXXX, where X is any base. In some embodiments the aforementioned sequence is located at or near the 5-prime position of a CIC, e.g., 5'^F-TCGXCGX, 5'^F-TCGXTCG, 5'^F-TCGXXCG, 5'^F-TCGCGXX, 5'^F-TCGXTCG, 5'^F-TCGTTCG, 5'^F-TCGATCG, 5'^F-TCGCTCG, 5'^F-TCGGTCG, 5'^F- TCGTXXX. CICs comprising these sequences have been discovered to be particularly effective for induction of IFN secretion and/or B cell activity.

[00255] In some embodiments, a nucleic acid moiety comprises the following sequences:

[00256] In some embodiments, a nucleic acid moiety comprises the sequence 5'-ACGTTCG-3'; 5'-TCGTCG-3'; 5'-AACGTTC-3'; 5'-GACGTTC-3'; 5'-AACGTT-3'; 5'-GACGTT-3'; 5'-TCGTT-3'; 5'-CGTTCG-3'; 5'-TCGTCGA-3'; 5'-TCGXXX-3'; 5'-XTCGXX-3'; 5'-XXTCGX-3'; 5'-TCGAGA-3'; 5'-TCGTTT-3'; 5'-TTCGAG-3'; 5'-TTCGT-3'; 5'-TTCGC-3'; 5'-GTCGT-3'; 5'-ATCGT-3'; 5'-ATCGAT-3'; 5'-GTCGTT-3'; 5'-GTCGAC-3'; 5'-ACCGGT-3'; 5'-AABGTT-3'; 5'-AABGUT-3\ 5'-TCGTBG-3' where X is any nucleotide.

[00257] In some embodiments, a nucleic acid moiety comprises a sequence that is 5'- purine, purine, C, G, pyrimidine, pyrimidine-3'; 5 '-purine, purine, C, G, pyrimidine, pyrimidine, C, G-3'; or 5 '-purine, purine, C, G, pyrimidine, pyrimidine, C, C-3'; for example (all 5'->3'), GACGCT; GACGTC; GACGTT; GACGCC; GACGCU; GACGUC; GACGUU; GACGUT; GACGTU; AGCGTT; AGCGCT; AGCGTC; AGCGCC; AGCGUU; AGCGCU; AGCGUC; AGCGUT; AGCGTU; AACGTC; AACGCC; AACGTT; AACGCT; AACGUC; AACGUU; AACGCU; AACGUT; AACGTU; GGCGTT; GGCGCT; GGCGTC; GGCGCC; GGCGUU; GGCGCU; GGCGUC; GGCGUT; GGCGTU, AACGTT, AGCGTC, GACGTT, GGCGTT, AACGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC, GGCGCC, AGCGCT, GACGCT, GGCGCT, GGCGTT, and AACGCC. In some embodiments, a nucleic acid moiety comprises the sequence: 5'-purine, purine, cytosine, guanine, pyrimidine, pyrimidine, cytosine, cytosine-3' or 5 '-purine, purine, cytosine, guanine, pyrimidine, pyrimidine, cytosine, guanine-3'. [00258] In some embodiments, a nucleic acid moiety comprises a sequence (all 5' -^3') AACGTTCG; AACGTTCC; GACGTTCG; GACGTTCC; AACGUTCG; AABGTTCG; AABGUTCG and/or AABGTTBG.

[00259] In various embodiments, a nucleic acid moiety comprises the motif 5'-Xi X₂ A X₃ C G X₄ T C G-3' wherein X_x is T, G, C or B, wherein X₂ is T, G, A or U, wherein X₃ is T, A or C, wherein X₄ is T, G or U and wherein the sequence is not 5'-TGAACGTTCG-3' or 5'-GGAACGTTCG-3'. Examples include (all 5'->3'): TGAACGUTCG; TGACCGTTCG; TGATCGGTCG; TGATCGTTCG; TGAACGGTCG; GTAACGTTCG; GTATCGGTCG; GTACCGTTCG; GAACCGTTCG; BGACCGTTCG; CGAACGTTCG; CGACCGTTCG; BGAACGTTCG; TTAACGUTCG; TUAACGUTCG and TTAACGTTCG.

[00260] In various embodiments, a nucleic acid moiety comprises a sequence:

5 ' -TCGTCGAACGTTCGTTAACGTTCG-3 ' ; 5 ' -TGACTGTGAACGUTCGAGATGA-S ' ; 5 ' -TCGTCGAUCGUTCGTTAACGUTCG-S ' ; 5 ' -TCGTCGAUCGTTCGTUAACGUTCG-S ' ; 5 ' -TCGTCGUACGUTCGTTAACGUTCG-S ' ; 5'-TCGTCGAa-ACGUTCGTTAACGUTCG-S' (SEQ ID NO:28); 5 ' -TGATCGAACGTTCGTTAACGTTCG-S' ; 5 ' -TGACTGTGAACGUTCGGTATGA-S ; 5 ' -TGACTGTGACCGTTCGGTATGA-S ' ; 5 ' -TG ACTGTGATCGGTCGGTATGA-3 ' ; 5 ' -TCGTCGAACGTTCGTT-3 ' ; 5 ' -TCGTCGTGAACGTTCGAGATGA-S ' ; 5 ' -TCGTCGGTATCGGTCGGTATGA-S ' ; 5 ' -CTTCGAACGTTCGAGATG-3 ' ; 5'-CTGTGATCGTTCGAGATG-S' (SEQ ID NO:37);

5'-TGACTGTGAACGGTCGGTATGA-S' ; 5'-TCGTCGGTACCGTTCGGTATGA-S' (SEQ ID NO:39); 5'-TCGTCGGAACCGTTCGGAATGA-S' (SEQ ID NO:40); 5'-TCGTCGAACGTTCGAGATG-S' (SEQ ID NO:41); 5'-TCGTCGTAACGTTCGAGATG-S' (SEQ ID NO:42); 5'-TGACTGTGACCGTTCGGAATGA-S' (SEQ ID NO:43); 5'-TCGTCGAACGTTCGAACGTTCG-S' (SEQ ID NO:44); 5'-TBGTBGAACGTTCGAGATG-S' (SEQ ID NO:45); 5'-TCGTBGAACGTTCGAGATG-S' (SEQ ID NO:46); 5'-TCGTCGACCGTTCGGAATGA-S' (SEQ ID NO:47); 5'-TBGTBGACCGTTCGGAATGA-S' (SEQ ID NO:48); 5'-TCGTBGACCGTTCGGAATGA-S' (SEQ ID NO:49); 5'-TTCGAACGTTCGTTAACGTTCG-S' (SEQ ID NO:50); 5'-CTTBGAACGTTCGAGATG-S' (SEQ ID NO:51); 5'-TGATCGTCGAACGTTCGAGATG-S' (SEQ ID NO:52).

[00261] In some embodiments, a nucleic acid moiety comprises the sequence: 5'-Xi X₂ A X₃ B G X₄ T C G-3' (SEQ ID NO:53), wherein Xi is T, G, C or B, wherein X₂ is T, G, A or U, wherein X₃ is T, A or C, wherein X₄ is T, G or U. In some embodiments, the nucleic acid moiety is not 5'-TGAABGTTCG-3' (SEQ ID NO:54). Examples include (all 5'->3'): TGAABGUTCG (SEQ ID NO:55); TGACBGTTCG (SEQ ID NO:56); TGATBGGTCG (SEQ ID NO:57); GTATBGGTCG (SEQ ID NO:58); GTACBGTTCG (SEQ ID NO:59); GAACBGTTCG (SEQ ID NO:60); GAAABGUTCG (SEQ ID NO:61); BGACBGTTCG (SEQ ID NO:62); CGAABGTTCG (SEQ ID NO:63); BGAABGTTCG (SEQ ID NO:64); BGAABGUTCG (SEQ ID NO:65); TTAABGUTCG (SEQ ID NO:66); TUAABGUTCG (SEQ ID NO:67) and TTAABGTTCG (SEQ ID NO:68).

[00262] In some embodiments, a nucleic acid moiety comprises the sequence:

5'-TGACTGTGAABGUTCGAGATGA-S' (SEQ ID NO:69); 5'-TCGTCGAABGTTCGTTAABGTTCG-S' (SEQ ID NO:70); 5'-TGACTGTGAABGUTCGGTATGA-S' (SEQ ID NO:71); 5'-TGACTGTGAABGUTCGGAATGA-S' (SEQ ID NO:72); 5'-TCGTCGGAAABGUTCGGAATGA-S' (SEQ ID NO:73); 5'-TCGTBGAABGUTCGGAATGA-S' (SEQ ID NO:74).

[00263] In some embodiments, a nucleic acid moiety comprises the sequence: 5'-Xi X₂ A X₃ C G X₄ T C G-3' (SEQ ID NO:75) wherein Xi is T, C or B, wherein X₂ is T, G, A or U, wherein X₃ is T, A or C, wherein X₄ is T, G or U. In some embodiments, the formula is not 5'-TGAACGTTCG-3'

[00264] In other embodiments, the nucleic acid moiety comprises the sequence:

5'-TGACTGTGAABGTTCGAGATGA-S' (SEQ ID NO:76); 5'-TGACTGTGAABGTTBGAGATGA-S' (SEQ ID NO:77); 5'-TGACTGTGAABGTTCCAGATGA-S' (SEQ ID NO:78);

5'-TGACTGTGAACGTUCGAGATGA-S' (SEQ ID NO:79); 5'-TGACTGTGAACGbUTCGAGATGA-S' (SEQ ID NO:80); 5'-TGACTGTGAABGTTCGTUATGA-S' (SEQ ID NO:81); 5'-TGACTGTGAABGTTCGGTATGA-S' (SEQ ID NO: 82); 5'-CTGTGAACGTTCGAGATG-S' (SEQ ID NO: 83); 5'-TBGTBGTGAACGTTCGAGATGA-S' (SEQ ID NO: 84); 5'-TCGTBGTGAACGTTCGAGATGA-S' (SEQ ID NO: 85); 5'-TGACTGTGAACGtTCGAGATGA-S' (SEQ ID NO: 86); 5'-TGACTGTGAACgTTCgAGATGA-S' (SEQ ID NO:87); 5'-TGACTGTGAACGTTCGTUATGA-S' (SEQ ID NO:88); 5'-TGACTGTGAACGTTCGTTATGA-S' (SEQ ID NO: 89); 5'-TCGTTCAACGTTCGTTAACGTTCG-S' (SEQ ID NO:90); 5'-TGATTCAACGTTCGTTAACGTTCG-S' (SEQ ID NO:91); 5'-CTGTCAACGTTCGAGATG-S' (SEQ ID NO:92); 5'-TCGTCGGAACGTTCGAGATG-S' (SEQ ID NO:93); 5'-TCGTCGGACGTTCGAGATG-S' (SEQ ID NO:94); 5'-TCGTCGTACGTTCGAGATG-S' (SEQ ID NO:95); 5'-TCGTCGTTCGTTCGAGATG-S' (SEQ ID NO:96).

[00265] In some embodiments, with respect to any of the sequences disclosed supra, the nucleic acid moiety further comprises one, two, three or more TCG and/or TBG and/or THG, sequences, preferably 5' to the sequence provided supra. The TCG(s) and/or TBG(s) may or may not be directly adjacent to the sequence shown. For example, in some embodiments, a nucleic acid moiety includes any of the following: 5'-TCGTGAACGTTCG-S' (SEQ ID NO:97); 5'-TCGTCGAACGTTCG-S' (SEQ ID NO:98); 5'-TBGTGAACGTTCG-S' (SEQ ID NO:99); 5-TBGTBGAACGTTCG-3' (SEQ ID NO: 100); 5'-TCGTTAACGTTCG-S' (SEQ ID NO: 101). In some embodiments, the additional TCG and/or TBG sequence(s) is immediately 5' and adjacent to the reference sequence. In other embodiments, there is a one or two base separation.

[00266] In some embodiments, a nucleic acid moiety has the sequence: 5'-(TCG)_w N_yA X₃ C G X₄ T C G-3' (SEQ ID NO: 102) wherein w is 1-2, wherein y is 0-2, wherein N is any base, wherein X₃ is T, A or C, wherein X₄ is T, G or U. [00267] In some embodiments, the nucleic acid moiety comprises any of the following sequences: TCGAACGTTCG (SEQ ID NO: 103); TCGTCGAACGTTCG (SEQ ID NO:98); TCGTGAACGTTCG (SEQ ID NO:97); TCGGTATCGGTCG (SEQ ID NO: 106); TCGGTACCGTTCG (SEQ ID NO: 107); TCGGAACCGTTCG (SEQ ID NO: 108); TCGGAACGTTCG (SEQ ID NO: 109); TCGTCGGAACGTTCG (SEQ ID NO: 110); TCGTAACGTTCG (SEQ ID NO: 111); TCGACCGTTCG (SEQ ID NO: 112); TCGTCGACCGTTCG (SEQ ID NO: 113); TCGTTAACGTTCG (SEQ ID NO: 101) ; TCGAACGTT; TCGAACGTTC; TCGAACGTTT.

[00268] In some embodiments, a nucleic acid moiety comprises any of the following sequences: 5'-(TBG)_z N_y A X₃ C G X₄ T C G-3' (SEQ ID NO: 115) wherein z is 1-2, wherein y is 0-2, wherein B is 5-bromocytosine, wherein N is any base, wherein X₃ is T, A or C, wherein X₄ is T, G or U.

[00269] In some embodiments, a nucleic acid moiety comprises: TBGTGAACGTTCG (SEQ ID NO:99); TBGTBGTGAACGTTCG (SEQ ID NO: 117); TBGAACGTTCG (SEQ ID NO: 118); TBGTBGAACGTTCG (SEQ ID NO: 100); TBGACCGTTCG (SEQ ID NO: 119); TBGTBGACCGTTCG (SEQ ID NO: 120).

[00270] In some embodiments, a nucleic acid moiety comprises any of the following sequences: 5'-T C G T B G NyA X₃ C G X₄T C G-3' (SEQ ID NO: 121) wherein y is 0-2, wherein B is 5-bromocytosine, wherein N is any base, wherein X₃ is T, A or C, wherein X₄ is T, G or U. In some embodiments, the nucleic acid moiety comprises any of the following sequences: TCGTBGTGAACGTTCG (SEQ ID NO: 122); TCGTBGAACGTTCG (SEQ ID NO: 123); TCGTBGACCGTTCG (SEQ ID NO: 124).

[00271] In some embodiments, a nucleic acid moiety comprises any of the following sequences: 5'-(TCG)_w N_y A X₃ B G X₄T C G-3' (SEQ ID NO:125) wherein w is 1-2, wherein y is 0-2, wherein N is any base, wherein X₃ is T, A or C, wherein X₄ is T, G or U. In some embodiments, the nucleic acid moiety comprises any of the following sequences: TCGGAAABGTTCG (SEQ ID NO: 126) or TCGAABGTTCG (SEQ ID NO: 127).

[00272] In some embodiments, a nucleic acid moiety comprises any of the following sequences: 5'-(TBG)_z N_yA X₃ B G X₄T C G-3' (SEQ ID NO:128) wherein z is 1-2, wherein y is 0-2, wherein B is 5-bromocytosine, wherein N is any base, wherein X₃ is T, A or C, wherein X₄ is T, G or U. In some embodiments, the nucleic acid moiety comprises any of the following sequences: TBGAABGUTCG (SEQ ID NO: 129) or TBGAABGTTCG (SEQ ID NO:130).

[00273] In some embodiments, a nucleic acid moiety comprises any of the following sequences: 5'-T C G T B G NyA X₃ B G X₄T C G-3' (SEQ ID NO:131) wherein y is 0-2, wherein B is 5-bromocytosine, wherein N is any base, wherein X₃ is T, A or C, wherein X₄ is T, G or U. In some embodiments, the nucleic acid moiety comprises any of the following sequences: TCGTBGAABGUTCG (SEQ ID NO: 132) or TCGTBGAABGTTCG (SEQ ID NO:133).

[00274] In some embodiments, a nucleic acid moiety comprises the sequence: AACGTTCC, AACGTTCG, GACGTTCC, GACGTTCG.

[00275] In some embodiments, a nucleic acid moiety comprises the sequence: GGCGTTCG; GGCGCTCG; GGCGTCCG; GGCGCCCG; GACGTTCC; GACGCTCC; GACGTCCC; GACGCCCC; AGCGTTCC; AGCGCTCC; AGCGTCCC; AGCGCCCC; AACGTTCC; AACGCTCC; AACGTCCC; AACGCCCC; GGCGTTCC; GGCGCTCC; GGCGTCCC; GGCGCCCC; GACGTTCG; GACGCTCG; GACGTCCG; GACGCCCG; AGCGTTCG; AGCGCTCG; AGCGTCCG; AGCGCCCG; AACGTTCG; AACGCTCG; AACGTCCG; AACGCCCG; GACGCTCC; GACGCCC; AGCGTTCC; AGCGCTCC; AGCGTCCC; AGCGCCCC; AACGTCCC; AACGCCCC; GGCGTTCC; GGCGCTCC; GGCGTCCC; GGCGCCCC; GACGCTCG; GACGTCCG; GACGCCCG; AGCGTTCG; AGCGTCCG; AGCGCCCG; AACGTCCG; AACGCCCG.

[00276] In some embodiments, a nucleic acid moiety comprises the sequence: (5' -^3') TCGTCGA; TCGTCG; TCGTTT; TTCGTT; TTTTCG; ATCGAT; GTCGAC; GTCGTT; TCGCGA; TCGTTTT; TCGTC; TCGTT; TCGT; TCG; ACGTTT; CCGTTT; GCGTTT; AACGTT; TCGAAAA; TCGCCCC; TCGGGGG; GACGTTC; GACGTCC; or AGCGCTC.

[00277] In some embodiments, a nucleic acid moiety comprises an RNA of the sequence AACGUUCC, AACGUUCG, GACGUUCC, and GACGUUCG.

[00278] In some embodiments, a nucleic acid moiety has a sequence comprising a sequence or sequence motif described in copending coassigned U.S. patent applications

09/802,685 (published as U.S. Application Publication No. 20020028784A1 on March 7, 2002 and as WO 01/68077 on September 20, 2001); 09/802,359 ( published as WO 01/68144 on September 20, 2001), and copending U.S. Application Serial No. 10/033,243, or in PCT publications WO 97/28259, WO 98/16247; WO 98/55495; WO 99/11275; WO 99/62923; and WO 01/35991. The nucleic acid moiety can also have a sequence comprising any or several of the sequences previously reported to be correlated with immuno stimulatory activity when administered as a polynucleotide greater (often substantially greater) than 8 nucleotides in length, e.g., Kandimalla et al., 2001, Bioorg. Med. Chem. 9:807-13; Krieg et al. (1989) J. Immunol. 143:2448-2451; Tokunaga et al. (1992) Microbiol. Immunol. 36:55-66; Kataoka et al. (1992) Jpn. J. Cancer Res. 83:244-247; Yamamoto et al. (1992) J. Immunol. 148:4072-4076; Mojcik et al. (1993) Clin. Immuno. and Immunopathol. 67:130-136; Branda et al. (1993) Biochem. Pharmacol. 45:2037-2043; Pisetsky et al. (1994) Life ScL 54(2): 101- 107; Yamamoto et al. (1994a) Antisense Research and Development. 4:119-122; Yamamoto et al. (1994b) Jpn. J. Cancer Res. 85:775-779; Raz et al. (1994) Proc. Natl. Acad. ScL USA 91:9519-9523; Kimura et al. (1994) /. Biochem. (Tokyo) 116:991-994; Krieg et al. (1995) Nature 374:546-549; Pisetsky et al. (1995) Ann. NY. Acad. ScL 772:152-163; Pisetsky (1996a) J. Immunol. 156:421-423; Pisetsky (1996b) Immunity 5:303-310; Zhao et al. (1996) Biochem. Pharmacol. 51:173-182; Yi et al. (1996) J. Immunol. 156:558-564; Krieg (1996) Trends Microbiol. 4(2):73-76; Krieg et al. (1996) Antisense Nucleic Acid Drug Dev. 6:133- 139; Klinman et al. (1996) Proc. Natl. Acad. ScL USA. 93:2879-2883; Raz et al. (1996); Sato et al. (1996) Science 273:352-354; Stacey et al. (1996) J. Immunol. 157:2116-2122; Ballas et al. (1996) J. Immunol. 157:1840-1845; Branda et al. (1996) J. Lab. Clin. Med. 128:329-338; Sonehara et al. (1996) J. Interferon and Cytokine Res. 16:799-803; Klinman et al. (1997) J. Immunol. 158:3635-3639; Sparwasser et al. (1997) Eur. J. Immunol. 27:1671-1679; Roman et al. (1997) Nat Med. 3:849-54; Carson et al. (1997) J. Exp. Med. 186:1621-1622; Chace et al. (1997) Clin. Immunol, and Immunopathol. 84:185-193; Chu et al. (1997) J. Exp. Med. 186:1623-1631; Lipford et al. (1997a) Eur. J. Immunol. 27:2340-2344; Lipford et al. (1997b) Eur. J. Immunol. 27:3420-3426; Weiner et al. (1997) Proc. Natl. Acad. ScL USA 94:10833- 10837; Macfarlane et al. (1997) Immunology 91:586-593; Schwartz et al. (1997) J. Clin. Invest. 100:68-73; Stein et al. (1997) Antisense Technology, Ch. 11 pp. 241-264, C. Lichtenstein and W. Nellen, Eds., IRL Press; Wooldridge et al. (1997) Blood 89:2994-2998; Leclerc et al. (1997) Cell. Immunol. 179:97-106; Kline et al. (1997) J. Invest. Med. 45(3):282A; Yi et al. (1998a) J. Immunol. 160:1240-1245; Yi et al. (1998b) J. Immunol. 160:4755-4761; Yi et al. (1998c) J. Immunol. 160:5898-5906; Yi et al. (1998d) J. Immunol. 161:4493-4497; Krieg (1998) Applied Antisense Oligonucleotide Technology Ch. 24, pp. 431-448, CA. Stein and A.M. Krieg, Eds., Wiley-Liss, Inc.; Krieg et al. (1998a) Trends Microbiol. 6:23-27; Krieg et al. (1998b) J. Immunol. 161:2428-2434; Krieg et al. (1998c) Proc. Natl. Acad. ScL USA 95:12631-12636; Spiegelberg et al. (1998) Allergy 53(45S):93-97; Homer et al. (1998) Cell Immunol. 190:77-82; Jakob et al. (1998) /. Immunol. 161:3042- 3049; Redford et al. (1998) /. Immunol. 161:3930-3935; Weeratna et al. (1998) Antisense & Nucleic Acid Drug Development 8:351-356; McCluskie et al. (1998) /. Immunol. 161(9):4463-4466; Gramzinski et al. (1998) Mol.Med. 4:109-118; Liu et al. (1998) Blood 92:3730-3736; Moldoveanu et al. (1998) Vaccine 16: 1216-1224; Brazolot Milan et al. (1998) Proc. Natl. Acad. ScL USA 95:15553-15558; Briode et al. (1998) /. Immunol. 161:7054- 7062; Briode et al. (1999) Int. Arch. Allergy Immunol. 118:453-456; Kovarik et al. (1999) /. Immunol. 162:1611-1617; Spiegelberg et al. (1999) Pediatr. Pulmonol. Suppl. 18:118-121; Martin-Orozco et al. (1999) Int. Immunol. 11:1111-1118; EP 468,520; WO 96/02555; WO 97/28259; WO 98/16247; WO 98/18810; WO 98/37919; WO 98/40100; WO 98/52581; WO 98/55495; WO 98/55609 and WO 99/11275. See also Elkins et al. (1999) /. Immunol. 162:2291-2298, WO 98/52962, WO 99/33488, WO 99/33868, WO 99/51259 and WO 99/62923. See also Zimmermann et al. (1998) /. Immunol. 160:3627-3630; Krieg (1999) Trends Microbiol. 7:64-65 and U.S. Patent Nos. 5,663,153, 5,723,335 and 5,849,719. See also Liang et al. (1996) /. Clin. Invest. 98:1119-1129; Bohle et al. (1999) Eur. J. Immunol. 29:2344-2353 and WO 99/56755. See also WO 99/61056; WO 00/06588; WO 00/16804; WO 00/21556; WO 00/54803; WO 00/61151; WO 00/67023; WO 00/67787 and U.S. Patent No. 6,090,791. In one embodiment, at least one nucleic acid moiety of a CIC comprises a TG sequence or a pyrimidine-rich (e.g., T-rich or C-rich) sequence, as described in PCT publication WO 01/22972.

[00279] In some embodiments, the nucleic acid moiety is other than one or more of the hexamers 5'-GACGTT-3\ 5'-GAGCTT-3\ 5'-TCCGGA-3\ 5'-AACGTT-3\ 5' -GACGTT- 3', 5'-TACGTT-3, '5'-CACGTT-S', 5'-AGCGTT-3', 5'-ATCGTT-3', 5'-ACCGTT-3', 5'- AACGGT-3', 5'-AACGAT-3', 5'-AACGCT-3', 5'-AACGTG-3', 5'-AACGTA-3', and 5'- AACGTC-3'.

[00280] In some embodiments, the CIC contains at least 3, at least 10, at least 30 or at least 100 nucleic acid moieties having a sequence described above. [00281] In some embodiments, a CIC of the present invention is a CISC, wherein the oligonucleotides contained in the CISC do not include an immunoregulatory sequence (IRS), such as those described herein. In some embodiments, a CIC of the present invention is a CIRC, wherein the oligonucleotides contained in the CIRC do not include an immuno stimulatory sequence (ISS), such as those described herein.

Nucleic Acid Moiety Sequences: Heterogeneity and Position

[00282] It is contemplated that in a CIC comprising multiple nucleic acid moieties, the nucleic acid moieties can be the same or different. In one embodiment, all of the nucleic acid moieties in a CIC have the same sequence. In one embodiment, a CIC comprises nucleic acid moieties with at least 2, at least 3, or at least 4 different sequences. In one embodiment, each of the nucleic acid moieties in a CIC has a different sequence.

[00283] In some embodiments, a single nucleic acid moiety contains more than one iteration of a sequence motif listed above or two or more different sequence motifs. The motifs within a single nucleic acid moiety can be adjacent, overlapping, or separated by additional nucleotide bases within the nucleic acid moiety. In an embodiment, a nucleic acid moiety includes one or more palindromic regions. In certain embodiments and in the context of single- stranded oligonucleotides, the term 'palindromic' refers to a sequence that is self- complementary and can form a double- stranded molecule with itself or with another strand of itself. In another embodiment, one nucleic acid moiety has a sequence that is complementary or partially complementary in relation to a second nucleic acid moiety in the CIC. In an embodiment of the invention, the sequence of one or more of the nucleic acid moieties of a CIC is not palindromic. In an embodiment of the invention, the sequence of one or more of the nucleic acid moieties of a CIC does not include a palindromic sequence greater than four bases, optionally greater than 6 bases. In an embodiment of the invention, none of the nucleic acid moieties of the CIC include a palindromic sequence greater than four bases, optionally greater than 6 bases. In certain embodiments and in the context of single- stranded oligonucleotides, the term 'palindromic' refers to a sequence that would be palindromic if the oligonucleotide were complexed with a complementary sequence to form a double- stranded molecule. In another embodiment, one nucleic acid moiety has a sequence that is palindromic or partially palindromic in relation to a second nucleic acid moiety in the CIC. In an embodiment of the invention, the sequence of one or more of the nucleic acid moieties of a CIC is not palindromic. In an embodiment of the invention, the sequence of one or more of the nucleic acid moieties of a CIC does not include a palindromic sequence greater than four bases, optionally greater than 6 bases.

[00284] As described supra, in various embodiments, one or more (e.g., all) of the nucleic acid moieties in a CIC comprises a 5'-CG-3' sequence, alternatively a 5'-TCG-3' sequence. In one embodiment, the nucleic acid moiety is 5, 6 or 7 bases in length. In an embodiment, the nucleic acid moiety has the formula 5'-TCG[(X)₂-₄]-3' or 5'- TCG(AyT) [(X)_1-3] or 5'-TCG(A/T)CG(A/T)-3' or 5'-TCGACGT-3' (where each X is an independently selected nucleotide). In one embodiment, the aformentioned nucleic acid moiety is a 5 -prime moiety.

[00285] In one embodiment, a nucleic acid moiety comprises a sequence 5 ' -TCGTCGA- 3'. In one embodiment, a nucleic acid moiety comprises a sequence selected from (all 5'->3): TCGXXXX, TCGAXXX, XTCGXXX, XTCGAXX, TCGACGT, TCGAACG, TCGAGAT, TCGACTC, TCGAGCG, TCGATTT, TCGCTTT, TCGGTTT, TCGTTTT, TCGTCGT, TCGTTCG, TCGGCGC, TCGCCGG, TCGATCG, ATCGATT, TTCGTTT, TTCGATT, ACGTTCG, AACGTTC, TGACGTT, TGTCGTT, TCGXXX, TCGAXX, GTCGTT, GACGTT, ATCGAT, TCGTCG; TCGTTT; TCGAGA; TTCGAG; TTCGTT; AACGTT; AACGTTCG; AACGUTCG, ABGUTCG, TCGXX, TCGAX, TCGAT, TCGTT, TCGTC; TCGA, TCGT, and TCGX (where X is A, T, G or C; U is2'-deoxyuridine and B is5-bromo-2'-deoxycytidine).

[00286] In one embodiment, the nucleic acid moiety is a 7-mer having the sequence TCGXXXX, TCGAXXX, XTCGXXX, XTCGAXX, TCGTCGA, TCGACGT, TCGAACG, TCGAGAT, TCGACTC, TCGAGCG, TCGATTT, TCGCTTT, TCGGTTT, TCGTTTT, TCGTCGT, ATCGATT, TTCGTTT, TTCGATT, ACGTTCG, AACGTTC, TGACGTT, or TGTCGTT; or is a 6-mer having the sequence TCGXXX, TCGAXX, TCGTCG, AACGTT, ATCGAT, GTCGTT, or GACGTT; or is a 5-mer having the sequence TCGXX, TCGAX, TCGAT, TCGTT, or TCGTC; or is a 4-mer having the sequence TCGA, TCGT, or TCGX, or is a 3-mer having the sequence TCG, where X is A, T, G or C.

[00287] In one embodiment, at least about 25%, preferably at least about 50%, or at least about 75%, and sometimes all of the nucleic acid moieties in the CIC comprise at least one of the aforementioned sequences. In one embodiment, at least one nucleic acid moiety does not comprise a CG motif. In other embodiments, at least about 25%, sometimes at least about 50%, and sometimes at least about 75% of the nucleic acid moieties in the CIC are nucleic acid moieties that do not have a CG motif or, alternatively, a TCG motif.

[00288] The position of a sequence or sequence motif in a CIC can influence the immunomodulatory activity of the CIC, as is illustrated in the Examples, infra. In referring to the position of a sequence motif in a nucleic acid moiety of a CIC, the following terminology can be used: (1) In a CIC containing multiple nucleic acid moieties, a moiety with a free-5' end is referred to as 'a 5-prime moiety.' It will be appreciated that a single CIC may have multiple 5-prime moieties. (2) Within any particular nucleic acid moiety, a sequence or motif is in 'the 5-prime position' of the moiety when there are no nucleotide bases 5' to the reference sequence in that moiety. Thus, in the moiety with the sequence 5'- TCGACGT-3', the sequences T, TC, TCG and TCGA, are in 'the 5-prime position,' while the sequence GAC is not. By way of illustration, a CIC containing the sequence TCG in the 5-prime position of a nucleic acid moiety can render the CIC more active than an otherwise similar CIC with a differently positioned TCG motif. A CIC with a TCG sequence in a 5- prime moiety, e.g., at the 5-prime position of the 5-prime moiety can render a CIC particularly active. A nucleic acid moiety with a free 5' end can be designated using the symbol '5'^F' to the left of the formula for the base sequence of the nucleic acid moiety (e.g., 5'^F-TACG-3'). As used herein, the term 'free 5' end' in the context of a nucleic acid moiety has its usual meaning and means that the 5' terminus of the nucleic acid moiety is not conjugated to a blocking group or a non-nucleotide spacer moiety. The free 5 '-nucleoside contains an unmodified 5'-hydroxy group or a 5'-phosphate, 5 '-diphosphate, or 5'- triphosphate group, or other common modified phosphate groups (such as thiophosphate, dithiophosphate, and the like) that is not further linked to a blocking or functional group.

[00289] Immuno stimulatory activity can also be influenced by the position of a CG motif in a nucleic acid moiety (e.g., in a 5'-moiety). For example, in one embodiment the CIC contains at least one nucleic acid moiety with the sequence 5'-X-CG-Y-3' where X is zero, one, or two nucleotides and Y is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 nucleotides in length. In an embodiment, the 5'-X-CG- Y-3' sequence is in a 5'-moiety of the CIC, e.g., the 5-prime position of the CIC. In an embodiment, the CIC contains 2, 3 or more nucleic acid moieties with a sequence having the formula 5' -X-CG- Y-3' sequence. For example, in an embodiment, all of the nucleic acid moieties of the CIC have sequences of the formula 5'-X-CG- Y-3' sequence.

[00290] Similarly, a CIC including the sequence TCGA (e.g., a sequence including TCGACGT) in a nucleic acid moiety has immunomodulatory activity, and is effective in IFN-alpha induction, particularly in humans. A TCGA (e.g., a sequence including TCGACGT) in a 5-prime moiety, e.g., at the 5-prime position of the 5-prime moiety, renders the CIC particularly active. Thus, in one embodiment, a CIC comprises a core structure with the formula (5'-N₁^)-S₁-N₂ (Ia) where Ni has the sequence 5'-TCGAX-3' and X is 0 to 20 nucleotide bases, often 0 to 30 bases. In one embodiment, X is CGT.

[00291] The sequences TCGTCGA and TCGTTCG are also particularly effective in IFN-alpha induction and/or B cell activation, particularly in humans. In some preferred embodiments, the nucleic acid moiety including the IFN-alpha induction and/or B cell activation sequence motif may further comprise one or more additional nucleotide residues that are 5'- and/or 3'- to the motif. Such additional nucleotides may include one or more C and/or T residues, preferably C and/or T that are 3' with respect to the sequence motif. Such additional C and/or T residues may improve the synthesis of the nucleic acid moiety.

[00292] In some preferred embodiments, the additional nucleotide residues may comprise one or more T residues that are 5'- to, 3'- to, and/or within the sequence motif. Such additional T residues may increase the B cell activity of the sequence motif. In some preferred embodiments, the additional nucleotides can be a 5'-GAT-3' or 5'-CTT-3' or 5'- AAT-3', which may also increase B cell activity, that are positioned 3' of the sequence motif. In such preferred embodiments, the nucleic acid moiety comprises one or more of the 10-mer sequences 5'-TCGTCGACTT-S', 5'-TCGTCGAGAT-S' , 5'-TCGTGATCGT-S' and 5'- TCGTTCGAAT-3'.

[00293] Similarly, a CIC including one or more of the sequences AACGTTC or GACGTTC (e.g., a sequence including one or more of the foregoing sequences) in a nucleic acid moiety can be immunomodulatory, and effective for strong B cell activition in mice and humans. Such additional nucleotides may include one or more C and/or T residues, preferably C and/or T that are 5'- and/or 3'- with respect to the sequence motif. Such additional C and/or T residues may improve the synthesis of the nucleic acid moiety. Such additional residues may also increase the B cell activation activity. Thus, in some preferred embodiments, a nucleic acid moiety further comprises a T nucleotide residue 5'- to the sequence and a 5'-GT-3' that is 3'- to the sequence, such that the nucleic acid moiety comprises one or more of the 10-mer sequences 5'-TAACGTTCGT-S' or 5'- TGACGTTCGT-3'.

[00294] In addition, the presence of free (unconjugated) nucleic acid 5' -ends can affect immuno stimulatory activity. In various embodiments, a CIC of the invention comprises at least 1, at least 2, at least 3, at least four 5'ends. In some embodiments, the number of free 5'-ends is from 1 to 10, from 2 to 6, from 3 to 5, or from 4-5. In one embodiment, the number of free 5' ends is at least about 50 or at least about 100.

'Isolated Immunomodulatory Activity '

[00295] One property of a nucleic acid moiety is the 'isolated immunomodulatory activity' associated with the nucleotide sequence of the nucleic acid moiety. In some embodiments, a nucleic acid moiety of a CIC does not have 'isolated immunomodulatory activity,' or has 'inferior isolated immunomodulatory activity,' (i.e., when compared to the CIC), as described below.

[00296] The 'isolated immunomodulatory activity' of a nucleic acid moiety is determined by measuring the immunomodulatory activity of an isolated polynucleotide having the primary sequence of the nucleic acid moiety, and having the same nucleic acid backbone (e.g., phosphorothioate, phosphodiester, chimeric). For example, a CIC having the structure 'S'-TCGTCG-S'-HEG-S'-ACGTTCG-S'-HEG-S'-AGATGAT-S" contains three nucleic acid moieties. To determine the independent immunomodulatory activity of, for example, the first nucleic acid moiety in the CIC, a test polynucleotide having the same sequence (i.e., 5'-TCGTCG-3') and same backbone structure (e.g., phosphorothioate) is synthesized using routine methods, and its immunomodulatory activity (if any) is measured. Immunomodulatory activity can be determined using standard assays which indicate various aspects of the immune response, such as those described supra. Preferably the human PBMC assay described supra is used. As discussed supra, to account for donor variation, typically the assay is carried out using cells obtained from multiple donors. A polynucleotide does not have immunomodulatory activity (and the corresponding nucleic acid moiety does not have 'isolated immunomodulatory activity') when the amount of IFN-alpha secreted by PBMCs contacted with the polynucleotide is not significantly greater (e.g., less than about 2-fold greater) in the majority of donors than in the absence of the test compound or, (in some embodiments) in the presence of an inactive control compound (e.g., 5'- TGACTGTGAACCTTAGAGATGA-3'.) Alternatively, a phosphorothioate DNA having the sequence 5'-TGCTTGCAAGCTTGCAAGCA-S' is used as a negative control in some embodiments.

[00297] To compare the immunomodulatory activity of a CIC and an isolated polynucleotide, immunomodulatory activity is measured, preferably using the human PBMC assay described supra. Usually, the activity of two compounds is compared by assaying them in parallel under the same conditions (e.g., using the same cells), usually at a concentration of about 20 ug/ml. Alternatively, in cell-based assays, CICs or polynucleotides can be tested in concentration ranges based on micromolar concentration of CIC or polynucleotide. As noted supra, typically, concentration is determined by measuring absorbance at 260 nm and using the conversion 0.5 OD₂₆o/ml = 20 ug/ml. This normalizes the amount of total nucleic acid in the test sample. Alternatively, concentration or weight can be measured by other methods known in the art. If desired, the amount of nucleic acid moiety can be determined by measuring absorbance at 260, and the weight of the CIC calculated using the molecular formula of the CIC. This method is sometimes used when the ratio of weight contributed by the spacer moiety(s) to weight contributed by the nucleic acid moieties in a CIC is high (i.e., greater than 1). Alternatively, a concentration of 3 uM may be used, particularly when the calculated molecular weights of two samples being compared differ by more than 20%.

[00298] A nucleic acid moiety of a CIC is characterized as having 'inferior immunomodulatory activity,' when the test polynucleotide has less activity than the CIC to which it is compared. Preferably the isolated immunomodulatory activity of the test polynucleotide is no more than about 50% of the activity of the CIC, more preferably no more than about 20%, most preferably no more than about 10% of the activity of the CIC, or in some embodiments, even less.

[00299] For CICs with multiple (e.g., multiple different) nucleic acid moieties, it is also possible to determine the immunomodulatory activity (if any) of a mixture of test polynucleotides corresponding to the multiple nucleic acid moieties. The assay can be carried out using a total amount of test polynucleotide (i.e., in the mixture) which equals the amount of CIC used. Alternatively, an amount of each test polynucleotide, or each different test polynucleotide, in the mixture can be equal to the amount of the CIC in the assay. As noted in herein, to account for donor variation, preferably assays and analysis use PMBCs from multiple donors.

[00300] In one embodiment, one or more (e.g., at least about 2, at least about 4, or at least about 25%, at least about 50%, or all, measured individually or, alternatively, in combination) of the nucleic acid moieties in a CIC do not have isolated immunomodulatory activity. In one embodiment, one or more (e.g., at least about 2, at least about 4, or at least about 25%, at least about 50%, or all, measured individually or, alternatively, in combination) has inferior isolated immunomodulatory activity compared to the CIC.

[00301] In a related embodiment, a CIC comprises one or more nucleic acid moieties with isolated immunomodulatory activity. For example, in some embodiments, all or almost all (e.g., at least 90%, preferably at least 95%) of the nucleic acid moieties has isolated immunomodulatory activity. For example, a CIC comprising a multivalent spacer(s) can comprise more than 4, often more than 10, frequently at least about 20, at least about 50, at least about 100, at least about 400 or at least about 1000 nucleic acid moieties (e.g., at least about 2500) with isolated immuno stimulatory activity (e.g., having the sequence 5'-TGA CTG TGA ACG TTC GAG ATG A-3').

[00302] Thus, in a particular CIC, the number of nucleic acid moieties that have isolated immunomodulatory activity can be zero (0), one (1), 2 or more, 3 or more, fewer than 3, 4 or more, fewer than 4, 5 or more, fewer than 5, at least 10, at least about 20, at least about 50, at least about 100, at least about 400 or at least about 1000, all, or less than all, of the nucleic acid moieties of the CIC.

Immunoregulatory Activity of CIRCs

[00303] The invention provides CICs, which include chimeric immunoregulatory compounds ('CIRCs') and methods of regulating immune responses in individuals, particularly humans, using these CIRCs. In some variations, the CIRCs of the present invention comprise a modified immunoregulatory sequence ('IRS'). In some variations, the CIRCs comprise an unmodified IRS. In some variations, the CIRCs comprise both modified and unmodified IRSs. The CIRCs of the invention particularly inhibit innate immune responses, including those responses that involve signaling through TLR7 and/or TLR9. [00304] The invention further provides CIRCs that efficiently regulate immune cells, including human cells, in a variety of ways. CIRCs of the invention can effectively suppress cytokine production, including IFN-alpha and/or IL-6, from human cells. CIRCs of the invention suppress cell responses, including cytokine production, stimulated through TLR7 and/or TLR9 receptors. CIRCs described herein also can effectively suppress proliferation and/or maturation of cells stimulated with an immunostimulatory nucleic acid, including B cells and plasmacytoid dendritic cells. In some variations, the CIRCs comprise at least one modified immunoregulatory compounds. Thus, the CIRCs described herein are of use in the suppression of immune responses to ISNA such as microbial DNA present due to an infection or suppression of nucleic acid vectors administered for gene therapy purposes.

[00305] Provide herein are also methods of treating and preventing autoimmune disorders and chronic inflammatory disorders in an individual by administering a CIRC described herein to the individual. In some variations, the CIRC is administered in combination with another therapeutic agent. In some variations, the other therapeutic agent is a corticosteroid. In some variations, the CIRCs comprise at least one modified immunoregulatory compounds.

[00306] CIRCs are provided herein for regulating immune responses in individuals. Each CIRC described herein comprises at least one IRS. In some variations, the IRS is modified. In some variations, the IRS is unmodified. In some variations, the CIRC described herein comprises both modified and unmodified IRSs.

[00307] Compositions provided herein comprise a CIRC alone (or a combination of two or more CIRCs). In some variations, the CIRCs comprise a modified IRS. In some variations, the CIRCs comprise an unmodified IRS. In some variations, the CIRCs comprise both modified and unmodified IRSs. Compositions provided herein may comprise a CIRC and a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients, including buffers, are described herein and well known in the art. Remington: The Science and Practice of Pharmacy, 20th edition, Mack Publishing (2000).

[00308] In accordance with the present invention, a CIRC contains at least one IRS. In some instances, an IRS comprises a 5'-GC-3' sequence. In some instances, an IRS includes at least one TGC trinucleotide sequence at or near the 5' end of the polynucleotide (i.e., 5'- TGC). In some variations, the TGC trinucleotide sequence is about any of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polynucleotide. In some variations, the TGC trinucleotide sequence is less than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polynucleotide. In some instances, an IRS comprises a 5'-GGGG-3' sequence. In some instances, an IRS does not comprise a 5'-GGGG-3' sequence. Accordingly, in some instances, a CIRC does not comprise a 5'-GGGG-3' sequence. In some instances, a CIRC comprising a 5'-GGGG-3' sequence is particularly effective when used in the single- stranded form. In some instances, a CIRC comprising a 5'-GGGG-3' sequence is particularly effective when made with a phosphothioate backbone.

[00309] As demonstrated herein, particular CIRCs inhibit TLR-7 dependent cell responses. Also, particular CIRCs inhibit TLR9 dependent cell responses. In some variations, particular CIRCs inhibit TLR7 dependent cell responses and TLR-9 dependent cell responses. Accordingly, as used herein, "TLR7/9" refers to "TLR7 and TLR9." In some variations, certain CIRCs do not inhibit TLR4 dependent cell responses.

[00310] As is clearly conveyed herein, it is understood that, with respect to formulae described herein, any and all parameters are independently selected. For example, if x=0-2, y may be independently selected regardless of the values of x (or any other selectable parameter in a formula).

[00311] As demonstrated herein, one class of IRS discovered is particularly effective in inhibiting TLR9 dependent cell stimulation. Accordingly, IRS with this activity are referred to as "TLR9 class" IRS.

[00312] In some variations, an IRS may comprise a sequence of the formula: X1GGGGX2X3 wherein X₁, X₂, and X₃ are nucleotides, provided that if Xi= C or A, then X₂X₃ is not AA. In some variations, an IRS may comprise a sequence of the formula XiGGGGX₂X₃ wherein Xi is C or A. In some variations, an IRS may comprise a sequence of the formula: XiGGGGX₂X₃ wherein X₁, X₂, and X₃ are nucleotides, provided that if Xi= C or A, then X₂X₃ is not AA and wherein Xi is C or A.

[00313] In some variations, an IRS may comprise a sequence of the formula: GGN_nXiGGGGX₂X₃, wherein n is an integer from 1 to about 100 (preferably from 1 to about 20), each N is a nucleotide, and X₁, X₂, and X₃ are nucleotides, provided that if Xi= C or A, then X₂X₃ is not AA. In some variations, an IRS may comprise a sequence of the formula GGN_nXiGGGGX₂X₃ wherein Xi is C or A.

[00314] In some variations, an IRS may comprise a sequence of the formula: N₁TCCN_j(GG)]JNmXiGGGGX₂X₃, wherein each N is a nucleotide, wherein i is an integer from 1 to about 50, wherein j is an integer from 1 to about 50, k is 0 or 1, m is an integer from 1 to about 20, and X₁, X₂, and X₃ are nucleotides, provided that if Xi= C or A, then X₂X₃ is not AA. In some variations, an IRS may comprise a sequence of the formula N₁TCCN_j(GGXNmXiGGGGX₂X₃ wherein Xi is C or A.

[00315] In some variations, an IRS may comprise a sequence of the formula: XiX₂X₃GGGGAA, wherein X₁, X₂, and X₃ are nucleotides, provided that if X₃= C or A, then XiX₂ is not GG.

[00316] In some variations, the IRSs XiGGGGX₂X₃, GGN_nXiGGGGX₂X₃, N₁TCCN_j(GGXNmXiGGGGX₂X₃ or XiX₂X₃GGGGAA as defined herein may each further comprise at least one 5'-TGC-3'. In some variations, the 5'-TGC-3' is about 0-10 nucleotides from the 5' end IRS and/or IRP. In some variations, the TGC trinucleotide sequence is about any of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polynucleotide. In some variations, the TGC trinucleotide sequence is less than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polynucleotide. In some variations, the 5'-TGC-3' is a 5'-TGC nucleotide sequence at the 5' end.

[00317] Examples of IRS include the following sequences: 5'- TCCTAACGGGGAAGT-3' (SEQ ID NO:_); 5'-TCCTAAGGGGGAAGT-S' (SEQ ID NO:_); 5'-TCCTAACGGGGTTGT-S' (SEQ ID NO:_); 5'-TCCTAACGGGGCTGT-S' (SEQ ID NO:_); 5'-TCCTCAAGGGGCTGT-S' (SEQ ID NO:_); 5'- TCCTCAAGGGGTTGT-3' (SEQ ID NO:_); 5'-TCCTCATGGGGTTGT-S' (SEQ ID NO:_); 5'-TCCTGGAGGGGTTGT-S' (SEQ ID NO:_); 5'-TCCTGGAGGGGCTGT-S' (SEQ ID NO:_); 5'-TCCTGGAGGGGCCAT-S' (SEQ ID NO:_); 5'- TCCTGGAGGGGTCAT-3' (SEQ ID NO:_); 5'-TCCGGAAGGGGAAGT-S' (SEQ ID NO:_); and 5'-TCCGGAAGGGGTTGT-S' (SEQ ID NO:_).

[00318] As shown herein, some IRS are particularly effective in inhibiting TLR7 dependent cell stimulation. Accordingly, IRS with this activity are referred to as "TLR7 class" IRS. For example, an oligonucleotide comprising the sequence 5'- TGCTTGCAAGCTTGCAAGCA- 3' (SEQ ID NO:27) inhibits TLR7 dependent cell stimulation.

[00319] In some variations, an IRS comprises a fragment of 5'- TGCTTGCAAGCTTGCAAGCA- 3' and includes at least a 10 base palindromic portion thereof. In some variations, the CIRC consists of 5'-TGCTTGCAAGCTTGCAAGCA- 3'. For example, such sequences include the following sequences: 5'-

TGCTTGC AAGCTTGC AAG-3' (SEQ ID NO:28); 5'-TGCTTGCAAGCTTGCA-S' (SEQ ID NO:29); 5'-GCTTGCAAGCTTGCAAGCA-S' (SEQ ID NO:30); 5'- CTTGCAAGCTTGCAAGCA-3' (SEQ ID NO:31); and 5'-TTGCAAGCTTGCAAGCA-S' (SEQ ID NO:32).

[00320] In some variations, a CIRC effective in inhibiting TLR7 dependent cell stimulation includes an IRS consisting of a sequence of the formula: 5'-TGCN_m-3' (SEQ ID NO: 126), where N is a nucleotide, m is an integer from 5 to about 50 and wherein the sequence N₁-N_m comprises at least one GC dinucleotide. In some variations, such an IRP consists of the sequence 5'-TGCNmA-3' (SEQ ID NO: 127), the sequence 5'-TGCNmCA- 3'(SEQ ID NO:128), or the sequence 5 '-TGCNmGCA- 3' (SEQ ID NO:129). For example, in some variations, the IRS may consist of the following sequences: 5'-

TGCTTGCAAGCTAGCAAGCA-3' (SEQ ID NO:33); 5'-TGCTTGCAAGCTTGCTAGCA- 3' (SEQ ID NO:34); 5'-TGCTTGACAGCTTGACAGCA-S' (SEQ ID NO:35); 5'- TGCTTAGCAGCTATGCAGCA-3' (SEQ ID NO:36); or 5'- TGCAAGCAAGCTAGCAAGCA-3' (SEQ ID NO:37).

[00321] In some variations, the IRP comprises a sequence of the formula: 5'-TGCN_m-3' (SEQ ID NO: 194), where each N is a nucleotide, m is an integer from 5 to about 50 and wherein the sequence Nl-Nm. In some variations, the IRP further comprises the nucleotide sequence 5'-SiS₂S₃S₄-S', wherein S₁, S₂, S₃, and S₄ are independently G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing disrupts or prevents formation of tetrameric/quadruplex structure of G-quadruplexes. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing is a nucleotide or derivative thereof. Examples of molecules that are capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing included, but are not limited to, I, 7-deaza-dG, 7-deaza-2'- deoxyxanthosine, 7-deaza-8-aza-2' -deoxyguanosine, 2' -deoxynebularine, isodeoxyguanosine, 8-oxo-2' -deoxyguanosine. In some variations, at least one, two, three, or four of S₁, S₂, S3, and S₄ are G. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are a molecule that are capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are I. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are 7-deaza- dG. In some variations, S₁, S₂, S₃, and S₄ are G.

[00322] Other IRS sequences which are also effective in inhibiting TLR7 dependent cell signaling include the following: 5'-TGCAAGCTTGCAAGCTTG CAA GCT T-3' (SEQ ID NO:38); 5'-TGCTGCAAGCTTGCAGAT GAT-3' (SEQ ID NO:39); 5'- TGCTTGC AAGCTTGC AAGC-3' (SEQ ID NO:40); 5'-

TGCAAGCTTGCAAGCTTGCAAT-3' (SEQ ID NO:41); 5'-TGCTTGCAAGCTTG-S' (SEQ ID NO:42); 5'-AGCTTGCAAGCTTGCAAGCA-S' (SEQ ID NO:43); 5'- TACTTGC AAGCTTGCAAGCA-3' (SEQ ID NO:44); 5' -TGATTGC AAGCTTGC AAGCA- 3' (SEQ ID NO:45); 5'-AAATTGCAAGCTTGCAAGCA-S' (SEQ ID NO:46); 5'- TGCTGGAGGGGTTGT-3' (SEQ ID NO:47); 5'-AAATTGACAGCTTGACAGCA-S' (SEQ ID NO:48); 5'-TGATTGACAGCTTGACAGCA-S' (SEQ ID NO:49); 5'- TGATTGACAGATTGACAGCA-3' (SEQ ID NO:50); and 5'- TGATTGACAGATTGACAGAC-3' (SEQ ID NO:51).

[00323] CIRCs comprising IRSs XiGGGGX₂X₃, GGN_nXiGGGGX₂X₃, N₁TCCN_j(GGXNmXiGGGGX₂X₃ or XiX₂X₃GGGGAA as defined herein, wherein at least one G is replaced by 7-deaza-dG potentially have a particular effectiveness in inhibiting TLR9 dependent cell stimulation. For example, in some variations, the IRS may comprise the sequence 5'-TCCTGGAGZOGTTGT-S' (Z'=7-deaza-dG; SEQ ID NO:23). Other IRS sequences which are also effective in inhibiting TLR9 dependent cell signaling include the following: 5'-TGACTGTAGGCGGGGAAGATGA-S' (SEQ ID NO:_); 5'- GAGC AAGCTGGACCTTCC AT-3' (SEQ ID NO:_); and 5'-CCTCAAGCTTGAGZOG- 3' (Z'=7-deaza-dG; SEQ ID NO:_). [00324] In some variations, CIRCs of the present invention contain an IRS comprising inosine such as wherein at least one G is replaced with an inosine. In some variations, the inosine is deoxy-inosine. In some variations, the IRS contained in the CIRCs may comprise the sequence 5'-TGC TGC TCC TTG AGI GGT TGT TTG T-3\ wherein I is deoxy-inosine (SEQ ID NO: 169). In some variations, the IRS contained in the CIRCs may comprise the sequence 5'TGC TCC TTG AGI GGT TGT TTG T-3', wherein I is deoxy-inosine (SEQ ID NO: 172).

[00325] Another class of CIRCs of the present invention contains an IRS that potentially has particular effectiveness in inhibiting both TLR7 and TLR9 dependent cell stimulation. Accordingly, a CIRC with this activity is referred to as "TLR7/9 class" CIRC. In some instances, a combination of a TLR7 class CIRC with a TLR9 class CIRC results in a CIRC of the TLR7/9 class.

[00326] The TLR7/9 class of CIRC include those comprising the sequence 5'- TGCN_mTCCTGG AGGGGTTGT-3' (SEQ ID NO:6) where each N is a nucleotide and m is an integer from 0 to about 100, in some instances from 0 to about 50, preferably from 0 to about 20.

[00327] In some variations, a CIRC containing an IRS that comprises 5'-

TGCN_mTCCTGGAGGGGTTGT-3', wherein the sequence Ni - N_m comprises a fragment of the sequence 5'-TTGACAGCTTGACAGCA-S' (SEQ ID NO:_). A fragment of 5'-

TTGAC AGCTTGAC AGCA-3' (SEQ ID NO:_) is any portion of that sequence, for example, TTGAC or GCTTGA. In some variations, the fragment of 5'-

TTGAC AGCTTGAC AGCA-3' (SEQ ID NO:_) is from the 5' end of 5'-

TTGAC AGCTTGAC AGCA-3' (SEQ ID NO:_), including, for example, TTGAC or TTG.

[00328] In some variations, the CIRC comprises an IRS that comprises sequence 5'-

TGCRRZNYY-3' (SEQ ID NO: ), wherein Z is any nucleotide except C, wherein N is any nucleotide, wherein when Z is not G or inosine, N is guanosine or inosine. In other variations, the IRS of the CIRC comprises the sequence 5'-TGCRRZNpoly(Pyrimidine)-3' (SEQ ID

NO: ), wherein Z is any nucleotide except C, wherein N is any nucleotide, wherein when Z is not G or inosine, N is guanosine or inosine. [00329] Examples of CIRCs of the present invention that contain IRSs that are also effective in inhibiting TLR7/9 dependent cell signaling include the following: 5'- TGCTCCTGGAGGGGTTGT-3' (SEQ ID NO:52); 5'-TGCTTGTCCTGGAGGGGTTGT-S' (SEQ ID NO:53); 5'-TGCTTGACATCCTGGAGGGGTTGT-S' (SEQ ID NO:54); 5'- TGCTTGACAGCTTGACAGTCCTGGAGGGGTTGT-3' (SEQ ID NO:55); 5'- TGCTTGACAGCTTGATCCTGGAGGGGTTGT-3' (SEQ ID NO:56); 5'- TGCTTGAC AGCTTCCTGGAGGGGTTGT-3' (SEQ ID NO:57); 5'- TGCTTGACAGCTTGCTCCTGGAGGGGTTGT-3' (SEQ ID NO:58); 5'- TGCTTGACAGCTTGCTTGTCCTGGAGGGGTTGT-3' (SEQ ID NO:59); 5'- TGCTTGACAGCTTGACAGCATCCTGGAGGGGTTGT-3' (SEQ ID NO:60); 5'- TGCTTGACAGCTTGACAGCATCCTGGAGGGGTTGT-3' (SEQ ID NO:61); 5'- TGCTTGACAGCTTGACAGCATCCTGGAGGGGT-3' (SEQ ID NO:62); 5'- TGCTTGACAGCTTGACAGCATCCTGGAGGGG-3' (SEQ ID NO:63); 5'- TGCTTGCAAGCTTGCTCCTGGAGGGGTTGT-3' (SEQ ID NO:64); 5'- TGCTTGC AAGCTTCCTGGAGGGGTTGT-3' (SEQ ID NO:65); and 5'- TGCTTGCAAGCTTGCAAGCATCCTGGAGGGGTTGT-3' (SEQ ID NO:66).

[00330] In some embodiments, the CIRC of the present invention comprises one or more oligonucleotides consisting of or comprising any one of the following sequences or oligonucleotides: 5¹- TGC TCC TGG AGG GGT TGT-3', wherein the 5' TTGT is modified with 2'-0Me; 5¹- TGC TCC TG AGG GGA AGT TTG T-3', wherein the 5' TTGT is modified with 2'-0Me; 5¹- CTC CTG GAG GGG TTG T-3'; 5'- TCC TGG AGG GGT TGT-3'; 5'- TGC-HEG-TGG AGG GGT TGT-3', wherein HEG is hexaethyleneglycol; 5'- TGC-TEG-TGG AGG GGT TGT-3', wherein HEG is hexaethyleneglycol; 5'- TGC TCC TGG AGG GGT TGT-3', wherein all internucleotide linkages are phosphoramidite; 5'- TGC TTG CAA GCT TGC AAG CA-3'.

[00331] In some embodiments, the CIRC of the present invention comprises one or more oligonucleotides consisting of or comprising any one of the following sequences or oligonucleotides shown in Table C (wherein Z is 7-dezaz-dG, U is uracil, HEG is hexaethyleneglycol spacer,TEG is triethyleneglycol spacer, LNA is locked nucleic acid; d is an abasic residue, C6 is (CH₂)ό spacer, C12 is (CH₂)i2 spacer, M is 5-methyl-dC). [00332] In some embodiments, the CIRC contains an IRS of any of the following sequences: 5'-TGC TGC TCC TGG AGG GGT TGT TTG T-3' (SEQ ID NO: 164) 5'-TGC TGC TCC TTG AGG GGT TGT TTG T-3' (SEQ ID NO: 165) 5'-TGC TGC TCC TTG AGG GGT TGT-3' (SEQ ID NO: 166); or 5'-TGC TGC TCC TGG AGG GGT TGT-3' (SEQ ID

NO: 167).

[00333] Exemplary examples of IRS that are effective in suppressing TLR7 and/or TLR9 are found, for example, in PCT/US2005/030494, which is hereby incorporated by reference in its entirety. Such IRS can be contained in CIRCs of the present invention.

[00334] IRPs used in the invention can comprise one or more ribonucleotides (containing ribose as the only or principal sugar component) and/or deoxyribonucleotides (containing deoxyribose as the principal sugar component). The heterocyclic bases, or nucleic acid bases, which are incorporated in the IRP can be the naturally-occurring principal purine and pyrimidine bases, (namely uracil, thymine, cytosine, adenine and guanine). An IRP may be single stranded or double stranded DNA, as well as single or double- stranded RNA. An IRP may be linear, may be circular or include circular portions and/or may include a hairpin loop.

[00335] In some variations, an immunoregulatory polynucleotide is less than about any of the following lengths (in bases or base pairs): 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175; 150; 125; 100; 75; 60; 50; 40; 30; 25; 20; 15; 14; 13; 12; 11; 10; 9; 8; 7; 6; 5; 4. In some variations, an immunoregulatory polynucleotide is greater than about any of the following lengths (in bases or base pairs): 4; 5; 6, 7, 8, 9, 10; 11; 12; 13; 14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000; 7500; 10000; 20000; 50000. Alternately, the immunoregulatory polynucleotide can be any of a range of sizes having an upper limit of 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175; 150; 125; 100; 75; 60; 50; 40; 30; 25; 20; 15; 14; 13; 12; 11; 10; 9; 8; 7; 6; 5; 4 and an independently selected lower limit of 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000; 7500, wherein the lower limit is less than the upper limit. In some variations, an IRP is preferably about 200 or less bases in length.

Modified immunoregulatory polynucleotides and modified immunoregulatory compounds [00336] The invention further provides CIRCs comprising at least one modified IRS. A modified IRS comprises at least one modified nucleotide. The modification of at least one nucleotide may be a modified base, a modified sugar, and/or a modified phosphate. In some variations, the modification of at least one nucleotide may be a naturally-occurring modified. In some variations, the modification of at least one nucleotide may be a synthetic modification. In some variations, the modifications may be imparted before or after assembly of the polynucleotide. In some variations, the modified nucleotide comprises one or more modified nucleosides. "Modified nucleotide" or "modified nucleosides" are herein defined as being synonymous with nucleoside or nucleotide "analogs."

[00337] In some variations, the modification of at least one nucleotide comprises a modified base. As used herein, the term "modified base" is synonymous with "base analog", for example, "modified cytosine" is synonymous with "cytosine analog." Examples of base modifications include, but are not limited to, addition of an electron- withdrawing moiety to C- 5 and/or C-6 of a cytosine of the IRP. Preferably, the electron- withdrawing moiety is a halogen, e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine. In some variations, the base modifications include, but are not limited to, addition of an electron- withdrawing moiety to C- 5 and/or C-6 of a uracil of the immunoregulatory polynucleotide. Preferably, the electron-withdrawing moiety is a halogen. Such modified uracils can include, but are not limited to, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil. In some variations, the base modifications include the addition of one or more thiol groups to the base including, but not limited to, 6-thio-guanine, 4-thio-thymine, and 4-thio-uracil. In some variations, the base modifications include, but are not limited to, N4-ethylcytosine, 7- deazaguanine, and 5-hydroxycytosine. See, for example, Kandimalla et al. (2001) Bioorg. Med. Chem. 9:807-813. In some variations, the IRS may include 2'-deoxyuridine and/or 2- amino-2'-deoxyadenosine. In some variations, the modified base comprises a methylation modification. In some variations, the methylation modification comprises a 5'-methyl- cytosine modification. In some variations, an IRS comprises multiple base modifications. In some variations, the base modifications are the same. In some variations, the base modifications are different. In some variations, the IRS comprises any of about 1, about 2, about 3, about 4, about 5 different base modifications. Base modifications may also be made and combined with any phosphate modification and/or sugar modification in the preparation of a modified IRS contained in a CIRC of the present invention.. [00338] In some variations, the modification of at least one nucleotide comprises a modified phosphate. In some variations, the modified phosphate is a phosphodiester linkage modification. For example, phosphate modifications may include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoamidates, phosphoramidate (bridging or non- bridging), phosphotriester and phosphorodithioate and may be used in any combination. In some variations, the modified phosphate is a 3 '-terminal internucleotide phosphodiester linkage modification. For example, the 3 '-terminal internucleotide phosphodiester linkage modifications include, but are not limited to, an alkyl or aryl phosphotriester, an alkyl or aryl phosphonate, a hydrogen phosphonate, a phosphoramidate, and/or a phosphoroselenate linkage modification. In some variations, the 3 '-terminal internucleotide phophodiester linkage modification is a phosphoramidate modification. In some variations, the modified phosphate includes, but is not limited to, variations wherein the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ('amidate"), P(O)R, P(R)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloaklyl, cycloalkenyl, or araldyl.

[00339] In some variations, a CIRC of the present invention contains an IRS that comprises at least one nucleotide comprising at least one phosphothioate backbone linkage. In some variations, polynucleotides of the IRS comprise only phosphorothioate backbones. In some variations, polynucleotides of the IRS comprise only phosphodiester backbones. In some variations, an IRS may comprise a combination of phosphate linkages in the phosphate backbone including, but not limited to, a combination of phosphodiester and phosphorothioate linkages.

[00340] The IRS can contain phosphate-modified polynucleotides, some of which may stabilize the polynucleotide. Accordingly, some variations include a stabilized immunoregulatory polynucleotides. In some variations, an IRS comprises multiple phosphate modifications. In some variations, the phosphate modifications are the same. In some variations, the phosphate modifications are different. In some variations, the IRS comprises any of about 1, about 2, about 3, about 4, about 5 different phosphate modifications. Phosphate modifications may also be made and combined with any base modification and/or sugar modification in the preparation of a modified IRS. [00341] In some variations, the modification of at least one nucleotide comprises a modified sugar. IRPs used in the invention may comprise one or more modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or in a furanosyl form. In the IRS, the sugar moiety is preferably the furanoside of ribose, deoxyribose, arabinose or 2'-O-alkylribose. In some variations, the sugar can be attached to the respective heterocyclic bases either in alpha or beta anomeric configuration. In some variations, the sugar is modified by replacing a hydroxyl group ordinarily present. The hydroxyl group ordinarily present in the sugar may be replaced by, for example, but not limited to, phosphonate groups or phosphate groups. The 5' and 3' terminal hydroxyl group can additionally be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. In some variations, the modified sugars are 2' -sugar modifications including, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA/DNA chimeras. In some variations, the modified sugars include, but are not limited to, 2'-O- methyl-, 2'-OaIIyI, or 2'-azido- sugar modification. In some variations, the 2'-modified sugar is 2'-O-methyl sugar modification. In some variations, the 2' -modified sugar is 2'-O- methoxyethyl sugar modification. For example, a CIRC may contain an IRS comprising a sugar modification, the modification includes, but is not limited to, 2'-O-methyl-uridine, T- O-methyl-thymidine, 2' -O-methyl- adenine, 2'-O-methyl-guanine, or 2'-O-methyl-cytidine. In some variations, the sugar-modified nucleotide comprises one or more sugar modified nucleosides. The preparation of these sugars or sugar analogs and the respective "nucleosides" wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base) per se is known, and need not be described here, except to the extent such preparation can pertain to any specific example. In some variations, an IRS comprises multiple sugar modifications. In some variations, the sugar modifications are the same. In some variations, the sugar modifications are different. In some variations, the IRS comprises any of about 1, about 2, about 3, about 4, about 5 different sugar modifications. Sugar modifications may also be made and combined with any base modification and/or phosphate modification in the preparation of a modified IRS.

[00342] As demonstrated herein, particular CIRCs comprising a modified IRS inhibit TLR7 dependent cell responses. In some variations, the CIRCs comprising a modified IRS inhibit TLR7 dependent cell responses independent of TLR9 dependent cell responses. In some variations, the CIRCs comprising a modified IRS inhibit TLR9 dependent cell responses independent of TLR7 dependent cell responses. In some variations, the CIRCs comprising a modified IRS inhibit TLR9 dependent cell responses. In some variations, the CIRCs comprising a modified IRS inhibit TLR7 dependent cell responses and TLR9 dependent cell responses. In some variations, particular CIRCs include one or more branches containing a first modified IRS that inhibits TLR9-dependent cell responses and one or more branches containing a second modified IRS that inhibits TLR7-dependent cell responses. In some embodiments of CIRCs having multiple IRSs that separately inhibit TLR7- dependent and TLR9-dependent cell responses, the CIRC overall is of the TLR7/9 class.

[00343] Any of the modified polynucleotides described herein may comprise a modification any where in the polynucleotide sequence. In some variations, the modification is a modification of the nucleotides at or near the 5' end of the polynucleotide sequence. In some variations, at the 5' end of the polynucleotide sequence, about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified. In some variations, at the 5' end of the polynucleotide sequence, at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified. In some variations, the modification is a modification of the nucleotides at or near the 3' end of the polynucleotide sequence. In some variations, at the 3' end of the polynucleotide sequence, about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified. In some embodiments, at the 3' end of the polynucleotide sequence, at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified. In some variations, both the nucleotides at or near the 5' end of the polynucleotide sequence and the nucleotides at or near the 3' end of the polynucleotide sequence are modified. In some variations, at the 5' end of the polynucleotide sequence and at the 3' end of the polynucleotide sequence, about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified. In some embodiments, at the 5' end of the polynucleotide sequence and at the 3' end of the polynucleotide sequence, at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified.

[00344] Immuno stimulatory nucleic acids and other stimulators of an innate immune response have been described in the art and their activity may be readily measured using standard assays which indicate various aspects of an innate immune response, such as cytokine secretion, antibody production, NK cell activation, B cell proliferation, T cell proliferation, dendritic cell maturation. See, e.g. Krieg et al. (1995) Nature 374:546-549; Yamamoto et al. (1992) J. Immunol. 148:4072-4076; Klinman et al. (1997) J. Immunol. 158:3635-3639; Pisetsky (1996) J. Immunol. 156:421-423; Roman et al. (1997) Nature Med. 3:849-854; Hemmi et al. (2000), Supra; Lee et al. (2003), Supra; WO 98/16247; WO 98/55495; WO 00/61151 and U.S. Pat. No. 6,225,292. Accordingly, these and other methods can be used to identify, test and/or confirm immunoregulatory sequences, polynucleotides and/or compounds. For example, the effect of a CIRC comprising a modified IRS can be determined when cells or individuals in which an innate immune response has been stimulated are contacted with the CIRC comprising a modified IRS.

[00345] In some variations, an IRS may comprise a sequence comprising 7-deaza-dG, such as wherein at least one G is replaced with a 7-deaza-dG. In some variations, the IRS may comprise the sequence 5'-TGC TGC TCC TTG AGZ' GGT TGT TTG T-3', wherein Z' is 7-deaza-dG (SEQ ID NO: 168).

[00346] As described herein, some IRPs comprising a modified IRS are particularly effective in suppressing TLR7 and/or TLR9 dependent cell responses.

[00347] The invention provides polynucleotides consisting of a nucleotide sequence of the formula: 5'-JGCN_z-3' (SEQ ID NO: 130), wherein J is U or T, the sequence 5'-JGC-3' comprises a modification, each N is a nucleotide, and z is an integer from about 1 to about 1000. In some embodiments, the polynucleotide is effective in suppressing TLR7 and/or TLR9 dependent cell responses. In some variations, the sequence 5'-JGC-3' is modified.

[00348] The modification may be any described above, for example, a modified base, a modified sugar, a modified phosphate. In some variations, modification includes a 2'-sugar modification, a 3'-terminal internucleotide phosphodiester linkage modification, and/or a 5'- methyl-cytosine modification. In some variations, the modification may be a phosphate or termini modification. In some variations, the phosphate or termini modification may be a 3'terminal internucletide phosphodiester linkage modification. In some variations, the 3'- terminal internucleotide phosphodiester linkage modification is selected from the group consisting of an alkyl or aryl phosphotriester, alkyl or aryl phosphonate, hydrogen phosphonate, phosphoramidate, and phosphoroselenate linkage modification. In some variations, 3'-terminal internucleotide phosphodiester linkage modification is a phosphoramidate modification. In some variations, the modification may be a sugar modification. In some variations, the sugar modification is a 2' -sugar modification as described herein. In some variations, the 2' -sugar modification is a 2'-O-methyl sugar modification or 2'-O-methoxyethyl sugar modification. In some variations, the modification is a base modified, for example, a 5'-methyl-cytosine modification.

[00349] In some variations, every nucleotide of the polynucleotide comprises at least one modification (i.e., nucleotide N comprises a modification). In some variations, the modification is the same modification for each nucleotide. In some variations, every nucleotide of the polynucleotide is modified and the modification is a 2'-O-methyl sugar modification (i.e., nucleotide N consists of a modification and said modification is a 2'-O- methyl sugar modification). In some variations, the modification comprises more than one different type of modification. In some variations, one or more nucleotides of the polynucleotide comprise a modification (i.e., sequence Nz comprises a modification).

[00350] In some variations, z is an integer of any of about between about 1 to about 750, between about 1 to about 500, between about 1 to about 250, between about 1 to about 200, between about 1 to about 150, between about 1 to about 125, between about 1 to about 100, between about 1 to about 75, between about 1 to about 50, between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5. In some variation, z is an integer between about 1 to about 100. In some variations, z is an integer between 1 and 100. In some variations, z is an integer less than any of about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 40, about 30, about 25, about 20, about 15 or about 10. In some variations, z is an integer less than 100. In some variations, z is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 10, about 15, or about 20.

[00351] In some variations, the polynucleotide, such as 5'-JGCN_z-3' (SEQ ID NO: 130), comprises a modification of the nucleotides at or near the 3' end of the polynucleotide sequence. In some variations, at the 3' end of the polynucleotide sequence, about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified. In some embodiments, at the 3' end of the polynucleotide sequence, at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified.

[00352] In some variations, the polynucleotide, such as 5'-JGCN_z-3' (SEQ ID NO: 130), further comprises a nucleotide sequence 5'-TGC-3', wherein 5'-TGC-3' is unmodified. In some variations, the TGC trinucleotide sequence is about any of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polynucleotide. In some variations, the TGC trinucleotide sequence is less than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polynucleotide. In some variations, the polynucleotide consists of a nucleotide sequence 5'-JGCTGC-3' (SEQ ID NO: 189), wherein J is U or T and the sequence 5'-JGC-3' comprises a modification. In some variations, the modification is any 2'-sugar modification described herein. In some variations, the 2' -sugar modification is a 2'O- methoxyethyl sugar modification.

[00353] In some variations, the polynucleotide, such as 5'-JGCN_z-3' (SEQ ID NO: 130), further comprises a nucleotide sequence of the formula: 5'-SiS₂S₃S₄-S', wherein S₁, S₂, S₃, and S₄ are independently G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing, each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing disrupts or prevents formation of tetrameric/quadruplex structure of G-quadruplexes. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing is a nucleotide or derivative thereof. Examples of molecules that are capable of preventing G- tetrad formation and/or preventing Hoogsteen base pairing included, but are not limited to, I, 7-deaza-dG, 7-deaza-2'-deoxyxanthosine, 7-deaza-8-aza-2'-deoxyguanosine, T- deoxynebularine, isodeoxyguanosine, 8-oxo-2'-deoxyguanosine. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are molecules that are capable of preventing G- tetrad formation and/or preventing Hoogsteen base pairing. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are I. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are 7-deaza-dG. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are G. In some variations, S₁, S₂, S₃, and S₄ are G. In some variations, S₁, S₂, S₃, and S₄are not modified and/or not further modified. In some variations, the polynucleotide comprises the nucleotide sequence of the formula: 5'-GS₅GGQ_yM_r-3' (SEQ ID NO: 187), wherein S₅ is G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing pairing such as I or 7-deaza-dG, each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000. In some variations, the polynucleotide comprises the nucleotide sequence of the formula: 5'-GGGGQ_yM_r-3' (SEQ ID NO: 131), wherein each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000. [00354] The modification of nucleotide M may be any described above, including, but not limited to, a modified base, a modified sugar, a modified phosphate. In some variations, the modification of nucleotide M is selected from the group consisting of a 2'- sugar modification, a 3'-terminal internucleotide phosphodiester linkage modification, and a 5'- methyl-cytosine modification. In some variations, the modification is any 2' -sugar modification described herein. In some variations, the 2' -sugar modification is a 2'O- methoxyethyl sugar modification.

[00355] In some variations, r is an integer of any of about between about 1 to about 750, between about 1 to about 500, between about 1 to about 250, between about 1 to about 200, between about 1 to about 150, between about 1 to about 125, between about 1 to about 100, between about 1 to about 75, between about 1 to about 50, between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5. In some variation, r is an integer between about 1 to about 50. In some variations, r is an integer between 1 and 50. In some variations, r is an integer less than any of about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 40, about 30, about 25, about 20, about 15 or about 10. In some variations, r is an integer less than 50. In some variations, r is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 10, about 15, or about 20.

[00356] In some variations, y is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In some variations, y is an integer any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15.

[00357] Provided herein are also polynucleotides consisting of the nucleotide sequence of the formula: 5'-M_aTGCN_β-3' (SEQ ID NO: 198), wherein each M is a nucleotide comprising a modification, a is an integer from about 1 to about 10, each N is a nucleotide, and β is an integer from about 1 to about 1000.

[00358] The modification may be any described above, for example, a modified base, a modified sugar, a modified phosphate. In some variations, modification includes a 2'-sugar modification, a 3'-terminal internucleotide phosphodiester linkage modification, and/or a 5'- methyl-cytosine modification. In some variations, the modification may be a phosphate or termini modification. In some variations, the phosphate or termini modification may be a 3'terminal internucletide phosphodiester linkage modification. In some variations, the 3'- terminal internucleotide phosphodiester linkage modification is selected from the group consisting of an alkyl or aryl phosphotriester, alkyl or aryl phosphonate, hydrogen phosphonate, phosphoramidate, and phosphoroselenate linkage modification. In some variations, 3'-terminal internucleotide phosphodiester linkage modification is a phosphoramidate modification. In some variations, the modification may be a sugar modification. In some variations, the sugar modification is any 2' -sugar modification described herein. In some variations, the 2' -sugar modification is a 2'O-methoxyethyl sugar modification.

[00359] In some variations, every nucleotide of the polynucleotide comprises at least one modification (i.e., nucleotide N comprises a modification). In some variations, the modification is the same modification for each nucleotide. In some variations, every nucleotide of the polynucleotide is modified and the modification is a 2'-O-methyl sugar modification (i.e., nucleotide N consists of a modification and said modification is a 2'-O- methyl sugar modification). In some variations, the modification comprises more than one different types of modification. In some variations, one or more nucleotides of the polynucleotide comprise a modification (e.g., sequence Ma and/or NB comprises a modification).

[00360] In some variations, a is an integer of any of about between about 1 to about 7, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some variations, a is an integer of about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[00361] In some variations, β is an integer of any of about between about 1 to about 750, between about 1 to about 500, between about 1 to about 250, between about 1 to about 200, between about 1 to about 150, between about 1 to about 125, between about 1 to about 100, between about 1 to about 75, between about 1 to about 50, between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5. In some variation, z is an integer between about 1 to about 100. In some variations, β is an integer between 1 and 100. In some variations, β is an integer less than any of about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 40, about 30, about 25, about 20, about 15 or about 10. In some variations, z is an integer less than 100. In some variations, β is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 10, about 15, or about 20.

[00362] In some variations, the polynucleotide, such as 5'-M_aTGCN_β-3' (SEQ ID NO: 198), comprises a modification of the nucleotides at or near the 3' end of the polynucleotide sequence. In some variations, at the 3' end of the polynucleotide sequence, about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified. In some embodiments, at the 3' end of the polynucleotide sequence, at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are modified.

[00363] In some variations, the polynucleotide, such as 5'-M_aTGCN_β-3' (SEQ ID NO:198), further comprises a nucleotide sequence of the formula: 5'-SiS₂S₃S₄-S', wherein S₁, S₂, S₃, and S₄ are independently G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing, each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing disrupts or prevents formation of tetrameric/quadruplex structure of G-quadruplexes. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing is a nucleotide or derivative thereof. Examples of molecules that are capable of preventing G- tetrad formation and/or preventing Hoogsteen base pairing included, but are not limited to, I, 7-deaza-dG, 7-deaza-2'-deoxyxanthosine, 7-deaza-8-aza-2'-deoxyguanosine, T- deoxynebularine, isodeoxyguanosine, 8-oxo-2'-deoxyguanosine. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are molecules that are capable of preventing G- tetrad formation and/or preventing Hoogsteen base pairing. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are I. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are 7-deaza-dG. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄are G. In some variations, Sl, S2, S3, and S4 are G. In some variations, Si, S₂, S₃, and S₄ are not modified and/or not further modified. In some variations, the polynucleotide comprises the nucleotide sequence of the formula: 5'-GSsGGQ_yM_r-3' (SEQ ID NO: 187), wherein S₅ is G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing such as I or 7-deaza-dG, each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000. In some variations, inosine is deoxy-inosine. In some variations, the polynucleotide comprises the nucleotide sequence of the formula: 5'- GGGGQ_yM_r-3' (SEQ ID NO: 131), wherein each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000.

[00364] The modification of nucleotide M may be any described above, including, but not limited to, a modified base, a modified sugar, a modified phosphate. In some variations, the modification of nucleotide M is selected from the group consisting of a 2'- sugar modification, a 3'-terminal internucleotide phosphodiester linkage modification, and a 5'- methyl-cytosine modification. In some variations, the sugar modification is any 2' -sugar modification described herein. In some variations, the 2' -sugar modification is a 2'O- methoxyethyl sugar modification.

[00365] In some variations, r is an integer of any of about between about 1 to about 750, between about 1 to about 500, between about 1 to about 250, between about 1 to about 200, between about 1 to about 150, between about 1 to about 125, between about 1 to about 100, between about 1 to about 75, between about 1 to about 50, between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5. In some variation, r is an integer between about 1 to about 50. In some variations, r is an integer between 1 and 50. In some variations, r is an integer less than any of about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 40, about 30, about 25, about 20, about 15 or about 10. In some variations, r is an integer less than 50. In some variations, r is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 10, about 15, or about 20.

[00366] In some variations, y is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In some variations, y is an integer any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15.

[00367] Provided herein are also polynucleotides consisting of a nucleotide sequence of the formula: 5'-JGCL_pK_wSiS₂S₃S₄Q_yM₁-S' (SEQ ID NO: 191), wherein J is U or T, the sequence 5'-JGC-3' comprises a modification, each L is a nucleotide, p is an integer from about 1 to about 1000, each K is an unmodified nucleotide, w is an integer greater than 1, S₁, S_2, S₃, and S₄ are independently G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000. In some variations, the sequence 5'-JGC-3' is modified. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing disrupts or prevents formation of tetrameric/quadruplex structure of G-quadruplexes. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing is a nucleotide or derivative thereof. Examples of molecules that are capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing included, but are not limited to, I, 7-deaza-dG, 7-deaza-2'- deoxyxanthosine, 7-deaza-8-aza-2' -deoxyguanosine, 2' -deoxynebularine, isodeoxyguanosine, 8-oxo-2' -deoxyguanosine. In some variations, at least one, two, three, or four of S₁, S_2, S₃, and S₄ are molecules that are capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing. In some variations, at least one, two, three, or four of S₁, S_2, S₃, and S₄ are I. In some variations, at least one, two, three, or four of S₁, S_2, S₃, and S₄ are 7-deaza-dG. In some variations, at least one, two, three, or four of S₁, S_2, S₃, and S₄ are G. In some variations, S₁, S_2, S₃, and S₄ are G. In some variations, S₁, S_2, S₃, and S₄ are not modified and/or not further modified. In some variations, the polynucleotide consists of a nucleotide sequence of the formula: 5'-JGCL_pK_wGS₅GGQ_yM_r-3' (SEQ ID NO: 188), wherein J is U or T, the sequence 5'-JGC-3' comprises a modification, each L is a nucleotide, p is an integer from about 1 to about 1000, each K is an unmodified nucleotide, w is an integer greater than 1, S₅ is G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing such as I or 7-deaza-dG, each Q is an unmodified nucleotide, each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000. In some variations, the inosine is deoxy-inosine. In some variations, the polynucleotide consists of a nucleotide sequence of the formula: 5'-JGCL_pK_wGGGGQ_yM_r-3' (SEQ ID NO: 132), wherein J is U or T, the sequence 5'-JGC-3' comprises a modification, each L is a nucleotide, p is an integer from about 1 to about 1000, each K is an unmodified nucleotide, w is an integer greater than 1, each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000.

[00368] In some variations, L is modified. The modification of nucleotide M and/or L may be any described above, for example, a modified base, a modified sugar, a modified phosphate. In some variations, the modification of nucleotide M and/or L is selected from the group consisting of a 2'-sugar modification, a 3'-terminal internucleotide phosphodiester linkage modification, and a 5'-methyl-cytosine modification. In some variations, the sugar modification is any 2'-sugar modification described herein. In some variations, the 2'-sugar modification is a 2'O-methoxyethyl sugar modification.

[00369] In some variations, r is an integer of any of about between about 1 to about 750, between about 1 to about 500, between about 1 to about 250, between about 1 to about 200, between about 1 to about 150, between about 1 to about 125, between about 1 to about 100, between about 1 to about 75, between about 1 to about 50, between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5. In some variation, r is an integer between about 1 to about 50. In some variations, r is an integer between 1 and 50. In some variations, r is an integer less than any of about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 40, about 30, about 25, about 20, about 15 or about 10. In some variations, r is an integer less than 50. In some variations, r is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 10, about 15, or about 20.

[00370] In some variations, p is an integer of any of about between about 1 to about 750, between about 1 to about 500, between about 1 to about 250, between about 1 to about 200, between about 1 to about 150, between about 1 to about 125, between about 1 to about 100, between about 1 to about 75, between about 1 to about 50, between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5. In some variation, p is an integer between about 1 to about 50. In some variations, p is an integer between 1 and 50. In some variations, p is an integer less than any of about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 40, about 30, about 25, about 20, about 15 or about 10. In some variations, p is an integer less than 50. In some variations, p is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 10, about 15, or about 20.

[00371] In some variations, y is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In some variations, y is an integer any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10, about 11, about 12, about 13, about 14, or about 15.

[00372] In some variations, w is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In some variations, w is an integer any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15.

[00373] Provided herein are also polynucleotides comprising a nucleotide sequence of the formula: 5'-SiS₂S₃S₄QyM₁-S' (SEQ ID NO: 192), wherein S₁, S₂, S₃, and S₄ are independently G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing, each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing disrupts or prevents formation of tetrameric/quadruplex structure of G-quadruplexes. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing is a nucleotide or derivative thereof. Examples of molecules that are capable of preventing G- tetrad formation and/or preventing Hoogsteen base pairing included, but are not limited to, I, 7-deaza-dG, 7-deaza-2'-deoxyxanthosine, 7-deaza-8-aza-2'-deoxyguanosine, T- deoxynebularine, isodeoxyguanosine, 8-oxo-2'-deoxyguanosine. In some variations, at least one, two, three, or four of Si, S_2, S₃, and S₄ are molecules that are capable of preventing G- tetrad formation and/or preventing Hoogsteen base pairing. In some variations, at least one, two, three, or four of S₁, S_2, S₃, and S₄ are I. In some variations, at least one, two, three, or four of S₁, S_2, S₃, and S₄ are 7-deaza-dG. In some variations, at least one, two, three, or four of S₁, S_2, S₃, and S₄ are G. In some variations, S₁, S_2, S₃, and S₄ are G. In some variations, S₁, S_2, S₃, and S₄ are not modified and/or not further modified. The nucleotide sequence of the formula: 5'-SiS₂S₃S₄Q_yM_r-3' (SEQ ID NO: 192) can be found any where in the polynucleotide sequence. In some variation, the nucleotide sequence of the formula: 5'- SiS₂S₃S₄Q_yM_r -3' (SEQ ID NO: 192) is found internally in the polynucleotide sequence, i.e., not at the 5' end or 3' end of the nucleotide sequence. In some variations, the polynucleotides comprising a nucleotide sequence of the formula: 5'-GSsGGQ_yM_r-3' (SEQ ID NO: 193), wherein S₅ is G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing such as I or 7-deaza-dG, each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000. In some variation, the polynucleotides comprising a nucleotide sequence of the formula: 5'-GGGGQ_yM_r-3' (SEQ ID NO: 133), wherein each Q is an unmodified nucleotide, each M is a nucleotide comprising a modification, y is an integer greater than 1, and r is an integer from 1 to about 1000.

[00374] The modification of nucleotide M may be any described above, for example, a modified base, a modified sugar, a modified phosphate. In some variations, the modification of nucleotide M is selected from the group consisting of a 2'-sugar modification, a 3'-terminal internucleotide phosphodiester linkage modification, and a 5'-methyl-cytosine modification. In some variations, the sugar modification is any 2'-sugar modification described herein. In some variations, the 2' -sugar modification is a 2'O-methoxyethyl sugar modification.

[00375] In some variations, r is an integer of any of about between about 1 to about 750, between about 1 to about 500, between about 1 to about 250, between about 1 to about 200, between about 1 to about 150, between about 1 to about 125, between about 1 to about 100, between about 1 to about 75, between about 1 to about 50, between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5. In some variation, r is an integer between about 1 to about 50. In some variations, r is an integer between 1 and 50. In some variations, r is an integer less than any of about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 40, about 30, about 25, about 20, about 15 or about 10. In some variations, r is an integer less than 50. In some variations, r is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 10, about 15, or about 20.

[00376] In some variations, y is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In some variations, y is an integer any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15.

[00377] In some variations, the polynucleotide further comprises at least one trinucleotide sequence 5'-TGC-3'. In some variations, the 5'-TGC-3' is about 0-10 nucleotides from the 5' end IRS and/or IRP. The 5'-TGC-3' may be between about any of 1- 7, 1-5, 1-3, or 1-2 nucleotides from the 5' end of the IRS and/or IRP. In some variations, the 5'-TGC-3' is a 5'-TGC nucleotide sequence at the 5' end.

[00378] Further provided herein are polynucleotides comprising the nucleotide sequence of the formula: 5'-L_pK_wSiS₂S₃S₄-3' (SEQ ID NO: 195), wherein each L is a nucleotide, p is an integer from about 1 to about 1000, each K is an unmodified nucleotide, w is an integer greater than 1, and S₁, S_2, S₃, and S₄ are independently G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing disrupts or prevents formation of tetrameric/quadruplex structure of G- quadruplexes. In some variations, the molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing is a nucleotide or derivative thereof. Examples of molecules that are capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing included, but are not limited to, I, 7-deaza-dG, 7-deaza-2'- deoxyxanthosine, 7-deaza-8-aza-2' -deoxyguanosine, 2' -deoxynebularine, isodeoxyguanosine, 8-oxo-2' -deoxyguanosine. In some variations, at least one, two, three, or four of S₁, S_2, S₃, and S₄ are molecules that are capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are I. In some variations, at least one, two, three, or four of S₁, S₂, S₃, and S₄ are 7-deaza-dG. In some variations, at least one, two, three, or four of S₁, S_2, S₃, and S₄ are G. In some variations, S₁, S_2, S₃, and S₄ are G. In some variations, polynucleotides comprising the nucleotide sequence of the formula: 5'-L_pK_wGSsGG-3' (SEQ ID NO: 196), wherein each L is a nucleotide, p is an integer from about 1 to about 1000, each K is an unmodified nucleotide, w is an integer greater than 1, and S5 is G or a molecule that is capable of preventing G-tetrad formation and/or preventing Hoogsteen base pairing such as I or 7-deaza-dG. In some variations the polynucleotides comprising a nucleotide sequence of the formula: 5'-L_pK_wGGGG-3' (SEQ ID NO: 134), wherein each L is a nucleotide, p is an integer from about 1 to about 1000, each K is an unmodified nucleotide, and w is an integer greater than 1.

[00379] In some variations, L is modified. The modification of nucleotide L may be any described above, for example, a modified base, a modified sugar, a modified phosphate. In some variations, the modification of nucleotide L is selected from the group consisting of a 2'-O-methyl sugar modification, a 3'-terminal internucleotide phosphodiester linkage modification, and a 5'-methyl-cytosine modification. In some variations, the sugar modification is any 2'-sugar modification described herein. In some variations, the 2'-sugar modification is a 2'0-methoxyethyl sugar modification.

[00380] In some variations, p is an integer of any of about between about 1 to about 750, between about 1 to about 500, between about 1 to about 250, between about 1 to about 200, between about 1 to about 150, between about 1 to about 125, between about 1 to about 100, between about 1 to about 75, between about 1 to about 50, between about 1 to about 25, between about 1 to about 20, between about 1 to about 15, between about 1 to about 10, or between about 1 to about 5. In some variation, p is an integer between about 1 to about 50. In some variations, p is an integer between 1 and 50. In some variations, p is an integer less than any of about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 40, about 30, about 25, about 20, about 15 or about 10. In some variations, p is an integer less than 50. In some variations, p is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 10, about 15, or about 20.

[00381] In some variations, w is an integer greater than any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In some variations, w is an integer any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15.

[00382] In some variations, the polynucleotide further comprises at least one trinucleotide sequence 5'-TGC-3'. In some variations, the 5'-TGC-3' is about 0-10 nucleotides from the 5' end IRS and/or IRP. The 5'-TGC-3' may be between about any of 1- 7, 1-5, 1-3, or 1-2 nucleotides from the 5' end of the IRS and/or IRP. In some variations, the 5'-TGC-3' is a 5'-TGC nucleotide sequence at the 5' end.

[00383] In some variations, the modified IRS is C999 (SEQ ID NO: 135) 5'-UGC UCC UGG AGG GGU UGU-3', wherein all nucleotides are modified with a 2'-0-Me modification, a sugar modification). In some variations, the modified IRS is DVO 17 (SEQ ID NO: 136) 5'-UGC UCC UGG AGG GGU UGU-3', wherein all nucleotides are modified with phosphoramidate modification, a phosphate modification). In some variations, the modified IRS is DV031 (SEQ ID NO: 137) 5'-UGC UCC UGG AGG GGU UGU-3', wherein all cytosines are modified with a 5-methyl dC (M) modification, a base modification). [00384] [0179] In some variations, the modified IRS is modified with a 2' -O-Me modification. In some variations, the modified IRS modified with a 2'-0-Me modification is any of: UGC TCC TGG AGG GGT TGT (SEQ ID NO: 138); TGC TCC TGG AGG GGU UGU (SEQ ID NO: 139); UGC TCC TGG AGG GGU UGU (SEQ ID NO: 140); TGC TCC TGG AGG GGT TGT (SEQ ID NO: 141); UGC TTG TCC TGG AGG GGT TGT (SEQ ID NO: 142); TGC TCC TGG AGG GGA AGT UUG U (SEQ ID NO: 143); UGC TTG TCC TGG AGG GGU UGU (SEQ ID NO: 144); UGC TTG TCC TGG AGG GGA AGT UUG U (SEQ ID NO: 145); UGC TG TCC TGG AGG GGA AGT UUG U (SEQ ID NO: 146); UGC G TCC TGG AGG GGA AGT UUG U (SEQ ID NO: 147); UGC TTG TCC TGG AGG GG TG UUG U (SEQ ID NO: 148); UGC TG TCC TGG AGG GG TG UUG U (SEQ ID NO:149); UGC G TCC TGG AGG GG TG UUG U (SEQ ID NO: 150); UGC TTG TCC TGG AGG GGT UGU (SEQ ID NO: 151); UGC TG TCC TGG AGG GGT UGU (SEQ ID NO:152); UGC G TCC TGG AGG GGT UGU (SEQ ID NO: 153); UGC TTG TCC TGG AGG GGT TGT UUG U (SEQ ID NO: 154); UGC TTG TCC TGG AGG GGT TGU UUG U (SEQ ID NO: 155); UGC TGC TCC TGG AGG GGT TGT UUG U (SEQ ID NO: 156); UGC TGC TCC TTG AGG GGT TGT UUG U (SEQ ID NO: 157); UGC TGC TCC TTG AGG GGT GUU GU (SEQ ID NO: 158); UGC TGC TCC TTG AGG GGT TGU UUG U (SEQ ID NO: 159); UGC UGC UCC UUG AGA GGU UGU (SEQ ID NO: 160); UGC TGC TCC TGG AGG GGT TGU UUG U (SEQ ID NO: 163); UGC TGC TCC TTG AGG GGT TGT TTG T (SEQ ID NO: 170); or UGC TGC TCC TGG AGG GGT TGT TTG T (SEQ ID NO:171); wherein the bolded and italicized nucleotides are modified with a 2'-0-Me sugar modification.

[00385] In some variations, the modified IRS is modified with a 2'-0-Me modification and further comprises the nucleoside inosine and/or deoxy-inosine. In some variations, the modified IRS is modified with a 2'-0-Me modification and further comprises 7-deaza-dG. In some variations, the modified IRS is any of: 5'-UGC TGC TCC TTG AGI GGT TGT TTG T-3', wherein I is deoxy-inosine (SEQ ID NO: 173); 5'-UGC TGC TCC TTG AGZ' GGT TGT TTG T-3', wherein Z' is 7-deaza-dG (SEQ ID NO: 174) 5'-UGC TGC TCC TTG AGI GGT TGT TTG-3', wherein I is deoxy-inosine (SEQ ID NO: 175); 5'-UGC TGC TCC TTG AGI GGT TGT TT-3', wherein I is deoxy-inosine (SEQ ID NO: 176); 5'-UGC TGC TCC TTG AGI GGT TGT T-3', wherein I is deoxy-inosine (SEQ ID NO: 177); 5'-UGC TGC TCC TTG AGI GGT TGT-3', wherein I is deoxy-inosine (SEQ ID NO: 178); 5'-UGC TGC TCC

TTG AGI GGT T-3', wherein I is deoxy-inosine (SEQ ID NO: 179); 5'-UGC TGC TCC TTG AGI GGT-3', wherein I is deoxy-inosine (SEQ ID NO: 180); 5'-UGC TGC TCC TTG AGI GG-3', wherein I is deoxy-inosine (SEQ ID NO: 181); 5'-UGC TGC TCC TTG AGI G-3\ wherein I is deoxy-inosine (SEQ ID NO: 182); 5'-UGC TGC TCC TTG AGI-3', wherein I is deoxy-inosine (SEQ ID NO:183); 5'-GC TGC TCC TTG AGI GGT TGT TTG T-3\ wherein I is deoxy-inosine (SEQ ID NO: 184); 5'-C TGC TCC TTG AGI GGT TGT TTG T-3\ wherein I is deoxy-inosine (SEQ ID NO:185); or 5'-UGC TGC TCC TTG AGI GGT TG-3', wherein I is deoxy-inosine (SEQ ID NO: 186); wherein the bolded and italicized nucleotides are modified with a 2'-0-Me sugar modification.

[00386] A CIRC of the present invention may comprise a modified IRS in the form of single stranded or double stranded DNA, as well as in the form of single or double- stranded RNA. A CIRC may comprise a modified IRS in the form of a linear polynucleotide, a circular polynucleotide or may include circular portions and/or may include a hairpin loop.

[00387] In some variations of any of the modified immunoregulatory sequences, a uridine (U) nucleoside of the modified IRS may be substituted with a thymidine (T) nucleoside. In some variations, all uridine (U) nucleoside of the modified IRS may be substituted with a thymidine (T) nucleoside. In some variations of any of the modified immunoregulatory sequences, a thymidine (T) nucleoside of the modified IRS may be substituted with a uridine (U) nucleoside. In some variations, all thymidine (T) nucleoside of the modified IRS may be substituted with a uridine (U) nucleoside. In some variations, the modified IRS may comprise both uridine (U) nucleosides and thymidine (T) nucleosides.

[00388] In some variations, a modified immunoregulatory polynucleotide is less than about any of the following lengths (in bases or base pairs): 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175; 150; 125; 100; 75; 60; 50; 40; 30; 25; 20; 15; 14; 13; 12; 11; 10; 9; 8; 7; 6; 5; 4. In some variations, a modified immunoregulatory polynucleotide is greater than about any of the following lengths (in bases or base pairs): 4; 5; 6, 7, 8, 9, 10; 11; 12; 13; 14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000; 7500; 10000; 20000; 50000. Alternately, the modified immunoregulatory polynucleotide can be any of a range of sizes having an upper limit of 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175; 150; 125; 100; 75; 60; 50; 40; 30; 25; 20; 15; 14; 13; 12; 11; 10; 9; 8; 7; 6; 5; 4 and an independently selected lower limit of 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000; 7500, wherein the lower limit is less than the upper limit. In some variations, a modified IRP is preferably about 200 or less bases in length.

[00389] In some variations,CIRCs comprising a modified IRS, as described herein, inhibit and/or suppress a measurable immune response as measured in vitro, in vivo, and/or ex vivo. In some variations, the immune response is an innate immune response. In some variations, the immune response is an adaptive immune response. In some variations, a CIRC comprising a modified IRS results in potentially increased inhibition of a measurable immune response as measured in vitro, in vivo, and/or ex vivo compared to a CIRC comprising an unmodified IRS. In some variations, the immune response is an innate immune response. In some variations, the immune response is an adaptive immune response. In some variations, the nucleotide sequence of the modified and unmodified IRS is the same, and the only difference is the modification of at least one nucleotide. In some variations, inhibition of a measurable immune response as measured in vitro, in vivo, and/or ex vivo by a CIRC comprising a modified IRS is increased by greater than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to a CIRC comprising an unmodified IRS. In some variations, inhibition of a measurable immune response as measured in vitro, in vivo, and/or ex vivo by a CIRC comprising a modified IRS is increased by any of about 10%, about 15%, about 20%, or about 25% compared to a CIRC comprising an unmodified IRS. In some variations, the nucleotide sequence of the modified and unmodified IRS is the same, and the only difference is the modification of at least one nucleotide.

[00390] In some variations, CIRCs comprising a modified IRS, as described herein, inhibit TLR7 dependent cell responses. In some variations, the CIRCs comprising a modified IRS, as described herein, TLR7 dependent cell responses independently of TLR9 dependent cell responses. In some variations, the IRPs and/or IRCs comprising a modified IRS, as described herein, inhibit TLR9 dependent cell responses. In some variations, the CIRCs comprising a modified IRS, as described herein, inhibit TLR7 dependent cell responses and TLR9 dependent cell responses. [00391] In some variations, a CIRC comprising a modified IRS result in increased inhibition of TLR7 and/or TLR9 dependent cell responses compared to a CIRC comprising an unmodified IRS. In some variations, the nucleotide sequence of the modified and unmodified IRS is the same, and the only difference is the modification of at least one nucleotide. In some variations, inhibition of TLR7 and/or TLR9 dependent cell responses by a CIRC comprising a modified IRS is increased by greater than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to a CIRC comprising an unmodified IRS. In some variations, inhibition of TLR7 and/or TLR9 dependent cell responses by a CIRC comprising a modified IRS is increased by any of about 10%, about 15%, about 20%, or about 25% compared to a CIRC comprising an unmodified IRS. In some variations, the nucleotide sequence of the modified and unmodified IRS is the same, and the only difference is the modification of at least one nucleotide.

Other Nucleic Acid Moiety Activity

[00392] In some embodiments of this invention, one or more of the nucleic acid moieties in the branched CIC have an activity other than an immunomodulatory activity, such as other than an immunostimulatory and/or immunoregulatory activity. Other types of nucleic acid moiety activity include (but are not limited to): antisense activity, aptameric activity (i.e., ability to bind specifically to a non-nucleic acid target), transcription factor decoy activity, micro-RNA activity and siRNA activity. In some embodiments, one or more of the nucleic acid moieties in the branched CIC have no significant activity. In some embodiments, all of the nucleic acid moieties in the branched CIC have an activity other than an immunomodulatory (i.e., other than an immunostimulatory and/or immunoregulatory activity), or may have some other activity, for example as an aptamer or antisense. Structural motifs and methods of making oligonucleotides with these activities are known in the art. Nucleic acid moieties with immunoinhibitory activity are described in U.S. Patent No. 6,225,292 and PCT Publication WO 06/28742.

Structure of the Nucleic Acid Moiety

[00393] A nucleic acid moiety of a CIC may contain structural modifications relative to naturally occurring nucleic acids. Modifications include any known in the art for polynucleotides, but are not limited to, modifications of the 3'OH or 5'OH group, modifications of the nucleotide base, modifications of the sugar component, and modifications of the phosphate group. Various such modifications are described below.

[00394] The nucleic acid moiety may be DNA, RNA or mixed DNA/RNA, single stranded, double stranded or partially double stranded, and may contain other modified polynucleotides. Double stranded nucleic acid moieties and CICs are contemplated, and the recitation of the term 'base' or 'nucleotide' is intended to encompass basepair or basepaired nucleotide, unless otherwise indicated. A nucleic acid moiety may contain naturally- occurring or modified, non-naturally occurring bases, and may contain modified sugar, phosphate, and/or termini. For example, phosphate modifications include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester and phosphorodithioate and may be used in any combination. Other non- phosphate linkages may also be used such as peptide nucleic acids (PNAs) or cationic oligos. Preferably, CICs, CIRCs and nucleic acid moieties of the present invention comprise phosphorothioate backbones. Sugar modifications known in the field, such as 2'-alkoxy- RNA analogs, 2'-amino-RNA analogs and 2'-alkoxy- or amino-RNA/DNA chimeras and others described herein, may also be made and combined with any phosphate modification. Examples of base modifications (discussed further below) include, but are not limited to, addition of an electron- withdrawing moiety to C- 5 and/or C-6 of a cytosine (e.g., 5- bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) and C- 5 and/or C-6 of a uracil (e.g., 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil). Other modified bases include N4 alkylations of a cytosine (e.g., N4-methylcytosine, N4-ethylcytosine), modified purines (e.g., 7-deazaguanosine, inosine). In certain embodiments, modified cytosines and guanosine can replace the cytosine and guanosine in an immuno stimulatory CpG motif. See, for example, PCT Application No. WO 99/62923.

[00395] The nucleic acid moiety can also contain phosphate-modified nucleotides. Synthesis of nucleic acids containing modified phosphate linkages or non-phosphate linkages is also know in the art. For a review, see Matteucci (1997) 'Oligonucleotide Analogs: an Overview' in Oligonucleotides as Therapeutic Agents, (DJ. Chadwick and G. Cardew, ed.) John Wiley and Sons, New York, NY. The phosphorous derivative (or modified phosphate group) which can be attached to the sugar or sugar analog moiety in the nucleic acids of the present invention can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here in detail. Peyrottes et al. (1996) Nucleic Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-2323; and Schultz et al. (1996) Nucleic Acids Res. 24:2966-2973. For example, synthesis of phosphorothioate oligonucleotides is similar to that described above for naturally occurring oligonucleotides except that the oxidation step is replaced by a sulfurization step (Zon (1993) 'Oligonucleoside Phosphorothioates' in Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190). Similarly the synthesis of other phosphate analogs, such as phosphotriester (Miller et al. (1971) JACS 93:6657-6665), non-bridging phosphoramidates (Jager et al. (1988) Biochem. 27:7247-7246), N3' to P5' phosphoramidiates (Nelson et al. (1997) JOC 62:7278- 7287) and phosphorodithioates (U.S. Patent No. 5,453,496) has also been described. Other non-phosphorous based modified nucleic acids can also be used (Stirchak et al. (1989) Nucleic Acids Res. 17:6129-6141). Nucleic acids with phosphorothioate backbones appear to be more resistant to degradation after injection into the host. Braun et al. (1988) J. Immunol. 141:2084-2089; and Latimer et al. (1995) MoI. Immunol. 32:1057-1064.

[00396] Nucleic acid moieties used in the invention can comprise ribonucleotides (containing ribose as the only or principal sugar component), and/or deoxyribonucleotides (containing deoxyribose as the principal sugar component). Modified sugars or sugar analogs can be incorporated in the nucleic acid moiety. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar 'analog' cyclopentyl group. The sugar can be in pyranosyl or in a furanosyl form. The sugar moiety is preferably the furanoside of ribose, deoxyribose, arabinose or 2'-0-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in alpha or beta anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs and 2'-alkoxy- or amino- RNA/DNA chimeras. For example, a sugar modification in the CIC includes, but is not limited to, 2'-amino-2'-deoxyadenosine. The preparation of these sugars or sugar analogs and the respective 'nucleosides' wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base) per se is known, and need not be described here, except to the extent such preparation can pertain to any specific example. Sugar modifications may also be made and combined with any phosphate modification in the preparation of a CIC. [00397] The heterocyclic bases, or nucleic acid bases, which are incorporated in the nucleic acid moiety can be the naturally- occurring principal purine and pyrimidine bases, (namely uracil, thymine, cytosine, adenine and guanine, as mentioned above), as well as synthetic modifications of said principal bases.

[00398] Those skilled in the art will recognize that a large number of 'synthetic' non- natural nucleosides comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and that as long as other criteria of the present invention are satisfied, the nucleic acid moiety can include one or several heterocyclic bases other than the principal five base components of naturally- occurring nucleic acids. Preferably, however, the heterocyclic base is, without limitation, uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo [2.3-d] pyrimidin-5-yl, 2- amino-4-oxopyrolo [2,3-d] pyrimidin-5-yl, or 2-amino-4-oxopyrrolo [2.3-d] pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the nucleic acid moiety via the 9-position, the pyrimidines via the 1 -position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1 -position.

[00399] The nucleic acid moiety may comprise at least one modified base. As used herein, the term 'modified base' is synonymous with 'base analog', for example, 'modified cytosine' is synonymous with 'cytosine analog.' Similarly, 'modified' nucleosides or nucleotides are herein defined as being synonymous with nucleoside or nucleotide 'analogs.' Examples of base modifications include, but are not limited to, addition of an electron- withdrawing moiety to C- 5 and/or C-6 of a cytosine of the nucleic acid moiety. Preferably, the electron-withdrawing moiety is a halogen. Such modified cytosines can include, but are not limited to, azacytosine, 5-bromocytosine, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, 5,6-dihydrocytosine, 5-iodocytosine, 5-nitrocytosine, and any other pyrimidine analog or modified pyrimidine. Other examples of base modifications include, but are not limited to, addition of an electron- withdrawing moiety to C- 5 and/or C-6 of a uracil of the nucleic acid moiety. Preferably, the electron- withdrawing moiety is a halogen. Such modified uracils can include, but are not limited to, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil. Also see, Kandimalla et al, 2001, Bioorg. Med. Chem. 9:807-13. [00400] Other examples of base modifications include the addition of one or more thiol groups to the base including, but not limited to, 6-thio-guanine, 4-thio-thymine and 4-thio- uracil.

[00401] The preparation of base-modified nucleosides, and the synthesis of modified oligonucleotides using said base-modified nucleosides as precursors, has been described, for example, in U.S. Patents 4,910,300, 4,948,882, and 5,093,232. These base-modified nucleosides have been designed so that they can be incorporated by chemical synthesis into either terminal or internal positions of an oligonucleotide. Such base-modified nucleosides, present at either terminal or internal positions of an oligonucleotide, can serve as sites for attachment of a peptide or other antigen. Nucleosides modified in their sugar moiety have also been described (including, but not limited to, e.g., U.S. Patents 4,849,513, 5,015,733, 5,118,800, 5,118,802) and can be used similarly.

Non-Nucleic Acid Spacer Moieties

[00402] The CIC compounds of the invention comprise one or more non-nucleic acid spacer moieties covalently bound to the nucleic acid moieties. For convenience, non-nucleic acid spacer moieties are sometimes referred to herein simply as 'spacers' or 'spacer moieties.'

[00403] Spacers are generally of molecular weight about 50 to about 500,000 (e.g. about 50 to about 50,000), sometimes from about 75 to about 5000, sometimes from about 75 to about 500, which are covalently bound, in various embodiments, to one, two, three, or more than three nucleic acid moieties. A variety of agents are suitable for connecting nucleic acid moieties. For example, a variety of compounds referred to in the scientific literature as 'non- nucleic acid linkers,' 'non-nucleotidic linkers,' or 'valency platform molecules' may be used as spacers in a CIC. A spacer moiety is said to comprise a particular spacer component (e.g., hexaethylene glycol) when the spacer includes the component (or a substituted derivative) as a subunit or portion of the spacer. As described infra, in certain embodiments, a spacer comprises multiple covalently connected subunits and may have a homopolymeric or heteropolymeric structure. Often the subunits are connected by a linker, phosphodiester linkage, and/or phosphorothioate ester linkage. See the Examples, infra. Nonnucleotide spacer moieties of a CIC comprising or derived from such multiple units can be referred to as 'compound spacers.' [00404] In one embodiment, for illustration and not limitation, the CIC comprises a compound spacer comprising any two or more (e.g., 3 or more, 4 or more, or 5 or more) of the following compounds in phosphodiester linkage and/or phosphorothioate ester linkage: oligoethylene glycol unit (e.g., triethylene glycol spacer; hexaethylene glycol spacer); alkyl unit (e.g., propyl spacer; butyl spacer; hexyl spacer); 2-(hydroxymethyl)ethyl spacer; glycerol spacer; trebler spacer; symmetrical doubler spacer.

[00405] It will be appreciated that mononucleotides and polynucleotides are not included in the definition of non-nucleic acid spacers, without which exclusion there would be no difference between nucleic acid moiety and an adjacent non-nucleic acid spacer moiety.

[00406] A variety of spacers are described herein, for illustration and not limitation. It will be appreciated by the reader that, for convenience, a spacer moiety (or component of a spacer moiety) is sometimes referred to by the chemical name of the compound (e.g., hexaethylene glycol) from which the spacer moiety or component is derived, with the understanding that the CIC actually comprises the conjugate of the compound(s) to nucleic acid moieties. As will be understood by the ordinarily skilled practitioner, the non-nucleic acid spacer can be (and usually is) formed from a spacer moiety precursor(s) that include reactive groups to permit coupling of one more nucleic acid (e.g., oligonucleotides) to the spacer moiety precursor to form the CIC and protecting groups may be included. The reactive groups on the spacer precursor may be the same or different.

[00407] Exemplary non-nucleic acid spacers comprise oligo-ethylene glycol (e.g., triethylene glycol, tetraethylene glycol, hexaethylene glycol spacers, and other polymers comprising up to about 10, about 20, about 40, about 50, about 100 or about 200 ethylene glycol units), alkyl spacers (e.g., propyl, butyl, hexyl , and other C2 - C 12 alkyl spacers, e.g., usually C2 - ClO alkyl, most often C2 - C6 alkyl), symmetric or asymmetric spacers derived from glycerol, pentaerythritol, 1,3,5-trihydroxycyclohexane or l,3-diamino-2-propanol (e.g., symmetrical doubler and trebler spacer moieties described herein). Optionally these spacer componants are substituted. For example, as will be understood by one of ordinary skill in the art, glycerol and l,3-diamino-2-propanol may be substituted at the 1, 2, and/or 3 position (e.g., replacement of one or more hydrogens attached to carbon with one of the groups listed below). Similarly, pentaerythritol may be substituted at any, or all, of the methylene positions with any of the groups described below. Substituents include alcohol, alkoxy (such as methoxy, ethoxy, and propoxy), straight or branched chain alkyl (such as C1-C12 alkyl, preferably Cl-ClO alkyl), amine, aminoalkyl (such as amino C1-C12 alkyl, preferably amino Cl-ClO alkyl), phosphoramidite, phosphate, phosphoramidate, phosphorodithioate, thiophosphate, hydrazide, hydrazine, halogen, (such as F, Cl, Br, or I), amide, alkylamide (such as amide C1-C12 alkyl, preferably Cl-ClO alkyl), carboxylic acid, carboxylic ester, carboxylic anhydride, carboxylic acid halide, ether, sulfonyl halide, imidate ester, isocyanate, isothiocyanate, haloformate, carbodiimide adduct, aldehydes, ketone, sulfhydryl, haloacetyl, alkyl halide, alkyl sulfonate, NR1R2 wherein R1R2 is -C(=O)CH=CHC(=O) (maleimide), thioether, cyano, sugar (such as mannose, galactose, and glucose), alpha,beta-unsaturated carbonyl, alkyl mercurial, alpha,beta-unsaturated sulfone.

[00408] In one embodiment, a spacer may comprise one or more abasic nucleotides (i.e., lacking a nucleotide base, but having the sugar and phosphate portions). Exemplary abasic nucleotides include l'2'-dideoxyribose, l'-deoxyribose, l'-deoxarabinose and polymers thereof.

[00409] Spacers can comprise heteromeric or homomeric oligomers and polymers of the nonnucleic acid components described herein (e.g., linked by a phosphodiester or phosphorothioate linkage or, alteratively an amide, ester, ether, thioether, disulfide, phosphoramidate, phosphotriester, phosphorodithioate, methyl phosphonate or other linkage). For example, in one embodiment, the spacer moiety comprises a branched spacer component (e.g., glycerol) conjugated via a phosphodiester or phosphorothioate linkage to an oligoethylene glycol such as HEG (see, e.g., C-94). Another example, is a spacer comprising a multivalent spacer component conjugated to an oligoethylene glycol such as HEG.

[00410] Other suitable spacers comprise substituted alkyl, substituted polyglycol, optionally substituted polyamine, optionally substituted polyalcohol, optionally substituted polyamide, optionally substituted polyether, optionally substituted polyimine, optionally substituted polyphosphodiester (such as poly(l-phospho-3-propanol), and the like. Optional substituents include alcohol, alkoxy (such as methoxy, ethoxy, and propoxy), straight or branched chain alkyl (such as C1-C12 alkyl, preferably C1-C12 alkyl), amine, aminoalkyl (such as amino C1-C12 alkyl, preferably C1-C12 alkyl), phosphoramidite, phosphate, thiophosphate, hydrazide, hydrazine, halogen, (such as F, Cl, Br, or I), amide, alkylamide (such as amide C1-C12 alkyl or C1-C12 alkyl), carboxylic acid, carboxylic ester, carboxylic anhydride, carboxylic acid halide, ether, sulfonyl halide, imidate ester, isocyanate, isothiocyanate, haloformate, carbodiimide adduct, aldehydes, ketone, sulfhydryl, haloacetyl, alkyl halide, alkyl sulfonate, NR1R2 wherein R1R2 is -C(=O)CH=CHC(=O) (maleimide), thioether, cyano, sugar (such as mannose, galactose, and glucose), alpha, beta-unsaturated carbonyl, alkyl mercurial, alpha, beta -unsaturated sulfone.

[00411] Other suitable spacers may comprise polycyclic molecules, such as those containing phenyl or cyclohexyl rings. The spacer may be a polyether such as polyphosphopropanediol, polyethylene glycol, polypropylene glycol, a bifunctional polycyclic molecule such as a bifunctional pentalene, indene, naphthalene, azulene, heptalene, biphenylene, asymindacene, sym-indacene, acenaphthylene, fluorene, phenalene, phenanthrene, anthracene, fluoranthene, acephenathrylene, aceanthrylene, triphenylene, pyrene, chrysene, naphthacene, thianthrene, isobenzofuran, chromene, xanthene, phenoxathiin, which may be substituted or modified, or a combination of the polyethers and the polycyclic molecules. The polycyclic molecule may be substituted or polysubstituted with C1-C5 alkyl, C6 alkyl, alkenyl, hydroxyalkyl, halogen or haloalkyl group. Nitrogen- containing polyheterocyclic molecules (e.g., indolizine) are typically not suitable spacers. The spacer may also be a polyalcohol, such as glycerol or pentaerythritol. In one embodiment, the spacer comprises (l-phosphopropane)₃-phosphate or (l-phosphopropane)₄- phosphate (also called tetraphosphopropanediol and pentaphosphopropanediol). In one embodiment, the spacer comprises derivatized 2,2'-ethylenedioxydiethylamine (EDDA).

[00412] Other examples of non-nucleic acid spacers that may be used in CICs include 'linkers' described by Cload and Schepartz, J. Am. Chem. Soc. (1991), 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. (1991), 113:5109; Ma et al, Nucleic Acids Research (1993), 21:2585; Ma et al., Biochemistry (1993), 32:1751; McCurdy et al., Nucleosides & Nucleotides (1991), 10:287; Jaschke et al., Tetrahedron Lett. (1993), 34:301; Ono et al., Biochemistry (1991), 30:9914; and Arnold et al., International Publication No. WO 89/02439 and EP0313219B1 entitled 'Non-nucleic acid Linking Reagents for Nucleotide Probes,' linkers described by Salunkhe et al., J. Am. Chem. Soc. (1992), 114:8768; Nelson et al., Biochemistry 35:5339-5344 (1996); Bartley et al., Biochemistry 36:14502-511 (1997); Dagneaux et al. Nucleic Acids Research 24:4506-12 (1996); Durand et al., Nucleic Acids Research 18:6353-59 (1990); Reynolds et al., Nucleic Acids Research, 24:760-65 (1996); Hendry et al. Biochemica et Biophysica Acta, 1219:405-12 (1994); Altmann et al., Nucleic Acids Research, 23:4827-35 (1995), and U.S. Patent No. 6,117,657 (Usman et al.).

[00413] Suitable spacer moieties can contribute charge and/or hydrophobicity to the CIC, contribute favorable pharmacokinetic properties (e.g., improved stability, longer residence time in blood) to the CIC, and/or result in targeting of the CIC to particular cells or organs. Spacer moieties can be selected or modified to tailor the CIC for desired pharmacokinetic properties, induction of a particular immune response, or suitability for desired modes of administration (e.g., oral administration).

[00414] Suitable spacer moieties can also be selected or modified to improve the stepwise and/or overall yield of CIC synthesis. As described herein, a platform molecule, a branch molecule or a CIC can be synthesized having one or more spacer moieties positioned between each of one or more of its nucleic acid moieties, its termini, its branch point, or any other functional moiety therein. In such molecules, the spacer moiety at a given position in the platform molecule may be selected or modified to improve the yield of one or more of the subsequent addition or conjugation steps. For example, the spacer can be more hydrophobic (e.g., divalent alkyls, such as propylene or hexylene, or any other suitable spacer described herein) or more hydrophilic (e.g., polyethylene glycols, such as diethylene glycol, TEG or HEG, or any other suitable spacer described herein). Independently, the length of the spacer can also be modified or selected. The character of a spacer can also be selected or modified by use of multiple spacers connected by linkers, such as phosphodiester linkages and/or phosphorothioate ester linkages.

[00415] Suitable spacer moieties can also be selected or modified to improve the overall yield of CIC synthesis in embodiments of the present invention where a CIC is synthesized by conjugating its precursors, e.g., a platform molecule with one or more branch molecules. As described herein, a platform molecule having one or more suitable reactive groups can be conjugated to one or more branch molecules, the branch molecules each having their respective suitable reactive groups. The yield of the conjugated product of the platform molecule and the one or more branch molecules can be affected by the composition of one or more of the precursor molecules. Thus, by selecting or modifying the one or more of the spacer moieties in a given precursor, the composition of the precursor can be affected. [00416] For example, in some preferred embodiments of the present invention, a branched platform molecule may include one or more spacers between its nucleic acid moiety and its branch point. Similarly, the branched platform molecule may include one or more spacers positioned between its branch point and one or more of its reactive termini. Selecting or modifying one or more of such spacers may improve the synthetic and/or recovery yield of the platform molecule. Similarly, selecting or modifying one or more of such spacers, particularly those between the branch point and the reactive termini, may improve the synthetic and/or recovery yield of the conjugation step between the branch molecule and one or more branch molecules.

[00417] Without intending to be bound by theory, spacer characteristics may affect overall and/or stepwise synthetic yield of a final or nascent product by determining steric accessibility, secondary structure, hydration environment, charge and polarity environment, and/or the relative energies of the starting, final or nascent products. One or more of the foregoing properties that may be affected by the spacer can thus affect the coupling efficiency, the efficiency of protection and/or deprotection, or the efficiency of termination and/or removal of undesirable side products. Furthermore, overall and/or stepwise yield can also be affected by spacer characteristics by affecting one or more of the subsequent purification and/or recovery steps. Determining and/or optimizing the suitable spacer moiety at one or more given positions for given molecules in a given reaction can be detemined empirically, experimentally and/or theoretically in any manner known in the art.

[00418] Similarly, suitable spacer moieties can also be selected or modified for a CIC to determine, improve and/or optimize of the biological activity and/or biocompatibility of the CIC. Such spacer selection and/or modification may be performed independently, or in conjunction with spacer selection and/or modification in order to affect synthetic yields, as described above.

[00419] For example, in some preferred embodiments of the present invention, a CIC may include one or more spacer moieties that have hydrophilic characteristics (e.g., polyethylene glycols, such as diethylene glycol, TEG or HEG, or any other suitable spacer described herein). In some embodiments, a CIC may preferably exclude spacers that have hydrophobic characteristics, such as longer alkyl spacers (e.g., divalent dodecylene). Similarly, the lengths of one or more of the spacer molecules can be modified and/or selected in order to effect the spacer property. For example, selecting and/or modifying the length of of one or more spacer moieties can be used to affect the distance between functional moieties (e.g., nucleic acid moieities) linked by the spacer. Likewise, selecting and/or modifying the spacer length can also be used to affect the relative chemical and physical properties of the spacer. For example, lengthening a short hydrophobic spacer (e.g., propylene to dodecylene) may increase its overall hydrophobicity, which may be undersirable.

[00420] A further advantage of modifying and/or selecting one or more of the spacer moieties is to provide further degrees of freedom to modify, improve, and/or optimize the CIC. For example, a CIC with given nucleic acid moieties may have demonstrable biological activity. The CIC can be further improved or optimized for one or more given properties (e.g., efficacy, clearance, bioavailability, side effects, toxicity, cross-reactivity, etc.) by modification and/or selection of one or more of its spacers. Such spacer optimization can be performed without need, if undesirable, to modify the already-efficacious nucleic acid moieties.

[00421] Without intending to be bound by theory, spacer characteristics may affect the biological activity and/or bioavailability of a CIC by determining steric accessibility, secondary structure, hydration environment, charge and polarity environment, and/or the relative energies of the starting, final or nascent products. In some embodiments, a CIC has spacers with sufficient hydrophilicity (as determined by, for example, empirically, experimentally and/or theoretically in any manner known in the art) to allow one or more of the immunomodulatory nucleic acid moieties of the CIC to bind and/or associate with its target site, for example, in an aqueous environment or solution. Selecting and/or modifying a spacer moiety at one or more given positions in a CIC to determine, improve, and/or optimize its biological activity and/or bioavailibity can be detemined empirically, experimentally and/or theoretically in any manner known in the art. For example, the biological activity to be determined, improved, and/or optimized can be any of the activities described herein, including immuno stimulatory activity in a mammal upon administration thereto, stimulation of interferon- alpha in a mammal upon administration thereto, or stimulation and/or activation of B cell activation in a mammal upon administration thereto.

[00422] In a CIC comprising more than one spacer moiety, the spacers may be the same or different. Thus, in one embodiment all of the non-nucleic acid spacer moieties in a CIC have the same structure. In one embodiment, a CIC comprises non-nucleic acid spacer moieties with at least 2, at least 3, at least 4, at least 5, or at least 6 or more different structures.

[00423] In some contemplated embodiments of the invention, the spacer moiety of a CIC is defined to exclude certain structures. Thus, in some embodiments of the invention, a spacer is other than an abasic nucleotide or polymer of abasic nucleotides. In some embodiments of the invention, a spacer is other than a oligo(ethyleneglycol) (e.g., HEG, TEG and the like) or poly(ethyleneglycol). In some embodiments a spacer is other than a C3 alkyl spacer. In some embodiments a spacer is other than an alkyl or substituted spacer. In some embodiments, a spacer is other than a polypeptide. Thus, in some embodiments, an immunogenic molecule, e.g., a protein or polypeptide, is not suitable as a component of spacer moieties. However, as discussed infra, it is contemplated that in certain embodiments, a CIC is a 'proteinaceous CIC, i.e., comprising a spacer moiety comprising a polypeptide (i.e., oligomer or polymer of amino acids). For example, as discussed infra, in some embodiments, a polypeptide antigen can be used as a platform (multivalent spacer) to which a plurality of nucleic acid moieties are conjugated. However, in some embodiments, the spacer moiety is not proteinaceous and/or is not an antigen (i.e., the spacer moiety, if isolated from the CIC, is not an antigen).

[00424] Suitable spacer moieties do not render the CIC of which they are a component insoluble in an aqueous solution (e.g., PBS, pH 7.0). Thus, the definition of spacers excludes microcarriers or nanocarriers. In addition, a spacer moiety that has low solubility, such as a dodecyl spacer (solubility < 5mg/ml when measured as dialcohol precursor 1,12- dihydroxydodecane) is not preferred because it can reduce the hydrophilicity and activity of the CIC. Preferably, spacer moieties have solubility much greater than 5 mg/ml (e.g., solubility at least about 20 mg/ml, at least about 50 mg/ml or at least about 100 mg/ml), e.g., when measured as dialcohol precursors. The form of the spacer moiety used for testing its water solubility is generally its most closely related unactivated and unprotected spacer precursor molecule. For example, a CIC containing a spacer moiety including a dodecyl group with phosphorothioate diester linkages at the C-I and C- 12 positons, thereby connecting the spacer moiety to the nucleic acid moieties. In this case, the water solubility of the dialcohol version of the dodecyl spacer, 1,12-dihydroxydodecane, was tested and found to be less than 5 mg/ml. Spacers with higher water solubility, when tested as their dialcohol precursors, resulted in more immuno stimulatory CICs. These higher water solubility spacers include, without limitation, propane 1,3 diol; glycerol; butane- 1, 4- diol; pentane-l,5-diol; hexane-l,6-diol; triethylene glycol, tetraethylene glycol and HEG.

Charged and Multiunit Spacer Moieties

[00425] The charge of a CIC is determined by phosphate, thiophosphate, or other groups in the nucleic acid moieties as well as groups in non-nucleic acid spacer moieties. In some embodiments of the invention, a non-nucleic acid spacer moiety carries a net charge (e.g., a net positive charge or net negative charge when measured at pH 7). In one embodiment, the CIC has a net negative charge. In some embodiments, the negative charge of a spacer moiety in a CIC is increased by derivatizing a spacer subunit described herein to increase its charge. For example, glycerol can be covalently bound to two nucleic acid moieties and the remaining alcohol can be reacted with an activated phosphoramidite, followed by oxidation or sulfurization to form a phosphate or thiophosphate, respectively. In certain embodiments the negative charge contributed by the non-nucleic acid spacer moieties in a CIC (i.e., the sum of the charges when there is more than one spacer) is greater than the negative charge contributed by the nucleic acid moieties of the CIC. Charge can be calculated based on molecular formula, or determined experimentally, e.g., by capillary electrophoresis (Li, ed., 1992, Capillary Electrophoresis, Principles, Practice and Application Elsevier Science Publishers, Amsterdam, The Netherlands, pp202-206).

[00426] As is noted supra, suitable spacers include polymers of smaller non-nucleic acid (e.g., non-nucleotide) compounds that may be used as spacers. The smaller non-nucleic acid compounds include compounds commonly referred to as non-nucleotide 'linkers' and other spacers described herein. Such polymers (i.e., 'multiunit spacers') may be heteromeric or homomeric, and often comprise monomeric units (e.g., oligoethylene glycols, [e.g., HO- (CH₂CH₂-O)_N-H, where N = 2-10; such as HEG and TEG], glycerol, l'2'-dideoxyribose, and the like) linked by an ester linkage (e.g., phosphodiester or phosphorothioate ester). Thus, in one embodiment the spacer comprises a polymeric (e.g., heteropolymeric) structure of non- nucleotide units (e.g., from 2 to about 100 units, alternatively 2 to about 50, e.g., 2 to about 5, alternatively e.g., about 5 to about 50, e.g., about 5 to about 20). [00427] For illustration, CICs containing multiunit spacers include

5'-TCGTCG-(C3)i₅-T 5'-TCGTCG-(glycerol)₁₅-T 5'-TCGTCG-(TEG)₈-T 5'-TCGTCG-(HEG)₄-T

where (C3)is means 15 propyl linkers connected via phosphorothioate esters; (glycerol) ₁₅ means 15 glycerol linkers connected via phosphorothioate esters; (TEG)g means 8 triethyleneglycol linkers connected via phosphorothioate esters; and (HEG)₄ means 4 hexaethyleneglycol linkers connected via phosphorothioate esters. It will be appreciated that certain multiunit spacers have a net negative charge, and that the negative charge can be increased by increasing the number of e.g., ester- linked monomeric units.

Multivalent Spacer Moiety

[00428] In certain embodiments, a spacer moiety is a multivalent non-nucleic acid spacer moiety (i.e., a 'multivalent spacer'). As used in this context, a CIC containing a multivalent spacer contains a spacer covalently bound to three (3) or more nucleic acid moieties. Multivalent spacers are sometimes referred to in the art as 'platform molecules.' Multivalent spacers can be polymeric or nonpolymeric. Examples of suitable molecules include glycerol or substituted glycerol (e.g., 2-hydroxymethyl glycerol, Ie vulinyl- glycerol); tetraaminobenzene, heptaaminobetacyclodextrin, 1 ,3,5-trihydroxycyclohexane, pentaerythritol and derivatives of pentaerythritol, tetraaminopentaerythritol, 1,4,8,11- tetraazacyclo tetradecane (Cyclam), 1,4,7, 10-tetraazacyclododecane (Cyclen), polyethyleneimine, l,3-diamino-2-propanol and substituted derivatives (e.g., 'symetrical doubler'), [propyloxymethyl] ethyl compounds (e.g., 'trebler'), polyethylene glycol derivatives such as so-called 'Star PEGs' and 'bPEG' (see, e.g., Gnanou et al., 1988, Makromol. Chem. 189:2885; Rein et al., 1993, Acta Polymer 44:225, Merrill et al., U.S. pat. no. 5,171,264; Shearwater Polymers Inc., Huntsville AL), dendrimers and polysaccharides.

[00429] Dendrimers are known in the art and are chemically defined globular molecules, generally prepared by stepwise or reiterative reaction of multifunctional monomers to obtain a branched structure (see, e.g., Tomalia et al., 1990, Angew. Chem. Int. Ed. Engl. 29:138-75). A variety of dendrimers are known, e.g., amine-terminated polyamidoamine, polyethyleneimine and polypropyleneimine dendrimers. Exemplary dendrimers for use in the present invention include 'dense star' polymers or 'starburst' polymers such as those described in U. S. Pat. Nos. 4,587,329; 5,338,532; and 6, 177 ',414, including so-called 'poly(amidoamine) ('PAMAM') dendrimers.' Still other multimeric spacer molecules suitable for use within the present invention include chemically-defined, non-polymeric valency platform molecules such as those disclosed in U.S. patent 5,552,391; and PCT applications PCT/USOO/15968 (published as WO 00/75105); PCT/US96/09976 (published as WO 96/40197), PCT/US97/10075 (published as WO 97/46251); PCT/US94/10031 (published as WO 95/07073); and PCT/US99/29339 (published as WO 00/34231). Many other suitable multivalent spacers can be used and will be known to those of skill in the art.

[00430] Conjugation of a nucleic acid moiety to a platform molecule can be effected in any number of ways, typically involving one or more crosslinking agents and functional groups on the nucleic acid moiety and platform molecule. Linking groups are added to platforms using standard synthetic chemistry techniques. Linking groups can be added to nucleic acid moieties using standard synthetic techniques.

[00431] Multivalent spacers with a variety of valencies may be used in the practice of the invention, and in various embodiments the multivalent spacer of a CIC is bound to between about 3 and about 400 nucleic acid moieties, sometimes about 100 to about 500, sometimes about 150 to about 250, sometimes 3-200, sometimes from 3 to 100, sometimes from 3-50, frequently from 3-10, and sometimes more than 400 nucleic acid moieties. In various embodiments, the multivalent spacer is conjugated to more than 10, more than 25, more than 50, more than 100 or more than 500 nucleic acid moieties (which may be the same or different). It will be appreciated that, in certain embodiments in which a CIC comprises a multivalent spacer, the invention provides a population of CICs with slightly different molecular structures. For example, when a CIC is prepared using a dendrimer, polysaccharide or other multivalent spacer with a high valency, a somewhat heterogeneous mixture of molecules is produced, i.e., comprising different numbers (within or predominantly within a determinable range) of nucleic acid moieties joined to the multivalent spacer moiety. When a dendrimer, polysaccharide or the like is used as an element of a multivalent spacer, the nucleic acid moieties can be joined directly or indirectly to the element (e.g., dendrimer). For example, a CIC can comprise nucleic acid moiety joined to a dendrimer via an oligoethyleneglycol element (where the dendrimer + oligoethyleneglycol constitute the spacer moiety). It will be recognized that the nucleic acid moieties may be conjugated to more than one spacer moiety.

[00432] Polysaccharides derivatized to allow linking to nucleic acid moieties can be used as multivalent spacers in CICs. Suitable polysaccharides may be naturally occurring polysaccharides or synthetic polysaccharides. Exemplary polysaccharides include, e.g., dextran, mannin, chitosan, agarose, and starch. Mannin may be used, for example, because there are mannin (mannose) receptors on immunologically relevant cell types, such as monocytes and alveolar macrophages, and so the polysaccharide spacer moiety may be used for targeting particular cell types. In an embodiment, the polysaccharide is cross-linked. One suitable compound is epichlorohydrin-crosslinked sucrose (e.g., FICOLL^®). FICOLL^® is synthesized by cross-linking sucrose with epichlorohydrin which results in a highly branched structure. The number of nucleic acid moieties in a CIC comprising a polysaccharide can be any range described herein for a CIC (e.g., a multivalent CIC). For example, in one embodiment, the polysaccharide comprises between about 150 and about 250 nucleic acid moieties. AECM-Ficoll can then be reacted with a heterobifunctional crosslinking reagent, such as 6-maleimido caproic acyl N-hydroxysuccinimide ester, and then conjugated to a thiol-derivatized nucleic acid moiety (see Lee, et al., 1980, MoI. Imm. 17:749-56). Other polysaccharides may be modified similarly.

Synthesis of CIC Precursors and Intermediates

[00433] It will be well within the ability of one of skill, guided by this specification and knowledge in the art, to prepare the precursor and intermediate molecules using routine methods. For example, techniques for making nucleic acid moieties (e.g., oligonucleotides and modified oligonucleotides) are known. Nucleic acid moieties can be synthesized using techniques including, but not limited to, enzymatic methods and chemical methods and combinations of enzymatic and chemical approaches. For example, DNA or RNA containing phosphodiester linkages can be chemically synthesized by sequentially coupling the appropriate nucleoside phosphoramidite to the 5 '-hydroxy group of the growing oligonucleotide attached to a solid support at the 3 '-end, followed by oxidation of the intermediate phosphite triester to a phosphate triester. Useful solid supports for DNA synthesis include Controlled Pore Glass (Applied Biosystems, Foster City, CA), polystyrene bead matrix (Primer Support, Amersham Pharmacia, Piscataway, NJ) and TentGel (Rapp Polymere GmbH, Tubingen, Germany). Once the desired oligonucleotide sequence has been synthesized, the oligonucleotide is removed from the support, the phosphate triester groups are deprotected to phosphate diesters and the nucleoside bases are deprotected using aqueous ammonia or other bases.

[00434] For instance, DNA or RNA polynucleotides (nucleic acid moieties) containing phosphodiester linkages are generally synthesized by repetitive iterations of the following steps: a) removal of the protecting group from the 5'-hydroxyl group of the 3'-solid support- bound nucleoside or nucleic acid, b) coupling of the activated nucleoside phosphoramidite to the 5'-hydroxyl group, c) oxidation of the phosphite triester to the phosphate triester, and d) capping of unreacted 5'-hydroxyl groups. DNA or RNA containing phosphorothioate linkages is prepared as described above, except that the oxidation step is replaced with a sulfurization step. Once the desired oligonucleotide sequence has been synthesized, the oligonucleotide is removed from the support, the phosphate triester groups are deprotected to phosphate diesters and the nucleoside bases are deprotected using aqueous ammonia or other bases. See, for example, Beaucage (1993) 'Oligodeoxyribonucleotide Synthesis' in PROTOCOLS FOR OLIGONUCLEOTIDES AND ANALOGS, SYNTHESIS AND PROPERTIES (Agrawal, ed.) Humana Press, Totowa, NJ; Warner et al. (1984) DNA 3:401; Tang et al. (2000) Org. Process Res. Dev. 4:194-198; Wyrzykiewica et al. (1994) Bioorg. & Med. Chem. Lett. 4:1519-1522; Radhakrishna et al. (1989) /. Org. Chem. 55:4693-4699 and U.S. Patent No. 4,458,066. Programmable machines that automatically synthesize nucleic acid moieties of specified sequences are widely available. Examples include the Expedite 8909 automated DNA synthesizer (Perseptive Biosystem, Framington MA); the ABI 394 (Applied Biosystems, Inc., Foster City, CA); and the OligoPilot II (Amersham Pharmacia Biotech, Piscataway, NJ).

[00435] Polynucleotides can be assembled in the 3' to 5' direction, e.g., using base- protected nucleosides (monomers) containing an acid-labile 5 '-protecting group and a 3'- phosphoramidite. Examples of such monomers include 5'-O-(4,4'-dimethoxytrityl)-protected nucleoside-3'-O-(N,N-diisopropylamino) 2-cyanoethyl phosphoramidite, where examples of the protected nucleosides include, but are not limited to, N6-benzoyladenosine, N4- benzoylcytidine, N2-isobutryrylguanosine, thymidine, and uridine. In this case, the solid support used contains a 3 '-linked protected nucleoside. Alternatively, polynucleotides can be assembled in the 5' to 3' direction using base-protected nucleosides containing an acid-labile 3'-protecting group and a 5'-phosphoramidite. Examples of such monomers include 3'-O- (4,4'-dimethoxytrityl)-protected nucleoside-5'-O-(N,N-diisopropylamino) 2-cyanoethyl phosphoramidite, where examples of the protected nucleosides include, but are not limited to, N6-benzoyladenosine, N4-benzoylcytidine, N2-isobutryrylguanosine, thymidine, and uridine (Glen Research, Sterling, VA). In this case, the solid support used contains a 5 '-linked protected nucleoside. Circular nucleic acid components can be isolated, synthesized through recombinant methods, or chemically synthesized. Chemical synthesis can be performed using any method described in the literature. See, for instance, Gao et al. (1995) Nucleic Acids Res. 23:2025-2029 and Wang et al. (1994) Nucleic Acids Res. 22:2326-2333.

[00436] In addition to the examples and embodiments described herein, conjugation of the nucleic acid moieties and spacer moieties can be carrried out in a variety of ways, depending on the particular CIC being prepared. Methods for addition of particular spacer moieties are known in the art and, for example, are described in the references cited supra. See, e.g., Durand et al., Nucleic Acids Research 18:6353-59 (1990). The covalent linkage between a spacer moiety and nucleic acid moiety can be any of a number of types, including phosphodiester, phosphorothioate, amide, ester, ether, thioether, disulfide, phosphoramidate, phosphotriester, phosphorodithioate, methyl phosphonate and other linkages. As noted supra, spacer moiety precursors can optionally be modified with terminal activating groups for coupling to nucleic acids. Other spacer moiety precursors include, for example and not for limitation, (1) HOCH₂CH₂O(CH₂CH₂O)_nCH₂CH₂OH, where n= 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 or is greater than 45; (2) HOCH₂CHOHCH₂OH; (3) HO(CH₂)_nOH, where n= 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[00437] In one embodiment, a spacer moiety precursor is used that includes first and second reactive groups to permit conjugation to nucleic acid moieties in a stepwise fashion, in which the first reactive group has the property that it can couple efficiently to the terminus of a growing chain of nucleic acids and the second reactive group is capable of further extending, in a step-wise fashion the growing chain of mixed nucleotide and non-nucleotide moieties. It will often be convenient to combine a spacer moiety(s) and a nucleic acid moiety(s) using the same phosphoramidite-type chemistry used for synthesis of the nucleic acid moiety. For example, such molecules can be conveniently synthesized using an automated DNA synthesizer (e.g., Expedite 8909; Perseptive Biosystems, Framington, MA) using phosphoramidite chemistry (see, e.g., Beaucage, 1993, supra; Current Protocols in Nucleic Acid Chemistry, supra). However, one of skill will understand that the same (or equivalent) synthesis steps carried out by an automated DNA synthesizer can also be carried out manually, if desired. The resulting linkage between the nucleic acid and the spacer precursors can be a phosphorothioate or phosphodiester linkage. In such a synthesis, typically, one end of the spacer (or spacer subunit for multimeric spacers) is protected with a 4,4'-dimethyoxytrityl group, while the other end contains a phosphoramidite group.

[00438] A variety of spacers with useful protecting and reacting groups are commercially available, for example:

triethylene glycol spacer or TEG spacer' 9-O-(4,4'-dimethoxytrityl)triethyleneglycol-l-O- [(2-cyanoethyl) N,N-diisopropylphosphoramidite] (Glen Research, 22825 Davis Drive, Sterling, VA);

hexaethylene glycol spacer or ΗEG spacer' 18-O-(4,4' -dimethoxytrityl)hexaethyleneglycol- l-O-[(2-cyanoethyl) N,N-diisopropylphosphoramidite] (Glen Research, Sterling, VA);

propyl spacer 3-(4,4'-dimethoxytrityloxy)propyloxy-l-O-[(2-cyanoethyl) N,N- diisopropylphosphoramidite] (Glen Research, Sterling, VA);

butyl spacer 4-(4,4'-dimethoxytrityloxy)butyloxy-l-O-[(2-cyanoethyl) N,N- diisopropylphosphoramidite] (Chem Genes Corporation, Ashland Technology Center, 200 Homer Ave, Ashland, MA);

Hexyl spacer: 6-(4,4'-dimethoxytrityloxy)hexyloxy-l-O-[(2-cyanoethyl) N,N- diisopropylphosphoramidite] (Biosearch Technologies, Novoto, CA)

2-(hydroxymethyl)ethyl spacer or ΗME spacer' l-(4,4'-dimethoxytrityloxy)-3- (levulinyloxy)-propyloxy-2-O-[(2-cyanoethyl) N,N-diisopropylphosphoramidite]; also called 'asymmetrical branched' spacer (see Fig. 2) (Chem Genes Corp., Ashland Technoklgy Center, Ashland MA.);

'abasic nucleotide spacer' or 'abasic spacer' 5-O-(4,4' -dimethoxytrityl)- 1 ,2-dideoxyribose- 3-O-[(2-cyanoethyl) N,N-diisopropylphosphoramidite] (Glen Research, Sterling, VA); "symmetrical branched spacer' or "glycerol spacer' l,3-O,O-bis(4,4'- dimethoxytrityl)glycerol-2-O- [(2-cyanoethyl) N,N-diisopropylphosphoramidite] (Chem Genes, Ashland, MA) (see Fig. 2);

'trebler spacer' (see Fig. 2) 2,2,2-O,O,O-tris[3-O-(4,4'- dimethoxy trityloxy )propyloxymethyl] ethyl- 1 - O- [ (2-cyanoethyl) N ,N- diisopropylphosphoramidite] (Glen Research, Sterling, VA);

"symmetrical doubler spacer' (see Fig. 2) 1,3-0, 0-bis[5-O-(4,4'- dimethoxytrityloxy)pentylamido]propyl-2-O- [(2-cyanoethyl) N,N- diisopropylphosphoramidite] (Glen Research, Sterling, VA);

'dodecyl spacer' 12-(4,4'-dimethoxytrityloxy)dodecyloxy- 1-0- [(2-cyanoethyl) N,N- diisopropylphosphoramidite] (Glen Research, Sterling, VA).

[00439] These and a large variety of other protected spacer moiety precursors (e.g., comprising DMT and phosphoramidite group protecting groups) can be purchased or can be synthesized using routine methods for use in preparing CICs disclosed herein. The instrument is programmed according to the manufacturer' s instructions to add nucleotide monomers and spacers in the desired order.

[00440] CICs prepared 'in situ' on a DNA synthesizer require protected nucleoside and protected spacer monomers, both containing reactive or activatable functional groups. The reactive and/or protected form of the spacer moiety can be described as a 'spacer precursor component.' It will be appreciated by those with skill in the art that the reactive groups in the spacer precursors form stable linkages after coupling and the protecting groups on the spacer precursor are removed in the resultant spacer moiety in the CIC. The protecting groups are generally removed during the step-wise synthesis of the CIC, in order to allow reaction at that site.

[00441] An example of a spacer precursor with no additional reactive functionality is 18- O-(4,4'-dimethoxytrityl)hexaethyleneglycol-l-O-[(2-cyanoethyl)N,N- diisopropylphosphoramidite], which contains a protecting group, the 4,4'-dimethoxytrityl group, and a reactive group, the (2-cyanoethyl)N,N-diisopropylphosphoramidite group. During preparation of the CIC using phosphoramidite chemistry on a DNA synthesizer, the

(2-cyanoethyl)N,N-diisopropylphosphoramidite group in the spacer precursor is activated by a weak acid, such as lH-tetrazole, and reacted with the free 5'-hydroxyl of the nucleobase- protected nucleic acid moiety to form a phosphite triester. The phosphite triester group is then either oxizided or sulfurized to a stable phosphotriester or phosphorothioate triester group, respectively. The resultant triester group is stable to the rest of the CIC synthesis and remains in that form until the final deprotection. In order to couple either another spacer precursor or an activated nucleoside monomer, which will become part of the next nucleic acid moiety, the 4,4'-dimethoxytrityl group on the spacer precursor is removed. After coupling and either oxidation or sulfurization, this group also becomes either a stable phosphotriester or phosphorothioate triester group, respectively. Once the protected CIC is fully assembled, the CIC is cleaved from the solid support, the cyanoethyl groups on the phosphotriester or phosphorothioate triester groups are removed, and the nucleobase protection is removed. In this example, the CIC contains spacer moieties including stable phosphodiester or phosphorothioate diester linkages to the nucleic acid moieties. Both the reactive phosphoramidite group and the protected hydroxyl group of the spacer precursor are converted to stable phosphodiester or phosphorothioate diester linkages in the spacer moiety. Because the reaction of each end of the spacer may be independent, one linkage may be phosphodiester and the other linkage phosphorothioate diester, or any combination thereof. CICs with other phosphate modifications, such as phosphorodithioate, methyl phosphonate, and phosphoramidate, may also be prepared in this manner by using a spacer precursor with the appropriate reactive group, the correct ancilliary reagents, and protocols designed for that type of linkage. These protocols are analogous to those described for preparing nucleic acid moieties with modified phosphate linkages.

Alternate Syntheses of CICs and Their Precursors

[00442] It will be appreciated that the CICs platform molecules, oligonucleotides and other molecules described herein are not limited to any particular method of synthesis or preparation. For example, oligonucleotide intermediates described herein may include nucleic acid moieties containing groups not compatible with DNA synthesis and deprotection conditions, such as (but not limited to) hydrazine or maleimide. Such oligonucleotide intermediates can be prepared by reacting a nucleic acid moiety containing an amino linker with the appropriate heterobifunctional crosslinking reagent, such as SHNH (succinimidyl hydraziniumnicotinate) or sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]- cyclohexame- 1-carboxylate). [00443] Methods for conjugating protein, peptides, oligonucleotides, and small molecules in various combinations are described in the literature and can be adapted to achieve conjugation of a nucleic acid moiety containing a reactive linking group to a spacer moiety precursor. See, for example, Bioconjugate Techniques, Greg T. Hermanson, Academic Press, Inc., San Diego, CA, 1996. In some embodiments, a nucleic acid moiety(s) is synthesized, and a reactive linking group (e.g., amino, carboxylate, thio, disulfide, and the like) is added using standard synthetic chemistry techniques. The reactive linking group (which is considered to form a portion of the resulting spacer moiety) is conjugated to additional non-nucleic acid compounds to form a portion of the spacer moiety. Reactive linking groups are added to nucleic acids using standard methods for nucleic acid synthesis, employing a variety of reagents known in the art. Examples include reagents that contain a protected amino group, carboxylate group, thiol group, disulfide group, aldehyde group, diol group, diene group and a phosphoramidite group. Once these compounds are incorporated into the nucleic acids, via the activated phosphoramidite group, and are deprotected, they provide nucleic acids with amino, carboxylate, aldehyde, diol, diene or thiol reactivity. Examples of reactive groups for conjugating a nucleic acid moiety containing a reactive linker group to a spacer moiety precursor that contains a reactive group are shown below.

Nucleic acid reactive Spacer moiety precursor Stable linkage formed group reactive group thiol maleimide, haloacetyl thioether maleimide thiol thioether thiol pyridine disulfide disulfide pyridine disulfide thiol disulfide amine NHS or other active ester amide amine carboxylate amide carboxylate amine amide aldehyde, ketone hydrazine, hydrazide hydrazone, hydrazide hydrazine, hydrazide aldehyde, ketone hydrazone, hydrazide diene dienophile aliphatic or heterocyclic ring

[00444] The reactive linking group and the spacer precursor react to form a stable bond and the entire group of atoms between the two (or more) nucleic acid moieties is defined as the spacer moiety. For example, a nucleic acid moiety synthesized with a mercaptohexyl group linked to the nucleic acid moiety via a phosphorothioate group can be reacted with a spacer precursor containing one (or more) maleimide group(s), forming a thioether linkage(s). The spacer moiety of this CIC includes the phosphorothioate group and hexyl group from the linker on the nucleic acid moiety, the new thioether linkage, and the rest of the spacer that was part of the spacer precursor.

[00445] Both linear and particularly branched CICs can be made using these conjugation strategies. Additionally, spacer precursor molecules can be prepared with several orthogonal reactive groups to allow for the addition of more than one type nucleic acid moiety (e.g., different sequence motif).

[00446] In one embodiment, CICs with multivalent spacers conjugated to more than one type of nucleic acid moiety are prepared. For instance, platforms containing two maleimide groups (which can react with thiol-containing polynucleotides), and two activated ester groups (which can react with amino-containing nucleic acids) have been described (see, e.g., PCT/US94/10031, published as WO 95/07073). These two activated groups can be reacted independently of each other. This would result in a CIC containing a total of 4 nucleic acid moieties, two of each sequence.

Linkers

[00447] Hydrophilic linkers of variable lengths may be used, for example to link nucleic acids moieties and platform molecules. A variety of suitable linkers are known. Suitable linkers include, without limitation, linear oligomers or polymers of ethylene glycol. Such linkers include linkers with the formula R¹S(CH₂CH₂O)_nCH₂CH₂O (CH₂)_mCO₂R² wherein n = 0-200, m = 1 or 2, R¹ = H or a protecting group such as trityl, R² = H or alkyl or aryl, e.g., 4-nitrophenyl ester. These linkers may be used in connecting a molecule containing a thiol reactive group such as haloaceyl, maleiamide, etc., via a thioether to a second molecule which contains an amino group via an amide bond. The order of attachment can vary, i.e., the thioether bond can be formed before or after the amide bond is formed. Other linkers include Sulfo-SMCC (sulfosuccinimidyl 4-[/V-maleimidomethyl]-cyclohexane-l-carboxylate) Pierce Chemical Co. product 22322; Sulfo-EMCS (/V-[ε-maleimidocaproyloxyl sulfosuccinimide ester) Pierce Chemical Co. product 22307; Sulfo-GMBS (N-[y- maleimidobutyryloxy] sulfosuccinimide ester) Pierce Chemical Co. product 22324 (Pierce Chemical Company, Rockford, IL), and similar compounds of the general formula maleimido-R-C(O)NHS ester, where R = alkyl, cyclic alkyl, polymers of ethylene glycol, and the like.

[00448] As described and discussed herein, CICs can be made from platform molecules and one or more branch molecules in relatively few steps instead of many discrete coupling steps which can lead to impurities which are difficult to remove from the final product. In particular, multimeric CICs manufactured on a large scale may be more difficult to purify to state of the art levels for drug development when synthesized by the previously described stepwise procedure. However, use of smaller branch and platform compounds that can be more easily purified prior to their conjugation to form the multimeric CIC, thereby leading to CICs of higher purity than those made by a stepwise procedure. The methods provided herein increase the purity of multimeric CICs manufactured on a large scale to the state of the art or greater levels by providing a purification of the small branch and platform compounds, followed by conjugation of the purified small branch and platform compounds to form the multimeric CICs, and then purification of the final CIC. Generally, increasing the purity of the branch and/or platform compounds results in an increase in the purity of the resulting multimeric CICs when manufactured by the methods described herein. CICs of increased purity may contribute to an improved biological activity and/or reduced unwanted effects.

[00449] In certain preferred embodiments, the conjugation of a platform molecule and one or more branch molecules can improve the purity and hence the yield of the desired full- length CIC by allowing purification and/or recovery of the platform and/or branch molecules (i.e., CIC precursor molecules) following their respective syntheses, but prior to conjugation therebetween. Side- or incomplete products can arise during the synthesis of a platform or branch molecule, particularly when using stepwise synthesis. Such products of stepwise synthesis can be identified and removed based on their differences in molecular weight (MW) and/or charge, both of which characteristics are typically lower when compared to the desired product. The ability to resolve molecules of different MWs and/or charges increases with the differences in these properties, yet decreases with the absolute MW and/or charge of the molecules. Thus, it may be easier to resolve and remove unreacted or side products upon completing synthesis of the CIC precursor molecules, e.g., the platform molecule and/or one or more of the branch molecules. Further, when one or more of the precursor molecules involves the stepwise synthesis of one or more nucleic acid motifs, such intermediate purification may improve the nucleic acid sequence homogeneity (including any other spacers added thereto) of the precursor, such that the purified, recovered precursor is substantially pure.

[00450] By the methodology disclosed herein, obtaining a substantially pure preparation of a given CIC of the present invention is made easier because of the greater difference in size when an entire branch is added to the platform molecule. For example, a CIC of the present invention can be more readily purified away from unreacted or side products, due to the greater size differences between the CIC and its precursor branch and platform molecules, and thus obtain a substantially pure preparation of the desired CIC. Correspondingly, such a purified preparation of the substantially pure CIC is substantially free of non-conforming side- or incomplete products, due to the greater difference between the latter non-conforming products and the former desired product. As a consequence, CICs made from such purified precursors may also be substantially pure with respect to sequence, spacer and/or molecular weight.

[00451] Further, by purifying and recovering one or more of the precursor molecules prior to their conjugation, the expected CIC from the reaction is readily resolvable, and thus can be purified, from, the unreacted precursors or side products or other non-conforming compounds due to their large relative differences in MW and charge. In addition, the prior removal of the undesired side products of stepwise syntheses prior to the conjugation reaction may improve the yield and/or recovery by reducing the undesired side products' ability to compete in or with the desired conjugation reaction.

[00452] An example of the foregoing improvement is depicted in Figs. 21 and 22 and described in Example 23, showing an exemplary ion exchange chromatogram of a product of a conjugation reaction of two purified 7-mer branch molecules and a purified 7-mer platform molecule. As indicated, the relatively large charge and mass differences between the desired 21-mer product and either the mono-adduct (14-mer) or excess branch molecules (7-mer) allows a good resolution of the molecules, and hence an improved purification of the desired 21-mer. Although the branch dimer (formed from disulfide bond formation) is less resolvable than the other non-comforming compounds, the disulfide functionality of the dimer side product is advantageously exploited by reducing the dimer to its constituent monomers prior to purification of the branched CIC, as shown in Fig. 22. The excess branch precursor molecules, as they retain their active thiol groups, may be recovered for further conjugation reactions. In addition, the thioether linkage formed in the CIC conjugation is shown to have long-term stability under a variety of neutral and basic pH conditions.

[00453] Similarly, as depicted in Figs. 23 and 24 and described in Example 24, showing an exemplary ion exchange chromatogram of a product of a conjugation reaction of two purified 10-mer branch molecules and a purified 10-mer platform molecule. As indicated, the relatively large charge and mass differences between the desired 30-mer product and either the mono-adduct (20-mer) or excess branch molecules (10-mer) allows a good resolution of the molecules, and hence an improved purification of the desired 30-mer. In addition, reduction of the dimer, as shown in Fig. 24, allows resolution between the dimer and the branched CIC.

[00454] Another example of the improved conjugation method is depicted in Fig. 19 and described in Example 25, which shows an exemplary reverse-phase HPLC chromatogram of an exemplary 30-mer branched CIC that is made and purified in accordance with the present invention. As depicted in Fig. 19, the desired 30-mer is about 92% pure, with little detectable amount of early-eluting impurities, such as the single phosphodiester impurity or the n-1 impurity. In comparison, in Fig. 20 (also an exemplary reverse-phase HPLC chromatogram), a 21-mer branched CIC that was made on a 200 umol scale by a stepwise method and purified by standard ion exchange procedures is recovered in 71% purity, despite its shorter length. Moreover, an early-eluting impurity, which is poorly resolved from the desired 25- mer, is present at about 22%, having been carried over with the 21-mer upon its purification.

[00455] In another aspect, the conjugation of a platform molecule and one or more branch molecules in accordance with the present invention allows synthesis of CIC compounds in good yield, as compared to synthesis of the same molecule using only stepwise synthesis, due to the fewer number of sequential synthetic steps required for the conjugation method compared to the stepwise method. As a result, the conjugation methods of the present invention may also allow synthesis of longer CIC molecules (i.e., CICs having longer oligonucleotide(s) and/or multiple spacers). In comparison, an attempt to synthesize such longer CIC compounds using exclusively stepwise synthesis may result in an impractically small yield of the desired product due to the larger number of sequential reactions.

[00456] In some embodiments, the conjugation of platform molecule and one or more of the branch molecule precursors may use particular reactive groups on the respective molecules which, when reacted, allow coupling of the platform and branch molecule(s) to form the CIC. The selection of suitable reactive groups may be based on certain considerations. As described herein, reactive groups include the group both in its reactive and (if necessary) its protected forms, as is suitable.

[00457] For example, it may be desirable to select reactive groups that are sufficiently reactive (e.g., nucleophilicity, electrophilicity) to result in a high product yield. It may also be desirable to select reactive groups that (either by itself, in a protected form, or upon formation of the linker) are stable under certain reaction environmental conditions (e.g., aqueous/non-polar, pH, temperature, ionic strength, redox potential). It may also be desirable to select reactive groups that are stable (either by itself or in a protected form) during the synthesis and/or purification of its precursor molecule. It may also be desirable to select reactive groups that, upon conjugation reaction, form a linker that is stable during the synthesis and/or purification of the final CIC molecule and/or during the formulation of the CIC molecule as a pharmaceutical composition. It may also be desirable to select reactive groups that, upon the conjugation reaction, form a linker that is stable during administration, delivery, or any other given in situ or in vivo environments in which the CIC molecule or composition thereof may be provided.

[00458] In addition, selection of the reactive groups can also involve determining the polarity to the reactive groups. For example, if the selected reactive groups are a nucleophile and an electrophile, it may be preferred, based on certain considerations, to have the nucleophile on the branch molecule rather than the platform molecule, or vice versa. If a precursor molecule is multivalent (i.e., having more than one reactive group), a reactive group may be selected that is unable to undergo undesirable intra- or intermolecular reactions involving the precursor. For example, a thiol reactive group can act as both a nucleophile and form a disulphide linkage. The latter reaction may be favored under high-concentration and/or intramolecular contexts, and thus, in certain embodiments, the use of a thiol (or protected form thereof) may be unsuitable in a multivalent platform molecule.

[00459] Another consideration for selecting polarity of the reactive groups on their respective precursors is the relative reactivity of the reactive groups. For example, in certain embodiments, it may be desirable to use a lower-reactive group due to its relative environmental stability, so as to preclude or minimize inactivation and/or side reaction. Such low reactivity can be offset by, for example, increasing the reaction stoichiometry in favor of the lower-reactive group so that it is excess over the other precursor compound. Thus, in such embodiments, the lower-reactive group is selected for the precursor that, for example, can be made or used more readily in excess, due to its cost, difficulty of synthesis, ability to purify, and the like. Moreover, the reactive group and its precursor to be used in excess may also be based on whether the excess precursor is suitable for recovery for use in other reactions.

[00460] For example, certain embodiments may use a nucleophilic reactive group on one precursor in reaction with a electrophilic reactive group on the other precursor. In certain embodiments, a haloacetyl electrophile may be preferred over a maleimide electrophile (both described herein) when the former forms a more stable linkage at neutral to basic pH, as may be desired in certain pharmaceutical applications.

[00461] In a further example, in certain embodiments a chloroacetyl electrophile may be preferred over a bromoacetyl electrophile (both described herein) when the latter is less stable during the isolation, purification, and use of its precursor molecule. In comparison, the chloroacetyl is preferred due to its greater stability under neutral, aqueous conditions. Chloroacetyl reactive groups may also be preferred over bromoacetyl due to its stability at low or freezing temperatures (e.g., -2O⁰C), thereby allowing storage of the activated precursor.

[00462] Platform and branch molecules of the present invention may be preferred due to their improved amenability to purification. In some embodiments, the platform or branch molecule requires fewer purification steps following their respective syntheses. For example, the platform and/or branch molecule may require only a single desalting step (using, e.g., C18 column on an AKTA System or an ultrafiltration system), compared to the typical use of one or more precipitations or chromatographies prior to desalting. In some embodiments, the platform molecule of the present invention that requires fewer purification steps includes a chloroacetyl electrophile reactive group or protected form thereof, as described herein. In some embodiments, the branch molecule of the present invention that requires fewer purification steps includes a thiol nucleophile reactive group or protected form thereof, as described herein. [00463] In a further example, in certain embodiments a thiol nucleophile may be preferred over a thiophosphate electrophile (both described herein) in reaction with a haloacetyl (e.g., chloroacetyl), when the latter is less nucleophilic at more basic pH values (e.g., about or greater than pH 9), thereby producing a greater yield of the conjugation product.

[00464] In some preferred embodiments, a platform molecule of the present invention can be conjugated with a single species of branch molecule. In such embodiments, the platform molecule comprises multiple PMRGs. In preferred embodiments, the multiple PMRGs of the multivalent platform molecule are substantially equivalent in their reactivity to a given branch molecule. A suitable number of equivalents of a single species of reactive branch molecule is reacted with the multivalent platform molecule. The resulting CIC can be considered substantially symmetrical with respect to its branch, as each branch comprises the same spacers and oligonucleotide moieties. Such symmetrical conjugations may be preferred due to the relatively high number of equivalents of immunomodulatory oligonucleotides in each CIC, and the relative ease of synthesis provided by the use of only a single branch molecule species during the synthesis.

Proteinaceous CICs

[00465] In certain embodiments, a polypeptide, such as a protein antigen or antigen fragment, is used as a multivalent spacer moiety to which a plurality of nucleic acid moieties are covalently conjugated, directly or via linkers, to form a 'proteinaceous CIC The polypeptide can be an antigen or immunogen to which an adaptive immune response is desired, or a carrier (e.g., albumin). Typically, a proteinaceous CIC comprises at least one, and usually several or many nucleic acid moieties that (a) are between 2 and 7, more often between 4 and 7 nucleotides in length, alternatively between 2 and 6, 2 and 5_, 4 and 6, or 4 and 5 nucleotides in length and/or (b) have inferior isolated immunomodulatory activity or do not have isolated immunomodulatory activity. Methods of making a proteinaceous CIC will be apparent to one of skill upon review of the present disclosure. A nucleic acid, for example, can be covalently conjugated to a polypeptide spacer moiety by art known methods including linkages between a 3' or 5' end of a nucleic acid moiety (or at a suitably modified base at an internal position in the a nucleic acid moiety) and a polypeptide with a suitable reactive group (e.g., an N-hydroxysuccinimide ester, which can be reacted directly with the N⁴ amino group of cytosine residues). As a further example, a polypeptide can be attached to a free 5 '-end of a nucleic acid moiety through an amine, thiol, or carboxyl group that has been incorporated into nucleic acid moiety. Alternatively, the polypeptide can be conjugated to a spacer moiety, as described herein. Further, a linking group comprising a protected amine, thiol, or carboxyl at one end, and a phosphoramidite can be covalently attached to a hydroxyl group of a polynucleotide, and, subsequent to deprotection, the functionality can be used to covalently attach the CIC to a peptide.

Purification

[00466] The CICs of the invention are purified using any conventional means, such as high performance liquid chromatography, electrophoretic methods, nucleic acid affinity chromatography, size exclusion chromatography, and ion exchange chromatography. In some embodiments, a CIC compound that is substantially pure is intended to mean a preparation of the compound that includes at least 80% to at least 99% of the compound by weight after correction of the total weight for water content, as described below. As used herein, the term 'compound' refers to a structurally-defined product, in which the defined product includes, for example, particular oligonucleotide sequence(s), spacer(s) and backbone configuration. In some embodiments, a preparation of a compound of the present invention that is substantially pure is at least 85% pure by weight, at least 86% pure by weight, at least 87% pure by weight, at least 88% pure by weight, at least 89% pure by weight, at least 90% pure by weight, at least 91% pure by weight, at least 92% pure by weight, at least 93% pure by weight, at least 94% pure by weight, at least 95% pure by weight, at least 96% pure by weight, at least 97% pure by weight, at least 98% pure by weight or at least 99% pure by weight on an anhydrous basis (e.g., after correction of the total weight for water content). The total weight is preferably corrected for water content because CICs and oligonucleotides isolated by lyophilization often contain high, and variable, levels of water, e.g., 3-20%. The water content can be determined on a weight percent basis by known methods, such as Karl Fischer analysis (U.S. Pharmacopoeia, vol. 23, 1995, method 921, U.S.P. Pharmacopeial Convention, Inc., Rockville, MD, USA). For instance, if 100 mg (total weight) of material were weighed and the water content was determined to be 10%, then the total weight corrected for the water content would be 90 mg (100 mg x (100- 10)/ 100). In this example, a compound with a purity of 90% by weight would contain 81 mg (90 mg x 90/100) of the compound after correction of the total weight for the water content. [00467] The purity of the compound on an area percent basis can be determined, for instance, by a HPLC method that resolves the compound from the compound-related impurities on a chromatography column and uses detection at a suitable characteristic wavelength where the compound absorbs light, e.g., at 260 nm. See Example 25 for an exemplary suitable HPLC method. As the response factor (area counts per weight) of the compound and compound-related impurities are highly similar, the area percent result can be taken as the weight percent result. For instance, if the area percent purity by HPLC is 90% and the total weight after correction for water content is 90 mg, then 81 mg of compound would be present in the sample (90 mg x 90/100).

[00468] In another aspect, a given compound of the present invention that is 'substantially pure' is intended to mean that a preparation of the compound is substantially free of non-conforming compound. As used herein, a 'non-conforming compound' of a given compound differs from the given compound with respect to one or more of the following exemplary characteristics: one or more of the compound's oligonucleotide sequences, one or more of the compound's spacers, the compound's backbone configuration, or any other stable attribute of the compound. Such non-conforming compounds may result from incomplete synthesis of the given compound, or other side products that arise during the synthesis of the given compound. For example, for compounds of the present invention that include phosphorothioate oligonucleotides, typical non-conforming compounds include, for example, deletions in one or more of the oligonucleotides (e.g., n-1, n-2, etc.) in which the non-conforming compound is missing one or more nucleotide -phosphorothioate groups with respect to the defined compound; PO defects, in which the non-conforming compound contains one or more phosphodiester backbone linkages instead of a phosphorothioate linkage as in the compound; hydrophobic modifications, in which the non-conforming compound contains one or more hydrophobic modifications, such as cyanoethyl, acetyl, t- butyl, etc., which are not present in the compound; additions in one or more of the oligonucleotides (e.g., n+1, n+2, etc.), in which the non-conforming compound contains one or more extra nucleotide-phosphorothioate groups than the compound; depurination, in which the non-conforming compound is missing one or more bases in at least one of the defined oligonucleotide sequences of the compound; depurination cleavage products, in which the non-conforming compound comprises or is missing one or more fragments of the compound that have resulted from cleavage at a depurination site. Accordingly, in some embodiments, a preparation of a compound of the present invention that is substantially pure includes less than 20% non-conforming compounds by weight, less than 14% non-conforming compounds by weight, less than 13% non-conforming compounds by weight, less than 12% non- conforming compounds by weight, less than 11% non-conforming compounds by weight, less than 15% non-conforming compounds by weight, less than 10% non-conforming compounds by weight, less than 9% non-conforming compounds by weight, less than 8% non-conforming compounds by weight, less than 7% non-conforming compounds by weight, less than 6% non-conforming compounds by weight, less than 5% non-conforming compounds by weight, less than 4% non-conforming compounds by weight, less than 3% by weight non-conforming compounds by weight, less than 2% by weight non-conforming compounds by weight or less than 1% by weight non-conforming compounds by weight, wherein all foregoing percentages are determined after correction of the total weight for the water content. The respective weights of the compound and non-conforming compounds can be determined as described herein. In certain aspects, the aforementioned aspects of a preparation of substantially pure compound of the present invention is intended to refer to a preparation of the compound following at least one step of purification or isolation. By the methodology disclosed herein, purification is made easier than in prior disclosures because of the greater difference in size when an entire branch is added to the platform molecule.

Compositions

[00469] In various embodiments, compositions of the invention comprise one or more CICs (i.e. a single CIC or a combination of two or more CICs) either optionally in conjunction with another immunomodulatory or immunoregulatory agent, such as a peptide, an antigen (described below) and/or an additional adjuvant. Compositions of the invention may comprise a CIC and pharmaceutically acceptable excipient. By 'pharmaceutically acceptable' it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutically acceptable excipients are well known in the art and include sterile water, isotonic solutions such as saline and phosphate buffered saline, and other excipients known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (19th edition, 1995, Gennavo, ed.). Adjuvants (an example of which is alum) are known in the art. CIC formulations may be prepared with other immunotherapeutic agents, such as cytokines and antibodies. In some embodiments the composition is isotonic and/or sterile, e.g., suitable for administration to a human patient, e.g., manufactured or formulated under GMP standards. CIC/MC Complexes

[00470] CICs may be administered in the form of CIC/microcarrier (CIC/MC) complexes. Accordingly, the invention provides compositions comprising CIC/MC complexes.

[00471] CIC/MC complexes comprise a CIC bound to the surface of a microcarrier (i.e., the CIC is not encapsulated in the MC), and preferably comprise multiple molecules of CIC bound to each microcarrier. In certain embodiments, a mixture of different CICs may be complexed with a microcarrier, such that the microcarrier is bound to more than one CIC species. The bond between the CIC and MC may be covalent or non-covalent (e.g. mediated by ionic and/or hydrophobic interactions). As will be understood by one of skill in the art, the CIC may be modified or derivatized and the composition of the microcarrier may be selected and/or modified to accommodate the desired type of binding desired for CIC/MC complex formation.

[00472] Covalently bonded CIC/MC complexes may be linked using any covalent crosslinking technology known in the art. Typically, the CIC portion will be modified, either to incorporate an additional moiety (e.g., a free amine, carboxyl or sulfhydryl group) or incorporate modified (e.g., phosphorothioate) nucleotide bases to provide a site at which the CIC portion may be linked to the microcarrier. The link between the CIC and MC portions of the complex can be made at the 3' or 5' end of the CIC, or at a suitably modified base at an internal position in the CIC. The microcarrier is generally also modified to incorporate moieties through which a covalent link may be formed, although functional groups normally present on the microcarrier may also be utilized. The CIC/MC is formed by incubating the CIC with a microcarrier under conditions which permit the formation of a covalent complex (e.g., in the presence of a crosslinking agent or by use of an activated microcarrier comprising an activated moiety which will form a covalent bond with the CIC).

[00473] A wide variety of crosslinking technologies are known in the art, and include crosslinkers reactive with amino, carboxyl and sulfhydryl groups. As will be apparent to one of skill in the art, the selection of a crosslinking agent and crosslinking protocol will depend on the configuration of the CIC and the microcarrier as well as the desired final configuration of the CIC/MC complex. The crosslinker may be either homobifunctional or heterobifunctional. When a homobifunctional crosslinker is used, the crosslinker exploits the same moiety on the CIC and MC (e.g., an aldehyde crosslinker may be used to covalently link a CIC and MC where both the CIC and MC comprise one or more free amines). Heterobifunctional crosslinkers utilize different moieties on the CIC and MC, (e.g., a maleimido-N-hydroxysuccinimide ester may be used to covalently link a free sulfhydryl on the CIC and a free amine on the MC), and are preferred to minimize formation of inter- microcarrier bonds. In most cases, it is preferable to crosslink through a first crosslinking moiety on the microcarrier and a second crosslinking moiety on the CIC, where the second crosslinking moiety is not present on the microcarrier. One preferred method of producing the CIC/MC complex complex is by 'activating' the microcarrier by incubating with a heterobifunctional crosslinking agent, then forming the CIC/MC complex by incubating the CIC and activated MC under conditions appropriate for reaction. The crosslinker may incorporate a 'spacer' arm between the reactive moieties, or the two reactive moieties in the crosslinker may be directly linked.

[00474] In one preferred embodiment, the CIC portion comprises at least one free sulfhydryl (e.g., provided by a 5'-thiol modified base or linker) for crosslinking to the microcarrier, while the microcarrier comprises free amine groups. A heterobifunctional crosslinker reactive with these two groups (e.g., a crosslinker comprising a maleimide group and a NHS-ester), such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate is used to activate the MC, then covalently crosslink the CIC to form the CIC/MC complex.

[00475] Non-covalent CIC/MC complexes may be linked by any non-covalent binding or interaction, including ionic (electrostatic) bonds, hydrophobic interactions, hydrogen bonds, van der Waals attractions, or a combination of two or more different interactions, as is normally the case when a binding pair is to link the CIC and MC.

[00476] Preferred non-covalent CIC/MC complexes are typically complexed by hydrophobic or electrostatic (ionic) interactions, or a combination thereof, (e.g., through base pairing between a CIC and a polynucleotide bound to an MC). Due to the hydrophilic nature of the backbone of polynucleotides, CIC/MC complexes which rely on hydrophobic interactions to form the complex generally require modification of the CIC portion of the complex to incorporate a highly hydrophobic moiety. Preferably, the hydrophobic moiety is biocompatible, nonimmunogenic, and is naturally occurring in the individual for whom the composition is intended (e.g., is found in mammals, particularly humans). Examples of

919 preferred hydrophobic moieties include lipids, steroids, sterols such as cholesterol, and terpenes. The method of linking the hydrophobic moiety to the CIC will, of course, depend on the configuration of the CIC and the identity of the hydrophobic moiety. The hydrophobic moiety may be added at any convenient site in the CIC, preferably at either the 5' or 3' end; in the case of addition of a cholesterol moiety to a CIC, the cholesterol moiety is preferably added to the 5' end of the CIC, using conventional chemical reactions (see, for example, Godard et al. (1995) Eur. J. Biochem. 232:404-410). Preferably, microcarriers for use in CIC/MC complexes linked by hydrophobic bonding are made from hydrophobic materials, such as oil droplets or hydrophobic polymers, although hydrophilic materials modified to incorporate hydrophobic moieties may be utilized as well. When the microcarrier is a liposome or other liquid phase microcarrier comprising a lumen, the CIC/MC complex is formed by mixing the CIC and the MC after preparation of the MC, in order to avoid encapsulation of the CIC during the MC preparation process.

[00477] Non-covalent CIC/MC complexes bound by electrostatic binding typically exploit the highly negative charge of the polynucleotide backbone. Accordingly, microcarriers for use in non-covalently bound CIC/MC complexes are generally positively charged at physiological pH {e.g., about pH 6.8-7.4). The microcarrier may intrinsically possess a positive charge, but microcarriers made from compounds not normally possessing a positive charge may be derivatized or otherwise modified to become positively charged. For example, the polymer used to make the microcarrier may be derivatized to add positively charged groups, such as primary amines. Alternately, positively charged compounds may be incorporated in the formulation of the microcarrier during manufacture {e.g., positively charged surfactants may be used during the manufacture of poly(lactic acid)/poly(glycolic acid) copolymers to confer a positive charge on the resulting microcarrier particles).

[00478] Non-covalent CIC/MC complexes linked by nucleotide base pairing may be produced using conventional methodologies. Generally, base-paired CIC/MC complexes are produced using a microcarrier comprising a bound, preferably a covalently bound, polynucleotide (the 'capture polynucleotide') that is at least partially complementary to the CIC. The segment of complementarity between the CIC and the capture nucleotide is preferably at least 6, 8, 10 or 15 contiguous base pairs, more preferably at least 20 contiguous base pairs. The capture nucleotide may be bound to the MC by any method known in the art, and is preferably covalently bound to the CIC at the 5' or 3' end. [00479] In other embodiments, a binding pair may be used to link the CIC and MC in a CIC/MC complex. The binding pair may be a receptor and ligand, an antibody and antigen (or epitope), or any other binding pair which binds at high affinity (e.g., K_d less than about 10^~8). One type of preferred binding pair is biotin and streptavidin or biotin and avidin, which form very tight complexes. When using a binding pair to mediate CIC/MC complex binding, the CIC is derivatized, typically by a covalent linkage, with one member of the binding pair, and the MC is derivatized with the other member of the binding pair. Mixture of the two derivatized compounds results in CIC/MC complex formation.

[00480] Many CIC/MC complex embodiments do not include an antigen, and certain embodiments exclude antigen(s) associated with the disease or disorder which is the object of the CIC/MC complex therapy. In further embodiments, the CIC is also bound to one or more antigen molecules. Antigen may be coupled with the CIC portion of a CIC/MC complex in a variety of ways, including covalent and/or non-covalent interactions. Alternately, the antigen may be linked to the microcarrier. The link between the antigen and the CIC in the CIC/MC complexe comprising an antigen bound to the CIC can be made by techniques described herein and known in the art.

Co -Administered Antigen

[00481] In some embodiments, the CIC is co-administered with an antigen. Any antigen may be co-administered with a CIC and/or used for preparation of compositions comprising a CIC and antigen.

[00482] In some embodiments, the antigen is an allergen. Examples of recombinant allergens are provided in Table 7. Preparation of many allergens is well-known in the art, including, but not limited to, preparation of ragweed pollen allergen Antigen E (Amb al) (Rafnar et al. (1991) J. Biol. Chem. 266:1229-1236), grass allergen LoI p 1 (Tamborini et al. (1997) Eur. J. Biochem. 249:886-894), major dust mite allergens Der pi and Der PII (Chua et al. (1988) J. Exp. Med. 167:175-182; Chua et al. (1990) Int. Arch. Allergy Appl. Immunol. 91:124-129), domestic cat allergen FeI d I (Rogers et al. (1993) MoI. Immunol. 30:559-568), white birch pollen Bet vl (Breiteneder et al. (1989) EMBO J. 8:1935-1938), Japanese cedar allergens Cry j 1 and Cry j 2 (Kingetsu et al. (2000) Immunology 99:625-629), and protein antigens from other tree pollen (Elsayed et al. (1991) Scand. J. Clin. Lab. Invest. Suppl. 204:17-31). Preparation of protein antigens from grass pollen for in vivo administration has been reported.

[00483] In some embodiments, the allergen is a food allergen, including, but not limited to, peanut allergen, for example Ara h I (Stanley et al. (1996) Adv. Exp. Med. Biol. 409:213- 216); walnut allergen, for example, Jug r I (Tueber et al. (1998) /. Allergy Clin. Immunol. 101:807-814); brazil nut allergen, for example, albumin (Pastorello et al. (1998) /. Allergy Clin. Immunol. 102:1021-1027; shrimp allergen, for example, Pen a I (Reese et al. (1997) Int. Arch. Allergy Immunol. 113:240-242); egg allergen, for example, ovomucoid (Crooke et al. (1997) /. Immunol. 159:2026-2032); milk allergen, for example, bovine beta-lactoglobin (Selot al. (1999) Clin. Exp. Allergy 29:1055-1063); fish allergen, for example, paralbumins (Van Do et al. (1999) Scand. J. Immunol. 50:619-625; Galland et al. (1998) /. Chromatogr. B. Biomed. ScL Appl. 706:63-71). In some embodiments, the allergen is a latex allergen, including but not limited to, Hev b 7 (Sowka et al. (1998) Eur. J. Biochem. 255:213-219). Table 7 shows a list of allergens that may be used.

TABLE 7 RECOMBINANTALLERGENS

[00484] In some embodiments, the antigen is from an infectious agent, including protozoan, bacterial, fungal (including unicellular and multicellular), and viral infectious agents. Examples of suitable viral antigens are described herein and are known in the art. Bacteria include Hemophilus influenza, Mycobacterium tuberculosis and Bordetella pertussis. Protozoan infectious agents include malarial plasmodia, Leishmania species, Trypanosoma species and Schistosoma species. Fungi include Candida albicans.

[00485] In some embodiments, the antigen is a viral antigen. Viral polypeptide antigens include, but are not limited to, HIV proteins such as HIV gag proteins (including, but not limited to, membrane anchoring (MA) protein, core capsid (CA) protein and nucleocapsid (NC) protein), HIV polymerase, influenza virus matrix (M) protein and influenza virus nucleocapsid (NP) protein, hepatitis B surface antigen (HBsAg), hepatitis B core protein (HBcAg), hepatitis e protein (HBeAg), hepatitis B DNA polymerase, hepatitis C antigens, and the like. References discussing influenza vaccination include Scherle and Gerhard (1988) Proc. Natl. Acad. ScL USA 85:4446-4450; Scherle and Gerhard (1986) J. Exp. Med. 164:1114-1128; Granoff et al. (1993) Vaccine 11:S46-51; Kodihalli et al. (1997) J. Virol. 71:3391-3396; Ahmeida et al. (1993) Vaccine 11:1302-1309; Chen et al. (1999) Vaccine 17:653-659; Govorkova and Smirnov (1997) Acta Virol. (1997) 41:251-257; Koide et al. (1995) Vaccine 13:3-5; Mbawuike et al. (1994) Vaccine 12:1340-1348; Tamura et al. (1994) Vaccine 12:310-316; Tamura et al. (1992) Eur. J. Immunol. 22:477-481; Hirabayashi et al. (1990) Vaccine 8:595-599. Other examples of antigen polypeptides are group- or sub-group specific antigens, which are known for a number of infectious agents, including, but not limited to, adenovirus, herpes simplex virus, papilloma virus, respiratory syncytial virus and poxviruses.

[00486] Many antigenic peptides and proteins are known, and available in the art; others can be identified using conventional techniques. For immunization against tumor formation or treatment of existing tumors, immunomodulatory peptides can include tumor cells (live or irradiated), tumor cell extracts, or protein subunits of tumor antigens such as Her-2/neu, Marti, carcinoembryonic antigen (CEA), gangliosides, human milk fat globule (HMFG), mucin (MUCl), MAGE antigens, BAGE antigens, GAGE antigens, gplOO, prostate specific antigen (PSA), and tyrosinase. Vaccines for immuno-based contraception can be formed by including sperm proteins administered with CICs. Lea et al. (1996) Biochim. Biophys. Acta 1307:263.

[00487] Attenuated and inactivated viruses are suitable for use herein as the antigen. Preparation of these viruses is well-known in the art and many are commercially available (see, e.g., Physicians' Desk Reference (1998) 52nd edition, Medical Economics Company, Inc.). For example, polio virus is available as IPOL® (Pasteur Merieux Connaught) and ORIMUNE® (Lederle Laboratories), hepatitis A virus as VAQTA® (Merck), measles virus as ATTENUV AX® (Merck), mumps virus as MUMPSVAX® (Merck) and rubella virus as MERUVAX®II (Merck). Additionally, attenuated and inactivated viruses such as HIV-I, HIV-2, herpes simplex virus, hepatitis B virus, rotavirus, human and non-human papillomavirus and slow brain viruses can provide peptide antigens. [00488] In some embodiments, the antigen comprises a viral vector, such as vaccinia, adenovirus, and canary pox. Antigens may be isolated from their source using purification techniques known in the art or, more conveniently, may be produced using recombinant methods.

[00489] Antigenic peptides can include purified native peptides, synthetic peptides, recombinant proteins, crude protein extracts, attenuated or inactivated viruses, cells, microorganisms, or fragments of such peptides. Immunomodulatory peptides can be native or synthesized chemically or enzymatically. Any method of chemical synthesis known in the art is suitable. Solution phase peptide synthesis can be used to construct peptides of moderate size or, for the chemical construction of peptides, solid phase synthesis can be employed. Atherton et al. (1981) Hoppe Seylers Z. Physiol. Chem. 362:833-839. Proteolytic enzymes can also be utilized to couple amino acids to produce peptides. Kullmann (1987) Enzymatic Peptide Synthesis, CRC Press, Inc. Alternatively, the peptide can be obtained by using the biochemical machinery of a cell, or by isolation from a biological source. Recombinant DNA techniques can be employed for the production of peptides. Hames et al. (1987) Transcription and Translation: A Practical Approach, IRL Press. Peptides can also be isolated using standard techniques such as affinity chromatography.

[00490] Preferably the antigens are peptides, lipids (e.g., sterols excluding cholesterol, fatty acids, and phospholipids), polysaccharides such as those used in H. influenza vaccines, gangliosides and glycoproteins. These can be obtained through several methods known in the art, including isolation and synthesis using chemical and enzymatic methods. In certain cases, such as for many sterols, fatty acids and phospholipids, the antigenic portions of the molecules are commercially available.

[00491] Examples of viral antigens useful in the subject compositions and methods using the compositions include, but are not limited to, HIV antigens. Such antigens include, but are not limited to, those antigens derived from HIV envelope glycoproteins including, but not limited to, gpl60, gpl20 and gp41. Numerous sequences for HIV genes and antigens are known. For example, the Los Alamos National Laboratory HIV Sequence Database collects, curates and annotates HIV nucleotide and amino acid sequences. This database is accessible via the internet, at http://hiv-web.lanl.gov/, and in a yearly publication, see Human Retroviruses and AIDS Compendium (for example, 2000 edition). [00492] Antigens derived from infectious agents may be obtained using methods known in the art, for example, from native viral or bacterial extracts, from cells infected with the infectious agent, from purified polypeptides, from recombinantly produced polypeptides and/or as synthetic peptides.

[00493] CICs can be administered in combination with antigen in a variety of ways. In some embodiments, a CIC and antigen are administered spatially proximate with respect to each other. As described below, spatial proximation can be accomplished in a number of ways, including conjugation, encapsidation, via affixation to a platform or adsorption onto a surface. In one embodiment, a CIC and antigen are administered as an admixture (e.g., in solution). It is specifically contemplated that, in certain embodiments, the CIC is not conjugated to an immunogen or antigen.

[00494] In some embodiments, the CIC is linked to a polypeptide, e.g., an antigen. The CIC portion can be linked with the antigen portion of a conjugate in a variety of ways, including covalent and/or non-covalent interactions, via the nucleic acid moiety or non- nucleic acid spacer moiety. In some embodiments, linkage is via a reactive group such as, without limitation, thio, amine, carboxylate, aldehyde, hydrizine, hydrizone, disulfide and the like.

[00495] The link between the CIC and antigen portions can be made at the 3' or 5' end of a nucleic acid moiety, or at a suitably modified base at an internal position in the a nucleic acid moiety. For example, if the antigen is a peptide and contains a suitable reactive group (e.g., an N-hydroxysuccinimide ester) it can be reacted directly with the N⁴ amino group of cytosine residues. Depending on the number and location of cytosine residues in the CIC, specific coupling at one or more residues can be achieved.

[00496] Alternatively, modified oligonucleosides, such as are known in the art, can be incorporated at either terminus, or at internal positions in the CIC. These can contain blocked functional groups which, when deblocked, are reactive with a variety of functional groups which can be present on, or attached to, the antigen of interest.

[00497] Where the antigen is a peptide, this portion of the conjugate can be attached to the nucleic acid moiety or spacer moiety through solid support chemistry. For example, a nucleic acid portion of a CIC can be added to a polypeptide portion that has been pre- synthesized on a support. Haralambidis et al. (1990) Nucleic Acids Res. 18:493-499; and Haralambidis et al. (1990) Nucleic Acids Res. 18:501-505.

[00498] Alternatively, the CIC can be synthesized such that it is connected to a solid support through a cleavable linker extending from the 3 '-end of a nucleic acid moiety. Upon chemical cleavage of the CIC from the support, a terminal thiol group or a terminal amino group is left at the 3'-end of the nucleic acid moiety (Zuckermann et al., 1987, Nucleic Acids Res. 15:5305-5321; Corey et al., 1987, Science 238:1401-1403; Nelson et al., 1989, Nucleic Acids Res. 17:1781-1794). Conjugation of the amino-modified CIC to amino groups of the peptide can be performed as described in Benoit et al. (1987) Neuromethods 6:43-72. Conjugation of the thiol-modified CIC to carboxyl groups of the peptide can be performed as described in Sinah et al. (1991) Oligonucleotide Analogues: A Practical Approach, IRL Press. Coupling of a nucleic acid moiety or spacer carrying an appended maleimide to the thiol side chain of a cysteine residue of a peptide has also been described. Tung et al. (1991) Bioconjug. Chem. 2:464-465.

[00499] The peptide portion of the conjugate can be attached to a free 5 '-end of a nucleic acid moiety through an amine, thiol, or carboxyl group that has been incorporated into nucleic acid moiety or spacer (e.g., via a free 5 '-end, a 3 '-end, via a modified base, and the like).

[00500] Conveniently, a linking group comprising a protected amine, thiol, or carboxyl at one end, and a phosphoramidite can be covalently attached to a hydroxyl group of a CIC. Agrawal et al. (1986) Nucleic Acids Res. 14:6227-6245; Connolly (1985) Nucleic Acids Res. 13:4485-4502; Kremsky et al. (1987) Nucleic Acids Res. 15:2891-2909; Connolly (1987) Nucleic Acids Res. 15:3131-3139; Bischoff et al. (1987) Anal. Biochem. 164:336-344; Blanks et al. (19SS) Nucleic Acids Res. 16:10283-10299; and U.S. Patent Nos. 4,849,513, 5,015,733, 5,118,800, and 5,118,802. Subsequent to deprotection, the amine, thiol, and carboxyl functionalities can be used to covalently attach the CIC to a peptide. Benoit et al. (1987); and Sinah et al. (1991).

[00501] A CIC-antigen conjugate can also be formed through non-covalent interactions, such as ionic bonds, hydrophobic interactions, hydrogen bonds and/or van der Waals attractions. [00502] Non-covalently linked conjugates can include a non-covalent interaction such as a biotin-streptavidin complex. A biotinyl group can be attached, for example, to a modified base of a CIC. Roget et al. (1989) Nucleic Acids Res. 17:7643-7651. Incorporation of a streptavidin moiety into the peptide portion allows formation of a non-covalently bound complex of the streptavidin conjugated peptide and the biotinylated oligonucleotide.

[00503] Non-covalent associations can also occur through ionic interactions involving a CIC and residues within the antigen, such as charged amino acids, or through the use of a linker portion comprising charged residues that can interact with both the oligonucleotide and the antigen. For example, non-covalent conjugation can occur between a generally negatively-charged CIC and positively-charged amino acid residues of a peptide, e.g., polylysine, polyarginine and polyhistidine residues.

[00504] Non-covalent conjugation between CIC and antigens can occur through DNA binding motifs of molecules that interact with DNA as their natural ligands. For example, such DNA binding motifs can be found in transcription factors and anti-DNA antibodies.

[00505] The linkage of the CIC to a lipid can be formed using standard methods. These methods include, but are not limited to, the synthesis of oligonucleotide-phospholipid conjugates (Yanagawa et al. (1988) Nucleic Acids Symp. Ser. 19:189-192), oligonucleotide- fatty acid conjugates (Grabarek et al. (1990) Anal. Biochem. 185:131-135; and Staros et al. (1986) Anal. Biochem. 156:220-222), and oligonucleotide-sterol conjugates. Boujrad et al. (1993) Proc. Natl. Acad. ScL USA 90:5728-5731.

[00506] The linkage of the oligonucleotide to an oligosaccharide can be formed using standard known methods. These methods include, but are not limited to, the synthesis of oligonucleotide-oligosaccharide conjugates, wherein the oligosaccharide is a moiety of an immunoglobulin. O'Shannessy et al. (1985) /. Applied Biochem. 7:347-355.

[00507] Additional methods for the attachment of peptides and other molecules to oligonucleotides can be found in U.S. Patent No. 5,391,723; Kessler (1992) 'Nonradioactive labeling methods for nucleic acids' in Kricka (ed.) Nonisotopic DNA Probe Techniques, Academic Press; and Geoghegan et al. (1992) Bioconjug. Chem. 3:138-146.

[00508] A CIC may be proximately associated with an antigen(s) in other ways. In some embodiments, a CIC and antigen are proximately associated by encapsulation. In other embodiments, a CIC and antigen are proximately associated by linkage to a platform molecule. A 'platform molecule' (also termed 'platform') is a molecule containing sites which allow for attachment of a CIC and antigen(s). In other embodiments, a CIC and antigen are proximately associated by adsorption onto a surface, preferably a carrier particle.

[00509] In some embodiments, the methods of the invention employ an encapsulating agent that can maintain the proximate association of a CIC and first antigen until the complex is available to the target (or compositions comprising such encapsulating agents). Preferably, the composition comprising a CIC, antigen and encapsulating agent is in the form of adjuvant oil-in-water emulsions, microparticles and/or liposomes. More preferably, adjuvant oil-in- water emulsions, microparticles and/or liposomes encapsulating a CIC are in the form of particles having an average diameter from about 0.04 um to about 100 um, preferably any of the following ranges of average diameters: from about 0.1 um to about 20 um; from about 0.15 um to about 10 um; from about 0.05 um to about 1.00 um; from about 0.05 um to about 0.5 um.

[00510] Colloidal dispersion systems, such as microspheres, beads, macromolecular complexes, nanocapsules and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles and liposomes can provide effective encapsulation of CIC-containing compositions.

[00511] The encapsulation composition further comprises any of a wide variety of components. These include, but are not limited to, alum, lipids, phospholipids, lipid membrane structures (LMS), polyethylene glycol (PEG) and other polymers, such as polypeptides, glycopeptides, and polysaccharides.

[00512] Polypeptides suitable for encapsulation components include any known in the art and include, but are not limited to, fatty acid binding proteins. Modified polypeptides contain any of a variety of modifications, including, but not limited to glycosylation, phosphorylation, myristylation, sulfation and hydroxylation. As used herein, a suitable polypeptide is one that will protect a CIC-containing composition to preserve the immunomodulatory activity thereof. Examples of binding proteins include, but are not limited to, albumins such as bovine serum albumin (BSA) and pea albumin. [00513] Other suitable polymers can be any known in the art of pharmaceuticals and include, but are not limited to, naturally-occurring polymers such as dextrans, hydroxyethyl starch, and polysaccharides, and synthetic polymers. Examples of naturally occurring polymers include proteins, glycopeptides, polysaccharides, dextran and lipids. The additional polymer can be a synthetic polymer. Examples of synthetic polymers which are suitable for use in the present invention include, but are not limited to, polyalkyl glycols (PAG) such as PEG, polyoxyethylated polyols (POP), such as polyoxyethylated glycerol (POG), polytrimethylene glycol (PTG) polypropylene glycol (PPG), polyhydroxy ethyl methacrylate, polyvinyl alcohol (PVA), polyacrylic acid, polyethyloxazoline, polyacrylamide, polyvinylpyrrolidone (PVP), polyamino acids, polyurethane and polyphosphazene. The synthetic polymers can also be linear or branched, substituted or unsubstituted, homopolymeric, co-polymers, or block co-polymers of two or more different synthetic monomers.

[00514] The PEGs for use in encapsulation compositions of the present invention are either purchased from chemical suppliers or synthesized using techniques known to those of skill in the art.

[00515] The term 'LMS', as used herein, means lamellar lipid particles wherein polar head groups of a polar lipid are arranged to face an aqueous phase of an interface to form membrane structures. Examples of the LMSs include liposomes, micelles, cochleates (i.e., generally cylindrical liposomes), microemulsions, unilamellar vesicles, multilamellar vesicles, and the like.

[00516] One colloidal dispersion system useful in the administration of CICs is a liposome. In mice immunized with a liposome-encapsulated antigen, liposomes appeared to enhance a ThI -type immune response to the antigen. Aramaki et al. (1995) Vaccine 13:1809- 1814. As used herein, a 'liposome' or 'lipid vesicle' is a small vesicle bounded by at least one and possibly more than one bilayer lipid membrane. Liposomes are made artificially from phospholipids, glycolipids, lipids, steroids such as cholesterol, related molecules, or a combination thereof by any technique known in the art, including but not limited to sonication, extrusion, or removal of detergent from lipid-detergent complexes. One type of liposome for use in delivering CICs to cells is a cationic liposome. A liposome can also optionally comprise additional components, such as a tissue targeting component. It is understood that a 'lipid membrane' or 'lipid bilayer' need not consist exclusively of lipids, but can additionally contain any suitable other components, including, but not limited to, cholesterol and other steroids, lipid-soluble chemicals, proteins of any length, and other amphipathic molecules, providing the general structure of the membrane is a sheet of two hydrophilic surfaces sandwiching a hydrophobic core. For a general discussion of membrane structure, see The Encyclopedia of Molecular Biology by J. Kendrew (1994). For suitable lipids see e.g., Lasic (1993) 'Liposomes: from Physics to Applications' Elsevier, Amsterdam.

[00517] Processes for preparing liposomes containing CIC -containing compositions are known in the art. The lipid vesicles can be prepared by any suitable technique known in the art. Methods include, but are not limited to, microencapsulation, microfluidization, LLC method, ethanol injection, freon injection, the 'bubble' method, detergent dialysis, hydration, sonication, and reverse-phase evaporation. Reviewed in Watwe et al. (1995) Curr. Sci. 68:715-724. Techniques may be combined in order to provide vesicles with the most desirable attributes.

[00518] The invention encompasses use of LMSs containing tissue or cellular targeting components. Such targeting components are components of a LMS that enhance its accumulation at certain tissue or cellular sites in preference to other tissue or cellular sites when administered to an intact animal, organ, or cell culture. A targeting component is generally accessible from outside the liposome, and is therefore preferably either bound to the outer surface or inserted into the outer lipid bilayer. A targeting component can be inter alia a peptide, a region of a larger peptide, an antibody specific for a cell surface molecule or marker, or antigen binding fragment thereof, a nucleic acid, a carbohydrate, a region of a complex carbohydrate, a special lipid, or a small molecule such as a drug, hormone, or hapten, attached to any of the aforementioned molecules. Antibodies with specificity toward cell type- specific cell surface markers are known in the art and are readily prepared by methods known in the art.

[00519] The LMSs can be targeted to any cell type toward which a therapeutic treatment is to be directed, e.g., a cell type which can modulate and/or participate in an immune response. Such target cells and organs include, but are not limited to, APCs, such as macrophages, dendritic cells and lymphocytes, lymphatic structures, such as lymph nodes and the spleen, and nonlymphatic structures, particularly those in which dendritic cells are found.

[00520] The LMS compositions of the present invention can additionally comprise surfactants. Surfactants can be cationic, anionic, amphiphilic, or nonionic. A preferred class of surfactants are nonionic surfactants; particularly preferred are those that are water soluble.

[00521] In some embodiments a CIC and antigen are proximately associated by linkage to a platform molecule, such as a proteinaceous or non-pro teinaceous (e.g., synthetic) valency platform. Examples of suitable platforms are described supra, in the discussion of valency platforms used as a spacer moiety in a CIC. Attachment of antigens to valency platforms can be carried out using routine methods. As an example, polypeptides contain amino acid side chain moieties with functional groups such as amino, carboxyl or sulfhydryl groups that serve as sites for coupling the polypeptide to the platform. Residues that have such functional groups may be added to the polypeptide if the polypeptide does not already contain these groups. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the peptide synthesis arts. When the polypeptide has a carbohydrate side chain(s) (or if the antigen is a carbohydrate), functional amino, sulfhydryl and/or aldehyde groups may be incorporated therein by conventional chemistry. For instance, primary amino groups may be incorporated by reaction of the oxidized sugar with ethylenediamine in the presence of sodium cyanoborohydride, sulfhydryls may be introduced by reaction of cysteamine dihydrochloride followed by reduction with a standard disulfide reducing agent, while aldehyde groups may be generated following periodate oxidation. In a similar fashion, the platform molecule may also be derivatized to contain functional groups if it does not already possess appropriate functional groups.

[00522] In another embodiment, a CIC and antigen are coadministered by adsorbing both to a surface, such as a nanoparticle or microcarrier. Adsorption of a CIC and/or antigen to a surface may occur through non-covalent interactions, including ionic and/or hydrophobic interactions. Adsorption of polynucleotides and polypeptides to a surface for the purpose of delivery of the adsorbed molecules to cells is well known in the art. See, for example, Douglas et al. (1987) Crit. Rev. Ther. Drug. Carrier Sy st. 3:233-261; Hagiwara et al. (1987) In Vivo 1:241-252; Bousquet et al. (1999) Pharm. Res. 16:141-147; and Kossovsky et al., U.S. Patent 5,460,831. Preferably, the material comprising the adsorbent surface is biodegradable.

[00523] In general, characteristics of nanoparticles, such as surface charge, particle size and molecular weight, depend upon polymerization conditions, monomer concentration and the presence of stabilizers during the polymerization process (Douglas et al., 1987, supra). The surface of carrier particles may be modified, for example, with a surface coating, to allow or enhance adsorption of the CIC and/or antigen. Carrier particles with adsorbed CIC and/or antigen may be further coated with other substances. The addition of such other substances may, for example, prolong the half-life of the particles once administered to the subject and/or may target the particles to a specific cell type or tissue, as described herein.

[00524] Nanocrystalline surfaces to which a CIC and antigen may be adsorbed have been described (see, for example, U.S. Patent 5,460,831). Nanocrystalline core particles (with diameters of 1 um or less) are coated with a surface energy modifying layer that promotes adsorption of polypeptides, polynucleotides and/or other pharmaceutical agents. As described in U.S. Patent 5,460,831, for example, a core particle is coated with a surface that promotes adsorption of an oligonucleotide and is subsequently coated with an antigen preparation, for example, in the form of a lipid-antigen mixture. Such nanoparticles are self- assembling complexes of nanometer sized particles, typically on the order of 0.1 um, that carry an inner layer of CIC and an outer layer of antigen.

[00525] Another adsorbent surface are nanoparticles made by the polymerization of alkylcyanoacrylates. Alkylcyanoacrylates can be polymerized in acidified aqueous media by a process of anionic polymerization. Depending on the polymerization conditions, the small particles tend to have sizes in the range of 20 to 3000 nm, and it is possible to make nanoparticles specific surface characteristics and with specific surface charges (Douglas et al., 1987, supra). For example, oligonucleotides may be adsorbed to polyisobutyl- and polyisohexlcyanoacrylate nanoparticles in the presence of hydrophobic cations such as tetraphenylphosphonium chloride or quaternary ammonium salts, such as cetyltrimethyl ammonium bromide. Oligonucleotide adsorption on these nanoparticles appears to be mediated by the formation of ion pairs between negatively charged phosphate groups of the nucleic acid chain and the hydrophobic cations. See, for example, Lambert et al. (1998) Biochimie 80:969-976, Chavany et al. (1994) Pharm. Res. 11:1370-1378; Chavany et al. (1992) Pharm. Res. 9:441-449. Polypeptides may also be adsorbed to polyalkylcyanoacrylate nanoparticles. See, for example, Douglas et al., 1987; Schroeder et al. (1998) Peptides 19:777-780.

[00526] Another adsorbent surface are nanoparticles made by the polymerization of methylidene malonate. For example, as described in Bousquet et al., 1999, polypeptides adsorbed to poly(methylidene malonate 2.1.2) nanoparticles appear to do so initially through electrostatic forces followed by stabilization through hydrophobic forces.

Additional Adjuvants

[00527] A CIC may also be administered in conjunction with an adjuvant. Administration of an antigen with a CIC and an adjuvant leads to a potentiation of a immune response to the antigen and thus, can result in an enhanced immune response compared to that which results from a composition comprising the CIC and antigen alone. Adjuvants are known in the art and include, but are not limited to, oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts), liposomes and microparticles, including but not limited to, polystyrene, starch, polyphosphazene and polylactide/polyglycosides. Other suitable adjuvants also include, but are not limited to, MF59, DETOX™ (Ribi), squalene mixtures (SAF-I), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873- 875, as well as, lipid-based adjuvants and others described herein. For veterinary use and for production of antibodies in animals, mitogenic components of Freund' s adjuvant (both complete and incomplete) can be used.

Methods of the Invention (Immunomodulation)

[00528] The invention provides methods of modulating an immune response of an animal or population of cells, e.g., mammalian, optionally human, blood cells (e.g., PBMCs, lymphocytes, dendritic cells), bronchial alveolar lavage cells, or other cells or cell populations containing cells responsive to immunostimulatory agents, by contacting the cells with a CIC or CIC-containing composition described herein (e.g., a composition containing a CIC, CIC and an antigen, a CIC-antigen conjugate, a CIC/microcarrier complex, etc.) The modulation can be accomplished by any form of contacting, including without limitation, co- incubation of cells and CIC in vitro, application of the CIC to skin of a mammal (e.g., of an experimental animal), intranasal administration of a suitable formulation, such as an aerosol,,and parenteral administration.

[00529] An immune response in animals or cell populations can be detected in any number of ways, including a increased expression of one or more of IFN-gamma, IFN-alpha, IL-2, IL-12, TNF-alpha, IL-6, IL-4, IL-5, IP-IO, ISG-54K, MCP-I, or a change in gene expression profile characteristic of immune stimulation as well as responses such as B cell proliferation and dendritic cell maturation, The ability to stimulate an immune response in a cell population has a number of uses, e.g., in an assay system for immunosuppressive agents, and other uses as described herein.

[00530] Thus, the invention provides methods of modulating an immune response in an individual, preferably a mammal, more preferably a human, comprising administering to the individual a CIC as described herein. Immunomodulation may include stimulating a ThI- type immune response and/or inhibiting or reducing a Th2-type immune response. For example, an immunomodulated immune response according to the present invention may also be characterized by an inhibition of the Th2-type immune response in conjunction with a low or absent Thl-type response. In another example, immuno stimulatory seqences (ISSs) that contain internal unmethylated cytosine CG dinucleotide(s), such as the CICs of the present invention, can activate TLR9 expressed on plasmacytoid dendritic cells and B -cells in humans. In plasmacytoid dendritic cells, IFN-alpha is induced in response to TLR9 agonist stimulating the production of immune modulators such as natural killer (NK)-cells, monocytes, etc. Stimulated dendritic cells also undergo maturation and migrate to secondary lymphoid tissues activating naive and memory T-cells. The CIC is administered in an amount sufficient to modulate an immune response. As described herein, modulation of an immune response may be humoral and/or cellular, and is measured using standard techniques in the art and as described herein.

[00531] In certain embodiments, the individual suffers from a disorder associated with a Th2-type immune response, such as (without limitation) allergies, allergy-induced asthma, atopic dermatitis, eosinophillic gastrointestinal inflammation, eosinophillic esophagitis, and allergic bronchopulmonary aspergillosis. Administration of a CIC results in immunomodulation, increasing levels of one or more ThI -type response associated cytokines, which may result in a reduction of the Th2-type response features associated with the individual's response to the allergen. In some embodiments, an immunomodulated immune response according to the present invention may be characterized by an inhibition of the Th2- type immune response in conjunction with a low or absent ThI -type response, which may also result in a reduction of the Th2-type response features associated with the individual's response to the allergen. Immunomodulation of individuals with Th2-type response associated disorders results in a reduction or improvement in one or more of the symptoms of the disorder. Where the disorder is allergy or allergy-induced asthma, improvement in one or more of the symptoms includes a reduction one or more of the following: rhinitis, allergic conjunctivitis, circulating levels of IgE, circulating levels of histamine and/or requirement for 'rescue' inhaler therapy (e.g., inhaled albuterol administered by metered dose inhaler or nebulizer).

[00532] In further embodiments, the individual subject to the immunomodulatory therapy of the invention is an individual receiving a vaccine. The vaccine may be a prophylactic vaccine or a therapeutic vaccine. A prophylactic vaccine comprises one or more epitopes associated with a disorder for which the individual may be at risk (e.g., M. tuberculosis antigens as a vaccine for prevention of tuberculosis). Therapeutic vaccines comprise one or more epitopes associated with a particular disorder affecting the individual, such as M. tuberculosis or M. bovis surface antigens in tuberculosis patients, antigens to which the individual is allergic (i.e., allergy desensitization therapy) in individuals subject to allergies, tumor cells from an individual with cancer (e.g., as described in U.S. Patent No. 5,484,596), or tumor associated antigens in cancer patients. The CIC may be given in conjunction with the vaccine (e.g., in the same injection or a contemporaneous, but separate, injection) or the CIC may be administered separately (e.g., at least 12 hours before or after administration of the vaccine). In certain embodiments, the antigen(s) of the vaccine is part of the CIC, by either covalent or non-covalent linkage to the CIC. Administration of CIC therapy to an individual receiving a vaccine results in an immune response to the vaccine that is shifted towards a ThI -type response as compared to individuals which receive vaccine not containing a CIC. Shifting towards a ThI -type response may be recognized by a delay ed- type hypersensitivity (DTH) response to the antigen(s) in the vaccine, increased IFN-gamma and other Thl-type response associated cytokines, production of CTLs specific for the antigen(s) of the vaccine, low or reduced levels of IgE specific for the antigen(s) of the vaccine, a reduction in Th2- associated antibodies specific for the antigen(s) of the vaccine, and/or an increase in Thl-associated antibodies specific for the antigen(s) of the vaccine. In the case of therapeutic vaccines, administration of CIC and vaccine also results in amelioration of one or more symptoms of the disorder which the vaccine is intended to treat. As will be apparent to one of skill in the art, the exact symptoms and manner of their improvement will depend on the disorder sought to be treated. For example, where the therapeutic vaccine is for tuberculosis, CIC treatment with vaccine results in reduced coughing, pleural or chest wall pain, fever, and/or other symptoms known in the art. Where the vaccine is an allergen used in allergy desensitization therapy, the treatment results in a reduction in the symptoms of allergy (e.g., reduction in rhinitis, allergic conjunctivitis, circulating levels of IgE, circulating levels of histamine, and/or reduction of eosinophilia/inflammation) or disease modification (e.g., a long absence of symptoms).

[00533] The compositions of the invention may also be used prophylactically to increase resistance to infection by a wide range of bacterial or viral pathogens, including natural of genetically modified organisms employed as agents of biological warfare or terrorism.

[00534] The present invention also provides compounds, compositions and methods for inducing cytokines, such as IFN-alpha, and promoting maturation of plasmacytoid dendritic cells. In some embodiments, and without intending to be bound by theory, it is believed that immuno stimulatory sequences (ISSs) that contain internal unmethylated cytosine CG dinucleotide(s) activate TLR9 expressed on plasmacytoid dendritic cells and B-cells in humans. In plasmacytoid dendritic cells, IFN-alpha is induced in response to TLR9 agonist stimulating the production of immune modulators such as natural killer (NK)-cells, monocytes, etc. Stimulated dendritic cells also undergo maturation and migrate to secondary lymphoid tissues activating naive and memory T-cells.

[00535] Other embodiments of the invention relate to immunomodulatory therapy of individuals having a pre-existing disease or disorder, such as cancer or an infectious disease. Cancer is an attractive target for immunomodulation because most cancers express tumor- associated and/or tumor specific antigens which are not found on other cells in the body. Stimulation of a ThI -type response against tumor cells results in direct and/or bystander killing of tumor cells by the immune system, leading to a reduction in cancer cells and a reduction in symptoms. Administration of a CIC to an individual having cancer results in stimulation of a Thl-type immune response against the tumor cells. Such an immune response can kill tumor cells, either by direct action of cellular immune system cells (e.g., CTLs) or components of the humoral immune system, or by bystander effects on cells proximal to cells targeted by the immune system including macrophages and natural killer (NK) cells. See, for example, Cho et al. (2000) Nat. Biotechnol. 18:509-514. In treatment of a pre-existing disease or disorder, the CIC can be administered in conjunction with other immunotherapeutic agents such as cytokines, adjuvants and antibodies. For example, a CIC can be administered as part of a therapeutic regimen that includes administration of a binding agent that binds an antigen displayed by tumor cells. Exemplary binding agents include polyclonal and monoclonal antibodies. Examples of target antigens include CD20, CD22, HER2 and others known in the art or to be discovered in the future. Without intending to be bound by theory, it is believed that the CIC enhances killing of tumor cells to which the binding agent is associated (e.g., by enhancing antibody dependent cellular cytotoxicity and/or effector function). The binding agent can optionally be labeled, e.g., with a radioisotope or toxin that damages a cell to which the binding agent is bound. The CIC may be given in conjunction with the agent {e.g., at the same time) or before or after (e.g., less than 24 hours before or after administration of the agent). For example, in the case of cancer, the CIC can be administered in conjunction with a chemotherapeutic agent known or suspected of being effective for the treatment of cancer. As another example, the CIC can be administered in conjunction with radiation therapy, gene therapy, or the like. The CIC may be any of those described herein.

[00536] Immunomodulatory therapy in accordance with the invention is also beneficial for individuals with infectious diseases, particularly infectious diseases which are resistant to humoral immune responses (e.g., diseases caused by mycobacterial infections and intracellular pathogens). Immunomodulatory therapy may be used for the treatment of infectious diseases caused by cellular pathogens (e.g., bacteria or protozoans) or by subcellular pathogens (e.g., viruses). CIC therapy may be administered to individuals suffering from mycobacterial diseases such as tuberculosis (e.g., M. tuberculosis and/or M. bovis infections), leprosy (i.e., M. leprae infections), or M. marinum or M. ulcerans infections. CIC therapy is also may also be used for the treatment of viral infections, including infections by influenza virus, respiratory syncytial virus (RSV), hepatitis virus B, hepatitis virus C, herpes viruses, particularly herpes simplex viruses, and papilloma viruses.

Diseases caused by intracellular parasites such as malaria (e.g., infection by Plasmodium vivax, P. ovale, P. falciparum and/or P. malariae), leishmaniasis (e.g., infection by Leishmania donovani, L. tropica, L. mexicana, L. braziliensis, L. peruviana, L. infantum, L. chagasi, and/or L. aethiopica), and toxoplasmosis (i.e., infection by Toxoplasmosis gondii) also benefit from CIC therapy. CIC therapy may also be used for treatment of parasitic diseases such as schistosomiasis (i.e., infection by blood flukes of the genus Schistosoma such as S. haematobium, S. mansoni, S. japonicum, and S. mekongi) and clonorchiasis (i.e., infection by Clonorchis sinensis). Administration of a CIC to an individual suffering from an infectious disease results in an amelioration of symptoms of the infectious disease. In some embodiments, the infectious disease is not a viral disease.

[00537] The invention further provides methods of increasing or stimulating at least one ThI -associated cytokine in an individual, including IL-2, IL- 12, TNF-alpha, TNF-beta, IFN- gamma and IFN-alpha. In certain embodiments, the invention provides methods of increasing or stimulating IFN-gamma in an individual, particularly in an individual in need of increased IFN-gamma levels, by administering an effective amount of a CIC to the individual. Individuals in need of increased IFN-gamma are those having disorders which respond to the administration of IFN-gamma. Such disorders include a number of inflammatory disorders including, but not limited to, ulcerative colitis. Such disorders also include a number of fibrotic disorders, including, but not limited to, idiopathic pulmonary fibrosis (IPF), scleroderma, cutaneous radiation-induced fibrosis, hepatic fibrosis including schistosomiasis-induced hepatic fibrosis, renal fibrosis as well as other conditions which may be improved by administration of IFN-gamma. An increase in IFN-gamma levels may result in amelioration of one or more symptoms, stabilization of one or more symptoms, or prevention of progression (e.g., reduction or elimination of additional lesions or symptoms) of the disorder which responds to IFN-gamma. The methods of the invention may be practiced in combination with other therapies which make up the standard of care for the disorder, such as administration of anti-inflammatory agents such as systemic corticosteroid therapy (e.g., cortisone) in IPF.

[00538] In certain embodiments, the invention provides methods of increasing IFN-alpha in an individual, particularly in an individual in need of increased IFN-alpha levels, by administering an effective amount of a CIC to the individual such that IFN-alpha levels are increased. Individuals in need of increased IFN-alpha are those having disorders which respond to the administration of IFN-alpha, including recombinant IFN-alpha, including, but not limited to, viral infections and cancer.

[00539] Administration of a CIC in accordance with certain embodiments of the invention results in an increase in IFN-alpha levels, and results in amelioration of one or more symptoms, stabilization of one or more symptoms, or prevention of progression (e.g., reduction or elimination of additional lesions or symptoms) of the disorder which responds to IFN-alpha. The methods of the invention may be practiced in combination with other therapies which make up the standard of care for the disorder, such as administration of antiviral agents for treating viral infections, inhalants for treating asthma and chronic obstructive pulmonary disease (COPD), and antihistamines, corticosteroids, and/or decongestants for treating allergic rhinitis.

[00540] As will be apparent upon review of this disclosure, the spacer composition of a CIC can affect the immune response elicited by administration of the CIC. Virtually all of the spacers tested (with the exception of dodecyl) can be used in CICs to efficiently induce IFN-γ in human PBMCs. However, the spacer composition of linear CICs has been observed to have differential effects on induction of IFN-alpha. For example, CICs containing, for example, HEG, TEG or C6 spacers tend to cause higher IFN-alpha induction (and reduced B cell proliferation) in PBMCs than did CICs containing C3, C4 or abasic spacers.

[00541] The invention also provides methods of reducing levels, particularly serum levels, of IgE in an individual having an IgE-related disorder by administering an effective amount of a CIC to the individual. In such methods, the CIC may be administered alone (e.g., without antigen) or administered with antigen, such as an allergen. An IgE-related disorder is a condition, disorder, or set of symptoms ameliorated by a reduction in IgE levels. Reduction in IgE results in an amelioration of symptoms of the IgE-related disorder. Such symptoms include allergy symptoms such as rhinitis, conjunctivitis, in decreased sensitivity to allergens, a reduction in the symptoms of allergy in an individual with allergies, or a reduction in severity of an allergic response.

[00542] Guided by the present disclosure, CICs can be designed to achieve specific desired physiological responses. For example, the IFN-alpha inducing activity, and B cell activating activities of CICs can be independently varied based on the structure of the CIC and selection of nucleic acid moieties (NAMs). [00543] Because the IFN-alpha inducing activity and B cell activation activities of CICs can be independently varied based on the structure of the CIC and selection of NAMs, it is possible to identify and produce CICs with different levels of each of these activities, using screening methods described herein and the information about B cell stimulating activity described herein. For example, CICs can be designed to exhibit different B cell activation, from insignificant up to levels equivalent to SEQ ID NO:25 (5'-

TGACTGTGAACGTTCGAGATGA-3'), independently of the amount of IFN-alpha induced by that CIC. Similarly, CICs with different levels of IFN-inducing activity can be identified and produced, independently of their B cell activation activity.

[00544] Thus, without limitation, in one aspect, the invention provides CICs that induce IFN-alpha production and do not induce human B cell proliferation, and methods of using such CICs. In a related aspect, the invention provides CICs that induce IFN-alpha production and little human B cell proliferation and methods of using such CICs. In a related aspect, the invention provides design algorithms and screening methods for identifying CICs with these properties. Human B cell activation can be measured, for example, by B cell proliferation or maturation, or by secretion of cytokines, e.g. IL-6, from B cells. The potency of CICs or polynucleotides to induce B cell proliferation or maturation or cytokine secretion (measured for example by an EC50), or the maximum level of B cell proliferation or maturation or cytokine secretion induced by CICs or polynucleotides can be used determine B cell activity .

[00545] A CIC is considered to not induce human B cell proliferation if B cell proliferation in the presence of the CIC is at 'background' levels, i.e., 0 to 15%, optionally 0 to about 10%, of the proliferation induced by an equal amount (e.g., 5 μg/ml) of SEQ ID NO:25 (5'-TGACTGTGAACGTTCGAGATGA-S'). A CIC is considered to induce 'little' human B cell proliferation if B cell proliferation in the presence of the CIC is between greater than 15 to about 30% of SEQ ID NO:25. Thus, in some embodiments a CIC of the invention induces less than about 30%, sometimes less then about 25%, sometimes less than about 20%, sometimes less than about 15% or less than about 10% of the level of B cell proliferation induced by SEQ ID NO:25 (5'-TGACTGTGAACGTTCGAGATGA-S'). Alternatively, a CIC is considered to not induce human B cell proliferation if B cell proliferation in the presence of the CIC is not statistically significantly greater than the B cell proliferation induced by an equal concentration (e.g., 5 ug/ml) of an inactive control compound using an in vitro assay. [00546] The ability to 'program' CICs to exhibit different biological properties allows for the assembly of CICs exhibiting a defined set of activities tailored for specific clinical applications. For example, CICs with high IFN-alpha production and little B cell activation may be particularly useful in cancer therapies, while CICs with moderate IFN-alpha production and little B cell activation are particularly useful for treatment of diseases such as asthma. For certain indications, including the treatment of allergic asthma and certain cancers, it may be desirable to avoid polyclonal B cell activation, which might result in the potentiation of asthma- mediating B cells or B cell lymphomas. In certain indications, such as asthma, it may be desirable to activate polyclonal B cells, particularly when such B cell activation can effect inhibition of the Th2-type response, as described herein. A variety of uses are known for CICs that preferentially stimulate B cell proliferation, including without limitation in vivo expansion to produce B cell clones for analysis.

[00547] Methods of the invention includes embodiments in which CICs are administered in the form of a CIC/microcarrier complex(s). In some embodiments, the invention provides methods of stimulating CTL production in an individual, comprising administering an effective amount of a CIC to the individual such that CTL production is increased.

[00548] As will be apparent to one of skill in the art, the methods of the invention may be practiced in combination with other therapies for the particular indication for which the CIC is administered. For example, CIC therapy may be administered in conjunction with antimalarial drugs such as chloroquine for malaria patients, in conjunction with leishmanicidal drugs such as pentamidine and/or allopurinol for leishmaniasis patients, in conjunction with anti-mycobacterial drugs such as isoniazid, rifampin and/or ethambutol in tuberculosis patients, or in conjunction with allergen desensitization therapy for atopic (allergy) patients.

[00549] In vitro-based cell assay can be used to compare CICs or polynucleotides. Such comparisons can, for example, use the maximum activities induced by respective CICs or polynucleotide. In some embodiments the maximum activity can be based on, for example, the levels of IFN-alpha induced by CICs or polynucleotides from human PBMC. A CIC or polynucleotide is characterized as having 'superior immunomodulatory or immuno stimulatory activity' when the test CIC or polynucleotide has more activity than the CIC or polynucleotide to which it is compared. Preferably, the isolated immunomodulatory or immuno stimulatory activity is statistically significantly improved (p < 0.05, or p < 0.01, or p < 0.001) over the CICs or polynucleotides to which the respective CICs or polynucleotides are being compared. Preferably, the isolated immunomodulatory or immuno stimulatory activity is statistically significantly improved (with p < 0.05, or smaller) over the CICs or polynucleotides to which the respective CICs or polynucleotides are being compared. The statistical analysis applied can be different per activity evaluated, but can be appropriately selected by someone skilled in the art.

Methods of the Invention (Immunoregulation)

[00550] Provided herein are methods of regulating an immune response in an individual, preferably a mammal, more preferably a human, comprising administering to the individual an IRS-containing CIRC as described herein. Methods of immunoregulation provided by the invention include those that suppress and/or inhibit an immune response, including, but not limited to, an immune response stimulated by immuno stimulatory nucleic acid molecules such as bacterial DNA. In some variations, the IRS is a modified IRS. In some variations, the IRS is an unmodified IRS. The invention also provides methods for inhibiting TLR7 and/or TLR9 induced cell response. The invention also provides methods for ameliorating symptoms associated with unwanted immune activation, including, but not limited to, symptoms associated with autoimmunity.

[00551] Provided herein are methods for regulating an immune response in an individual, comprising administering to an individual a CIRC described herein in an amount sufficient to regulate an immune response in said individual. In some variations, the CIRC comprise a modified IRS. In some variations, the CIRC comprise an unmodified IRS. In some variations, the CIRC comprise both modified and unmodified IRSs. Immuno-regulation according to the methods described herein may be practiced on individuals including those suffering from a disorder associated with an unwanted activation of an immune response. In some variations, the immune response is an innate immune response. In some variations, the immune response is an adaptive immune response.

[00552] Further provided herein are methods for inhibiting an immune response comprising contacting a cell of the immune system with a polynucleotide comprising an immunoregulatory sequence (IRS), wherein the cell is contacted with the polynucleotide in an amount effective to inhibit a response from the cell that contributes to an immune response. In some variations, the IRS comprises a modification. In some variations, the IRS does not comprise a modification (i.e., an unmodified IRS).

[00553] Methods are provided herein for ameliorating one or more symptoms of an autoimmune disease, comprising administering an effective amount of a CIRC described herein to an individual having an autoimmune disease. In some variations, administration of a CIRC ameliorates one or more symptoms of the autoimmune disease, including SLE and rheumatoid arthritis. In some variations, the CIRC effective for suppressing a symptom of SLE comprises an immunoregulatory sequence of the TLR7 class or TLR9 class or TLR7/9 class. In some variations, the IRP and/or IRC comprise a modified IRS. In some variations, the CIRC comprise an unmodified IRS. In some variations, the CIRC comprise both modified and unmodified IRSs.

[00554] Methods are also provided herein for preventing or delaying development of an autoimmune disease, comprising administering an effective amount of a CIRC described herein to an individual at risk of developing an autoimmune disease. In some variations, administration of a CIRC prevents or delays development of the autoimmune disease. In some variations, the IRP and/or IRC comprise a modified IRS. In some variations, the CIRC comprise an unmodified IRS. In some variations, the CIRC comprise both modified and unmodified IRSs.

[00555] Methods of combination therapy are also provided herein. In some variations, methods are provided for ameliorating one or more symptoms of an autoimmune disease, comprising administering an effective amount of a CIRC described herein and an other therapeutic agent to an individual having an autoimmune disease. In some variations, the other therapeutic agent is a corticosteroid. In some variations, administration of the combination ameliorates one or more symptoms of the autoimmune disease, including SLE and rheumatoid arthritis. In some variations, the CIRC used in combination therapy effective for suppressing a symptom of SLE comprises an immunoregulatory sequence of the TLR7 class or TLR9 class or TLR7/9 class. In some variations, the CIRC used in combination therapy comprise a modified IRS. In some variations, the CIRC used in combination therapy comprise an unmodified IRS. In some variations, the CIRC used in combination therapy comprise both modified and unmodified IRSs. [00556] In some variations, methods are provided for preventing or delaying development of an autoimmune disease, comprising administering an effective amount of a CIRC described herein and an other therapeutic agent to an individual at risk of developing an autoimmune disease. In some variations, the other therapeutic agent is a corticosteroid. In some variations, administration of the combination prevents or delays development of one or more symptoms of the autoimmune disease, including SLE and rheumatoid arthritis. In some variations, the CIRC used in combination therapy comprise a modified IRS. In some variations, the CIRC used in combination therapy comprise an unmodified IRS. In some variations, the CIRC used in combination therapy comprise both modified and unmodified IRSs.

[00557] In certain variations, the individual suffers from a disorder associated with unwanted immune activation, such as autoimmune disease and inflammatory disease. An individual having an autoimmune disease or inflammatory disease is an individual with a recognizable symptom of an existing autoimmune disease or inflammatory disease.

[00558] Autoimmune diseases include, without limitation, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type I diabetes mellitus, type II diabetes mellitus, multiple sclerosis (MS), immune-mediated infertility such as premature ovarian failure, scleroderma, Sjogren's disease, vitiligo, alopecia (baldness), polyglandular failure, Grave's disease, hypothyroidism, polymyositis, pemphigus vulgaris, pemphigus foliaceus, inflammatory bowel disease including Crohn's disease and ulcerative colitis, autoimmune hepatitis including that associated with hepatitis B virus (HBV) and hepatitis C virus (HCV), hypopituitarism, graft- versus-host disease (GvHD), myocarditis, Addison's disease, autoimmune skin diseases, uveitis, pernicious anemia, and hypoparathyroidism.

[00559] Autoimmune diseases may also include, without limitation, Hashimoto's thyroiditis, Type I and Type II autoimmune polyglandular syndromes, paraneoplastic pemphigus, bullus pemphigoid, dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita, erythema nodosa, pemphigoid gestationis, cicatricial pemphigoid, mixed essential cryoglobulinemia, chronic bullous disease of childhood, hemolytic anemia, thrombocytopenic purpura, Goodpasture's syndrome, autoimmune neutropenia, myasthenia gravis, Eaton-Lambert myasthenic syndrome, stiff-man syndrome, acute disseminated encephalomyelitis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, multifocal motor neuropathy with conduction block, chronic neuropathy with monoclonal gammopathy, opsonoclonus-myoclonus syndrome, cerebellar degeneration, encephalomyelitis, retinopathy, primary biliary sclerosis, sclerosing cholangitis, gluten-sensitive enteropathy, ankylosing spondylitis, reactive arthritides, polymyositis/dermatomyositis, mixed connective tissue disease, Bechet's syndrome, psoriasis, polyarteritis nodosa, allergic anguitis and granulomatosis (Churg-Strauss disease), polyangiitis overlap syndrome, hypersensitivity vasculitis, Wegener's granulomatosis, temporal arteritis, Takayasu's arteritis, Kawasaki's disease, isolated vasculitis of the central nervous system, thromboangiutis obliterans, sarcoidosis, glomerulonephritis, and cryopathies. These conditions are well known in the medical arts and are described, for example, in Harrison's Principles of Internal Medicine, 14th ed., Fauci A S et al., eds., New York: McGraw-Hill, 1998.

[00560] The systemic disease SLE is characterized by the presence of antibodies to antigens that are abundant in nearly every cell, such as anti-chromatin antibodies, anti- splicesosome antibodies, anti-ribosome antibodies and anti-DNA antibodies. Consequently, the effects of SLE are seen in a variety of tissues, such as the skin and kidneys. Autoreactive T cells also play a role in SLE. For example, studies in a murine lupus model have shown that non-DNA nucleosomal antigens, e.g. histones, stimulate autoreactive T cells that can drive anti-DNA producing B cells. Increased serum levels of IFN-alpha has been observed in SLE patients and shown to correlate with both disease activity and severity, including fever and skin rashes, as well as essential markers associated with the disease process (e.g., anti- dsDNA antibody titers). It has also been shown that immune complexes present in the circulation could trigger IFN-alpha in these patients and, thus, maintain this chronic presence of elevated IFN- alpha. Two different types of immune complexes have been described to trigger IFN- alpha from human PDC: DNA / anti-DNA antibody complexes and RNA / anti- ribonucleoprotein-RNA antibody complexes. Because DNA is a ligand of TLR-9 and RNA a ligand for TLR7, it is expected that these two pathways utilize TLR-9 and TLR-7/8 signaling, respectively, in order to chronically induce IFN-alpha and thus participate in the etiopathogenesis of SLE. Accordingly, CIRC compositions which are effective in inhibiting TLR7 and TLR-9 responses may be particularly effective in treating SLE.

[00561] In certain variations, an individual is at risk of developing an autoimmune disease and an IRP or IRC is administered in an amount effective to delay or prevent the autoimmune disease. Individuals at risk of developing an autoimmune disease includes, for example, those with a genetic or other predisposition toward developing an autoimmune disease. In humans, susceptibility to particular autoimmune diseases is associated with HLA type with some being linked most strongly with particular MHC class II alleles and others with particular MHC class I alleles. For example, ankylosing spondylitis, acute anterior uveitis, and juvenile rheumatoid arthritis are associated with HLA-B27, Goodpasture's syndrome and MS are associated with HLA-DR2, Grave's disease, myasthenia gravis and SLE are associated with HLA-DR3, rheumatoid arthritis and pemphigus vulgaris are associated with HLA-DR4 and Hashimoto's thyroiditis is associated with HLA-DR5. Other genetic predispositions to autoimmune diseases are known in the art and an individual can be examined for existence of such predispositions by assays and methods well known in the art. Accordingly, in some instances, an individual at risk of developing an autoimmune can be identified.

[00562] As described herein, IRPs described herein may particularly inhibit production of a cytokine, including, but not limited to, IL-6, IL- 12, TNF-alpha and/or IFN-alpha and may suppress B cell proliferation and/or activation of plasmacytoid dendritic cells to differentiate. In some variations, the CIRCs comprise a modified IRS. In some variations, the CIRCs comprise an unmodified IRS. In some variations, the CIRCs comprise both a modified IRS and an unmodified IRS.

[00563] Animal models for the study of autoimmune disease are known in the art. For example, animal models which appear most similar to human autoimmune disease include animal strains which spontaneously develop a high incidence of the particular disease. Examples of such models include, but are not limited to, the nonobeses diabetic (NOD) mouse, which develops a disease similar to type 1 diabetes, and lupus-like disease prone animals, such as New Zealand hybrid, MRL-Faslpr and BXSB mice. Animal models in which an autoimmune disease has been induced include, but are not limited to, experimental autoimmune encephalomyelitis (EAE), which is a model for multiple sclerosis, collagen- induced arthritis (CIA), which is a model for rheumatoid arthritis, and experimental autoimmune uveitis (EAU), which is a model for uveitis. Animal models for autoimmune disease have also been created by genetic manipulation and include, for example, IL-2/IL-10 knockout mice for inflammatory bowel disease, Fas or Fas ligand knockout for SLE, and IL- lreceptor antagonist knockout for rheumatoid arthritis. [00564] Accordingly, animal models standard in the art are available for the screening and/or assessment for activity and/or effectiveness of the methods and compositions described herein for the treatment of autoimmune disorders.

[00565] Provided herein are methods for treating and/or ameliorating one or more symptoms of an inflammatory disease or disorder, comprising administering an effective amount of a CIRC described herein to an individual having an inflammatory disease or disorder. In some variations, administration of a CIRC ameliorates one or more symptoms of the inflammatory disease or disorder. In some variations, the compositions described herein are effective in ameliorating a symptom of chronic inflammatory disease or disorder. In some variations, the inflammatory disease or disorder is an autoimmune disease discussed above. In some variations, the CIRC comprise a modified IRS. In some variations, the CIRC comprise an unmodified IRS. In some variations, the CIRC comprise both modified and unmodified IRSs.

[00566] In some variations, provided herein are methods of suppressing and/or inhibiting an inflammatory response using any of the CIRCs described herein. In certain variations, the individual suffers from a disorder associated with a chronic inflammatory response. Administration of a CIRC results in immunoregulation, decreasing levels of one or more immune response associated cytokines, which may result in a reduction of the inflammatory response. Immunoregulation of individuals with the unwanted immune response associated the described disorders results in a reduction or improvement in one or more of the symptoms of the disorder. In some variations, the inflammatory response inhibited and/or suppressed is drug-induced inflammation. In some variations, the drug-induced inflammation is drug- induced inflammation of the liver. In some variations, the inflammatory response inhibited and/or suppressed is infection-induced inflammation. In some variations, the disorder is an inflammatory liver disease or an inflammatory pancreatic disorder. Examples of inflammatory liver disorders include, for example, ligalactosemia, Alagille's syndrome, alpha 1 -antitrypsin deficiency, neonatal hepatitis, tyrosinemia, hemorrhagic telangiectasia, Reye's syndrome, Wilson's disease, thalassemia, biliary atresia, chronic active hepatitis such as hepatitis A, hepatitis B, or hepatitis C, cancer of the liver, cirrhosis, type I glycogen storage disease, porphyria, hemochromatosis, primary sclerosing cholangitis, sarcoidosis, gallstones, fatty liver disease, alcoholic hepatitis, or alcoholic cirrhosis. Examples of inflammatory pancreatic disorders include, for example, pancreatitis or pancreatic cancer. [00567] Other variations provided herein relate to immunoregulatory therapy of individuals having been exposed to or infected with a virus. Administration of a CIRC to an individual having been exposed to or infected with a virus results in suppression of virus induced cytokine production. In some variations, the CIRCs comprise a modified IRS. In some variations, the CIRCs comprise an unmodified IRS. In some variations, the CIRCs comprise both a modified IRS and an unmodified IRS. Cytokine produced in response to a virus can contribute to an environment favorable for viral infection. Suppression of the virus- induced cytokine production may serve to limit or prevent the viral infection.

[00568] In some variations, methods are provided for suppressing chronic pathogen stimulation, comprising administering an effective amount of a CIRC described herein to an individual having a chronic pathogen infection or disease. In some variations, administration of a CIRC suppresses chronic pathogen stimulation in the individual, including that associated with malaria and chronic viral infections. CIRC compositions which are effective in inhibiting TLR7 responses may be particularly effective in treating disease and symptoms related to chronic pathogen stimulation. In some variations, the CIRC effective for suppressing chronic pathogen stimulation comprises an immunoregulatory sequence of the TLR7 class. In some variations, the CIRC comprise a modified IRS. In some variations, the CIRC comprise an unmodified IRS. In some variations, the CIRC comprise both modified and unmodified IRSs.

[00569] In some situations, peripheral tolerance to an autoantigen is lost (or broken) and an autoimmune response ensues. For example, in an animal model for EAE, activation of antigen presenting cells (APCs) through the immune receptor TLR9 or TLR4 was shown to break self-tolerance and result in the induction of EAE (Waldner et al. (2004) J. Clin. Invest. 113:990-997).

[00570] In any of the methods described herein the IRS-containing CIRC may be administered in an amount sufficient to regulate an immune response. As described herein, regulation of an immune response may be humoral and/or cellular, and is measured using standard techniques in the art and as described herein.

[00571] Accordingly, in some variations, provided herein are methods for suppressing, reducing, and/or inhibiting TLR9 dependent cell stimulation. Administration of a CIRC results in suppression of TLR9 dependent cell responses, including decreased levels of one or more TLR9-associated cytokines. CIRCs appropriate for use in suppressing TLR9 dependent cell stimulation are those CIRC that inhibit or suppress cell responses associated with TLR9. In some variations, the CIRCs comprise a modified IRS. In some variations, the CIRCs comprise an unmodified IRS. In some variations, the CIRCs comprise both a modified IRS and an unmodified IRS.

[00572] In some variations, provided herein are methods for suppressing, reducing, and/or inhibiting TLR7 dependent cell stimulation. Administration of a CIRC results in suppression of TLR7 dependent cell responses, including decreased levels of one or more TLR7- associated cytokines. CIRCs appropriate for use in suppressing TLR7 dependent cell stimulation are those CIRC that inhibit or suppress cell responses associated with TLR7. In some variations, the CIRCs comprise a modified IRS. In some variations, the CIRCs comprise an unmodified IRS. In some variations, the CIRCs comprise both a modified IRS and an unmodified IRS.

[00573] In some variations, methods are provided for inhibiting a TLR7 dependent immune response independently of TLR9 dependent immune response in an individual, comprising administering to an individual an a CIRC described herein in an amount sufficient to suppress TLR7 dependent cytokine production independently of TLR9 dependent cytokine production in said individual. In some variations, the TLR7 and/or TLR9 dependent immune response is an innate immune response. In some variations, the TLR7 and/or TLR9 dependent immune response is an adaptive immune response. In some variations, the CIRC comprise a modified IRS. In some variations, the CIRC comprise an unmodified IRS. In some variations, the CIRC comprise both modified and unmodified IRSs.

[00574] As demonstrated herein, some CIRC suppress both TLR9 dependent cell responses and TLR7 dependent cell responses. In some variations, methods are provided for inhibiting a TLR9 dependent immune response and a TLR7 dependent immune response in an individual, comprising administering to an individual a CIRC described herein in an amount sufficient to suppress TLR9 dependent cytokine production and TLR7 dependent cytokine production in said individual, wherein the CIRC comprises an IRS of the TLR7/9 class. In some variations, the TLR7 and/or TLR9 dependent immune response is an innate immune response. In some variations, the TLR7 and/or TLR9 dependent immune response is an adaptive immune response. In some variations, the CIRC comprise a modified IRS. In some variations, the CIRC comprise an unmodified IRS. In some variations, the CIRC comprise both modified and unmodified IRSs.

[00575] In some variations, the compositions described herein inhibit a response of a B cell or a plasmacytoid dendritic cell. In some variations, immune responses inhibited by the compositions described herein include inhibition of cytokine production, such as IL-6 and/or IFN-alpha, by the cell, inhibition of cell maturation and/or inhibition of cell proliferation. In some variations, the compositions described herein inhibit a TLR9 dependent cell response, a TLR7 dependent cell response, and/or a TLR7/9 dependent cell response.

Administration and Assessment of the Immune Response

[00576] The CIC can be administered in combination with pharmaceutical and/or immunogenic and/or other immuno stimulatory agents, as described herein, and can be combined with a physiologically acceptable carrier thereof. For example, a CIC or composition of the invention can be administered in conjunction with other immunotherapeutic agents such as cytokines, adjuvants and antibodies. The CIC may be given in conjunction with the agent (e.g., at the same time, or before or after (e.g., less than 24 hours before or after administration of the agent). The CIC may be any of those described herein.

[00577] As with all immuno stimulatory compositions, the immunologically effective amounts and method of administration of the particular CIC formulation can vary based on the individual, what condition is to be treated and other factors evident to one skilled in the art. Factors to be considered include the presence of a coadministered antigen, whether or not the CIC will be administered with or covalently attached to an adjuvant or delivery molecule, route of administration and the number of immunizing doses to be administered. Such factors are known in the art and it is well within the skill of those in the art to make such determinations without undue experimentation. A suitable dosage range is one that provides the desired modulation of immune response to the antigen. Generally, dosage is determined by the amount of CIC administered to the patient, rather than the overall quantity of CIC. Exemplary dosage ranges of the CIC, given in amounts of CIC delivered, may be, for example, from about any of the following: 1 to 500 μg/kg, 100 to 400 μg/kg, 200 to 300 μg/kg, 1 to 100 μg/kg, 100 to 200 μg/kg, 300 to 400 μg/kg, 400 to 500 μg/kg, 0.5 to 10 mg/kg, 1 to 9 mg/kg, 2 to 8 mg/kg, 3 to 7 mg/kg, 4 to 6 mg/kg, 5 mg/kg, 1 to 10 mg/kg, or 5 to 10 mg/kg. The absolute amount given to each patient depends on pharmacological properties such as bioavailability, clearance rate and route of administration.

[00578] The effective amount and method of administration of the particular CIC formulation can vary based on the individual patient and the stage of the disease and other factors evident to one skilled in the art. The route(s) of administration suited for a particular application will be known to one of skill in the art. Routes of administration include but are not limited to topical, dermal, transdermal, transmucosal, epidermal, parenteral, gastrointestinal, and naso-pharyngeal and pulmonary, including transbronchial and transalveolar. A suitable dosage range is one that provides sufficient CIC-containing composition to attain a tissue concentration of about 1-10 μM or 1-50 μM as measured by blood levels. The absolute amount given to each patient depends on pharmacological properties such as bioavailability, clearance rate and route of administration.

[00579] As described herein, APCs, dendritic cells and tissues with high concentration of APCs and dendritic cells are preferred targets for the CIC. Thus, administration of CIC to mammalian skin and/or mucosa, where APCs and dendritic cells are present in relatively high concentration, is preferred.

[00580] As described herein, tissues in which unwanted immune activation is occurring or is likely to occur are preferred targets for the CIRC. Thus, administration of CIRC to lymph nodes, spleen, bone marrow, blood, as well as tissue exposed to virus, are preferred sites of administration.

[00581] The present invention provides CIC formulations suitable for topical application including, but not limited to, physiologically acceptable implants, ointments, creams, rinses and gels. Topical administration is, for instance, by a dressing or bandage having dispersed therein a delivery system, by direct administration of a delivery system into incisions or open wounds, or by transdermal administration device directed at a site of interest. Creams, rinses, gels or ointments having dispersed therein a CIC are suitable for use as topical ointments or wound filling agents.

[00582] Preferred routes of dermal administration are those which are least invasive. Preferred among these means are transdermal transmission, epidermal administration and subcutaneous injection. Of these means, epidermal administration is preferred for the greater concentrations of APCs and dendritic cells expected to be in intradermal tissue.

[00583] Transdermal administration is accomplished by application of a cream, rinse, gel, etc. capable of allowing the CIC to penetrate the skin and enter the blood stream. Compositions suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams and ointments applied directly to the skin or incorporated into a protective carrier such as a transdermal device (so-called 'patch'). Examples of suitable creams, ointments etc. can be found, for instance, in the Physician's Desk Reference.

[00584] For transdermal transmission, iontophoresis is a suitable method. Iontophoretic transmission can be accomplished using commercially available patches which deliver their product continuously through unbroken skin for periods of several days or more. Use of this method allows for controlled transmission of pharmaceutical compositions in relatively great concentrations, permits infusion of combination drugs and allows for contemporaneous use of an absorption promoter.

[00585] An exemplary patch product for use in this method is the LECTRO PATCH trademarked product of General Medical Company of Los Angeles, CA. This product electronically maintains reservoir electrodes at neutral pH and can be adapted to provide dosages of differing concentrations, to dose continuously and/or periodically. Preparation and use of the patch should be performed according to the manufacturer' s printed instructions which accompany the LECTRO PATCH product; those instructions are incorporated herein by this reference. Other occlusive patch systems are also suitable.

[00586] For transdermal transmission, low-frequency ultrasonic delivery is also a suitable method. Mitragotri et al. (1995) Science 269:850-853. Application of low- frequency ultrasonic frequencies (about 1 MHz) allows the general controlled delivery of therapeutic compositions, including those of high molecular weight.

[00587] Epidermal administration essentially involves mechanically or chemically irritating the outermost layer of the epidermis sufficiently to provoke an immune response to the irritant. Specifically, the irritation should be sufficient to attract APCs and dendritic cells to the site of irritation. [00588] An exemplary mechanical irritant means employs a multiplicity of very narrow diameter, short tines which can be used to irritate the skin and attract APCs and dendritic cells to the site of irritation, to take up CIC transferred from the end of the tines. For example, the MONO-VACC old tuberculin test manufactured by Pasteur Merieux of Lyon, France contains a device suitable for introduction of CIC-containing compositions.

[00589] The device (which is distributed in the U.S. by Connaught Laboratories, Inc. of Swiftwater, PA) consists of a plastic container having a syringe plunger at one end and a tine disk at the other. The tine disk supports a multiplicity of narrow diameter tines of a length which will just scratch the outermost layer of epidermal cells. Each of the tines in the MONO-VACC kit is coated with old tuberculin; in the present invention, each needle is coated with a pharmaceutical composition of a CIC formulation. Use of the device is preferably according to the manufacturer' s written instructions included with the device product. Similar devices which can also be used in this embodiment are those which are currently used to perform allergy tests.

[00590] Another suitable approach to epidermal administration of CIC is by use of a chemical which irritates the outermost cells of the epidermis, thus provoking a sufficient immune response to attract APCs and dendritic cells to the area. An example is a keratinolytic agent, such as the salicylic acid used in the commercially available topical depilatory creme sold by Noxema Corporation under the trademark NAIR. This approach can also be used to achieve epithelial administration in the mucosa. The chemical irritant can also be applied in conjunction with the mechanical irritant (as, for example, would occur if the MONO-VACC type tine were also coated with the chemical irritant). The CIC can be suspended in a carrier which also contains the chemical irritant or co-administered therewith.

[00591] Parenteral routes of administration include but are not limited to electrical (iontophoresis) or direct injection such as direct injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. Formulations of CIC suitable for parenteral administration are generally formulated in USP water or water for injection and may further comprise pH buffers, salts bulking agents, preservatives, and other pharmaceutically acceptable excipients. CICs for parenteral injection may be formulated in pharmaceutically acceptable sterile isotonic solutions such as saline and phosphate buffered saline for injection. [00592] Gastrointestinal routes of administration include, but are not limited to, ingestion and rectal. The invention includes formulations CIC suitable for gastrointestinal administration including, but not limited to, pharmaceutically acceptable powders, pills or liquids for ingestion and suppositories for rectal administration. As will be apparent to one of skill in the art, pills or suppositories will further comprise pharmaceutically acceptable solids, such as starch, to provide bulk for the composition.

[00593] Naso-pharyngeal and pulmonary administration include are accomplished by inhalation, and include delivery routes such as intranasal, transbronchial and transalveolar routes. The invention includes formulations of CIC suitable for administration by inhalation including, but not limited to, liquid suspensions for forming aerosols as well as powder forms for dry powder inhalation delivery systems. Devices suitable for administration by inhalation of CIC formulations include, but are not limited to, atomizers, vaporizers, nebulizers, and dry powder inhalation delivery devices.

[00594] The choice of delivery routes can be used to modulate the immune response elicited. For example, IgG titers and CTL activities were identical when an influenza virus vector was administered via intramuscular or epidermal (gene gun) routes; however, the muscular inoculation yielded primarily IgG2a, while the epidermal route yielded mostly IgGl. Pertmer et al. (1996) /. Virol. 70:6119-6125. Thus, one skilled in the art can take advantage of slight differences in immunogenicity elicited by different routes of administering the immunomodulatory oligonucleotides of the present invention.

[00595] The above-mentioned compositions and methods of administration are meant to describe but not limit the methods of administering the formulations of CIC of the invention. The methods of producing the various compositions and devices are within the ability of one skilled in the art and are not described in detail here.

[00596] Analysis (both qualitative and quantitative) of the immune response to CIC can be by any method known in the art, including, but not limited to, measuring antigen- specific antibody production (including measuring specific antibody subclasses), activation of specific populations of lymphocytes such as CD4+ T cells, B cells, NK cells or CTLs, production of cytokines such as IFN-gamma, IFN-alpha, IL-2, IL-4, IL-5, IL-10, IL- 13 or IL- 12 and/or release of histamine. Methods for measuring specific antibody responses include enzyme- linked immunosorbent assay (ELISA) and are well known in the art. Measurement of numbers of specific types of lymphocytes such as CD4+ T cells can be achieved, for example, with fluorescence-activated cell sorting (FACS). Cytotoxicity and CTL assays can be performed for instance as described in Raz et al. (1994) Proc. Natl. Acad. ScL USA 91:9519-9523 and Cho et al. (2000). Cytokine concentrations can be measured, for example, by ELISA. These and other assays to evaluate the immune response to an immunogen are well known in the art. See, for example, SELECTED METHODS IN CELLULAR IMMUNOLOGY (1980) Mishell and Shiigi, eds., W.H. Freeman and Co.

[00597] Preferably, a ThI -type response is stimulated, i.e., elicited and/or enhanced. With reference to the invention, stimulating a ThI -type immune response can be determined in vitro or ex vivo by measuring cytokine production from cells treated with a CIC as compared to control cells not treated with CIC. Methods to determine the cytokine production of cells include those methods described herein and any known in the art. The type of cytokines produced in response to CIC treatment indicate a ThI -type or a Th2-type biased immune response by the cells. As used herein, the term 'ThI -type biased' cytokine production refers to the measurable increased production of cytokines associated with a ThI- type immune response in the presence of a stimulator as compared to production of such cytokines in the absence of stimulation. Examples of such ThI -type biased cytokines include, but are not limited to, IL-2, IL- 12, IFN-gamma and IFN-alpha. In contrast, Th2- type biased cytokines' refers to those associated with a Th2-type immune response, and include, but are not limited to, IL-4, IL-5, and IL- 13. Cells useful for the determination of CIC activity include cells of the immune system, primary cells isolated from a host and/or cell lines, preferably APCs, dendritic cells and lymphocytes, even more preferably macrophages, dendritic cells and T cells.

[00598] Stimulating a ThI -type immune response can also be measured in a host treated with a CIC can be determined by any method known in the art including, but not limited to: (1) a reduction in levels of IL-4, IL- 13 or IL-5 measured before and after antigen-challenge; or detection of lower (or even absent) levels of IL-4, IL- 13 or IL-5 in a CIC treated host as compared to an antigen-primed, or primed and challenged, control treated without CIC; (2) an increase in levels of IL- 12, IL- 18 and/or IFN (alpha, beta or gamma) before and after antigen challenge; or detection of higher levels of IL- 12, IL- 18 and/or IFN (alpha, beta or gamma) in a CIC treated host as compared to an antigen-primed or, primed and challenged, control treated without CIC; (3) 'Thl-type biased' antibody production in a CIC treated host as compared to a control treated without CIC; and/or (4) a reduction in levels of antigen- specific IgE as measured before and after antigen challenge; or detection of lower (or even absent) levels of antigen- specific IgE in a CIC treated host as compared to an antigen-primed, or primed and challenged, control treated without CIC. A variety of these determinations can be made by measuring cytokines made by APCs, dendritic cells and/or lymphocytes, preferably macrophages, dendritic cells and/or T cells, in vitro or ex vivo using methods described herein or any known in the art. Some of these determinations can be made by measuring the class and/or subclass of antigen- specific antibodies using methods described herein or any known in the art.

[00599] The class and/or subclass of antigen- specific antibodies produced in response to CIC treatment indicate a ThI -type or a Th2-type biased immune response by the cells. As used herein, the term 'ThI -type biased' antibody production refers to the measurable increased production of antibodies associated with a ThI -type immune response (i.e., ThI- associated antibodies). One or more ThI associated antibodies may be measured. Examples of such ThI -type biased antibodies include, but are not limited to, human IgGl and/or IgG3 (see, e.g., Widhe et al. (1998) Scand. J. Immunol. 47:575-581 and de Martino et al. (1999) Ann. Allergy Asthma Immunol. 83:160-164) and murine IgG2a. In contrast, 'Th2-type biased antibodies' refers to those associated with a Th2-type immune response, and include, but are not limited to, human IgG2, IgG4 and/or IgE (see, e.g., Widhe et al. (1998) and de Martino et al. (1999)) and murine IgGl and/or IgE.

[00600] The ThI -type biased cytokine induction which occurs as a result of administration of CIC produces enhanced cellular immune responses, such as those performed by NK cells, cytotoxic killer cells, ThI helper and memory cells. These responses are particularly beneficial for use in protective or therapeutic vaccination against viruses, fungi, protozoan parasites, bacteria, allergic diseases and asthma, as well as tumors.

[00601] In some embodiments, a Th2 response is suppressed. Suppression of a Th2 response may be determined by, for example, reduction in levels of Th2- associated cytokines, such as IL-4, IL- 13 and IL-5, as well as IgE reduction and reduction in histamine release, such as in an airway or other local area, in response to allergen and/or eosiniphilia.

Kits of the Invention [00602] The invention provides kits. In one aspect, the invention provides kits that contain a library of highly pure oligonucleotides for conjugation to the platform molecules. Optionally, the platform molecules are included or material needed to synthesize the platform molecules. In another aspect, the kit comprises at least one platform molecule (or the materials needed to synthesize it) and at least one material comprising nucleic acid moiety, optionally including instructions for use.

[00603] In certain embodiments, the kits of the invention comprise one or more containers comprising a CIC. The kits may further comprise a suitable set of instructions, generally written instructions, relating to the use of the CIC for the intended treatment (e.g., immunomodulation, immunoregulation, ameliorating symptoms of an infectious disease, increasing IFN-gamma levels, increasing IFN-alpha levels, or ameliorating an IgE-related disorder, suppression of a TLR7 and/or TLR9 dependent response, ameliorating one or more symptoms of an autoimmune disease, ameliorating a symptom of chronic inflammatory disease, decreasing cytokine production in response to a virus).

[00604] The kits may comprise CIC packaged in any convenient, appropriate packaging. For example, if the CIC is a dry formulation (e.g., freeze dried or a dry powder), a vial with a resilient stopper is normally used, so that the CIC may be easily resuspended by injecting fluid through the resilient stopper. Ampoules with non-resilient, removable closures (e.g., sealed glass) or resilient stoppers are most conveniently used for liquid formulations of CIC. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.

[00605] The instructions relating to the use of CIC generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers of CIC may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. [00606] In some embodiments, the kits further comprise an antigen (or one or more antigens), which may or may not be packaged in the same container (formulation) as the CIC(s). Antigen have been described herein.

[00607] In certain embodiments, the kits of the invention comprise a CIC in the form of a CIC/microcarrier complex (CIC/MC), and may further comprise a set of instructions, generally written instructions, relating to the use of the CIC/MC complex for the intended treatment (e.g., immunomodulation, ameliorating symptoms of an infectious disease, increasing IFN-gamma levels, increasing IFN-alpha levels, or ameliorating an IgE-related disorder).

[00608] In some embodiments, kits of the invention comprise materials for production of CIC/MC complex generally include separate containers of CIC and MC, although in certain embodiments materials for producing the MC are supplied rather than preformed MC. The CIC and MC are preferably supplied in a form which allows formation of CIC/MC complex upon mixing of the supplied CIC and MC. This configuration is preferred when the CIC/MC complex is linked by non-covalent bonding. This configuration is also preferred when the CIC and MC are to be crosslinked via a heterobifunctional crosslinker; either CIC or the MC is supplied in an 'activated' form (e.g., linked to the heterobifunctional crosslinker such that a moiety reactive with the CIC is available).

[00609] Kits for CIC/MC complexes comprising a liquid phase MC preferably comprise one or more containers including materials for producing liquid phase MC. For example, a CIC/MC kit for oil-in- water emulsion MC may comprise one or more containers containing an oil phase and an aqueous phase. The contents of the container are emulsified to produce the MC, which may be then mixed with the CIC, preferably a CIC which has been modified to incorporate a hydrophobic moiety. Such materials include oil and water, for production of oil-in-water emulsions, or containers of lyophilized liposome components (e.g., a mixture of phospholipid, cholesterol and a surfactant) plus one or more containers of an aqueous phase (e.g., a pharmaceutically- acceptable aqueous buffer).

VI. Examples

[00610] The following Examples are provided to illustrate, but not limit, the invention. Example 1 Synthesis of a Symmetrical Tri Ann Platform Molecule [00611] As depicted in Fig. 1, a Tri Arm platform molecule was made using a DNA synthesizer (Expedite 8909 DNA synthesizer) starting with compound (I) which is covalently bound to a solid support (e.g., glass beads). Compound (I) is commercially available from Glen Reearch. Compound (I) was coupled with a doubler compound (II) to generate compound III. Coupling with 5 '-amino modifier (compound IV) was used to generate compound V. The Tri Arm platform molecule was then cleaved from the solid support using excess ammonia to generate Compound (VI). In this manner, a symmetrical Tri Arm platform molecule was made in two steps and can be used without any substantial purification. This Tri Arm platform molecule is exemplary of, inter alia, formula (16).

Example 2 Conjugation Using Tri Arm Symmetrical Platform Molecule [00612] As indicated in Fig. 2, Compound (VII) is made separately using a uridine residue support then treating the highly pure oligonucleotide with NaIO₄ to open one of the rings for reacting with the symmetrical Tri Arm platform molecule of Example 1 (Compound VI). This generates a Tri Arm branched CIC with three branches comprising the same polynucleotide sequence 5'-XXXXXXX-3'. As described above, the polynucleotide sequence 5'-XXXXXXX-3' can be any 7-mer desired and can be selected from a readily available library of 7-mers. This Tri Arm branched CIC is exemplary of, inter alia, formula (18).

Example 3 Synthesis of a Tri Arm Platform Molecule with One Unique Branch [00613] As shown in Fig. 3, a unique arm is first synthesized from Compound (IX) (commercially available from Glen Research) on a DNA synthesizer to generate Compound (X) which is then coupled with a doubler, Compound (II) to generate Compound (XI). Compound (XI) is then coupled with a 5 '-amino modifider Compound (IV) to generate Compound (XII). The Tri Arm platform molecule with a unique branch was then cleaved from the solid support using excess ammonia to generate Compound (XIII). Various unique arms, similar to Compound (X), with different polynucleotide sequences for 5'-XXXXXXX- 3' are made on a DNA synthesizer and kept as a library for easy access to generate a multitude of Tri Arm Platform Molecules, each of which has a unique branch of polynucleotides. Compound (XXIII) is exemplary of, inter alia, formulae (19) and (22). Example 4 Conjugation of a Tri Arm Platform Molecule with One Unique Branch [00614] As shown in Fig. 4, the Tri Arm platform molecule with a unique branch Compound (XIII) is used for conjugation. Compound (XIII) is reacted with Compound

(XIV) with a different polynucleotide sequence 5'-YYYYYYY-3' that has been treated to make it reactive (e.g., treating with NaIO₄) to form Compound (XV). Compound (XV) is a Tri Arm molecule that contains branches comprising two different 7-mer polynucleotide sequences, indicated in the figure as 5'-XXXXXXX-3' and 5'-YYYYYYY-3'. Compound

(XV) is exemplary of, inter alia, formulae (20- A) and (71).

Example 5 Synthesis of a Tri Arm Platform Molecule with AU Unique Branches [00615] In this Example, a Tri Arm platform molecule is made that has one unique arm made on a DNA synthesizer and two other unique arms grafted onto the structure. As depicted in Fig. 5, starting material Compound (IX) is used for DNA synthesis of the first arm to make Compound (X). Compound (X) is reacted with an asymmetrical doubler Compound (XVI) to make Compound (XVII), which is then coupled with a 5 '-amino modifier Compound (IV) to form Compound (XVIII). Compound (XVIII) is then coupled with a 5'-thio modifier to form Compound (XIX) that has two reactive groups at the end of each branch. Compound (XIX) is exemplary of, inter alia, formula (64).

Example 6 Conjugation Using Tri Arm Platform Molecule with AU Unique Branches The Tri Arm platform molecule with all unique branches is used to react with two entities, indicated in Fig. 6 as Compound (XIV) and Compound (XX) to produce Compound (XXI). The reaction is performed at the same time for simultaneous conjugation in some experiments. In other experiments, the reaction is performed by conjugating through the amine group first and the thiol group second. The reaction is optionally also performed at the same temperature and/or at the same pH. In some cases, the temperature is about from standard room temperature to about 37 degrees C. In other cases, the reaction is optionally performed at the same pH of about 4-10, preferably between pH 6-8. Compound (XXI) is exemplary of, inter alia, formula (64-C). Example 7 Synthesis of a Symmetrical Tetra Arm Platform Molecule [00616] As depicted in Fig. 7, the same starting material as used to make a symmetrical Tri Arm platform molecule, Compound (I), is used. Compound (I) is coupled with a trebler Compound (XXII) to generate Compound (XXIII). Compound (XXIII) in turn is coupled with a 5 '-amino modifier Compound (IV) to generate Compound (XXIV). The solid support is removed by using excess ammonia to make Compound (XXV). In this manner, a symmetrical Tetra Arm platform molecule is made in two steps and is able to be used without substantial purification. Compound (25) is exemplary of formula XXX. Compound (XXV) is exemplary of, inter alia, formula (28).

Example 8 Conjugation Using a Symmetrical Tetra Arm Platform Molecule [00617] A chimeric immunomodulatory molecule with four arms comprising the same polynucleotide sequence represented by 5'-XXXXXXX-3' is made as shown in Fig. 8. The symmetrical Tetra Arm platform molecule, as described in Example 7 and indicated in Fig. 8 as Compound (XXV) is combined with Compound (VII) to form Compound (XXVI). The resultant product has four arms that comprise the same polynucleotide sequence represented by 5'-XXXXXXX-3'. Compound (XXVI) is exemplary of, inter alia, formula (27).

Example 9 Synthesis of a Tetra Arm Platform Molecule with One Unique Branch [00618] As shown in Fig. 9, a unique arm is first synthesized starting with Compound (IX) and using a DNA synthesizer to generate Compound (X). Compound (X) is then coupled with a trebler (Compound XXII) to form Compound (XXVII), which is coupled with 5'-amino modifier Compound (IV) to form Compound (XXVIII). The solid support is cleaved off using excess ammonia to generate Compound (XXIX). Compound (XXIX) is exemplary of, inter alia, formula (66).

Example 10 Conjugation Using a Tetra Arm Platform Molecule with One Unique Branch [00619] As shown in Fig. 10, a CIC is made starting with the Tetra Arm platform molecule with one unique branch, Compound (XXIX), as described in Example 9 and Fig. 9. Compound (XXIX) is conjugated with to Compound (XIV) to form Compound (XXX) that has four arms comprising two different polynucleotide sequences, indicated by 5'- XXXXXXX-3' and 5'-YYYYYYY-3'. Compound (XXX) is exemplary of, inter alia, formula (65).

Example 11 Synthesis of a Tetra Arm Platform Molecule with Two Unique Branches [00620] As depicted in Fig. 11, one unique arm, Compound (X) is made on a DNA synthesizer starting from Compound (IX). Compound (X) is then coupled with an asymmetrical doubler, Compound (XVI) to form Compound (XVII). Compound (XVII) is then coupled with a doubler Compound (II) to form Compound (XXXI). Compound (XXXI) is then coupled with a 5'-amino modifier (Compound IV) to form Compound (XXXII), which is then treated with a mild base, subjected to one coupling with a 5 '-thiol modifier and treatment with ammonia to remove the solids support to generate a Tetra Arm platform molecule with two unique branches, Compound (XXXIII).

Example 12 Conjugation Using a Tetra Arm platform molecule with two unique branches [00621] As depicted in Fig. 12, a Tetra Arm platform molecule with two unique branches, Compound (XXXIII), was used and reacted with two entities, Compound (XIV) and Compound (XXXIV). In some experiments, the reaction is performed at the same time for simultaneous conjugation. In other experiments, the reaction is performed by conjugating through the amine group first and the thiol group second. The reaction is optionally also performed at the same temperature and/or at the same pH. In some cases, the temperature is about from standard room temperature to about 37 degrees C. In other cases, the reaction is optionally performed at the same pH of about 4-10, preferably between pH 6-8. The resultant product, Compound (XXXV) has two unique branches that where each unique branch comprises a different polynucleotide. As such, the CIC made using Compound (XXXV) comprises two polynucleotides of different sequences, indicated by 5' -XXXXXXX-3' and 5'-YYYYYYY-3'.

Example 13 Synthesis of a Tetra Arm Platform Molecule with AU Unique Branches [00622] As shown in Fig. 13, the first unique arm comprising a first polynucleotide sequence 5' -XXXXXXX-3' is synthesized from Compound (IX) using a DNA synthesizer to generate Compound (X). Compound (X) is then coupled with an asymmetrical doubler Compound (XVI) to form Compound (XVII). A second arm comprising a second polynucleotide sequence, 5'-YYYYYYY-3' is added to the -ODMT part of Compound (XVII) by using a DNA synthesizer. To form Compound (XXXVI). A cap is added to end of the polynucleotide sequence to prevent reactivity in the next step which involves another coupling with an asymmetrical doubler Compound (XVI) to form Compound (XXXVII). Compound (XXXVII) is coupled with 5 '-amino modifer Compound (IV) followed by coupling with a thiol modifier and treatment with ammonia to cleave away from the solid support. This results in Compound (XXXVIII).

Example 14 Conjugation Using a Tetra Arm Platform Molecule with AU Unique Branches [00623] As shown in Fig. 14, a Tetra Arm platform molecule with all unique branches, Compound (XXXVIII), was used and reacted with two entities, Compound (XXXIX) and Compound (XX). In some experiments, the reaction is performed at the same time for simultaneous conjugation. In other experiments, the reaction is performed by conjugating through the amine group first and the thiol group second. The reaction is optionally also performed at the same temperature and/or at the same pH. In some cases, the temperature is about from standard room temperature to about 37 degrees C. In other cases, the reaction is optionally performed at the same pH of about 4-10, preferably between pH 6-8. The resultant product, Compound (XXXX) has four unique branches that where each unique branch comprises a different polynucleotide. As such, the CIC made using Compound (XXXX) comprises four polynucleotides of different sequences, indicated by 5'-XXXXXXX-3'; 5'- YYYYYYY-3'; 5'-QQQQQQQ-3' and 5'-ZZZZZZZ-3'.

Example 15 Synthesis of a Click Platform Molecule with One Unique Branch [00624] As shown in Fig. 15, the first unique arm comprising a first polynucleotide sequence 5'-XXXXXXX-3' is synthesized from Compound (IX) using a DNA synthesizer to generate Compound (X). Compound (X) is then coupled with a doubler, Compound (II) to generate Compound (III). Compound (III) is coupled with a 5'-alkynyl modifier, Compound (XXXXI) to form Compound (XXXXII). Treatment with ammonia to remove the solid support yields a product of (XXXXIII). Example 16 Conjugation Using Click Platform Molecule

[00625] As shown in Fig. 16, Compound (XXXXIII) is reacted with Compound

(XXXXIV) and subjected to heat or copper to generate Compound (XXXXV).

Example 17 Synthesis of Click Hexavalent Platform Molecule

[00626] As shown in Fig. 17, Compound (IX) is coupled with a doubler Compound (XXXXVI) to form (XXXXVII) which is then coupled with a trebler, Compound (XXII). This generates Compound (XXXXVIII). Treatment of Compound (XXXXVIII) with 5'- alkynyl modifer Compound (XXXXI) yields Compound (IL).

Example 18 Conjugation Using Click Hexavalent Platform Molecule

[00627] As shown in Fig. 18, Compound (L) is treated with Compound (XXXXIV) and heat and/or copper to generate a hexavalent compound (LI).

Example 19: Synthesis of a Bis- Amino Tri Arm Platform Molecule with One Unique Branch Oligonucleotide (Formula (22)-Bis-Amino)

[00628] The following describes the synthesis of P-I, a tri arm platform as a fully modified phosphorothioate 2' -deoxyribonucleic acid with the structure: (5'-NH2-CH2CH2OCH2CH2-OPSO2-HEG-OPSO2-CH2)2-CH-OPSO2-HEG-OPSO2- TAACGTTCGT (SEQ ID NO:22) -3', where each nucleotide A, C, G, or T also is linked by a phosphorothioate group.

[00629] The platform was synthesized in the 3' to 5' direction by standard solid phase DNA methods on an AKTA DNA synthesizer using polystyrene containing the 3'-T nucleoside as the solid support (scale 450 umol). Protected nucleoside (DMT- A^Bz, DMT-C^Bz, DMT-G^lBu, DMT-T), spacer (e.g., DMT-hexaethylene glycol), brancher (bis-DMT-glycerol) and amino linker (e.g., MMT-amino-diethylene glycol) cyanoethylphosphoramidites and synthesis reagents were purchased from Pierce Milwaukee, ChemGenes, or Glen Research. Each protected nucleoside, spacer or amino linker phosphoramidite was added in successive order using the following synthesis cycle: 1) removal of the 5'-dimethoxytrityl protecting group with dichloroacetic acid in toluene, 2) coupling of the next protected phosphoramidite with tetrazole in acetonitrile, 3) sulfurization of the phosphite trimester intermediate with xanthane hydride in pyridine/acetonitrile, and 4) capping of any unreacted 5'-hydroxyl groups with acetic acid/l-methylimidazole/acetonitrile. The synthesis cycle was repeated until the 5'- protected platform was assembled. At the end of the chain assembly, the monomethyoxytrityl (MMT) group was removed from the amino linker with dichloroacetic acid in toluene.

[00630] The on- support protected platform was treated with te/t-butylamine in acetonitrile to remove the cyanoethyl phosphate protecting groups. Overnight treatment with concentrated aqueous ammonium hydroxide at 55 ⁰C removed base protecting groups and the crude product from the solid support. The isolated product was concentrated in vacuo and purified by preparative anion exchange HPLC. The preparative anion exchange HPLC system used a 60 mL column filled with IEX Q-Sepharose Fast Flow media (GE Healthcare) with elution using an increasing sodium chloride salt gradient in aqueous buffer at pH 12. The fractions containing the product were concentrated and desalted on a 50 mL Cl 8 column using water followed by an acetonitrile/water step gradient for elution. The product was isolated by concentration in vacuo to remove the acetonitrile, followed by lyophilization to a powder.

[00631] Table 8 shows the tri arm platforms prepared using the procedure outlined above, the synthesis scale, the amount isolated and molecular weight found, compared to the theoretical value. Synthesis scales varied for these compounds, but the procedure is representative for all. The molecular weight (MW) of the compounds was determined by electrospray mass spectrometry and compared to the theoretical value to confirm incorporation of the nucleotides, spacers and amino linker.

Table 8: Bis-Amino Tri Arm Platform Synthesis Summary Results

[00632] Additional tri ami platforms were manufactured using similar procedures by TriLink Biotechnologies.

Example 20: Synthesis of Branch Oligonucleotide Molecule with Disulfide Group (Formula (17)-Disurfide)

[00633] The following describes the synthesis of B-I, a branch oligonucleotide as a fully modified phosphorothioate 2' -deoxyribonucleic acid with the structure: 5'-TCGTCGACTT (SEQ ID NO:1) -OPSO₂-(CH₂)₆-S-S-(CH2)₆-OH-3\ where each nucleotide A, C, G, or T also is linked by a phosphorothioate group.

[00634] The branch oligonucleotide was synthesized in the 3' to 5' direction by standard solid phase DNA methods on an AKTA DNA synthesizer using DMT-O-(CH₂)₆-S-S-(CH2)₆- O- Controlled Pore Glass (CPG) as the solid support (scale 140 umol). The CPG support was purchased from ChemGenes. The protected nucleoside cyanoethylphosphoramidites (DMT- A^Bz, DMT-C^Bz, DMT-G^lBu, DMT-T), spacer cyanoethylphosphoramidites (e.g., DMT- hexaethylene glycol), if required, and synthesis reagents were purchased from Pierce Milwaukee, ChemGenes, or Glen Research. Each protected nucleoside phosphoramidite and spacer phosphoramidite, if required, was added in successive order using the following synthesis cycle: 1) removal of the 5'-dimethoxytrityl protecting group with dichloroacetic acid in toluene, 2) coupling of the next protected phosphoramidite with tetrazole in acetonitrile, 3) sulfurization of the phosphite triester intermediate with xanthane hydride in pyridine/acetonitrile, and 4) capping of any unreacted 5'-hydroxyl groups with acetic acid/1- methylimidazole/tetrahydrofuran. The synthesis cycle was repeated until the 5'- protected branch oligonucleotide was assembled. At the end of the chain assembly, the dimethyoxytrityl (DMT) group was removed from the amino linker with dichloroacetic acid in toluene.

[00635] The on- support protected branch oligonucleotide was treated with tert- butylamine in acetonitrile to remove the cyanoethyl phosphate protecting groups. Overnight treatment with concentrated aqueous ammonium hydroxide at 55 ⁰C removed base protecting groups and the crude product from the solid support. The isolated product was concentrated in vacuo and purified by preparative anion exchange HPLC. The preparative anion exchange HPLC system used a 60 mL column filled with IEX Q-Sepharose Fast Flow media (GE Healthcare) with elution using an increasing sodium chloride salt gradient in aqueous buffer at pH 12. The fractions containing the product were concentrated and desalted on a 50 rnL C18 column using water followed by an acetonitrile/water step gradient for elution. The product was isolated by concentration in vacuo to remove the acetonitrile, followed by lyophilization to a powder.

[00636] Table 9 shows the branch oligonucleotides prepared, the synthesis scale, the amount isolated and molecular weight found compared to the theoretical value. Synthesis scales varied for these compounds, but the procedure is representative for all. The molecular weight (MW) of the compounds was determined by electrospray mass spectrometry and compared to the theoretical value to confirm incorporation of the nucleotides, spacers and disulfide linker.

Table 9: Disulfide Branch Oligonucleotide Synthesis Summary Results

[00637] Additional branch oligonucleotides were manufactured using similar procedures by TriLink Biotechnologies.

Example 21: Reduction of a Branch Oligonucleotide Molecule with a Disulfide Group to a

Thiol Group (Formula (17VThJoI)

[00638] B-8, 5'-TCGTTCG (SEQ ID NO:6) -OPSO₂-(CH₂)6-S-S-(CH2)6-OH-3' (150 mg,

55.9 umol) was reduced with 1.8 mL of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-

HCl) (88 mg, 307 umol, 5.5 equivalents) in 0.1 M sodium acetate buffer (7.4 mL) adjusted to pH 5.3, at 4O⁰C for 2 hours. The reaction was monitored by RP-HPLC on a PLRP-S column

(0.46 x 25 cm, 5 um) using an elution method with an increasing gradient of acetonitrile in

0.1 M triethylammonium acetate/pH 7 buffer and detection at 260 nm.

[00639] The reduced branch oligonucleotide, B-17, was desalted on a C18 column (30 mL) using water followed by a 40% acetonitrile/water step gradient for elution. The product was concentrated in vacuo to remove the acetonitrile and either used immediately or stored frozen until use.

[00640] Table 10 shows the thiol branch oligonucleotides prepared, and the amounts isolated. Reduction scales varied for these compounds, but the procedure is representative for all. Mass spectrometry was not performed on these compounds due to their relative instability to the mass spectrometry conditions.

Table 10: Thiol Branch Oligonucleotide Synthesis Summary Results

Example 22: Activation of a Bis-Amino Tri Arm Platform Molecule Containing One Unique Branch Oligonucleotide with Chloroacetic Anhydride (Formula (22)-Bis-Chloroacetyl) [00641] P-S₅ (S^NH₂-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)_I-CH-OPSO₂-HEG- OPSO₂-AACGTTC (SEQ ID NO:23)-3' (72 mg, 17.7 umol) was dissolved in 0.1 M sodium phosphate/pH 7.0 buffer (4.9 mL) and triethylamine (49 uL, 353 umol, 20 equivalents) was added. A 0.4 M solution of chloroacetic anhydride was prepared in acetonitrile and 442 uL (177 umol, 20 equivalents) was added to the platform solution at ambient temperature, while mixing continuously for 5 min. The reaction was stored at 4⁰C while it was checked for completion by RP-HPLC on a PLRP-S column (0.46 x 25 cm, 5 um) using an elution method with an increasing gradient of acetonitrile in 0.1 M triethylammonium acetate/pH 7 buffer and detection at 260 nm.

[00642] The activated platform oligonucleotide, P-7, was desalted on a C18 column (30 mL) using water followed by an acetonitrile/water step gradient for elution. The product was concentrated in vacuo to remove the acetonitrile and either used immediately or stored frozen until use. [00643] Table 11 shows the activated platform oligonucleotides prepared, and the amount isolated. Activation scales varied for these compounds, but the procedure is representative for all. Mass spectrometry was not performed on these compounds due to their relative instability to the mass spectrometry conditions.

Table 11 : Bis-Chloroacetyl Tri Arm Platform Synthesis Summary Results

Example 23: Conjugation of Thiol Branch Oligonucleotide (4 equivalents) with Bis- Chloroacetyl Platform (Formula (21)-Thioether) - Preparation of D-12 by Conjugation of Activated P-7 and B- 17

[00644] P-7 (50 mg, 11.8 umol, 1 equivalent) and B-17 (120.1 mg, 47.1 umol, 4 equivalents) were dissolved in 5.0 mL of water and 2.7 mL of 1 M sodium borate / 10 mM EDTA / pH 10 buffer was added. The reaction was degassed for 5min in vacuo and then frozen at -25⁰C overnight.

[00645] The reaction was monitored by anion exchange HPLC on a Dionex DNA-Pac column (4 x 250 mm) using elution with an increasing sodium chloride gradient in 10 mM sodium perchlorate/pH 10/20% acetonitrile buffer at 6O⁰C and detection at 260 nm. The resulting D-12 conjugation anion exchange chromatogram is shown in Fig. 21. [00646] As some of the excess B-17 thiol oxidizes to the disulfide and elutes near the product peak, it is convenient to reduce the disulfide back to the thiol form prior to purification. Therefore, a 48 mg/mL solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) was prepared in 0.2 M sodium phosphate/pH 7.5 buffer and 2.81 mL (135 mg, 471 umol) was added to the reaction mixture. The reaction was heated at 4O⁰C for 2 hours, or until the reduction was complete as monitored by anion exchange HPLC. The reduced conjugation anion exchange chromatogram for D-12 is shown in Fig. 22 and demonstrates the excellent resolution of the D- 12 conjugate product from the reaction byproducts and excess reagents.

[00647] The D- 12 conjugate was purified on an AKTA purifier by anion exchange chromatography using an increasing gradient of sodium chloride in 20% ACN/ water pH 7.0, with detection at 260 nm. The appropriate fractions were combined and desalted on a 30 mL C18 column using water followed by an acetonitrile/water step gradient for elution. The product was isolated by concentration in vacuo to remove the acetonitrile, followed by lyophilization to a powder.

Example 24: Conjugation of Thiol Branch Oligonucleotide (6 equivalents) with Bis- Chloroacetyl Platform (Formula (21)-Thioether Linkage) - Preparation of D-I by Conjugation of P-5 and B-IO

[00648] P-5 (5.9 mg, 1.13 umol, 1 equivalent) and B- 10 (24.1 mg, 6.76 umol, 6 equivalents) were dissolved in 2.6 mL of water and 0.87 mL of 1 M sodium borate / 10 mM EDTA / pH 10 buffer was added. The reaction was degassed for 5min in vacuo and then frozen at -25⁰C overnight.

[00649] The reaction was monitored by anion exchange HPLC on a Dionex DNA-Pac column (4x250 mm) using elution with an increasing sodium chloride gradient in 10 mM sodium perchlorate/pH 10/20% acetonitrile buffer at 6O⁰C and detection at 260 nm. The resulting D-I conjugation anion exchange chromatogram is shown in Fig. 23. [00650] As some of the excess B- 10 thiol oxidizes to the disulfide and elutes near the product peak, it is convenient to reduce the disulfide back to the thiol form prior to purification. Therefore, a 48 mg/mL solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) was prepared in 0.2 M sodium phosphate/pH 7.5 buffer and 0.45 mL (21.6 mg, 75 umol) was added to the reaction mixture. The reaction was heated at 4O⁰C for 2 hours, or until the reduction was complete as monitored by anion exchange HPLC. The reduced D-I conjugation chromatogram is shown in Fig. 24 and demonstrates the excellent resolution of the D-lconjugate product from the reaction byproducts and excess reagents. [00651] The D-lconjugate was purified on an AKTA purifier by anion exchange chromatography using an increasing gradient of sodium chloride (1.25 M in 20% ACN/ water pH 7.0),with detection at 260 nm. The appropriate fractions were combined and desalted on a 30 mL C18 column using water followed by an acetonitrile/water step gradient for elution. The product was isolated by concentration in vacuo to remove the acetonitrile, followed by lyophilization to a powder.

[00652] Table 12 shows the branched CIC conjugates prepared, the amount isolated and molecular weight found compared to the theoretical value. The reverse phase HPLC (RP- HPLC) purities are also reported in Table 5 and show purities ranging from 83% to 97%. Conjugation scales varied for these compounds, but the procedures described in Examples 5 and 6 are representative for all. Generally, 4 equivalents of branch was used for the conjugation reactions, rather than 6 equivalents.

Table 12: Branched CIC Conjugate Synthesis Summary Results

Example 25: Purity of Branched CICs

[00653] Fig. 19 shows an exemplary chromatogram for D-I synthesized using 6 equivalents of branch and purified as described in Example 24 (RP-HPLC purity 92%). Table 5 shows the RP-HPLC purities obtained compounds synthesized using 4 equivalents of branch and purified as described in Example 23, which ranged from 83% to 97%. For comparison, an exemplary reverse phase chromatogram of C- 1 synthesized on a 200 umol scale by a stepwise approach and ion exchange purification, described below in Example 26, is shown in Fig. 20 (purity 71%). These results demonstrate the significantly higher purity of branched CICs made by the present invention. C-I has the following structure: (5'- TCGTCGA (SEQ ID NO:5)-OPSO2-HEG-OPSO₂-CH₂)2-CH-OPSθ2-HEG-OPSθ2- TAACGTTCGT (SEQ ID Noillys'.

[00654] The RP-HPLC method used for these analyses was performed on a Waters X- Terra column (0.46 x 15 mm) using an increasing acetonitrile gradient in 1% hexafluoroisopropanol/0.2% triethylamine/water at 6O⁰C and detection at 260 nm. Example 26: Preparation and purity of a Branched CIC prepared by the stepwise approach

[00655] The branched CIC, C-I, was synthesized in the 3' to 5' direction by standard solid phase DNA methods on an AKTA DNA synthesizer using polystyrene containing the 3'-C nucleoside as the solid support (scale 200 umol). Protected nucleoside (DMT-ABz, DMT-CBz, DMT-GiBu, DMT-T), spacer (e.g., DMT-hexaethylene glycol) and branching group (e.g., glycerol) cyanoethylphosphoramidites and synthesis reagents were purchased from Pierce Milwaukee, ChemGenes, or Glen Research. Each protected nucleoside phosphoramidite, spacer or branching group was added in successive order using the following synthesis cycle: 1) removal of the 5'-dimethoxytrityl protecting group with dichloroacetic acid in toluene, 2) coupling of the next protected nucleoside phosphoramidite with tetrazole in acetonitrile, 3) sulfurization of the phosphite trimester intermediate with xanthane hydride in pyridine/acetonitrile, and 4) capping of any unreacted 5'-hydroxyl groups with acetic acid/1-methylimidazole/acetonitrile. The synthesis cycle was repeated until the 5'- protected branch oligonucleotide was assembled. After the addition of the branching group, double coupling was employed.

[00656] The on- support protected C-I was treated with tert-butylamine in acetonitrile to remove the cyanoethyl phosphate protecting groups. Twenty-four hour treatment with concentrated aqueous ammonium hydroxide at 55 ⁰C removed base protecting groups and the crude product from the solid support. The crude product was filtered and concentrated in vacuo and purified by preparative anion exchange HPLC. The preparative anion exchange HPLC system used a 60 mL column filled with IEX Q-Sepharose Fast Flow media (GE Healthcare) with elution using a three-part program: 1) a step gradient with sodium chloride in aqueous buffer at pH 12, 2) a step gradient with acetic acid to detritylate the compound, and 3) and increasing gradient 3M sodium chloride in aqueous buffer at pH 12. The fractions containing the product were concentrated and desalted on a 50 mL Cl 8 column using water followed by an acetonitrile/water step gradient for elution. The product was isolated by concentration in vacuo to remove the acetonitrile, followed by lyophilization to a powder. The isolated yield was 294 mg. [00657] Fig. 20 shows the reverse phase HPLC purity of C-lprepared by the stepwise method. The purity is only 71%, which is significantly lower than the purities obtained for the branched CICs prepared by the present invention.

Example 27: Synthesis of a Tris- Amino Tri Arm Platform Molecule (Formula (lβ)-Tris- Amino)

[00658] The following describes the synthesis of a tri arm platform as a fully modified phosphorothioate compound with the structure: ((5'-NH₂-CH₂CH₂OCH₂CH₂-OPSO₂-HEG- OPSO₂-T-OPSO₂-CH₂)₂-CH-OPSO₂-T-OPSO₂-HEG-OPSO₂-(CH₂)3-NH2-3' (P-9). [00659] The platform was synthesized in the 3' to 5' direction by standard solid phase DNA methods on an Perseptive Biosystems DNA synthesizer using phthalimido-(CH₂)3- Controlled Pore Glass containing (2 x 15 umol) purchased from Glen Research. DMT-T nucleoside, spacer (e.g., DMT-hexaethylene glycol), brancher (e.g., bis-DMT-glycerol) and amino linker (e.g., MMT-amino-diethylene glycol) cyanoethylphosphoramidites and synthesis reagents were purchased from Glen Research. Each protected nucleoside phosphoramidite, spacer, brancher or amino linker was added in successive order using the following synthesis cycle: 1) removal of the 5'-dimethoxytrityl protecting group with dichloroacetic acid in dichloromethane, 2) coupling of the next protected phosphoramidite with tetrazole in acetonitrile, 3) sulfurization of the phosphite triester intermediate with xanthane hydride in pyridine/acetonitrile, and 4) capping of any unreacted 5'-hydroxyl groups with acetic acid/1-methylimidazole/tetrahydrofuran. The synthesis cycle was repeated until the 5'- protected platform was assembled. At the end of the chain assembly, the monomethyoxytrityl (MMT) group was removed from the amino linker with dichloroacetic acid in dichloroacetic acid.

[00660] The on- support protected platform was treated with diethylamine in acetonitrile to remove the cyanoethyl phosphate protecting groups. Overnight treatment with concentrated aqueous ammonium hydroxide at 55 ⁰C removed protecting groups and the crude product from the solid support. The product was purified by RP-HPLC on a PLRP-S column (7.5 x 300 cm, 8 um) using an elution method with an increasing gradient of acetonitrile in 0.1 M triethylammonium acetate/pH 7 buffer and detection at 260 nm The product was isolated by concentration in vacuo, followed by lyophilization to a powder. Example 28: Activation of a Tris- Amino Tri Ann Platform Molecule (Formula (lβ)-Tris- Amino) with Chloroacetic Anhydride

[00661] P-9, ((5'-NH₂-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-T-OPSO₂-CH₂)₂-CH- OPSO₂-T-OPSO₂-HEG-OPSO₂-(CH₂)₃-NH₂-3', PM018, (0.53 mg, 0.2 umol) was dissolved in 0.05 M sodium phosphate/pH 7.0 buffer (0.25 mL) and triethylamine (0.56 uL, 20 equivalents) was added. A 0.4 M solution of chloroacetic anhydride was prepared in acetonitrile and 15 uL (6 umol, 30 equivalents) was added to the platform solution at ambient temperature, while mixing continuously for 5 min. The reaction was stored at 4⁰C while it was checked for completion by RP-HPLC on a PLRP-S column (0.46 x 25 cm, 5 um) using an elution method with an increasing gradient of acetonitrile in 0.1 M triethylammonium acetate/pH 7 buffer and detection at 260 nm.

[00662] The activated platform oligonucleotide, P-10, was desalted on a C18 column (2 mL) using water followed by a 40% acetonitrile/water step gradient for elution. The product was concentrated in vacuo to remove the acetonitrile and either used immediately or stored frozen until use.

Example 29: Human PBMC IFN-alpha Secretion

[00663] Peripheral blood was drawn from healthy volunteer subjects, and PBMC were isolated by Ficoll-Hypaque gradient centrifugation. PBMC were incubated at a concentration of 2.5 x 10e6 cells/mL in media (RPMI 1640, 10% fetal bovine serum, 50 U/ml Penicillin, 50 ug/ml Streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM Sodium Pyruvate )). Nucleic acids were tested at concentrations of 5.0000 uM, 2.5000 uM, 1.2500 uM, 0.6250 uM, 0.3125 uM, 0.1563 uM, 0.0781 uM, 0.0391 uM, 0.0195 uM, 0.0098 uM, 0.0049 uM and 0.0024 uM. All samples were tested in triplicate wells. After 20-24 hours, supernatant was collected and frozen for cytokine analysis. Supernatants were tested for IFN-alpha levels by capture ELISA. Data for PBMC IFN-alpha are shown in Fig. 25.

[00664] Fig. 25 shows data of IFN-alpha induction by human PBMC in the presence of immuno stimulatory nucleic acid sequences SEQ ID NO:25 (5'- TGACTGTGAACGTTCGAGATGA-3'; SEQ ID NO:25) and SEQ ID NO:26(5'- TCGTCGAACGTTCGAGATGAT-3'; SEQ ID NO:26) and branched CIC D-I, vertical axis in pg/mL of IFN-alpha, horizontal axis micromolar concentration of sequence. Solid diamond (♦ ) is data for D-I ((5'-TCGTCGACTT-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT - 3'). Solid box (!) is data for SEQ ID NO 25. Solid triangle (A) is data for SEQ ID NO 26 Mean and SEM are shown based on data from four donors. Medium control induced < 100 pg/ml IFN-alpha.

Example 30: Human PBMC IFN-alpha Secretion

[00665] Peripheral blood was drawn from healthy volunteer subjects, and PBMC were isolated by Ficoll-Hypaque gradient centrifugation. PBMC were incubated at a concentration of 2.5 x 10e6 cells/mL in media (RPMI 1640, 10% fetal bovine serum, 50 U/ml Penicillin, 50 Dg/ml Streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM Sodium Pyruvate )). Nucleic acids were tested at concentrations of 2.5000 uM, 1.2500 uM, 0.6250 uM, 0.3125 uM, 0.1563 uM, 0.0781 uM, 0.0391 uM, 0.0195 uM, 0.0098 uM, and 0.0049 uM. AU samples were tested in triplicate wells. After 20-24 hours, supernatant was collected and frozen for cytokine analysis. Supernatants were tested for IFN-alpha levels by capture ELISA. The EC50 was defined as the oligonucleotide concentration (μM) required to give a value equal to half the maximum pg/mL IFN-alpha level. This value was interpolated from individual donors using an Excel formula sheet. The IFN-alpha maximum values were determined from individual donors.

[00666] Fig. 26 shows data of IFN-alpha induction (EC50 and IFN-alpha maximum) by human PBMC in the presence of immuno stimulatory nucleic acid sequences SEQ ID NO 25 (5'-TGACTGTGAACGTTCGAGATGA-S'), SEQ ID NO 26 (5'-

TCGTCGAACGTTCGAGATGAT-3'), branched CIC D-I ((5 '-TCGTCGACTT-OPSO₂- (CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG- OPSO₂-TAACGTTCGT -3'), branched CIC D-2 ((5'-TCGTGATCGT-OPSO₂-(CH₂)6-S- CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- TAACGTTCGT -3'), branched CIC D-3 ((5'-TCGTCGA -OPSO₂-(CH₂)_O-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC -3'), branched CIC D-4 ((5'-TCGTCGA-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂- OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT -3'), branched CIC D-5 ((5'-TCGTCGACTT-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂- CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT -3'), branched CIC D-6 ((5'- TCGTCGACTT-OPSO₂-(CH₂)3-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂- CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT -3'), branched CIC D-7 ((5'- TCGTCGACTT-OPSO₂-HEG-OPSO₂-(CH₂)₃-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂- HEG-OPSO₂-CH₂)_I-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT -3'), branched CIC D-8 ((5'- TCGTCGA-OPSO₂-(CH2)6-S-CH2-C(O)-NH-CH2CH2OCH2CH2-OPSO₂-CH2)₂-CH-OPSO₂- HEG-OPSO₂-AACGTTC -3'), branched CIC D-9 ((5'-TCGTCGA-OPSO₂-(CH₂)₃-S-CH₂- C(O)-NH-CH2CH2OCH2CH2-OPSO2-HEG-OPSO2-CH2)2-CH-OPSO2-HEG-OPSO2- AACGTTC -3'), branched CIC D-IO ((5'-TCGTCGA-OPSO2-HEG-OPSO2-(CH2)3-S-CH2- C(O)-NH-CH2CH2OCH2CH2-OPSO2-HEG-OPSO2-CH2)2-CH-OPSO2-HEG-OPSO2- AACGTTC -3'), branched CIC D-I l ((5'-TCGTTCGAAT-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT - 3'), and branched CIC D-12 ((5'-TCGTTCG-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH2CH2OCH2CH2-OPSO2-HEG-OPSO2-CH2)2-CH-OPSO2-HEG-OPSO2-AACGTTC -3'). Mean and SEM are shown based on data from nine donors except SEQ ID NO 25 (six or nine donors).

Example 31 : Human B Cell Activation

[00667] Peripheral blood was drawn from healthy volunteer subjects, and PBMC were isolated by Ficoll-Hypaque gradient centrifugation. B cells were isolated by incubating PBMC with CD 19+ MACS beads (anti-CD 19 antibody conjugated to magnetic beads) followed by magnetic selection. Purified B cells were incubated at a concentration of 0.75 x 10e6 cells/mL in media (RPMI 1640, 10% fetal bovine serum, 50 U/ml Penicillin, 50 Dg/ml Streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM Sodium Pyruvate )). Nucleic acids were tested at concentrations of 5.5000 uM, 1.3750 uM, 0.3438 uM, 0.0859 uM, 0.0215 uM, 0.0054 uM. In a single donor, 0.0013 uM was included. All samples were tested in duplicate wells. After 87-91 hours, 150 uL of supernatant was collected and frozen. Medium containing thymidine methyl-3H was added back to each culture well. Incubation continued for an additional 8 hours, at which time the culture plates were frozen for proliferation analysis. Samples containing thymidine methyl-3H were harvested and read using a TopCount NXT microplate scintillation and luminescence counter. The EC50 was defined as the oligonucleotide concentration (μM) required to give a value equal to half the maximum proliferation (cpm) level. This value was interpolated from individual donors using an Excel formula sheet. The proliferation (cpm) maximum values were determined from individual donors.

[00668] Fig. 27 shows data of B cell proliferation (EC50 and proliferation maximum values) of immuno stimulatory nucleic acid sequences SEQ ID NO 25 (5'- TGACTGTGAACGTTCGAGATGA-S'), SEQ ID NO 26 (5'-

TCGTCGAACGTTCGAGATGAT-3'), branched CIC D-I ((5 '-TCGTCGACTT-OPSO₂- (CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG- OPSO₂-TAACGTTCGT -3'), branched CIC D-2 ((5'-TCGTGATCGT-OPSO₂-(CH₂)6-S- CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- TAACGTTCGT -3'), branched CIC D-3 ((5'-TCGTCGA -OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC -3'), branched CIC D-4 ((5'-TCGTCGA (SEQ ID NO:5)-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT - 3'), branched CIC D-5 ((5'-TCGTCGACTT-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT), branched CIC D-6 ((5'-TCGTCGACTT-OPSO₂-(CH₂)3-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG- OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCG -3'), branched CIC D-7 ((5'- TCGTCGACTT-OPSO₂-HEG-OPSO₂-(CH₂)3-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂- HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT -3'), branched CIC D-8 ((5'- TCGTCGA-OPSO₂-(CH₂)6-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-CH₂)₂-CH-OPSO₂- HEG-OPSO₂-AACGTTC -3'), branched CIC D-9 ((5'-TCGTCGA-OPSO₂-(CH₂)₃-S-CH₂- C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- AACGTTC -3'), branched CIC D-IO ((5'-TCGTCGA-OPSO₂-HEG-OPSO₂-(CH₂)₃-S-CH₂- C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- AACGTTC -3'), branched CIC D-I l ((5'-TCGTTCGAAT-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT - 3'), and branched CIC D-12 ((5'-TCGTTCG-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC -3'). Mean and SEM are shown based on data from six donors (SEQ ID NO 25, SEQ ID NO 26, D-2, D-3, D-8, D-IO), four donors (D-I), three donors (D-4, D-5, D-6, D-7), or two donors (D-9, D-I l, D-12).

Example 32: Human PBMC IFN-alpha Secretion

[00669] Peripheral blood was drawn from healthy volunteer subjects, and PBMC were isolated by Ficoll-Hypaque gradient centrifugation. PBMC were incubated at a concentration of 2.5 x 10e6 cells/mL in media (RPMI 1640, 10% fetal bovine serum, 50 U/ml Penicillin, 50 ug/ml Streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM Sodium Pyruvate)). Nucleic acids were tested at concentrations of 10.0000 μM, 5.0000 μM, 2.5000 μM, 1.2500 μM, 0.6250 μM, 0.3125 μM, 0.1563 μM, 0.0781 μM, 0.0391 μM, 0.0195 μM, 0.0098 μM, and 0.0049 μM. All samples were tested in triplicate wells. After 20-24 hours, supernatant was collected and frozen for cytokine analysis. Supernatants were tested for IFN-alpha levels by capture ELISA. The IFN-alpha maximum values were determined from individual donors.

[00670] Fig. 28 shows data of IFN-alpha induction by human PBMC in the presence of immuno stimulatory nucleic acid sequences branched CIC D-I ((5'-TCGTCGACTT-OPSO₂- (CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG- OPSO₂-TAACGTTCGT -3'), branched CIC C-I ((5'-TCGTCGA-OPSO₂-HEG-OPSO₂- CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC-3'), branched CIC C-2 ((5'-TCGTCGA-OPSO₂- HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TCGTCGA-3'), and branched CIC C-3 ((5¹- TCGTCGA-OPSO₂-(CH₂)3-OPSO₂-CH₂)₂-CH-OPSO₂-(CH₂)₃-OPSO₂-TCGTCGA-3'). Geomeans are shown based on data from nine or ten donors.

[00671] Fig. 29 shows data of IFN-alpha induction by human PBMC in the presence of immuno stimulatory nucleic acid sequences branched CIC D-7 ((5'-TCGTCGACTT-OPSO₂- HEG-OPSO₂-(CH₂)₃-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH- OPSO₂-HEG-OPSO₂-TAACGTTCGT -3'), branched CIC C-I ((5'-TCGTCGA-OPSO₂-HEG- OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC-3'), branched CIC C-2 ((5'-TCGTCGA- OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TCGTCGA-3'), and branched CIC C- 3 ((5'-TCGTCGA-OPSO₂-(CH₂)₃-OPSO₂-CH₂)₂-CH-OPSO₂-(CH₂)₃-OPSO₂-TCGTCGA-3'). Geomeans are shown based on data from nine or ten donors.

[00672] Fig. 30 shows data of IFN-alpha induction by human PBMC in the presence of immuno stimulatory nucleic acid sequences branched CIC D-3 ((5'-TCGTCGA -OPSO₂- (CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG- OPSO₂-AACGTTC -3'), branched CIC C-I ((5'-TCGTCGA-OPSO₂-HEG-OPSO₂-CH₂)₂- CH-OPSO₂-HEG-OPSO₂- AACGTTC-3'), branched CIC C-2 ((5'-TCGTCGA-OPSO₂-HEG- OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TCGTCGA-3'), and branched CIC C-3 ((5¹- TCGTCGA-OPSO₂-(CH₂)₃-OPSO₂-CH₂)₂-CH-OPSO₂-(CH₂)₃-OPSO₂-TCGTCGA-3'). Geomeans are shown based on data from nine or ten donors.

[00673] Fig. 31 shows data of IFN-alpha induction by human PBMC in the presence of immuno stimulatory nucleic acid sequences branched CIC D-IO ((5'-TCGTCGA-OPSO₂- HEG-OPSO₂-(CH₂)₃-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH- OPSO2-HEG-OPSO2-AACGTTC -3'), branched CIC C-I ((5'-TCGTCGA-OPSO₂-HEG- OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC-3'), branched CIC C-2 ((5'-TCGTCGA- OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TCGTCGA-3'), and branched CIC C- 3 ((5'-TCGTCGA-OPSO₂-(CH₂)3-OPSO₂-CH₂)₂-CH-OPSO₂-(CH₂)3-OPSO₂-TCGTCGA-3'). Geomeans are shown based on data from nine or ten donors.

[00674] Table 13 shows fold changes for IFN-alpha maximum. A geomean was calculated for all donors tested for a respective sequence, and fold changes determined by dividing geomeans. Data from nine to ten donors was available for analysis. Paired t test was used to determine statistical significance. * p < 0.05; ** p < 0.01; *** p < 0.001

Table 13: Fold change for IFN-alpha Maximum

[00675] Analysis of human PBMC IFN-alpha production demonstrated that the maximum levels of secreted IFN-alpha induced by the CICs D-I, D-7, D-3 and D-IO are, in general, statistically significantly improved compared to molecules C-I, C-2, and C-3 (Table 13). IFN-alpha production by CIC D-IO was maintained at high ISS concentrations. This extended dose curve may allow it to maintain a pharmacological effective IFN-alpha level over a broad CIC D-IO concentration range.

Example 33: Modulation of Maturation Markers in Enriched Human Plasmacytoid Dendritic Cells (PDC)

[00676] Peripheral blood was drawn from volunteer subjects, and PBMC were isolated by Ficoll-Hypaque gradient centrifugation. Plasmacytoid dendritic cells (PDC) were isolated by incubating PBMC with BDCA-4+ MACS beads (anti-BDCA-4 antibody conjugated to magnetic beads), and then separating through magnetic selection. Purity of BDCA-4 positive cells was greater than 90%(in three donors) or approximately 80% (in a single donor ). PDC were incubated at a concentration of 2.25 x 10e5 cells/mL in media (RPMI 1640, 10% fetal bovine serum, 50 U/ml Penicillin, 50 ug/ml Streptomycin, 2 mM L-glutamine, 10 mM

HEPES, 1 mM Sodium Pyruvate). Nucleic acids were tested at concentrations of 1.0000 μM, 0.2500 μM, 0.0625 μM, 0.0156 μM, 0.0039 μM, and 0.0010 μM (0.0010 μM concentration was tested for two out of four total donors). Samples were tested in single (for a single donor only) or duplicate wells. After 21-24 hours, cells were collected and stained with antibodies specific for cell maturation markers (CD80, CD86, and CD40). Flow cytometry analysis (FACS) was performed to determine expression levels of these markers.

[00677] Fig. 32 shows data for modulation of expression of maturation markers CD80, CD86, and CD40 by human PDC in the presence of immuno stimulatory nucleic acid sequences branched CIC D-I ((5'-TCGTCGACTT-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT - 3') (notated as open triangle, Δ), branched CIC D-3 ((5'-TCGTCGA -OPSO₂-(CH₂)₆-S-CH₂- C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- AACGTTC -3') (notated as open diamond, 0), branched CIC D-10 ((5'-TCGTCGA-OPSO₂- HEG-OPSO₂-(CH₂)₃-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH- OPSO₂-HEG-OPSO₂-AACGTTC -3') (notated as open square, D), and branched CIC C-I ((5'-TCGTCGA-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC-3') (notated as open circle, o). Mean and SEM are shown based on data from four donors (two donors for 0.0010 μM concentration). Analysis of enriched human PDC demonstrated that the CICs D-I, D-3, D-7, and D-IO of the present invention induce PDC maturation (Fig. 32). CIC D-I induced the most substantial PDC maturation

Example 34: Induction Of IL-6 From Mouse Splenocvtes

[00678] Spleens from BALB/c mice were harvested and splenocytes isolated. Splenocytes were incubated at a concentration of 3.5 x 10e6 cells/mL in media (RPMI- 1640, 10% Fetal Bovine Serum, 50 U/mL Penicillin, 50 mg/mL Streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM Sodium Pyruvate, 55 uM 2-Mercaptoethanol )). Nucleic acids were tested at concentrations of 22 uM, 5.5 uM, 1.375 uM, 0.3438 uM, 0.0856 uM, 0.0215 uM, 0.0053 uM, 0.0013 uM, and 0.0003 uM. All samples were tested in triplicate wells. After 20-24 hours, supernatant was collected and triplicates pooled before being frozen for cytokine analysis. Supernatants were tested in duplicate for IL-6 levels by capture ELISA.

[00679] Fig. 33 shows IL-6 induction in the presence of immuno stimulatory nucleic acid sequences: control ISS sequence SEQ ID NO 25 (5'-TGACTGTGAACGTTCGAGATGA- 3'), SEQ ID NO 26 (5'-TCGTCGAACGTTCGAGATGAT-S'), branched CIC D-I ((5'- TCGTCGACTT-OPSO₂-(CH₂)6-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂- CH₂)₂-CH-OPSO₂-HEG-OPSθ₂-TAACGTTCGT -3'), branched CIC D-2 ((5'- TCGTGATCGT-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂- CH₂)₂-CH-OPSO₂-HEG-OPSθ₂-TAACGTTCGT -3¹), branched CIC D-3 ((5'-TCGTCGA - OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH- OPSO₂-HEG-OPSO₂-AACGTTC -3'), branched CIC D-4 ((5'-TCGTCGA (SEQ ID NO:5)- OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH- OPSO₂-HEG-OPSO₂-TAACGTTCGT -3'), branched CIC D-5 ((5' -TCGTCGACTT-OPSO₂- (CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- TAACGTTCGT -3'), branched CIC D-6 ((5'-TCGTCGACTT-OPSO₂-(CH₂)3-S-CH₂-C(O)- NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- TAACGTTCG -3'),branched CIC D-7 ((5'-TCGTCGACTT-OPSO₂-HEG-OPSO₂-(CH₂)3-S- CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- TAACGTTCGT -3'), branched CIC D-8 ((5'-TCGTCGA-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- AACGTTC -3'), branched CIC D-9 ((5'-TCGTCGA-OPSO₂-(CH₂)3-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG- OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- AACGTTC -3¹), branched CIC D-IO ((5'- TCGTCGA-OPSO₂-HEG-OPSO₂-(CH₂)3-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG- OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC -3¹), branched CIC D-I l ((5'- TCGTTCGAAT-OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂- CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TAACGTTCGT -3'), and branched CIC D-12 ((5'- TCGTTCG-OPSO₂-(CH₂)6-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂- CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC -3'). Data from one experiment is shown. Analysis of mouse splenocyte demonstrated that CIC D-I, D-2, D-3, D-4, D-5, D-6, D-7, D- 8, D-9, D-IO, D-I l and D-12 of the current intervention induced IL-6.

Example 35: Human PBMC IL-IO Secretion

[00680] Peripheral blood was drawn from healthy volunteer subjects, and PBMC were isolated by Ficoll-Hypaque gradient centrifugation. PBMC were incubated at a concentration of 1.0 x 10e6 cells/mL in media (cRPMI 1640, 10% fetal bovine serum). Nucleic acids were tested at concentrations of 1.000 μM, 0.333 μM, 0.111 μM, 0.037 μM and 0.012 μM. AU samples were tested in duplicate wells. After 5 days, supernatant was collected and frozen for cytokine analysis. Supernatants were tested for IL-IO by capture ELISA. [00681] Fig. 34 shows data of IL-IO induction by human PBMC in the presence of immunostimulatory nucleic acid sequences branched CIC D-I ((5'-TCGTCGACTT-OPSO₂- (CH₂)₆-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG- OPSO₂-TAACGTTCGT -3'), D-3 ((5'-TCGTCGA -OPSO₂-(CH₂)₆-S-CH₂-C(O)-NH- CH₂CH₂OCH₂CH₂-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC -3¹), D-7 ((5'-TCGTCGACTT-OPSO₂-HEG-OPSO₂-(CH₂)₃-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂- OPSOrHEG-OPSOrCH^-CH-OPSOrHEG-OPSOrTAACGTTCGT -3¹), D-IO ((5'- TCGTCGA-OPSO₂-HEG-OPSO₂-(CH₂)3-S-CH₂-C(O)-NH-CH₂CH₂OCH₂CH₂-OPSO₂-HEG- OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂- AACGTTC -3¹), branched CIC C-I ((5¹- TCGTCGA-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-AACGTTC-3¹), branched CIC C-2 ((5'-TCGTCGA-OPSO₂-HEG-OPSO₂-CH₂)₂-CH-OPSO₂-HEG-OPSO₂-TCGTCGA- 3'), and branched CIC C-3 ((5'-TCGTCGA-OPSO₂-(CH₂)₃-OPSO₂-CH₂)₂-CH-OPSO₂- (CH₂)₃-OPSO₂-TCGTCGA-3'). Data is representative of 2 donors and shows mean ± range.

Example 36: Exemplary IRS Assays

[00682] Immunoregulatory compounds, such as CIRCs of the present invention that contain at least one immunoregulatory sequence, may be assayed for immunoregulatory (IR) activity. Examples of such assays are described in, for example, PCT International Publication Nos. WO 2006/028742, WO 2006/066003 and WO 2009/055076. [00683] In an exemplary assay, spleens from 6-12 week-old BALB/c mice spleen are harvested and mechanically dispersed by forcing the digested fragments through metal screens. The dispersed splenocytes are pelleted by centrifugation, then resuspended in fresh medium (RPMI 1640 with 10% fetal calf serum, plus 50 units/mL penicillin, 50 μg/mL streptomycin, 2 mM glutamine, and 0.05 mM β-mercaptoethanol). In a dose-dependent manner, the cells are then stimulated with an immunostimulatory compound, such as 0.7-1 mM of 1018 ISS (TLR9 ligand; 5'-TGACTGTGAACGTTCGAGATGA-S') or 1 μM of R848 (TLR7 ligand; a small molecule, an imidazoquinoline also called resiquimod) either alone or in the presence of the tested CIRCs. At 48 hours, supernatants are collected and cytokine levels, IL-6, are measured using immunoassays.

In some embodiments, a CIRC of the present invention inhibits a TLR7-dependent IL-6 production. In some embodiments, a CIRC of the present invention inhibits TLR9 -dependent IL-6 production. In some embodiments, a CIRC of the present invention inhibits TLR9- and TLR7- dependent IL-6 production. [00684] In another exemplary assay to determine the effect of CIRC on TLR7 and TLR9 activation, CIRCs can be assayed for immunoregulatory (IR) activity of innate immune responses on human plasmacytoid dendritic cells (PDCs). For example, human PDCs infected with herpes simplex virus type 1 (HSV- 1 KOS strain) respond by producing IFN- alpha and this response is dependent on TLR-9 signaling. Human PDCs infected with influenza virus (HlNl, A/PR/8/34 from a patient in Puerto Rico 1934. See ATCC catalog VR-95) also respond by producing IFN- alpha, however, this response is dependent on TLR- 7 signaling and independent of TLR-9. The effect of IRPs on innate immune response cytokine production by infected cells can be examined. In a dose-dependent manner, the cells are stimulated with HSV-I (5 or 2 MOI) or Influenza (2 MOI), either alone or in the presence of the tested IRPs. At 24 hours, supernatants are collected and cytokine levels, IFN-alpha, are measured using immunoassay. In some embodiments, a CIRC of the present invention inhibits a TLR7-dependent IFN-alpha production. In some embodiments, a CIRC of the present invention inhibits TLR9-dependent IFN-alpha production. In some embodiments, a CIRC of the present invention inhibits TLR9- and TLR7- dependent IFN-alpha production. [00685] In another exemplary assay to determine the effect of CIRC on TLR7 and TLR9 activation, CIRCs can be assayed for immunoregulatory (IR) activity of innate immune responses on human B-cells. For example, in a human B-cell assay, B-cells are purified from total blood cells obtained from healthy donors using magnetic beads (CD19 positive). Cells are resuspended in fresh medium (RPMI 1640 with 10% fetal calf serum, 50 units/mL penicillin, 50 μg/mL streptomycin, and 2 mM glutamine). In a dose-dependent manner, the cells are then stimulated with 0.7-1 μM of 1018 ISS (TLR9 ligand; 5'-

TGACTGTGAACGTTCGAGA TGA-3' (SEQ ID NO: 122)) or 1 μM of R848 (TLR7 ligand; a small molecule, an imidazoquinoline also called resiquimod), either alone or in the presence of the tested IRPs. At 48 hours, supernatants are collected and cytokine levels, IL-6, were measured using immunoassay. In some embodiments, a CIRC of the present invention inhibits a TLR7-dependent IL-6 production by human B-cells. In some embodiments, a CIRC of the present invention inhibits TLR9-dependent IL-6 production by human B-cells. In some embodiments, a CIRC of the present invention inhibits TLR9- and TLR7-dependent IL-6 production by human B-cells.

CIRCs in Combination with Corticosteroids to Treat Autoimmune Diseases

[00686] Corticosteroids are the most widely used anti-inflammatory drugs in lupus. A

SLE patient with a severe disease flare can receive up to lg/day of corticosteroids for about a week. Patients with "milder flares" are treated with about 0.5-lmg/kg/day of corticosteroids while patients with chronic mild, non- organ-threatening disease are treated with about 10-40 mg/day of corticosteroids for long periods of time. The side effects associated with corticosteroid administration depend on the dose and length of treatment. [00687] Some studies in both adult and pediatric SLE patients showed 70% decrease in circulating plasmacytoid dendritic cells (PDCs) in response to high dosages of corticosteroids. PBMC from healthy donors treated for 3 days with low dose of corticosteroids (30 mg/day) are unresponsive to TLR9 stimulation (HSV) and PDCs are reduced. However, corticosteroid treatment represses the IFN-signature in lupus patients. TLR7 and TLR9 by activating PDC may reduce the potency of corticosteroid to repress the IFN-signature. PDC activated by TLR7/9 are more resistant to corticosteroid-induced apoptosis. Blocking TLR7 and/or TLR9 could thus facilitate the corticosteroid effect on PDC resulting in increased PDC depletion and reduction of the IFN-signature [00688] The present invention includes methods and assays using said methods for administering a CIRC of the present invention to increase the killing of PDC by corticosteroids via the inhibition of TLR signaling and thereby reduce doses of corticosteroid required in the treatment of lupus. In an exemplary assay, the dosage of corticosteroid required to induce apoptosis in TLR-activated PDC in the presence of a CIRC of the present invention was evaluated.

[00689] Purified PDC (50-100,000 cells) are cultured for 24-48H either alone or in the presence of TLR7 or TLR9 ligands as depicted either alone or in the presence of a CIRC. Hydroxycortisone is added at various concentrations and cell survival measured at the end of the culture. The number of viable cells is evaluated by flow cytometry by comparing to a fixed amount of microbeads that is added in equal amount in all samples prior to the measure on the flow cytometer.

[00690] The percentage PDC death in the presence of influenza virus or HSV is determined at varying concentrations of the corticosteroid, hydroxycortisone, was increased in the presence of a CIRC. In some embodiments, a CIRC of the present invention decreases the amount of corticosteroid required for TLR7-dependent death of PDCs. In some embodiments, a CIRC of the present invention decreases the amount of corticosteroid required for TLR9-dependent death of PDCs. In some embodiments, a CIRC of the present invention decreases the amount of corticosteroid required for TLR9- and TLR7- dependent death of PDCs. In some embodiments, a CIRC of the present invention increases the percentage of TLR7-dependent PDC death in the presence of corticosteroid. In some embodiments, a CIRC of the present invention increases the percentage of TLR9- dependent PDC death in the presence of corticosteroid. In some embodiments, a CIRC of the present invention increases the percentage of TLR9- and TLR7-dependent PDC death in the presence of corticosteroid.

[00691] In another exemplarly assay, the effect of CIRCs on the inhibition of NF-kB transcriptional activity in human PDC stimulated with CpG-C can be determined. For example, 1x10⁶ PDC are stimulated for three hours in the presence of glucocorticoid, hydroxycortisone, and an immuno stimulatory sequence CpG-C ISS C274 ("ISS"; 5'-TCG TCG AAC GTT CGA GAT GAT-3' (SEQ ID NO:99)), CpG-C (1 μM) plus hydroxycortisone (IxIO^"6 M), CpG-C (1 μM) plus IRS (1 μM; SEQ ID NO: 123), CpG-C (1 μM) plus NF-kB inhibitor IKK (1 μM), or left untreated. Cells are then collected by centrifugation and nuclear extract is prepared using the nuclear extraction kit (Manufacture from Chemicon). Level of p65 transcriptional activity is evaluated using a sandwich based assay from Active motif that measure level of binding of the p65 NF-kB complex to a consensus DNA motif. In some embodiments, a CIRC of the present invention inhibits a TLR7- dependent NF-kB transcriptional activity in human PDCs. In some embodiments, a CIRC of the present invention inhibits TLR9-dependent NF-kB transcriptional activity in human PDCs. In some embodiments, a CIRC of the present invention inhibits TLR9- and TLR7- dependent NF-kB transcriptional activity in human PDCs.

CIRCs restoring in vivo PDC sensitivity to Glucocorticoid in mice treated with CpG- TLR9 ligand

[00692] In another exemplary assay, the effect of CIRCs of the present invention on restoring in vivo PDC sensitivity to glucocorticoid in mice treated with CpG- TLR9 ligand can be determined. In some embodiments, the mice are autoimmune prone (e.g., lupus-prone mice). For example, 129 mice are treated with the glucocorticoid Dexthametasone (DEX; 1000 μg) alone or DEX (1000 μg) plus a immuno stimulatory sequence CpG-C ISS C274 ("ISS"; 5'-TCG TCG AAC GTT CGA GAT GAT-3' (SEQ ID NO:99)) (50 μg) and DEX (1000 μg) plus CpG (50 μg) plus IRS (SEQ ID NO: 123) (100 μg). Viability of the different cell type is evaluated as described herein. Cells are identified to be PDC by the surface presence of B220, CDl Ic, and PDCAl marker. Myeloid dendritic cells are identified as to be CDl Ic positive and B220 negative. B-cells are identified as B220 positive cells and CDl Ic negative. Monocytes are identified as CDl Ib positive. PDC viability from the spleen or blood is evaluated under various conditions. Cell viability of cells from spleen tissue under various conditions is also evaluated. Similar results are obtained using blood.

CIRC can target skin inflammation in vivo.

[00693] PDC infiltrate the inflamed mouse skin. 129 mice are mechanical stripped 12 times with tape or left untreated as control. After 16 hr mice are euthanized and the skin was collected and digested for 1 hr with a solution containing Liberase 0.28 u/ml and the cellular content was analyzed by flow cytometry. Cells infiltrating inflamed skin show an IFN-alpha inflammatory gene signature which is preventable by treatment with IRS. 76. Mice are stripped only or stripped and treated with IRS (SEQ ID NO: 123) (100 μg) either administered s.c. or i.v. or locally on the inflamed skin. A group of mice is left completely untreated to serve as controls. Skin is processed and RNA is extracted from the infiltrating cells using an RNA kit from Quiagen according to manufacture instruction. Gene expression of specific genes was measured by Taqman.

[00694] Male mice of the C57BL/6 strain (15 mice/group) are injected intraperitoneally with 750 ug of acetaminophen (APA) either alone or in the presence of a single injection of 200 μg of the IRS of SEQ ID NO: 173 given s.c. Mice are surveyed overtime and percentage survival evaluated. The tape stripping model closely mimics aspects of human skin autoimmune disease including abundant infiltration of plasmacytoid dendritic cells and neutrophils, the upregulation of Type I IFN-a inducible genes, and inflammatory cytokines such as TNF-a, IL1A/B, and IP-10.

[00695] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, descriptions and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

[00696] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be so incorporated by reference.

Claims

What Is Claimed Is:

1. An immunomodulatory compound of Formula I:

wherein:

Ni is a first oligonucleotide comprising at least one immunomodulatory sequence and N₂ is a second oligonucleotide comprising at least one immunomodulatory sequence; Ri if present is poly₍₁.₁₂)ethyleneglycol-OPSO₂ or (CH₂)i-₈-OPSO₂; R₂ is poly(i_i₂)ethyleneglycol or (CH₂)i-s; R3 is poly(i πjethyleneglycol or (CH₂)I s;

R₄ if present is poly(i.i₂)ethyleneglycol-OPSθ2 or (CH₂)I-S-OPSO₂; and R₅ is poly₍i.₁₂₎ethyleneglycol-OPSO₂ or (CH₂)^₈-OPSO₂.

2. The immunomodulatory compound of claim 1 according to Formula II:

3^'

wherein:

HEG is hexaethyleneglycol;

Ri if present is hexaethylene glycol-OPSO₂;

R₂is (CH₂)₆ or (CHz)₃;

R₃ is CH₂CH₂OCH₂CH₂ or (CH₂)₆ or (CH₂)₃; and

R₄ if present is HEG-OPSO₂.

3. The immunomodulatory compound of claim 1 or claim 2, wherein: each Ni comprises one or more human motifs and each Ni may independently and optionally further comprise one or more rodent motifs; N₂ comprises one or more motifs each independently selected from the group consisting of: a human motif and a rodent motif; and wherein the immunomodulatory compound is an immunostimulatory compound.

4. The immunomodulatory compound of claim 3 according to Formula III:

5. The immunomodulatory compound of any one of claims 1-4, wherein: Ni is TCGT(N3)CG(N4)(N5); and

N₂ is (N6)(N7)ACGTTC(N8), wherein

N3 if present is GAT or T,

N4 if present is A or T,

N5 if present is CTT or GAT or AT,

N6 if present is T,

N7 is G or A, and

N8 if present is GT.

6. The immunomodulatory compound of any one of claims 1-5, wherein: Ni is selected from the group consisting of: 5'-TCGTCGACTT-3\

5'-TCGTCGAGAT-S' , 5'-TCGTGATCGT-S' , 5'- TCGTTCG-3', 5'- TCGTTCGAAT-3' and 5'-TCGTCGA-3'; and

N₂ is selected from the group consisting of: 5'-TAACGTTCGT-S', 5'-TGACGTTCGT-S' , 5'-AACGTTC-3' and 5'-GACGTTC-3'.

7. The immunomodulatory compound of claim 6, wherein the compound is selected from the group consisting of:

(5'-TCGTCGACTT-S'- OPSO₂- R_I - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂-CH- OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3', (5'- TCGTCGAGAT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂V CH-OPSO₂-HEG-OPSO₂- 5'-TGACGTTCGT -3',

(5'- TCGTGATCGT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3',

(5'- TCGTGATCGT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂- 5'-TGACGTTCGT -3',

(5'- TCGTCGA-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)^CH- OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3',

(5'- TCGTCGA-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂-CH- OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3'.

(5'- TCGTTCG-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂-CH- OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3' and

(5'- TCGTTCGAAT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3'; wherein

Ri is absent;

R₂ is (CH₂)₆ ,

R₃ is CH₂CH₂OCH₂CH₂, and and R₄ is HEG-OPSO₂.

8. The immunomodulatory compound of any one of claims 1-3 according to Formula

IV:

9. The immunomodulatory compound of claim 8, wherein: Ni is selected from the group consisting of: 5'-TCGTCGACTT-S' , 5'-TCGTCGAGAT-S' , 5'-TCGTGATCGT-S' , 5'- TCGTTCG-3', 5'- TCGTTCGAAT-3' and 5'-TCGTCGA-S'; and

10. The immunomodulatory compound of claim 9, wherein the compound is selected from the group consisting of:

(5'-TCGTCGACTT-S'- OPSO₂- R_I -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)^CH- OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3',

(5'- TCGTGATCGT-3' - OPSO₂- Ri -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂- CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3' and

(5'- TCGTCGA-3' - OPSO₂- Ri -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂-CH- OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3';

Ri is absent;

R₂ is (CH₂)₆ ;

R₃ is CH₂CH₂OCH₂CH₂; and R₄ is absent..

11. The immunomodulatory compound of any one of claims 1-3 according to Formula V:

-3¹

12. The immunomodulatory compound of claim 11, wherein:

Ni is selected from the group consisting of: 5'-TCGTCGACTT-S' ,

5'-TCGTCGAGAT-S' , 5'-TCGTGATCGT-S' , 5'- TCGTTCG-3', 5'- TCGTTCGAAT-3' and 5' -TCGTCGA-3'; and

N₂ is selected from the group consisting of: 5'-TAACGTTCGT-S',

5'-TGACGTTCGT-S' , 5'-AACGTTC-3' and 5'-GACGTTC-3'.

13. The immunomodulatory compound of claim 12, wherein the compound is selected from the group consisting of:

(5'-TCGTCGACTT-S'- OPSO₂- R_I - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂-CH- OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3' ,

(5'- TCGTGATCGT-3' - OPSO₂- Ri - R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CHl)₂- CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3', and

(5'-TCGTCGA-S'- OPSO₂- Ri-R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)^CH- OPSO₂-HEG-OPSO₂-5'-AACGTTC-3' ; wherein

Ri is absent;

R₂ is (CH₂)₃ ;

R₃ is CH₂CH₂OCH₂CH₂ ; and R₄ is HEG-OPSO₂.

14. The immunomodulatory compound of any one of claims 1-3 according to Formula VI:

15. The immunomodulatory compound of claim 14, wherein:

Ni is selected from the group consisting of: 5'-TCGTCGACTT-S' , 5'-TCGTCGAGAT-S' , 5'-TCGTGATCGT-S' , 5'- TCGTTCG-3', 5'- TCGTTCGAAT-3' and 5'-TCGTCGA-3'; and

16. The immunomodulatory compound of claim 15, wherein the compound is selected from the group consisting of: (5'-TCGTCGACTT-S'- OPSO₂- R_I -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)^CH- OPSO₂-HEG-OPSO₂-5'-TAACGTTCGT-3',

(5'- TCGTCGA-3' - OPSO₂- Ri -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CH₂)₂-CH- OPSO₂-HEG-OPSO₂- 5'-AACGTTC -3' and

(5'- TCGTGATCGT-3' - OPSO₂- Ri -R₂ -S-CH₂C(O)NH- R₃ -OPSO₂- R₄ -CHl)₂- CH-OPSO₂-HEG-OPSO₂- 5'-TAACGTTCGT -3'; wherein:

Ri is HEG-OPSO₂;

R₂ is (CH₂)₃ ;

R₃ is CH₂CH₂OCH₂CH₂ ; and R₄ is HEG-OPSO₂.

17. An immunomodulatory compound of Formula VII:

5'

OPSO₂ R₅ R₄ R₃-NHCOCH₂-S R₂ R₁-OPSO₂ — N₁ 5' wherein

Ni is an oligonucleotide comprising at least one immunomodulatory sequence; each Ri if present is independently OPSO₂-hexaethylene glycol, each R₂ is independently (CH₂)6 or (CH₂)₃, each R₃ if present is independently CH₂CH₂OCH₂CH₂ or (CH₂)₆ or (CH₂)₃, each R₄ if present is hexaethylene glycol-OPSO₂, and each R5 is independently 5'- ribo- or deoxyribonucleoside-3'-OPSO₂ or 3'- ribo-or deoxyribonucleoside-5'-OPSO₂.

18. The compound of claim 17, wherein: each Ni oligonucleotide comprises the same sequence,

Ri is absent,

R₂ is (CH₂)₆, each R₃ is independently CH₂CH₂OCH₂CH₂ or (CH₂)₃, R₄ is hexaethylene glycol-OPSO2, and and each R₅ is independently 5'-thymidine-3'-OPSθ₂ θr 3'-thymidine-5'- OPSO₂

19. The immunomodulatory compound of claim 17 or claim 18, wherein: each Ni independently comprises one or more human motifs and each Ni may independently and optionally further comprise one or more rodent motifs.

20. The immunomodulatory compound of any one of claims 17-19, wherein: each Ni is independently selected from the group consisting of: 5 ' -TCGTCG ACTT- 3', 5'-TCGTCGAGAT-S', 5'-TCGTGATCGT-S', 5'- TCGTTCG-3', 5'- TCGTTCGAAT- 3', 5'-TCGTCGA-S'; 5'-TAACGTTCGT-S' , 5'-TGACGTTCGT-S', 5'-AACGTTC-3', 5'- GACGTTC-3', 5'-TCGAACGTTT-3' and 5'-TCGGACGTTT-3'.

21. The immunomodulatory compound of claim 17 or claim 18, wherein: each Ni independently comprises one or more immunoregulatory sequences.

22. An immunomodulatory compound of Formula VIII:

wherein:

N_z is a linear oligonucleotide of z units, each N is an independently selected nucleotide; each N'_z' is a linear oligonucleotide of z' units, each N' is an independently selected nucleotide, each z and z' is independently an integer from 1 to 30;

Sp is selected from the group consisting of: a disulfide, a thioether, an amine, a phosphorothioate-ether and a phosphordithioate-ether;

BP is CR₇ or nitrogen; each Ro, R₂, R3, R₄, R5, RO, R7, R_x, Ry and R_z is independently a bond or a spacer moiety; each Y is independently S or O; each Z is independently S or O; and each m is independently 0, 1, 2 or 3.

23. The immunomodulatory compound of any one of claims 1-22, wherein the compound has sufficient hydrophilicity to promote availability of oligonucleotides to bind to a target site in an aqueous solution.

24. The immunomodulatory compound of any one of claims 1-23, wherein each oligonucleotide comprises phosphorothioate linkages.

25. The immunomodulatory compound of any one of claims 1-20 and 22-24, wherein: the immunomodulatory compound is an immuno stimulatory compound, the immuno stimulatory compound has sufficient hydrophilicity to promote immuno stimulatory activity in a mammal upon administration to said mammal, and the immuno stimulatory compound optionally stimulates the production of interferon alpha upon administration to said mammal.

26. The immunomodulatory compound of claim 1 or claim 2, wherein: each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences, and

N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences.

27. The immunomodulatory compound of claim 26, wherein at least one of the immunoregulatory sequences of Ni and N₂ is independently selected from the group consisting of: a TGC trinucleotide sequence at or near the 5' end of the oligonucleotide; a GGGG tetranucleotide;

XiGGGGX₂X₃ wherein X₁, X₂, and X₃ are nucleotides, provided that if Xi= C or A, then X₂X₃ is not AA; X1GGGGX2X3 wherein X₁, X₂, and X₃ are nucleotides, provided that if Xi= C or A, then X₂X₃ is not AA and wherein Xi is C or A;

GGN_nXiGGGGX₂X₃, wherein n is an integer from 1 to about 100 (preferably from 1 to about 20), each N is a nucleotide, and X₁, X₂, and X₃ are nucleotides, provided that if Xi= C or A, then X₂X₃ is not AA;

N₁TCCN_j(GG)IjNmXiGGGGX₂X₃, wherein each N is a nucleotide, wherein i is an integer from 1 to about 50, wherein j is an integer from 1 to about 50, k is 0 or 1, m is an integer from 1 to about 20, and X₁, X₂, and X₃ are nucleotides, provided that if Xi= C or A, then X₂X₃ is not AA; and

JGCN_z, wherein J is U or T, the sequence JGC comprises a modification, wherein each N is a nucleotide, and z is an integer from about 1 to about 1000, wherein the modification is selected from the group consisting of: a 2'-sugar modification, a 3'-terminal internucleotide phosphodiester linkage modification, and/or a 5'-methyl-cytosine modification.

28. The immunomodulatory compound of any one of claim 26 or claims 27, wherein each oligonucleotide comprises phosphorothioate linkages.

29. A composition comprising the immunomodulatory compound of any one of claims 1- 28, wherein the immunomodulatory compound is substantially pure.

30. A platform molecule of Formula IX:

wherein each R₃ if present is independently poly(i_i₂)ethyleneglycol-OPSO₂ or (CH₂)_1-8 - OPSO₂, each R₄ if present is poly(i_i2)ethyleneglycol-OPSO₂, each R₅ if present is independently 5'- ribo- or deoxyribonucleoside-3' or 3'- ribo- or deoxyribonucleoside-5' .

31. A method of inducing interferon- alpha in an individual, the method comprising: administering to the individual an effective amount of the immunomodulatory compound of any one of claims 1-20 and 22-25, or administering to the individual an effective amount of a composition comprising the substantially pure immunomodulatory compound of any one of claims 1-20 and 22-25; wherein the immunomodulatory compound is an immunostimulatory compound.

32. A method of treating one or more of an allergy, an allergy-induced asthma, atopic dermatitis, eosinophillic gastrointestinal inflammation, eosinophillic esophagitis and allergic bronchopulmonary aspergillosi in an individual in need of such treatment, the method comprising: administering to the individual an effective amount of the immunomodulatory compound of any one of claims 1-20 and 22-25, or administering to the individual an effective amount of a composition comprising the substantially pure immunomodulatory compound of any one of claims 1-20 and 22-25; wherein the immunomodulatory compound is an immunostimulatory compound.

33. A method of preparing an immunomodulatory compound of Formula I:

(D, the method comprising: activating a platform molecule of Formula X with ALG-C(O)-Rx-W

reacting the activated platform molecule with at least two equivalents of oligonucleotide XI:

5'^" N₁ OPSO₂-R₁ R₂ SH (χ[^ wherein: each Ni is a first oligonucleotide and N₂ is independently a second oligonucleotide; ALG is the leaving group of an activated carboxylic acid,; W is an electrophilic group;

W is a halide;

Ri if present is poly_(1-12)ethyleneglycol-OPSO₂ or (CH₂)i_₈-OPSO₂; R₂ is poly₍i_i₂₎ethyleneglycol or (CH₂)i_₈; R₃ is poly₍i_i₂₎ethyleneglycol or (CH₂)i_₈;

R₄ if present is poly₍i_i₂₎ethyleneglycol-OPSO₂ or (CH₂)i_₈-OPSO₂; R₅ is poly_(1-12)ethyleneglycol-OPSO₂ or (CH₂)i_₈-OPSO₂.

34. The method of claim 33, wherein each oligonucleotide comprises phosphorothioate linkages.

35. The method of claim 33 or claim 34, wherein each Ni is a first oligonucleotide comprising one or more human motifs and each Ni may independently and optionally further comprise one or more rodent motifs and N₂ is independently a second oligonucleotide comprising one or more motifs each independently selected from the group consisting of: a human motif and a rodent motif.

36. The method of claim 33 or claim 34, wherein each Ni is a first oligonucleotide comprising one or more immunoregulatory sequences, and N₂ is independently a second oligonucleotide comprising one or more immunoregulatory sequences.

37. An immunomodulatory compound of Formula I:

(D, wherein: each Ni is a first oligonucleotide and N₂ is independently a second oligonucleotide; Ri if present is poly₍i_i₂)ethyleneglycol-OPSO₂ or (CH₂)i_₈-OPSO₂;

R₂ is poly₍i_i₂₎ethyleneglycol or (CH₂)i_₈;

R₃ is poly₍i_i₂₎ethyleneglycol or (CH₂)i_₈;

R₄ if present is poly(i_i₂)ethyleneglycol-OPSO₂ or (CH₂)i_g-OPSO₂; and

R₅ is poly₍i_i₂₎ethyleneglycol-OPSO₂ or (CH₂)i_₈-OPSO₂.

38. A platform molecule of Formula X:

wherein:

N₂ is an oligonucleotide;

R3 is poly(i_i₂₎ethyleneglycol or (CH₂)i_₈;

R₄ if present is poly(i_i₂)ethyleneglycol-OPSO₂ or (CH₂)i_₈-OPSO₂; and

R₅ is poly₍i_i₂₎ethyleneglycol-OPSO₂ or (CH₂)i_₈-OPSO₂.