WO2016112963A1

WO2016112963A1 - Delivery of biomolecules into cells

Info

Publication number: WO2016112963A1
Application number: PCT/EP2015/050519
Authority: WO
Inventors: Jacques Rohayem
Original assignee: RiboxX GmbH
Current assignee: RiboxX GmbH
Priority date: 2015-01-13
Filing date: 2015-01-13
Publication date: 2016-07-21
Anticipated expiration: 2017-07-13

Abstract

The present invention generally relates to compositions or complexes, respectively, containing a biologically active substance or biomolecule and at least one nucleotide, oligonucleotide and polynucleotide forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions, compositions containing the same, methods for their production and their use as delivery reagents and/or transfection reagents in vitro and in vivo. The present invention also relates to specific embodiments of G-quadruplex forming oligonucleotides or polynucleotides, respectively, per se.

Description

Delivery of biomolecules into cells

The present invention generally relates to compositions or complexes, respectively, containing a biologically active substance or biomolecule and at least one nucleotide, oligonucleotide and polynucleotide forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions,

pharmaceutical compositions containing the same, methods for their production and their use as delivery reagents and/or transfection reagents in vitro and in vivo. The present invention also relates to specific embodiments of G-quadruplex forming oligonucleotides or polynucleotides, respectively, per se. Reagents for delivery of nucleic acids and other compounds into cells are known in the art as transfection agents and are based, e.g. on lipids (e.g. Lipofectamine^® and derivatives; see WO-A-00/27795), proteins (e.g. TurboFect™), other polymers and/or cell penetrating peptides (e.g. Xfect™; see WO-A-2014/10641 1 ) as well as saccharides (see, e.g. US-A- 2009/175905; for an overview of (poly)saccharides used for in vivo gene delivery, see Khan et al. (2012) Acta Biomaterialia 8, 4224-4232).

The technical problem underlying the present invention is to provide a novel system for delivery of biomolecules, in particular nucleic acid molecules peptides or proteins, into cells. The solution to the above technical problem is provided by the embodiments of the present invention as described herein and in the claims.

In particular, the present invention provides compositions or complexes, respectively, containing a biologically active substance or biomolecule, respectively, together with one or more nucleotide(s), oligonucleotide(s) and/or polynucleotide(s) forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures (i.e., in general, intermolecular or intramolecular complexes of or within one or more of such nucleotides, oligonucleotides or polynucleotides, typically based on interactions other than Watson-Crick base pairing) under appropriate conditions. The resent inventions also relates to the use or these compositions or complexes as well as the G-quadruplex forming entities as such for delivery of said biologically active substance or said biomolecule, respectively, in particular nucleic acids such as single or double stranded RNA and single or double stranded DNA, into cells in vitro or in vivo.

In the context of the present invention, the term "forming G-quadruplexes and/or

intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions" relates to assemblies of said nucleotides (preferably G, C, U, T, or A or analogues thereof), oligonucleotides or polynucleotides through inter and/or intramolecular interactions between and/or within said species under permissive conditions including buffer, salt components, temperature etc., more preferably under physiological conditions. The conditions required for the formation of said G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures are generally known in the art. Reference is given in this context, e.g. to Haider et al. "RNA Quadruplexes" in Sigel (ed.): Structural and catalytic roles of metal ions in RNA, RSC Publishing, Cambridge, 201 1 , pp. 125-139) and also to Neidle and Balasubramanian (eds.): Quadruplex Nucleic Acids, RSC Publishing, 2005.

The quadruplex forming entities of the invention typically represent complexes of the mentioned nucleotide, oligonucleotide or polynucleotide species with monovalent and/or divalent cations, in particular monovalent cations of alkaline metals such as K⁺ and Na⁺ and/or divalent cations of alkaline earth metals such as Ca²⁺ or Mg²⁺, and/or divalent cations of transition metals such as Mn². According to the present invention, the terms "biologically active substance" and

"biomolecule" are used synonymously and refer to any molecule present in biological or biochemical structures such as cells, tissues, organisms, viruses or viroids, which molecules include macromolecules such as proteins, polysaccharides, lipids and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites and other natural products. It is to be understood that the "biologically active substance" or "biomolecule" is not confined to naturally occurring molecules but also includes artificial substances not found in nature but being typically composed of naturally occurring building blocks and/or building blocks being chemically similar to the naturally occurring ones. According to the present invention, the biologically active substance or biomolecule is typically different from the G- quadruplex forming component of the inventive composition or complex, respectively. Various sequence species allow for the formation of such assemblies (see the above reference to Neidle and Balasubramanian (eds.): Quadruplex Nucleic Acids, RSC Publishing, 2005). Preferred embodiments of the invention and their use for delivery of biomolecules such as nucleic acids, peptides and proteins (as well as mixtures thereof) are compositions or complexes, respectively, containing oligonucleotides or polynucleotides that contain or have a sequence of the following formula (I): nGpXm (I) wherein X_n and X_m is any nucleotide sequence of length n or m, respectively, preferably being m = n, and G_p is any number of guanines involved in tedrad formation of length p.

Further preferred embodiments of species of the invention and their use for delivery of biomolecules into cells are oligonucleotides or polynucleotides, respectively, represented by sequences containing two guanine repeats separated by non-guanine nucleotides, for example oligonucleotides or polynucleotides containing or having a sequence of the following formula (II):

X_nGolXpG₀2Xm (II) wherein X_n and X_m is any non-guanine nucleotide sequence of length n or m, respectively, preferably being n=m, G₀i and G₀2 is any number of guanines involved in tetrad formation of length o1 and o2, respectively, preferably being o1 = o2, and X_p is any nucleotide sequence of length p involved in loop formation.

Further preferred embodiments of the invention, preferably useful as delivery agents for biomolecules into cells, are oligonucleotides or polynucleotides, respectively, represented by sequences forming intramolecular G-quadruplexes containing or having a sequence of the following formula (III):

X_nGolXpl Go2X 2Go3X 3G₀4Xm (HI) wherein X_n and X_m is any non-guanine nucleotide sequence of length n or m, respectively, preferably being n=m, G₀i , G₀2, G₀3 and G_o4 are independently any number of guanines involved in tetrad formation of length o1 , o2, o3 and o4, respectively, preferably being o1 = o2 = o3 = o4, and X_pi , X_p2 and X_p3 are independently any nucleotide sequence of length p1 , p2 and p3, respectively, involved in loop formation, preferably being p1 =p2=p3. According to the invention, not only species comprising four G-repeats (present in one molecule as is the case for above formula (III) or provided by more than one molecule) but any number of G-repeats can be included into a run of guanines and fold into an

intramolecular or intermolecular quadruplex.

Further embodiments of the invention are compositions or complexes, especially useful as delivery agents for biomolecules into cells, including species that form self-association of Watson-Crick duplex sequences, G:C tetrads formed, e.g. from (CCC)n:CCG repeats or other tetrad forming sequences such as the human quadruplex structures formed from sequences such as (TAGGGTTAGGGT)₂ (SEQ ID NO: 1 ) repeats and GAGCCAGT.

In general, the length of the oligonucleotides or polynucleotides as described herein is not critical. In some embodiments, nucleic acids, in particular ribonucleic acids, contained in the compositions and complexes as disclosed herein such as those of the above and the below formulas have a length of up to 1 1 nucleotides.

Further preferred embodiments of the compositions or complexes, respectively, according to the invention, in particular useful as reagents for delivery of biomolecules into cells, include poly and oligonucleotides, respectively, having the following formula (IV)

wherein n is an integer of at least 2 or at least 3, preferably 2 or 3 to 40, more preferably 2 or 3 to 20. In the following, molecules included in the complexes of the invention according to formula (I) will be referred to as "oligonucleotides", independent of their length.

The person skilled in the art understands that sequences of formulas (IV), (III) and (II) form sub-groups of the species according to formula (I).

According to preferred embodiments of the compositions or complexes, respectively, according to the invention, the above nucleotide, oligonucleotide or polynucleotide species contain a free 5'-triphosphate group. In this context, a "5'-triphosphated nucleotide, oligonucleotide or polynucleotide" as defined herein has a free triphosphate group bound to the 5' position of the (or at least one, in case of double stranded molecules) 5'-terminal ribose or deoxyribose, respectively. If embodiments of such species are initially produced, in particular through enzymatic synthesis using, e.g. viral nucleotidyltransferases such as RNA polymerases, typically as outlined below using RNA-dependent RNA-polymerases (RdRps) of caliciviruses, so that said species contain a free 5'-triphosphate group, it is desirable in certain embodiments of the invention to remove this 5'-triphosphate group. This can be done easily, if desired, especially for in vivo applications, e.g. by incubation of the resulting 5'-triphosphated molecule with a suitable phosphatase such as calf intestinal alkaline phosphatase (CIAP), (preferably recombinant) shrimp alkaline phosphatase (rSAP) or Antarctic phosphatase under appropriate buffer, salt and temperature conditions in a known fashion (e.g. for CIAP: 37°C for 30 to 60 min in 50mM Tris-HCI (pH 9.3 at 25°C), 1 mM MgCI₂, 0.1 mM ZnCI₂ and 1 mM spermidine).

It has been shown according to the invention that inventive G-quadruplex forming entities are very efficient delivery reagents for biomolecules, in particular nucleic acids, peptides and/or proteins, into cells, which need almost no incubation step with the cargo, i.e. the biomolecule, in particular nucleic acids such as RNA, to be delivered into the desired cell.

In preferred oligonucleotides of the invention, n in the above formula (IV) is 2, 3, 4, 5 or 6 to 14, particularly preferred 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or 14..Especially preferred oligonucleotides are those in which n in the above formula is 6 to 14, most preferred 6 to 1 1 .

As noted above, according to particularly preferred embodiments of the present invention, the nucleotides, poly or oligonucleotides as defined herein form G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures, typically in complex with mono and/or divalent cations under appropriate conditions, (including buffer, salt components, temperature; see, e.g. the review by Haider et al. "RNA Quadruplexes" in Sigel (ed.): Structural and catalytic roles of metal ions in RNA, Royal Society of Chemistry, Cambridge, 201 1 , pp. 125-139). Particularly preferred quadruplex-forming oligonucleotides and polynucleotides contained in the compositions or complexes, respectively, of the invention are single stranded and at least comprise ribonucleotides. Mixed RNA DNA species are contemplated as well, but especially preferred oligonucleotides are RNA oligonucleotides. The invention also relates to compositions or complexes, respectively, containing a biologically active substance or biomolecule and at least two different nucleotides, oligonucleotides or polynucleotides as defined above, more preferably of oligonucleotides according to above formula (I). Especially preferred compositions contain at least two different oligonucleotides of above formula (IV), most preferred selected from the group consisting of (G)₆ , (G)₇, (G)₈, (G)₉, (G)i₀ (SEQ ID NO: 2), (G)n (SEQ ID NO: 3), (G)_i2 (SEQ ID NO: 4), (G)i3 (SEQ ID NO: 5) and (G)_i4 (SEQ ID NO: 6). It is to be understood that the present invention is expressly directed to compositions with all possible combinations of at least two of the above oligonucleotides. More preferably, the oligonucleotides in such compositions are, or, in other embodiments, comprise (G)₈, (G)₉ and (G)i₀ (SEQ ID NO: 2). In other preferred embodiments, the compositions contain, as oligonucleotide species (or at least as the majority of oligonucleotide species) (G)₆, (G)₇, (G)₈, (G)₉, (G)i₀ (SEQ ID NO: 2), (G)ii (SEQ ID NO: 3), (G)_i2 (SEQ ID NO: 4), (G)_i3 (SEQ ID NO: 5) and (G)_i4 (SEQ ID NO: 6). In further preferred embodiments of this type, one ore more, preferably all of, the species (G)₆ to (G)i4 as outlined before contain a free 5'-triphosphate group. According to a further aspect, the present invention also relates to compositions of at least two different

nucleotides, oligonucleotides or polynucleotides as defined above per se.

According to certain embodiments of the invention the nucleotides, oligonucleotides or polynucleotides are preferably free of chemical modifications or other nucleotide analogues.

In further embodiments of the invention, however, the nucleotides, oligonucleotides or polynucleotides contained in the compositions or complexes, respectively, of the present invention can comprise at least one chemically modified and/or labeled nucleotide.

The chemical modification of the nucleotide analogue in comparison to the natural occurring nucleotide may be at the ribose, phosphate, and/or base moiety. With respect to molecules having an increased stability, especially with respect to RNA degrading enzymes, modifications at the backbone, i.e. the ribose and/or phosphate moieties, are especially preferred.

Preferred examples of ribose-modified ribonucleotides are analogues wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, or CN with R being CrC₆ alkyl, alkenyl or alkynyl and halo being F, CI, Br or I. It is clear for the person skilled in the art that the term "modified ribonucleotide" also includes 2'- deoxyderivatives, such as 2'-0-methyl derivatives, which may at several instances also be termed "deoxynucleotides".

As mentioned before, the at least one modified nucleotide may be selected from analogues having a chemical modification at the base moiety. Examples of such analogues include, but are not limited to, 5-aminoallyl-uridine, 6-aza-uridine, 8-aza-adenosine, 5-bromo-uridine, 7- deaza-adenine, 7-deaza-guanine, N⁶-methyl-adenine, 5-methyl-cytidine, pseudo-uridine, and 4-thio-uridine. Additionally, inosine can be used as base analogue in a ribonucleotide or deoxyribonucleotide contained in a composition or complex, respectively, according to the invention.

Examples of backbone-modified nucleotides, wherein the phosphoester group between adjacent ribonucleotides is modified, are phosphothioate groups. The "label" that may be included in the nucleotide analogue can be any chemical entity which enables the detection of the oligonucleotide in question via physical, chemical, and all biological means. Examples of typical labels linked to or integrated into one or more of the nucleotides or added to one end of the molecules as disclosed herein are radioactive labels, chromophores, and fluorophores (e. g. fluorescein, TAM etc.).

In general, the nucleotides, oligonucleotides and polynucleotides for use in the present invention can be prepared chemically using appropriate DNA or RNA, respectively, solid- phase chemistry well known in the art. Also, 5'-triphosphated nucleotides, oligonucleotides and polynucleotides for use in the invention may be prepared chemically (see, e.g., Zlatev et al. (2010) Org. Lett. 12 (10), pp. 2190-2193).

In other embodiments, oligonucleotides and polynucleotides contained in the compositions or complexes, respectively, of the present invention may be prepared enzymatically, or by a combination of chemical and enzymatic steps. Methods for the enzymatic preparation of the oligonucleotides are preferably those that use RNA-dependent RNA polymerases (RdRps) of caliciviruses as disclosed in WO-A-2007/012329. With respect to enzymatic synthesis of chemically modified RNAs using such RdRps the methods referred to in WO-A-2009/150156 are preferred. Enzymes of this type are also capable of using DNA templates; see WO-A- 2012/038448.

Thus, a further aspect of the present invention relates to a method for preparing the above compositions containing oligonucleotides or polynucleotides comprising the steps of (i) incubating a corresponding oligonucleotide or polynucleotide template with an RNA polymerase of a calicivirus, preferably in the presence of mono and/or divalent cations, typically cations of alkaline metals such as Na⁺ and/or K⁺, and/or of alkaline earth metals and/or of transition metals, preferably Mg²⁺ and/or Mn²⁺, and in the presence of appropriate rNTPs, in particular rGTP, rATP, rCTP, rUTP, and/or modified analogues thereof including, but not limited to, rITP; and (ii) contacting the thus-obtained reaction mixture with a biologically active substance or biomolecule, respectively. In case of the compounds according to above formula (IV) only GTP (and/or modified analogue thereof) is needed in step (i). Still a further aspect of the present invention relates to a corresponding method for preparing the above oligonucleotides or polynucleotides as such comprising the step of incubating a corresponding oligonucleotide or polynucleotide template with an RNA polymerase of a calicivirus, preferably in the presence of mono and/or divalent cations, typically cations of alkaline metals such as Na⁺ and/or K⁺, and/or of alkaline earth metals and/or of transition metals, more preferably Mg²⁺ and/or Mn²⁺, and in the presence of appropriate rNTPs, in particular rGTP, rATP, rCTP, rUTP, and/or modified analogues thereof including, but not limited to, rITP. In case of the compounds according to above formula (IV) only GTP (and/or modified analogue thereof) is needed.

For preparing preferred compositions of the inventive oligonucleotides as defined in formula (IV), as well as their complexes or compositions with biomolecules as outlined herein, in particular oligonucleotides selected from the group consisting of (G)_6, (G)₇, (G)₈, (G)₉, (G)i₀ (SEQ ID NO: 2), (G)n (SEQ ID NO: 3), (G)_i2 (SEQ ID NO: 4), (G)_i3 (SEQ ID NO: 5), and (G)i4 (SEQ ID NO: 6), more preferred a composition containing (as oligonucleotide species) (G)₈, (G)₉ and (G)₁₀ (SEQ ID NO: 2), or (G)₆ , (G)₇, (G)₈, (G)₉, (G)₁₀ (SEQ ID NO: 2), (G)n (SEQ ID NO: 3), (G)i₂ (SEQ ID NO: 4), (G)i₃ (SEQ ID NO: 5) and (G)i₄ (SEQ ID NO: 6), optionally having a free 5'-triphosphate group, it is preferred to incubate a (C) >₅ template, more preferably a rC5 or dC5, rC6 or dC6 template, with an RdRp of a calicivirus, typically in the presence of Mn²⁺ and/or Mg²⁺, and in the presence of rGTP (and/or chemically modified and/or labeled analogue(s) thereof) wherein the template may be RNA, DNA or mixed RNA/DNA.

The result is a mixture of the above preferred oligonucleotides and, depending on the amount of template used, the (C) >₅ template, optionally with minor contributions of the type (C)>₅(G)x (x being an integer of about 1 to 5), and complexes of the aforementioned oligonucleotides with mono and/or divalent cations. The length and mixture of

oligonucleotides prepared in this manner can be controlled by the reaction conditions and reagent concentrations used, in particular the reaction time. Optionally, complexes or compositions of the invention containing individual oligonucleotide species, as well as the individual oligonucleotides as such can also be isolated from these compositions by gel electrophoresis or chromatography, especially HPLC, in a known manner. Without being bound to a specific theory, the mechanism underlying this preferred manufacturing method for certain oligonucleotide compositions as disclosed herein is that the calicivirus RNA polymerase transcribes the (C)>₅ template into a corresponding homopolymeric polyG and then adds one or several G residues to the 3' end of template and/or the homopolymeric polyG though its terminal transferase activity (see WO-A-2007/012329; WO-A- 2012/038448). Since, according to preferred embodiments of the invention, the template is rather short, the melting temperature of the oligo(G:C) product of the transcription process is rather low so that the strands separate easily and a new round of transcription of the template can start. The co-production of by-products such as, e.g. (C)>₅(G)_X can be avoided by adjusting, preferably lowering, the concentration of the template. The oligonucleotides/polynucleotides, complexes and compositions thereof according to the present invention are particularly useful as reagents for delivery of biomolecules into cells (also referred to herein as "cell delivery reagents" or "delivery reagents", respectively). The invention therefore generally relates to the use of the compositions or complexes, respectively, containing a biologically active substance or biomolecule, respectively, and the nucleotides, oligonucleotides and/or polynucleotides as well as their compositions as disclosed herein, as well as the use of the nucleotides, oligonucleotides or polynucleotides and their compositions as such, for introducing said biologically active substance or biomolecule, respectively, preferably nucleic acids, peptides and/or proteins, into a cell. Especially in the case of delivery of nucleic acids into cells, the complexes or compositions, respectively of the invention are referred to as "transfection reagents". The same applies tor the nucleotides, oligonucleotides and polynucleotides as such as well as their compositions as defined herein.

Further subject matter of the present invention is a composition comprising one or more of the above-described compositions containing a biologically active substance or biomolecule, respectively, together with at least one nucleotide, oligonucleotide or polynucleotide species as described herein and at least one additional component, preferably selected from cells, cell culture media, and one ore more transfection enhancers and/or cell delivery enhances. In certain embodiments of the invention, compositions containing nucleotides,

oligonucleotides or polynucleotides as defined herein and the cargo molecule, in particular a biomolecule such as a nucleic acids, peptide or protein, are present in the form of complexes The invention also relates to corresponding compositions comprising one or more of the above described nucleotides, oligonucleotides and/or polynucleotides as such, as well as compositions thereof as outlined above, and at least one additional component as outlined before. Cells into which a biomolecule, preferably a nucleic acid, peptide or protein, can be introduced by using the reagents disclosed herein are prokaryotic cells such as bacteria, or eukaryotic cells. Preferred target cells are eukaryotic cells, e.g. cells of epithelial, neuronal, hematopoietic or any type originating or derived from primary cells, tumor and/or cancer cells or immortalized culture cell lines. The cell may be from any source including bacteria, animals (e.g. canine, mouse, hamster, cattle, sheep, goat, pig, guinea pig, fish, insect etc.), humans, fungi and plants.

Compositions according to the invention comprising a delivery or transfection, respectively, reagent as disclosed herein together with the desired cargo, e.g. present in the form of compositions or complexes as described above, may take the form of liquid compositions such as an aqueous solution containing the components, preferably in a suitable buffer or medium (e.g. a cell culture medium), but also extend to solid, typically dried compositions or complexes, respectively, e.g. a lyophilized mixture of the delivery reagent with the cargo, e.g. a nucleic acid, preferably examples as outlined below, or a lyophilized complex of these components.

Suitable mass ratios of the delivery or transfection, respectively, reagent according to the invention to the cargo can be determined easily using routine experimentation using the desired cell. Examples of useful ratios with respect to nucleic acid transfection using the transfection reagents according to the invention (e.g. for transfection of HeLa cells) range from 1 :1 to 3: 1 , preferably 1.5:1 to 2.5:1 , most preferred 2:1 .

Polyanions, preferably nucleic acids, which may be delivered or transfected, respectively, into cells using the delivery reagents of the invention (which may therefore be part of the compositions or complexes, respectivelym comprising the delivery reagent and the cargo, as described above), are preferably selected from DNA and RNA which may each be single stranded or double stranded as well as mixed forms thereof, i.e. DNA RNA hybrids.

Particularly preferred examples include, but are not limited to, plasmids, cosmids, oligonucleotides of DNA or RNA type, siRNA, sgRNA (small-guide RNA) (RNAs and DNAs useful in the CRISPR/Cas system; cf. US Patent No. 8,697,359), microRNA and microRNA mimetics, long non-coding RNAs as well as any combination thereof.

The nucleic acids to be delivered or transfected, respectively, according to the invention may contain nucleotide analogues as outlined before, and/or may be coupled to entities such as dyes, fluorophores, and proteins such as antibodies as further described below for the transfection agents of the invention. Proteins such as antibodies or other macromolecules may be equally used for delivery into cells using the complexes according to the invention. Biomolecule delivery enhancers and/or transfection enhancers useful in the present invention include any known aids for delivery of cargo molecules into cells, such as proteins, peptides, growth factors, lipids, (poly)saccharides and the like enhancing cell-targeting, uptake, internalization, nuclear targeting and expression. Such cell delivery and/or transfection enhancers include specific ligands such as insulin, transferrin, fibronectin for targeting cell surfaces. Further cell delivery and/or transfection enhancers include molecules or substances targeting cellular receptors, e.g. peptides targeting integrin receptors. Further transfection enhancers useful in the present invention include polycationic compounds such as protamine or poly-L-lysine, and liposomes, in particular cationic liposomes. The present invention is also directed to a pharmaceutical composition comprising at least one composition or complex containing a nucleotide, oligonucleotide or polynucleotide together with at least one pharmacologically active ingredient to be delivered into a cell or cells, respectively, of a, preferably mammalian, more preferably human, patient, optionally in combination with at least one pharmaceutically acceptable carrier, excipient, and/or diluent. In certain embodiments, the pharmaceutical composition of the invention thus includes a complex as described above in combination with at least one pharmaceutically acceptable carrier, excipient, and/or diluent. The preparation of pharmaceutical compositions in the context of the present invention, their dosages and their routes of administration are known to the skilled person, and general guidance can be found in the latest edition of Remington's Pharmaceutical Sciences (Mack publishing Co., Eastern, PA, USA).

Pharmaceutical compositions according to the invention are particularly applied for treatment and/or prevention of cancer, infections, degenerative diseases, and are foreseen for application in humans or animals. The invention also relates to corresponding botanical formulations for application in plants such as for the use of siRNAs as pesticides or agents used for the treatment of plant diseases.

The nucleotides, oligonucleotides and polynucleotides as disclosed herein may also be conjugated with other chemical entities, in particular with chemical groups which can be used to target the nucleotide, oligonucleotide or polynucleotide, respectively, to a cell in which said nucleotide, oligonucleotide or polynucleotide, or the cargo (biologically active substance or biomolecule) is to exert its properties. Chemical entities may be coupled to the inventive species through any kind of bonds such as covalent bonds, hydrogen bonds or Van der Waals bonds. Chemical entities to which the inventive species can be coupled include aptamers, polyethylenglycol such as PEG groups having an average molecular weight of from about 500 to about 1000 Da, e.g. about 750 Da, peptides, a palmitoyi group, cholesterol groups, phospholipids, proteins such as antibodies, and partners of non-covalent binding pairs such as biotin or digoxigenin. In the case of biotin, it can serve for associating the construct to a streptavidin group present on an antibody, or the biotin may serve as the ligand of an anti-biotin antibody which may, e.g. in turn have a second affinity to a target cell or tissue such as by having an affinity to a target receptor and/or ligand expressed intracellulary and/or on the surface of the target cell and/or tissue.

Antibodies in the context of the present invention may be selected from polyclonal, monoclonal, humanized, chimeric, single-chain antibodies and antibody fragments which antibodies or antibody fragments may be single-specific or bispecific species. The antibody fragment may be a Fab fragment, a F(ab')₂ fragment, or any fragment that retains the antigen-binding specificity of the intact antibody. Especially preferred antibody ligands that may be coupled to the molecules as disclosed herein are selected from antibodies or fragments thereof directed against cancer and/or tumor antigens, cancer- and/or tumor- associated antigens or oncoproteins, or antigens present on non-cancer and/or non-tumoral cells.

Further subject matter of the present invention relates methods for the delivery of molecules, in particular biomolecules, into cells in vitro and in vivo. In certain embodiments of the invention where the cargo to be delivered into a cell is a nucleic acid, such methods are also referred to as "transfection methods".

Thus, the present invention provides a method for introducing a biologically active substance into a cell in vitro comprising the step of contacting a complex or composition, respectively, as defined above comprising a nucleotide, oligonucleotide or polynucleotide as disclosed above and the biologically active substance (preferably as defined above) with the cell.

According to a preferred embodiment, the cell is present in culture medium in a reaction container and the composition or complex, respectively, according to the inventions is added to said culture medium. According to an alternative embodiment, the composition of cell delivery reagent and biologically active substance (or, in certain embodiments, the complex) is present in a suitable container, and the cell, preferably in culture medium, is added thereto. Especially for the letter kind of delivery of the desired molecule into the cell, a solid, i.e. dry or substantially dry, e.g. lyophilized, mixture of delivery regent of the invention and the desired biologically active substance (representing, in certain embodiments, a complex as described above), e.g. a nucleic acid (preferably as selected above) may be present in a suitable reaction or culture container, e.g. a well of a multiple-well plate (e.g. dry mixtures of transfection reagent with different molecules to be transfected such as nucleic acids of different sequence, and each different mixture present in an individual well). The invention therefore also extends to such reaction or culture containers containing a delivery

composition (delivery reagent of the invention plus cargo, or, in certain embodiments of the invention, the complex of delivery reagent with cargo) either in liquid or dry state.

Still further the invention also provides a method for introducing a biologically active substance (preferably as outlined above) into a cell in vivo comprising the step of

administering a composition comprising a biologically active substance or biomolecule, respectively, together with at least one nucleotide, oligonucleotide or polynucleotide as defined above to a tissue or organism, preferably a mammalian, more preferably a human, containing said cell. Alternatively, a cell into which a biologically substance is to be delivered by the embodiments according to the invention may be taken from said tissue or organism, the biologically active substance is delivered into the cell in vitro as outlined above, and the thus-treated cell is re-introduced into the tissue or organism.

The mechanism of action through which the compositions or complexes, respectively, of the invention or the nucleotides, oligonucleotides or polynucleotides as well as their

compositions as outlined herein are able to deliver cargo substances such as biologically active substances, preferably nucleic acids, peptides and/or proteins, into a target cell is currently under investigation. However, without being bound to any particular theory, it is plausible, and at least in part suggested by the Examples outlined below, that the G- quadruplex forming entities according to the invention represent negatively charged moieties which quite tightly attract cations which in turn can bind to negatively charged positions of the cargo (see Figs. 20 and 22 for nucleic acid as cargo molecule). These assemblies of G- quadruplex/cations with the cargo are presumably taken up by the target cell through a clathrin-dependent pathway and transported to endosomes. Due to the acidic milieu in endosomes the bonds in the internalized assemblies of G-quadruplexes/cations with the cargo are weakened such that the cargo molecule is released from the assemblies. Previous work on the stability of G-quadruplexes suggests that the dissociation of the cargo from the delivery reagent could also be at least in part due to a destabilization of the quadruplexes in an acidic environment (cf. Phong Lan Thao Tran et al. (2013) "Tetramolecular Quadruplex Stability and Assembly" in: Chaires, J. B., and Graves, D. (eds.) Topics in Current Chemistry 330, pages 243-274). The thus-dissociated cargo molecule is then released to the cytoplasm from late endosomes. The above-proposed mechanism is schematically illustrated for a nucleic acid cargo in Fig. 21 .

The complexes and compositions of the invention, i.e. the reagents for delivery of molecules, typically biomolecules such as nucleic acids peptides and proteins, are not only

characterized by their delivery efficiency and easy-to-use properties (making one-step delivery of cargo molecules into cells feasible), but also protect the cargo from environments influencing the stability and/or integrity of the cargo molecule(s). Accordingly, the invention also provides the use of delivery reagents as disclosed herein for protecting biologically active substances or molecules, respectively, against destabilizing and/or degrading environments such as solutions containing enzymes that can degrade or otherwise destabilize the biologically active substances or biomolecules, respectively (generally referred to herein as the "cargo" or "cargo substance" or "cargo molecule"). A preferred application of this kind is the use of the delivery agents as disclosed herein for protecting a nucleic acid against nucleic acid degrading enzymes such as exo- and endonucleases, e.g. DNAses and RNAses, more preferred the use of the delivery agents of the invention for protecting RNA against RNAses, e.g. present in an RNAse-containing solution or medium such as a cell culture medium or an animal, preferably mammalian, more preferably human, body fluid such as blood serum.

The figures show: Fig. 1. Synthesis of a delivery reagent of the invention using an RNA polymerase (RdRp) of a calicivirus. An ssRNA template (rC5; 5^'-CCCCC-3') or an ssDNA template (dC6; 5'-CCCCCC-3') was incubated with an RdRp of a calicivirus for 15 min, 45 min or 90 min. After purification of the reaction products using size exclusion chromatography, the amount of single stranded RNA was measured. (A) Graphical representation of the amount of ssRNA produced using the ssRNA template. (B) Amount of ssRNA produced using the ssDNA template.

Fig. 2. Synthesis of a delivery reagent of the invention using an RdRp of a calicivirus and a single stranded RNA (rC5; 5^'-CCCCC-3^') as a template. All reactions were performed in a total volume of 100 μΙ at 37°C. The reaction mix contained 8 μg of the template, 10 μΜ RdRp, 2 mM GTP, 20 μΙ reaction buffer (HEPES 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), and RNAse-DNAse free water to a total volume of 100 μΙ. The reaction was stopped at 15 min, 45 min and 90 min by adding 10 μΙ EDTA 0.3 M. The reaction products were analyzed using ion exchange HPLC and the area under the curve (AUC) of the eluted peak was measured. (A) Elution profile of the product of reaction using IEX-HPLC at 15 min, 45 min and 90 min. (B) Plot of AUC of eluted product as a function of time.

Fig. 3. Analysis of oligonucleotides produced according to the invention. All reactions were performed in a total volume of 100 μΙ at 37°C using a single stranded RNA (rC5; 5^'- CCCCC-3') or a single stranded DNA (dC6; 5^'-CCCCCC-3^') as a template. The reaction mix contained 8 μg of the template, 10 μΜ RdRp, 2 mM GTP, 20 μΙ reaction buffer (HEPES 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), and RNAse-DNAse free water to a total volume of 100 μΙ. The reaction was stopped at 15 min, 30 min, 45 min, 60 min, 90 min and 120 min by adding 10 μΙ EDTA 0.3 M. After purification of the reaction products by size exclusion chromatography using the riboxxPURE kit according to instructions of manufacturer (RiboxX GmbH, Radebeul, Germany), 15 μΙ of each reaction was analyzed on 20% PAGE and visualized after staining of nucleic acids. RNA marker corresponds to double stranded RNA of 19 bp, 21 bp and 25 bp, respectively, in length, as well as a single stranded RNA of 24 nt in length, as indicated. (A) Time course of synthesis of reaction products. The products of the reaction are shown. The visible products are isolated single stranded 5'-triphosphated poly(G) and/or single stranded 5'-triphosphated poly(G) forming complexes and G- quadruplexes (PPPrGx) as well as suprastructures. The single stranded RNA template has been used as a marker (rC5; 5^'-CCCCC-3^'). (B) Analysis of the reaction product resulting from a reaction as outlined above using a single stranded RNA (rC5; 5^'-CCCCC-3^') or a single stranded DNA (dC6; 5^'-CCCCCC-3^') as a template, as indicated. The reaction was allowed to proceed for 45 min, then stopped and analyzed by PAGE as described above.

Fig. 4. Characterisation of the synthesized products according to the reaction as described for Fig. 3B using a single stranded RNA (rC5; 5 -CCCCC-3 ) as a template by

HPLC. The HPLC elution profile of the products resulting from the reaction as described in Fig.3B is shown.

Fig. 5. Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution product of the peak as indicated in panel A was analyzed by ESI/MS, as shown in panel B. It displays a molecular weight (MW) of 1463.22 Da corresponding to the template rC5 (RNA molecule; 5^'-CCCCC-3^'). Fig. 6. Characterisation of the products synthesized as described above for Fig. 4B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution product of the peak as indicated in panel A was analyzed by ESI/MS, as shown in panel B. It displays a MW of 1808.27 Da corresponding to the MW of the product rC5G1 (RNA molecule; 5^'-CCCCCG-3^'). This product is expected to result from a terminal transferase activity of the RdRp on the rC5 template.

Fig. 7. Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution product of the peak as indicated in panel A was analyzed by ESI/MS, as shown in panel B. It displays a MW of 2499.43 Da corresponding to the MW of the product rC5G3 (i.e. RNA molecule 5^'-CCCCCGGGG-3^'). This product is also expected to result from a terminal transferase activity of the RdRp on the rC5 template.

Fig. 8. Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution products of the peak as indicated in panel A were analyzed by ESI/MS, as shown in panel B. They display MWs of 2249 Da, 2594 Da, 2939 Da, 3284 Da, 3630 Da, 3975 Da, 4320 Da, and 4665 Da, respectively, as indicated, corresponding to the respective MW of the following products:

PPPG6, i.e. RNA molecule 5^'-GGGGGG-3^', with a triphosphate bound to the 5' end of the molecule;

PPPG7, i.e. RNA molecule 5^'-GGGGGGG-3^', with a triphosphate bound to the 5^' end of the molecule;

PPPG8, i.e. RNA molecule 5^'-GGGGGGGG-3^', with a triphosphate bound to the 5^' end of the molecule;

PPPG9, i.e. RNA molecule; 5^'-GGGGGGGGG-3^', with a triphosphate bound to the 5^' end of the molecule;

PPPG10, i.e. RNA molecule 5^'-GGGGGGGGGG-3^' (SEQ ID NO: 2), with a triphosphate bound to the 5^' end of the molecule;

PPPG1 1 , i.e. RNA molecule 5^'-GGGGGGGGGGG-3^' (SEQ ID NO: 3), with a triphosphate bound to the 5^' end of the molecule;

PPPG12, i.e. RNA molecule 5^'-GGGGGGGGGGGG-3^' (SEQ ID NO: 4), with a triphosphate bound to the 5^' end of the molecule; PPPG13, i.e. RNA molecule 5^'-GGGGGGGGGGGGG-3^' (SEQ ID NO: 5), with a triphosphate bound to the 5^' end of the molecule.

Fig. 9. Characterisation of the products synthesized as described above for Fig. 4B by ESI/MS. The single stranded RNA template (rC5; 5^'-CCCCC-3^') was used for the reaction as described above (see Fig. 3B). The product was denatured and characterized by ESI/MS. The elution products of the peak as indicated in panel A were analyzed by ESI/MS, as shown in panel B. They display MWs of 3630 Da, 3975 Da, 4320 Da, 4665 Da, and 501 1 Da, respectively, as indicated, corresponding to the respective MW of the following products: PPPG10, i.e. RNA molecule 5^'-GGGGGGGGGG-3^' (SEQ ID NO: 2), with a triphosphate bound to the 5^' end of the molecule;

PPPG12, i.e. RNA molecule 5^'-GGGGGGGGGGGG-3^' (SEQ ID NO: 4), with a triphosphate bound to the 5^' end of the molecule;

PPPG13, i.e. RNA molecule 5^'-GGGGGGGGGGGGG-3^' (SEQ ID NO: 5), with a

triphosphate bound to the 5^' end of the molecule;

PPPG14, i.e. RNA molecule 5^'-GGGGGGGGGGGGGG-3^' (SEQ ID NO: 6), with a triphosphate bound to the 5^' end of the molecule.

Fig. 10. Silencing of GAPDH gene expression in HeLa cells by an siRNA targeting GAPDH in the presence of a cell delivery reagent of the invention. An siRNA ( iBONi^® siRNA -P4H; Riboxx GmbH, Radebeul, Germany; antisense strand: 5^'- AUGAGUCCUUCCACGAUACCCCC-3^' (SEQ ID NO: 7); sense strand:

5'-pppGGGGGUAUCGUGGAAGGACUCAU-3^' (SEQ ID NO: 8)) targeting the GAPDH gene was added in a 24-well plate at a concentration of 40 nM in the presence of a cell delivery reagent of the invention (0.3 μg/μl, volumes as indicated), then HeLa cells were added at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 72h. Gene silencing was assessed by western blot. As a control, gene expression of Hsp90 (Heat shock protein 90), as well as cells cultivated in the absence of both the siRNA and the inventive

transfection reagent are shown. The silencing of GAPDH is evidenced in dose-dependent manner on the addition of the cell delivery reagent according to the invention to the siRNA. The delivery reagent does not induce gene silencing by itself as shown in (B). Fig. 11. Uptake of siRNA by HeLa cells in the presence of a cell delivery reagent according to the invention. An siRNA ( iBONi^® siRNA transfection control T1 -H; Riboxx GmbH, Radebeul, Germany; same strands as the siRNA as in Fig. 10, but labelled with chromophore riboxx570) was added in a 24-well plate at a concentration of 20 nM in the presence of a cell delivery reagent of the invention (30 μΙ of a 0.3 μg/μl solution), then added to the cells seeded at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 4 h. Uptake of the siRNA was visualized by confocal microscopy. The cell nuclei were stained with DAPI (in blue, here in dark gray). The siRNA was labelled with a red chromophore (here in white to light gray).

Fig. 12. Silencing of GAPDH gene expression in 293T cells by an siRNA targeting GAPDH in the presence of a cell delivery reagent of the invention. An siRNA ( iBONi^® siRNA -P4H; Riboxx GmbH, Germany; antisense strand: 5^'-

AUGAGUCCUUCCACGAUACCCCC-3^' (SEQ ID NO: 7); sense strand:

5'-pppGGGGGUAUCGUGGAAGGACUCAU-3^' (SEQ ID NO: 8)) targeting the GAPDH gene was added in a 24-well plate at a concentration of 40 nM in the presence of a cell delivery reagent of the invention (0.3 μg/μl, volumes as indicated). Then, 293T cells were added at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 72 h. Gene silencing was assessed by western blot. As a control, gene expression of Hsp90 (Heat shock protein 90), as well as cells cultivated in the absence of both the iBONi^® siRNA -P4H and the inventive cell delivery reagent are shown. The silencing of GAPDH is evidenced in dose- dependent manner on the addition of the cell delivery reagent according to the invention to the siRNA.

Fig. 13. Uptake of siRNA by 293T cells in the presence of a cell delivery reagent according to the invention. Same experiment as in Fig. 1 1 , but using 293T cells. Fig. 14. Uptake of ssDNA by HeLa cells in the presence of a cell delivery reagent according to the invention. A fluorescein-labelled single stranded DNA was added to a 24- well plate at a concentration of 20 nM in the presence of a cell delivery reagent of the invention (10 μΙ of a 0.3 Mg/μΙ solution), then added to the cells seeded at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 4 h. Uptake was visualized by confocal microscopy. The cell nuclei are stained with DAPI (blue color; here in dark gray). The single stranded DNA bears a green chromophore (here in white to light gray).

Fig. 15. Uptake of ssDNA by 293 Tcells in the presence of a cell delivery reagent according to the invention. Same experiment as in Fig. 14, but using 293T cells.

Fig. 16. Uptake of dsDNA by HeLa cells in the presence of a cell delivery reagent according to the invention. A fluorescein-labelled double stranded DNA was added to a 24-well plate at a concentration of 20 nM in the presence of a cell delivery reagent of the invention (10 μΙ of a 0.3 Mg/μΙ solution), then added to the cells seeded at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 4h. Uptake was visualized by confocal microscopy. The cell nuclei are stained with DAPI (blue color; here in dark gray). The double stranded DNA bears a green chromophore (here in white to light gray).

Fig. 17. Uptake of dsDNA by 293T cells in the presence of a cell delivery reagent according to the invention. Same experiment as in Fig. 16, but using 293T cells. Fig. 18. Characterization of a composition containing a delivery reagent of the invention and siRNA cargo by ESI/MS. An exemplary delivery reagent of the invention was synthesized as previously described (see Fig. 3B using the rC5 ssRNA template). The complex consisting of the delivery reagent and siRNA was mixed as previously described (see above with respect to Fig. 10; 56 μg of delivery reagent with 28 μg of siRNA) and analysed by HPLC (A) and (C). The elution fractions corresponding to the area as indicated in (A) and (C), respectively, were denatured and analysed by ESI/MS (B), (D). The detected masses as indicated in (B) of 2939 Da, 3284 Da, 3630 Da, 3975 Da, 4320 Da and 4665 Da, respectively, correspond to the MW of the following products:

PPPG8; RNA molecule; 5^'-GGGGGGGG-3^', with a triphosphate bound to the 5^' end of the molecule

PPPG9; RNA molecule; 5^'-GGGGGGGGG-3^', with a triphosphate bound at 5^'-End of the molecule

PPPG10; RNA molecule; 5^'-GGGGGGGGGG-3^' (SEQ ID NO: 2), with a triphosphate bound to the 5^' end of the molecule

PPPG1 1 ; RNA molecule; 5^'-GGGGGGGGGGG-3^' (SEQ ID NO: 3), with a triphosphate bound to the 5^' end of the molecule

PPPG12; RNA molecule; 5^'-GGGGGGGGGGGG-3^' (SEQ ID NO: 4), with a triphosphate bound to the 5^' end of the molecule

PPPG13; RNA molecule; 5^'-GGGGGGGGGGGGG-3^' (SEQ ID NO: 5), with a triphosphate bound to the 5^' end of the molecule.

Furthermore, the elution fractions corresponding to the area as indicated in (C) was denatured and analysed by ESI/MS (D). The detected masses as indicated in (D) of 4665 Da, 501 1 Da, 7202 Da and 7482 Da correspond to the following products:

PPPG13; RNA molecule; 5^'-GGGGGGGGGGGGG-3^' (SEQ ID NO: 5), with a triphosphate bound to the 5^' end of the molecule

PPPG14; RNA molecule; 5^'-GGGGGGGGGGGGG-3^' (SEQ ID NO: 6), with a triphosphate bound to the 5^' end of the molecule ssRNA (antisense strand); 5^'-AUGAGUCCUUCCACGAUACCCCC-3^' (SEQ ID NO: 7) ssRNA (sense strand); 5^'-GGGGGUAUCGUGGAAGGACUCAU-3^' (SEQ ID NO: 8), with a triphosphate bound to the 5^' end of the molecule. Fig. 19. Serum stability of complexes comprising a delivery reagent of the invention and siRNA. An siRNA ( iBONi^® siRNA -P4H; Riboxx GmbH, Germany; antisense strand: 5^'- AUGAGUCCUUCCACGAUACCCCC-3^' (SEQ ID NO: 7); sense strand: 5 - pppGGGGGUAUCGUGGAAGGACUCAU-3^' (SEQ ID NO: 8)) was incubated at a

concentration of 2 μΜ for 37°C for the time as indicated in panel (A) in mouse serum (80%). In parallel, the siRNA was mixed as previously described (see above with respect to Fig. 10; 56 μg of delivery reagent with 28 μg of siRNA) and incubated at a concentration of 2 μΜ for 37°C for the indicated time (h) as indicated in panel (B) in mouse serum (80%). The siRNA incubated alone and in admixture with a delivery reagent of the invention were run on 20% native PAGE and visualized by UV transillumination.

Fig. 20. Schematic representation of the putative interaction of quadruplex-forming delivery reagents with nucleic acids. G-quadruplex forming delivery reagents of the invention are represented by gray dots. (A) Interaction of G-quadruplex forming delivery reagents with double stranded RNA or DNA. (B) Interaction of G-quadruplex forming delivery reagents with single stranded RNA or DNA. (C) Interaction of G-quadruplex forming delivery reagents with double stranded RNA or DNA bearing a functional group such as a chromo-, fluorophore, peptide, or a tag. (D) Interaction of G-quadruplex forming delivery reagents with single stranded RNA or DNA bearing a functional group such as a chromo-, fluorophore, peptide, or tag. (E) Interaction of G-quadruplex forming delivery reagents with circular double-stranded DNA (e.g. a plasmid).

Fig. 21. Schematic representation of a putative mechanism of uptake and release of nucleic acids associated to G-quadruplex forming delivery reagents of the invention into cells. A double-stranded RNA or DNA formulated with G-quadruplex forming delivery reagents of the invention is taken up through a clathrin-dependent pathway, and

subsequently transported to an endosome. After dissociation of the G-quadruplex forming delivery reagent from the nucleic acid, release of the nucleic acid cargo from the late endosome into the cytoplasma occurs. Fig. 22. Schematic representation of the putative interaction of nucleic acid with G- quadruplex forming delivery reagents of the invention. Interaction occurs through cations (mono- or divalent, e.g. K⁺, Na⁺, Mg²⁺ and/or Mn²⁺) present in the formulation. G-quadruplex forming reagents of the invention are present in complex with cations which in turn attract nucleic acid cargo molecule (negatively charged), thus forming a ternary complex G- quadruplex-cation-cargo nucleic acid. The present invention is further illustrated by the following non-limiting examples:

EXAMPLES

Example 1 : Synthesis of cell delivery reagents of the invention by RNA-dependent

RNA polymerase of a calicivirus using ssRNA or ssDNA template

All reactions were performed in a total volume of 100 μΙ at 37°C using a single stranded RNA (rC₅: 5'-CCCCC-3') or a single stranded DNA (dC₆: 5'-CCCCCC-3') as template. The reaction mix contained 8 μg of the template, 10 μΜ calicivirus RdRp, 2 mM GTP, 20 μΙ reaction buffer (HEPES 250 mM, MnCI₂ 25 mM, DTT 5 mM, pH 7.6), and RNAse-DNAse free water to a total volume of 100 μΙ. The reaction was stopped at 15 min, 45 min and 90 min by adding 10 μΙ EDTA 0.3 M. After purification of the reaction products using size exclusion

chromatography, the amount of single stranded RNA was measured at the indicated time points. The results are shown in Figs. 1 A and B.

In parallel experiments, the reaction products were also analyzed using ion exchange HPLC and the area under the curve (AUC) of the eluted peak was calculated. Results for the reaction using the rC5 template are shown in Fig. 2A (elution profile of reaction stopped after 15 min), Fig. 2B (elution profile of reaction stopped after 45 min) and Fig. 2C (elution profile of reaction stopped after 90 min). Fig. 2D shows the plot of the results of the AUC

measurements.

Example 2: Analysis of reaction products by PAGE RdRp reactions were set up ad carried out as in Example 1 using the rC₅ template or the dC₆ template, and the reactions were stopped at 15 min, 30 min, 45 min, 60 min, 90 min and 120 min by adding 10 μΙ EDTA 0.3 M. After purification of the reaction products by size exclusion chromatography using the riboxxPURE kit according to the instructions of the manufacturer (RiboxX GmbH, Radebeul, Germany), 15 μΙ of each reaction were analyzed by 20% PAGE and visualized through UV transillumination. Result of the time course experiment using the rC5 ssRNA template is shown in Fig. 3A. Fig, 3B shows the products of reactions using the rC₅ or the rC₆ template each allowed to proceed for 45 min. The bands visible correspond to isolated single stranded 5'-triphosphated poly(G) and/or single stranded 5'-triphosphated poly(G) forming complexes and G-quadruplexes (PPPrGx) as well as higher suprastructures.

Example 3: Characterization of RdRp reaction products using rC₅ template by

HPLC/mass spectroscopy

The reaction product of Example 2 using the rC₅ template was subjected to HPLC. The elution profile is shown in Fig. 4. The peak fractions as indicated in Figs. 5A, 6A, 7A, 8A and 9A were denatured and further analyzed by ESI/MS. The corresponding mass spectra are shown in Fig. 5B, 6B, 7B, 8B and 9B, respectively. The peak(s) in the mass spectra correspond to the molecular weight of the species indicated in Fig. 5B, 6B, 7B, 8B and 9B, respectively.

Example 4: Gene-silencing in eukaryotic cells by siRNA using transfection with a delivery reagent of the invention

An siRNA (iBONi^® siRNA-P4H; RiboxX GmbH, Radebeul, Germany; antisense strand: 5^'- AUGAGUCCUUCCACGAUACCCCC-3^' (SEQ ID NO: 7); sense strand:

5'-pppGGGGGUAUCGUGGAAGGACUCAU-3^' (SEQ ID NO: 8)) targeting the GAPDH gene was added in a 24-well plate at a concentration of 40 nM in the presence of 20 μΙ, 10 μΙ, 5 μΙ or 2.5 μΙ of a 0.3 μg/μl solution of the reaction of Example 1 using the rC5 ssRNA template. HeLa or 293T cells were added at 25.000 cells/well, and the plates incubated at

37°C/5%C02 for 72h. Gene silencing was assessed by western blot. As a control, gene expression of Hsp90 (Heat shock protein 90), as well as cells cultivated in the presence of siRNA - PH4 but no cell delivery reagent of the invention and in the absence of both siRNA - P4H and the cell delivery reagent of the invention were analyzed. The results for HeLa cells (Fig. 10A) as well as for 293T cells (Fig. 12) show that the silencing of GAPDH is evidenced in dose-dependent manner on the addition of the cell delivery reagent of the invention to the siRNA. The cell delivery reagent of the invention does not induce gene silencing by itself as shown for HeLa cells in Fig. 10B.

The results further demonstrate the surprising finding that no pre-incubation of cell delivery reagent of the invention with the nucleic acid to be transfected (here: siRNA) is necessary. Example 5: Transfection of RNA and DNA into eukaryotic cells using a delivery reagent of the invention An siRNA (iBONi^® siRNA transfection control T1 -H; antisense strand: 5^'- AUGAGUCCUUCCACGAUACCCCC-3^' (SEQ ID NO: 7); sense strand:

5'-pppGGGGGUAUCGUGGAAGGACUCAU-3^' (SEQ ID NO: 8); coupled with the

chromophore riboxx570 (RiboxX GmbH, Radebeul, Germany) was added to a 24-well plate at a concentration of 20 nM in the presence of the cell delivery reagent of the invention (30 μΙ of a 0.3 Mg/μΙ solution), then added to the cells seeded at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 4h. Uptake was visualized by confocal microscopy. The cell nuclei were stained with DAPI (blue color). The transfected siRNA is visible through its bound red fluorophore at 570 nm. Fig. 1 1 shows a photograph of a representative section of the plate wherein the cell nuclei are visible as darker gray structures, and the uptake of the siRNA is clearly visible as white to light gray dots. Larger areas in white to light gray indicate larger amounts of cell delivery reagent/siRNA complexes within the cells. The same experiment was repeated with 293T cells (epithelial cells which are not easy to transfect with hitherto known transfection agents), and the results are shown in Fig. 13.

Using the same conditions as above, a fluorescein-labelled single stranded DNA was successfully delivered into HeLa (Fig. 14) and 293T cells (Fig. 15). In further experiments using the same conditions as above, a fluorescein-labelled double-stranded DNA was delivered into HeLa (Fig. 16) and 293T cells (Fig. 17).

Summing up, oligonucleotides of the invention are potent cell delivery reagents for all kinds of nucleic acids. As shown by the siRNA experiments, the cell delivery reagent of the invention does not interfere with the function of the transfected nucleic acid in the cell.

Surprisingly, no pre-incubation step of cell delivery reagent with cargo is required for successful delivery of the nucleic acids into the cells. Example 6: Cell delivery reagents of the invention form complexes with a nucleic acid cargo

A cell delivery reagent was produced as outlined in Example 1 using the rC5 ssRNA template. 56 μg of cell delivery reagent were mixed with 28 μg of an siRNA (iBONi^® siRNA- P4H; RiboxX GmbH, Radebeul, Germany; antisense strand: 5^'- AUGAGUCCUUCCACGAUACCCCC-3^' (SEQ ID NO: 7); sense strand:

5'-pppGGGGGUAUCGUGGAAGGACUCAU-3^' (SEQ ID NO: 8)). The mixture was subjected to HPLC. The elution profile is shown in Figs. 18A and 18C, respectively. The peak fractions as indicated in Figs. 18A, and 18C were denatured and further analyzed by ESI/MS. The corresponding mass spectra are shown in Fig. 18B and 18D, respectively. The peak(s) in the mass spectra correspond to the molecular weight of the species indicated in the legend for Fig. 19. The co-elution of pppG13, pppG14, and the sense and anti-sense strand of the siRNA suggest a complex formed between the delivery reagent of the invention and the siRNA cargo molecule.

Example 7: Cell delivery reagent of the invention protects RNA from degradation in mouse serum

An siRNA ( iBONi^® siRNA -P4H; Riboxx GmbH, Germany; antisense strand: 5^'- AUGAGUCCUUCCACGAUACCCCC-3^' (SEQ ID NO: 7); sense strand:

5'-pppGGGGGUAUCGUGGAAGGACUCAU-3^' (SEQ ID NO: 8)) was incubated at a concentration of 2 μΜ for 37°C for 48 h in mouse serum (80%). In a parallel experiment, the siRNA was mixed with a cell delivery reagent of the invention as described in Example 6 and incubated at a concentration of 2 μΜ for 37°C for 48 h mouse serum (80%). Samples were taken from both reactions before incubation and after 0.5 h, 2 h, 4 h, 24 h and 48 h of incubation. The samples were run on 20% native PAGE and visualized by UV

transillumination. The results are shown in Figs. 19A and B. Whereas the siRNA is almost completely degraded already after 2 h of incubation in mouse serum, the same siRNA is stable even after 48 h of incubation in mouse serum when the delivery reagent of the invention is present.

Claims

A composition or complex containing a biologically active substance together with a nucleotide, oligonucleotide or polynucleotide forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions, in particular under appropriate buffer, salt and temperature conditions, preferably physiological conditions.

The composition or complex of claim 1 wherein the oligonucleotide or polynucleotide comprises a sequence of formula (I) nGpXm (I) wherein X_n and X_m is any nucleotide sequence of length n or m, respectively, preferably being m = n, and G_p is any number of guanines involved in tedrad formation of length p.

The composition or complex of claim 1 or 2 wherein the oligonucleotide or polynucleotide comprises or has a sequence selected from the group consisting of formulas (II), (III) and (IV):

X_nGolXpG₀2Xm (II) wherein X_n and X_m is any non-guanine nucleotide sequence of length n or m, respectively, preferably being n=m, G₀i and G₀2 is any number of guanines involved in tetrad formation of length o1 and o2, respectively, preferably being o1 = o2, and X_p is any nucleotide sequence of length p involved in loop formation;

X_nGolXpl Go2X 2Go3X 3G₀4Xm (HI) wherein X_n and X_m is any non-guanine nucleotide sequence of length n or m, respectively, preferably being n=m, G₀i , G₀2, G₀3 and G_o4 are independently any number of guanines involved in tetrad formation of length o1 , o2, o3 and o4, respectively, preferably being o1 = o2 = o3 = o4, and X_pi , X_p2 and X_p3 are independently any nucleotide sequence of length p1 , p2 and p3, respectively, involved in loop formation, preferably being p1 =p2=p3;

wherein n is an integer of at least 2 or 3, preferably 3 to 40, more preferably 3 to 20.

4. The composition or complex of claim 3 wherein n in formula (IV) is an integer of from 2 or 3 to 14, more preferably 6 to 14.

5. The composition or complex according to any one of the preceding claims wherein the oligonucleotide or polynucleotide is single stranded.

6. The compositions or complex according to any one of the preceding claims wherein the nucleotide, oligonucleotide or polynucleotide consists of ribonucleotides.

7. The composition or complex according to any one of the preceding claims comprising at least one chemically modified and/or labeled nucleotide.

8. The composition or complex according to any one of the preceding claims wherein the oligonucleotide or polynucleotide comprises a free 5'-triphosphate group.

9. The composition or complex according to any one of the preceding claims containing cations being selected from the group consisting of alkaline metals, alkaline earth metals and transition metals.

10. The composition or complex of claim 9 wherein the cation is selected from the group consisting of K⁺, Na⁺, Mg²⁺ and Mn²⁺.

1 1 . The composition or complex according to any one of the preceding claims wherein the biologically active substance is selected from the group consisting of nucleic acids, proteins, lipids and polysaccharides.

12. The composition or complex of claim 1 1 wherein the nucleic acid is DNA or RNA, preferably selected from plasmid DNA, siRNA, sgRNA, microRNA and microRNA mimetics.

13. The composition or complex of claim 1 1 or 12 wherein the nucleic acid is coupled to a chromophore, fluorophore, protein, peptide, cholesterol group or amino acid moiety.

14. A composition or complex according to any one of the preceding claims containing at least two different nucleotides, oligonucleotide(s) and/or polynucleotide(s) as defined in any one of claims 1 to 8.

15. The composition or complex of claim 14 containing at least two different

oligonucleotides selected from the group consisting of (G)₆, (G)₇, (G)₈, (G)₉, (G)i₀ (SEQ ID NO: 2), (G)n (SEQ ID NO: 3), (G)_i2 (SEQ ID NO: 4), G)_i3 (SEQ ID NO: 5)

16. A method for preparing a composition or complex of claim 6, optionally containing at least one chemically modified and/or labeled nucleotide, comprising the steps of (i) incubating a corresponding oligonucleotide or polynucleotide template with an

RNA polymerase of a calicivirus in the presence of appropriate rNTPs and/or chemically modified and/or labeled analogues thereof; and

(ii) contacting the reaction mixture obtained in step (i) with a biologically active substance.

17. A method for preparing a composition or complex as defined in claim 15 comprising the steps of:

(a) incubating a (C) >₅ template with an RNA polymerase of a calicivirus in the

presence of rGTP wherein the template may be RNA, DNA or mixed RNA DNA; and

(b) contacting the reaction mixture obtained in step (i) with a biologically active substance.

18. A reaction container comprising the composition or complex according to any one of claims 1 to 15.

19. The reaction container of claim 18 being a well of a cell culture plate.

20. The reaction container of claim 18 or 19 comprising the composition or complex in dried form.

21 . The reaction container of claim 20 wherein the composition or complex is lyophilized.

22. A pharmaceutical composition comprising a composition or complex according to any one of claims 1 to 15 together with at least one pharmaceutically acceptable carrier, vehicle and/or diluent.

23. A cell delivery or transfection kit comprising, in separate containers, a nucleotide, oligonucleotide and/or polynucleotide as defined in any one of claims 1 to 8 and at least one additional component selected from the group consisting of biologically active substances, cells, cell culture media, cell delivery enhancers and transfection enhancers.

24. A method for introducing a biologically active substance into a cell in vitro comprising the step of contacting a composition or complex according to any one of claims 1 to 15 with the cell.

25. The method of claim 24, wherein the cell is present in culture medium and the

composition or complex is added to said culture medium.

26. The method of claim 24, wherein the composition or complex is present in a reaction container and the cell, preferably in culture medium, is added to said composition or complex, respectively.

27. A method for introducing a biologically active substance into a cell in vivo comprising the step of administering a composition or complex according to any one of claims 1 to 15 and/or the pharmaceutical composition of claim 22 to a tissue or organism containing said cell.

28. A composition containing at least two different oligonucleotides selected from the group consisting of (G)₆, (G)₇, (G)₈, (G)₉, (G)_i0 (SEQ ID NO: 2), (G)n (SEQ ID NO: 3), (G)i2 (SEQ ID NO: 4), G)_i3 (SEQ ID NO: 5) and (G)_i4 (SEQ ID NO: 6).

29. A method for preparing the composition of claim 28 comprising the step of incubating a (C) >5 template with an RNA polymerase of a calicivirus in the presence of rGTP wherein the template may be RNA, DNA or mixed RNA DNA. claims 1 to 8 or the composition of claim 28 for introducing a biologically active substance into a cell. 31 . Use of the nucleotide, oligonucleotide or polynucleotide as defined in any one of claims 1 to 8 or the composition of claim 28 for protecting a biologically active substance against degradation or destabilization in a degrading or destabilizing environment. 32. The use of claim 30 or 31 wherein the biologically active substance is as defined in any one of claims 1 1 to 13.