WO1998050078A1

WO1998050078A1 - Lipophilic and/or lytic peptides for specific delivery of nucleic acids to cells

Info

Publication number: WO1998050078A1
Application number: PCT/US1998/008849
Authority: WO
Inventors: Manpreet S. Wadwha; Alain Rolland; Louis C. Smith; Mark Logan; James T. Sparrow
Original assignee: Baylor College of Medicine; Genemedicine Inc
Current assignee: Baylor College of Medicine; Genemedicine Inc
Priority date: 1997-05-02
Filing date: 1998-04-30
Publication date: 1998-11-12
Anticipated expiration: 1999-11-02
Also published as: AU7173698A

Abstract

The invention relates in part to molecular complexes for delivering macromolecules (e.g., nucleic acid molecules) into cells. These molecular complexes can comprise lipophilic peptides that condense and stabilize the macromolecule. These lipophilic binding peptides can also comprise moieties that target the macromolecule to specific cells in an organism. In addition, the molecular complexes can comprise lipophilic peptides that can lyse endosomal structures within cells and thereby enhance delivery of the macromolecule into cells. The invention also relates in part to methods of delivering macromolecules to cells using the molecular complexes described herein. Thus, the present invention provides for efficient and specific delivery of nucleic acid molecules into cells.

Description

DESCRIPTION

LIPOPHILIC AND/OR LYTIC PEPTIDES FOR SPECIFIC DELIVERY OF NUCLEIC ACIDS TO CELLS

Field Of The Invention The invention relates to synthetic molecules designed for the delivery of macromolecules, preferably nucleic acid molecules, to cells.

Background Of The Invention

The following description of the background of the invention is provided to aid in understanding the claimed invention, but it is not admitted to constitute or describe prior art to the claimed invention, and should in no way be construed as limiting the claimed invention.

Recombinant retroviral vectors have been used for delivery of genes to cells of living animals. Morgan et al., Annu . Rev. Biochem . , 62:191-217 (1993). Retroviral vectors permanently integrate the transferred gene into the host chromosomal DNA. In addition to retroviruses, other viruses have been used for gene delivery. Adenoviruses have been developed as a means for gene transfer into epithelial derived tissues. Stratford-Perricaudet et al . , Hum . Gene . Ther. , 1:241-256 (1990); Gilardi et al . , FEBS, 267:60-62 (1990); Rosenfeld et al . , Science, 252:4341-4346 (1991); Morgan et al . , Annu . Rev. Biochem . , 62:191-217 (1993). Recombinant adenoviral vectors have the advantage over retroviruses of being able to transduce nonproliferating cells, as well as an ability to produce purified high titer viruses .

In addition to viral-mediated gene delivery, a more recent means for DNA delivery has been receptor-mediated endocytosis. U.S. patent 5,166,320 describes soluble nucleic acid delivery systems that include ligands for receptor-mediated endocytosis. Endocytosis is the process by which eukaryotic cells continually ingest segments of the plasma membrane in the form of small endocytotic vesicles. Alberts et al . , Mol . Biol . Cell , Garland Publishing Co., New York, 1983. Extracellular fluid and material dissolved in it becomes trapped in the vesicle and is ingested into the cell. Id. This process of bulk fluid-phase endocytosis can be visualized and quantified using a tracer such as enzyme peroxidase introduced into the extracellular fluid. Id. The rate of constitutive endocytosis varies from cell type to cell type. Coated pits are specialized regions of the plasma membrane that include membrane receptors and their high affinity ligands, which cluster and are internalized during endocytotic events. Calvaloe are other specialized regions of the plasma membrane containing receptors which provide cell specific uptake of ligands.

Endocytotic vesicles exist in a variety of sizes and shapes and are usually enlarged by fusing with one another and/or with other intracellular vesicles. Stryer, Bioch . , Freeman and Co., New York (1988) . In most cells the great majority of endocytotic vesicles, also named endosomes, ultimately fuse with small vesicles called primary lysosomes to form secondary lysosomes which are specialized sites of intra-cellular degradation. Id. The lysosomes are acidic and contain a wide variety of degradative enzymes to digest the macromolecular contents of the vesicles . Silverstein et al., Annu . Rev. Biochem . , 46:669-722 (1977); Simianescu et al., J. Cell Biol . , 64:586-607 (1975).

Once nucleic acid molecules or other macromolecules are targeted to a cell for delivery and internalized by endocytosis, the nucleic acid molecules or macromolecule must be released from the endosome to function as a therapeutic agent. If not, the delivery of the nucleic acid molecules or macromolecules will be degraded by lysosomes . Studies have analyzed the endosomal/lysosomal degradation process. It has been determined that organisms which are internalized via receptor-mediated endocytosis or receptor/ligand systems, such as viruses and other microorganisms, escape lysosomal degradation in order to function. The entry mechanism of some viruses have been studied extensively. For some viruses outer membrane proteins have been demonstrated to be important for endosomal escape. Marsh et al., Adv. Virus Res . , 36:107-151 (1989) . Other studies have focused on methods to prevent lysosomal degradation. These studies have used substances which perturb endosomal/lysosomal function. Mellmann et al., Ann . Rev. Biochem . , 55:663-700 (1986). Most of these substances have only been used in vi tro .

In addition, studies show that the entire virus-shell is necessary for efficient endosomal lysis. Marsh et al., Adv. Virus Res . , 36:107-151 (1989). Studies have also demonstrated that adenovirus will enhance transferrin- polylysine mediated gene delivery. Curiel, P. N. A . S . , 88:8850-8854 (1991). These studies improved gene expression in vitro by using a replication defective adenovirus incorporated into DNA complexes. The effect of the adenovirus is to lyse the endosome before its contents can either be routed to the lysosome or recycled to the cell surface. To reduce viral induced cell death, adenovirus has been coupled enzymatically to polylysine through the ε-amino moiety of lysine and the γ-carboxyl moiety of glutamic acid. Wagner et al . , P. N. A. S . , 89:6099-03 (1992). Chemical coupling of polylysine with the acidic residues of adenovirus also accomplishes the same objective. In addition to adenoviruses, peptide sequences from other viruses, such as influenza, have been used to achieve endosome rupture. Wagner et al . , P. N. A. S . , 89:7934-7938 (1992). A lytic peptide from influenza hemagglutinin has been used to augment gene transfer by transferrin- polylysine-DNA complexes. Id. This virus-like genetic transfer vehicle has been shown to be functional in vi tro but 100-fold less effective than adenovirus, based on the delivery and expression of the luciferase reporter construct. Id. These influenza virus hemagglutinin complexes, however, have not been shown to promote effective gene transfer in in vivo studies.

In addition, numerous studies have analyzed the role of polyamines in the intracellular processes involving nucleic acids. In particular, studies show that polyamines enhance both transcription and translation, and are involved in maintaining tRNA structure and activity. Tabor, et al., Annu . Rev. Biochem . , 171:15-42 (1970); Cohen, Na ture, 274:209-210 (1978). Furthermore, polyamines have been shown to condense nucleic acids which may be utilized in the cell for packaging processes. Gosule, et al., J. Mol . Biol . , 121:311-326 (1978); Chattoraj , et al., J. Mol . Biol . , 121:327-337 (1978); Riemer, et al . , Biopolymers, 17:785-794 (1970) . Additional studies have demonstrated that polyamines are active in ribosome stabilization and in the packaging of DNA into phage heads. Stevens, et al . , Ann . N. Y. Acad. Sci . , 171:827-837 (1970); Wilson, et al . , Biochemistry, 18:2192-2196 (1979).

The above studies have investigated the electrostatic component in the interaction of the polyamines, such as large poly-L-lysine molecules, with nucleic acids. The average chain length of these poly-L-lysines ranged from 50- 200 residues. Use of the poly-L-lysines, however, have been shown to be toxic to cells in nM concentrations thereby limiting their applicability. Such studies have also been performed with spermine, spermidine and putrescine. Braunlin, et al., Biopolymers, 21:1301-1314 (1982).

Summary Of The Invention

The invention provides novel lipophilic peptides as well as their use for delivering macromolecules (e.g., nucleic acid molecules) into cells, complexes formed between the macromolecules to be delivered and the lipophilic peptides, and cells transformed by such complexes. Thus, the present invention allows for enhanced delivery of nucleic acid molecules into cells in vivo and in vi tro .

The lipophilic peptides of the invention comprise a peptide portion and one or more hydrophobic moieties. The amino acid sequences of several suitable peptides are set forth herein and those skilled in the art would be able to make and use many others given the methods described herein. The hydrophobic moiety or moieties render the peptide lipophilic, and examples of suitable modifications are provided herein. Examples of suitable hydrophobic moieties are cholesterol and cholesterol derivatives. In addition, fatty acid moieties are preferably utilized as the hydrophobic moieties of the invention. Again, however, those skilled in the art would be able to make and use lipophilic peptides having different hydrophobic, cholesterol, cholesterol derivative, and fatty acid moieties .

In particular, the invention relates to two types of lipophilic peptides: lipophilic lytic peptides and lipophilic binding peptides. Lipophilic binding peptides simply bind to macromolecules and localize the macromolecule to a particular cell compartment. In addition, lipophilic binding peptides can condense polymeric macromolecules, preferably nucleic acid molecules, which facilitates the delivery of these macromolecules to cells. Lipophilic binding peptides can form non-covalent complexes with macromolecules, preferably nucleic acid molecules, and thereby condense these molecules into small particles via electrostatic interactions. The small diameter particles are then capable of being transported into cells. In addition, these smaller particles can render nucleic acid molecules more stable with respect to degradation within the circulation system of animals and in cells. In this manner, lipophilic binding peptides facilitate the delivery of nucleic acid molecules from the outside of cells, through the plasma membrane, and, ultimately, to the nucleus of cells.

The present invention also relates in part to lipophilic lytic peptides to avoid the problems of endosomal/lysosomal degradation in the delivery of macromolecules, preferably nucleic acid molecules, to cells. Like the lipophilic binding peptides, the lipophilic lytic peptides also harbor a peptide moiety and one or more hydrophobic moieties, preferably fatty acids or other alkyl- containing moieties . The peptide moiety of the lipophilic lytic peptides confer a endosomal lysis property to nucleic acid complexes such that they are not degraded by lysosomes in the cell.

In addition to the lipophilic lytic peptides and lipophilic binding peptides, the present invention relates in part to molecular complexes containing one or more molecules which target the molecular complexes to specific tissues. Examples of these targeting molecules are surface ligands as well as nuclear ligands . The surface ligands are capable of binding to a cell surface receptor and entering a cell via internalization mechanisms (e.g., endocytosis, potocytosis, pinocytosis) . By using surface ligands specific to certain cells, nucleic acid molecules can be delivered directly to the desired tissue using the molecular complexes of the invention.

Thus, in a first aspect, the present invention features a molecular complex for delivering a macromolecule into the cells of an organism. The molecular complex comprises (a) a lipophilic lytic peptide comprising a peptide moiety and one or more hydrophobic moieties; (b) a lipophilic binding peptide comprising a peptide moiety and one or more hydrophobic moieties; (c) a multifunctional peptide comprising a peptide moiety and optionally comprising one or more hydrophobic moieties; and (d) the macromolecule. The molecular complex can be composed of any combination of these components in varying ratios.

The term "molecular complex" as used herein refers to a complex capable of transporting macromolecules through the lipid membranes of cells. This molecular complex is preferably bound to a macromolecule in a non-covalent manner. The molecular complex should be capable of transporting macromolecules in a stable and condensed state and of releasing the noncovalently bound macromolecule into the cellular interior. In addition, the molecular complex may prevent lysosomal degradation of the macromolecule by endosomal lysis. Furthermore, the molecular complex can also efficiently transport the macromolecule through the cytoplasm of the cell to the nuclear membrane, as discussed herein. The term "delivering" refers to transportation of a macromolecule to a desired cell or any cell. The macromolecule can be delivered to the cell surface, cell membrane, cell endosome, within the cell membrane, nucleus or within the nucleus, or any other desired area of the cell. Delivery includes transporting macromolecules such as nucleic acid molecules, proteins, lipids, carbohydrates, and various other molecules . The term "macromolecule" as used herein preferably refers to polymeric compounds. Biological polymers include polypeptides, proteins, glycogen, and nucleic acid molecules. The term "macromolecule" as used herein refers to lipids and carbohydrates, preferably peptidomimetics and organic compounds, more preferably proteins and peptides, and most preferably nucleic acid molecules.

The term "peptidomimetic" as used herein refers to peptide-like molecules which contain non-hydrolyzable chemical moieties in place of those which exist in naturally occurring peptides. Thus, regions of a peptide which are hydrolyzable, such as carboxyl moieties, are replaced by non-hydrolyzable moieties, such as methylene moieties, in a peptidomimetic .

The components of the molecular complexes can be non-covalently bound to the macromolecule at the same time, i.e., simultaneously, and in various proportions. The binding molecules can be the same molecule or a combination of different molecules as discussed above. Alternatively, components of the molecular complex may be covalently linked to the macromolecule by a variety of chemical linkages. Furthermore, the surface ligand, nuclear localization ligands, and lysis agent can be directly linked to the binding molecule or linked to the binding molecule by a spacer moiety as defined herein. The term "nucleic acid molecule" as used herein refers to DNA or RNA. Nucleic acid molecules include naked DNA, DNA complexed with other molecules, oligodeoxynucleotides, a nucleic acid cassette, naked RNA, RNA complexed with other molecules, or nucleic acid molecules contained within vectors, plasmids, or viruses or the vectors, and viral nucleic acid themselves. The RNA molecules can be ribozymes or antisense RNA molecules. The nucleic acid molecules can exist in single stranded, double stranded, and triple helix forms. These are examples and are not meant to be limiting.

The term "lipophilic binding peptide" as used herein refers to a peptide which is capable of binding to a macromolecule. The lipophilic binding peptide can target the macromolecule to a particular compartment within cells. In addition, lipophilic binding peptides may bind to macromolecules and condense the macromolecules . Methods of determining the degree to which a lipophilic binding peptide condenses macromolecules can be accomplished using light scattering techniques known by persons of ordinary skill in the art.

The lipophilic binding peptide also harbors one or more hydrophobic moieties which enable the peptide to associate with lipid membranes of cells. Lipophilic binding peptides include, but are not limited to, components capable of stabilizing and/or condensing nucleic acid molecules by electrostatic binding, hydrophobic binding, hydrogen binding, intercalation or forming helical structures with the macromolecule, preferably a nucleic acid molecule, including interaction with the major and/or minor grove of DNA. The lipophilic binding peptide can be capable of noncovalently binding to macromolecules, preferably nucleic acid molecules. The lipophilic binding peptides are also capable of associating with a surface ligand, a nuclear ligand, and/or a lysis agent.

The term "peptide" as used herein refers to a polymer of amino acids that is preferably less than 40 residues in length, more preferably less than 30 amino acids in length, and most preferably less than 20 amino acids in length.

The term "hydrophobic" as used herein refers to a chemical moiety or moieties which partition into non-polar environments with a higher probability than they partition into polar environments. Hydrophobic moieties can be attached to peptides of the invention to confer a lipophilic character to the peptides. An example of a general formula for a lipophilic peptide of the invention is:

N-CH(R ₃ ) -CO- peptide

R,

where the R__., R₂, and R₃ are hydrophobic moieties. A lipophilic peptide of the invention may include Ri, R₂, and R₃, or include only Ri and R₃, or include only Ri and R₂. Ri, R₂, and R₃ may be of the same or different chemical formula. Ri, R₂, and R₃ can comprise alkyl and acyl moieties of varying length. Ri and R₂ are preferably of the same chemical formula when no R₃ is present. These alkyl and acyl moieties can exist in straight chain and/or branched forms. The alkyl and acyl moieties can exist in saturated and unsaturated forms. Thus, R__., R₂, and R₃ can harbor alkyl moieties, alkene moieties, and alkyne moieties. The alkene moieties may be conjugated within the chain. The stereochemical orientations of the atoms around any unsaturations within an acyl or alkyl moiety can exist in trans or cis conformations. The acyl and alkyl moieties can also comprise heteroatoms. Examples of acyl and alkyl moieties of the invention are: CH₃-(CH₂)n-CO- CH₃-(CH₂)n-CH₂-

where n = 0 through 30. n is preferably less than 30, more preferably less than 25, and most preferably less than 20. Other examples of moieties that comprise RI, R2, and R3 are:

-NH-CO-S-CB,- -NH-CO-0-CH -NH-CO-NH-

-NH-C(NH)-NH-

Therefore, lipophilic peptides of the invention can have the following structural formulas:

CHHCI^n-CIi CO-peptide

CH₃-(CH₂)n-CO-NH-(CH₂)q-CH-CO-peptide

I

NH I

CO

I

(CH₂)m

CH₃ where n, m, and q are from 0 through 30. n, m, and q are preferably less than 30, more preferably less than 25, and most preferably less than 20. n, m, and q may be the same or different. The hydrophobic moieties of the lipophilic peptides (Ri, R₂, and R₃) of the invention are preferably fatty acid moieties such as laurate, myristate, palmitate, stearate, arachidate, behenate, lignocerate, palmitoleate, oleate, linoleate, linolenate, and arachidonate moieties. In addition, the hydrophobic regions of lipophilic peptides (Ri_/ FA and R₃) preferably comprise cholesterol moieties as well as cholesterol derivative moieties .

The term "lipophilic lytic peptide" as used herein refers to a molecule which is capable of fusing with an endosomal membrane or breaking down an endosomal membrane and freeing the contents into the cytoplasm of the cell. The lipophilic lytic peptide can contain the following elements: (1) a peptide region capable of lysing .endosome complexes; and (2) an hydrophobic region or regions capable of conferring a hydrophobic character to the peptide. The hydrophobic region or regions can enable the peptide to associate with lipid membranes of cells. The hydrophobic moieties of the lipophilic lytic peptide are preferably fatty alkyl moieties such as lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, palmitolyl, olyl, linolyl, linolyl, and arachidonyl moieties. It is preferred that lipophilic lytic peptides are pH selective, meaning that the lytic properties of the peptides are inactive at neutral pH, e.g., pH 6 through 9, while active at acidic pH, e.g., pH 6 and below. The lipophilic lytic peptide may also exist in a constitutively active state. It is also preferred that lipophilic lytic peptides interact with other components of the invention, such as macromolecules and lipophilic binding peptides.

The term "multifunctional peptide" as used herein refers to a peptide comprising one or more functional regions. The term "functional region" as used herein refers to a stretch of amino acids that confer a specific function to the peptide. Examples of functional regions within a multifunctional peptide are (a) macromolecule binding regions, which can condense the macromolecules, (b) lytic regions, (c) cell surface targeting regions, (d) nuclear targeting regions, (e) intracellular targeting regions, (f) intracellular trafficking regions, (g) complex stabilization regions, (h) complex destabilization regions that function after delivery into cells. Hence, a multifunctional peptide may comprise one or more lytic regions and one or more condensing regions. These distinct functional regions may exist within the multifunctional peptides of the invention in an overlapping or non-overlapping manner. These regions may be connected by other stretches of amino acids or by linker moieties as described herein. In addition, the peptide regions may be fused to hydrophobic moieties. These hydrophobic moieties can be selected from preferably cholesterol or cholesterol derivatives and more preferably fatty acid moieties. These hydrophobic regions can be linked to the peptide region of the multifunctional peptide via direct linkages by the peptide N-terminus or by epsilon amino regions of lysine residues. Alternatively, the hydrophobic regions may be linked to the peptide region via a linker molecule. Examples of linker molecules are described herein. Furthermore, the multifunctional peptide may comprise saccharide moieties or surface ligands which may act as targeting molecules that direct the complex to a desired group of cells or a desired region within those cells. Examples of the peptide regions from multifunctional peptides are selected from the group consisting of PKKKRKVGLFKLLEEWLE; PKKKRKVGLFEALEELWEA; PKKKRKVGELGLFKLLEEWLE; PKKKRKVGELLFKLLEWLE;

GLFKLLEEWLEGCKKKKK; (GLFKLLEEWLEGCKKKKK) ₂; GLFKLLEEWLEK-ε- ( CVKRKKKP) ; (GLFKLLEEWLEK-ε- (CVKRKKKP) ) ₂; PKKKRKVCGLFKLLEEWLE; ( PKKKRKVCGLFKLLEEWLE) ₂; GLFKLLEEWLEGCPKKKRKV; (GLFKLLEEWLEGCPKKKRKV) ₂; PKKKRKVGLFKLLEEWLEK-ε-G-Pam₂; PKKKRKVGLFKLLEEWLEK-ε-Pam; YKA (K) _nWKVGLFKLLEEWLE; YKA (K) _nWKVGLFKLLEEWLEK-ε-G-Pam₂; YKA(K)_nWKVGLFKLLEEWLEK-ε-Pam; (K) _nWKGLFKLLEEWLE; (K)_nWKGLFKLLEEWLEK-ε-G-Pam₂; and (K) _nWKGLFKLLEEWLEK-ε-Pam, where pam is a sixteen carbon moiety and n = 0 through 40. These peptide sequences are not meant to be exhaustive or limiting.

In another aspect, the invention relates to a molecular complex for delivering a macromolecule into cells. The molecular complex comprises (a) a lipophilic lytic peptide comprising a peptide moiety and one or more hydrophobic moieties; and (b) the macromolecule molecule.

In yet another aspect, the invention relates to a molecular complex for delivering a macromolecule into cells which comprises (a) a lipophilic binding peptide comprising a peptide moiety and one or more hydrophobic moieties; and (b) the macromolecule.

A preferred embodiment of the invention relates to the molecular complexes, where the macromolecule is a nucleic acid molecule.

In another preferred embodiment, the invention relates to a molecular complex, where the lipophilic binding peptide further comprises one or more saccharide moieties.

The term "saccharide" as used herein refers to a mono- or poly-hydroxylated hydrocarbon that can exist as a straight chain or a ring. Saccharides can exist as monosaccharides, polymeric forms of saccharides (such as disaccharides) and ketoses . Examples of monosaccharides are glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxsose, allose, altrose, glucose, mannose, gulose, idose, galactose, and talose. Examples of ketoses are erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose . Examples of disaccharides are sucrose, lactose, and maltose. The chiral carbon atoms of the saccharide moieties can exist as D and L isomers. Saccharides are also referred to as carbohydrates in the art. The saccharide moiety or moieties can be non-covalently associated with the molecular complex of the invention. Alternatively, the saccharide moiety can be covalently attached to one of the components of the molecular complex. The saccharide moiety can help target the molecular complex of the invention to specific cells within an organism's tissues. By targeting the nucleic acid molecule to a specific cell type in tissues, the saccharide moiety acts as a surface ligand which can bind to the surface of cellular receptors.

The targeting molecules of the invention are not limited to saccharide moieties. Surface ligands are also preferred types of targeting molecules.

The term "surface ligand" as used herein refers to a chemical compound or structure which will bind to a surface receptor of a cell. The term "cell surface receptor" as used herein refers to a specific chemical grouping on the surface of a cell to which the ligand can attach with high affinity and specificity. Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one cell rather than in another type of cell. For example, low density lipoproteins and asialoglycoprotein receptors are specific for hepatocytes . The receptor facilitates the internalization of the ligand and attached molecules. The term "nuclear ligand" as used herein refers to a ligand which will bind a nuclear receptor. The term "nuclear receptor" as used herein refers to a chemical grouping on the nuclear membrane which will bind a specific ligand and facilitate transport of the ligand through the nuclear membrane. Nuclear receptors can be, but are not limited to, those receptors which bind nuclear localization sequences. Non-limiting examples of nuclear ligands include those disclosed in PCT publication WO 93/18759, hereby incorporated by reference herein in its entirety including any figures and drawings. A nuclear localization signal accomplishes the entry of a macromolecule into the nucleus through its interactions, first with cytoplasmic components for directional movement to the nuclear membrane, and second with nuclear membrane components for transport through the nuclear pore into the nucleus .

The surface ligand, the nuclear ligand and/or the lipophilic lytic peptide can be covalently linked directly to the lipophilic binding peptide or can be covalently linked to the binding molecule via a spacer moiety.

In yet another preferred embodiment, the invention relates to the molecular complex, where the saccharide moiety or moieties of the lipophilic binding peptide are connected to the peptide by N-, S-, or 0- linkages. These types of linkages refer to a nitrogen atom, sulfur atom, or oxygen atom existing in the peptide or alternatively a spacer moiety between the peptide and the saccharide. For example, an N-linked sugar can be attached to a peptide moiety by virtue of a covalent link between the D-amino group of lysine and an atom within the sugar. Many examples of N-, S- or 0- linked sugars exist in glycoproteins isolated from a number of organisms.

In another preferred embodiment, the invention relates to a molecular complex, where the saccharide moiety or moieties are connected to the lipophilic binding peptide by one or more spacer moieties.

The term "spacer moiety" as used herein refers to any molecule that separates the saccharide moiety from the peptide moiety of the lipophilic binding peptide. A spacer moiety can be as short as a glycine moiety, or can exist as a longer organic based molecule. In yet another preferred embodiment, the invention relates to the molecular complex, where the spacer moiety or moieties are selected from the group consisting of:

and

rn f. o

where n = 0 through 20. n is preferably less than 20, more preferably less than 15, and most preferably less than 10.

In the latter spacer moiety, saccharide moieties may be covalently attached to the ε-amino groups of either lysine moieties as well as the ε-amino moiety of the terminal lysine.

The hydrophobic moieties may also be connected to the lipophilic peptides of the invention by cleavable bonds . Examples of cleavable bonds well known to those skilled in the art are (a) glutathione sensitive bonds, e.g., those that are disulfide bonds; and (b) acid labile bonds, e.g., cis-acylnitrile bonds. These types of bonds can render the peptides of the invention more soluble once they are released inside of the cell by allowing the peptides to release their hydrophobic components. Specifically, because the cellular environment is reducing, peptides comprising glutathione sensitive linkages to hydrophobic moieties will lose these hydrophobic moieties. In addition, acid labile linkages may cause lipophilic peptides to lose their hydrophobic moieties once the molecular complexes fuse to acidic endosomes. Thus, this feature likely provides more efficient macromolecule delivery once the molecular complex is delivered to cells.

In a preferred embodiment, the invention relates to a molecular complex where the peptide moiety of the lipophilic binding peptide consists of the amino acid sequences selected from the group consisting of KKKKKKKKKWK, (K)_nW, and YKA(K)_nWK, where n = 0 through 40. Preferably n is equal to 4, 5, 6, 7, 8, 10, 12, and 40. In addition, the amino acid sequence of the lipophilic binding peptide can consist essentially of KKKKKKKKKWK, (K)„W, and YKA(K)_nWK, where n = 0 through 40.

In yet another preferred embodiment, the invention relates to the molecular complex, where the hydrophobic moiety or moieties of the lipophilic binding peptide are selected from the group consisting of N-palmitoyl; Nα- palmitoyl; Nε-palmitoyl; Nα, Nε-dipalmitoyl; Nα, α-dipalmityl; Nε, Nε-dipalmityl; and N, N-dipalmityl-glycyl .

The term "N-palmitoyl" as used herein refers to a palmitoyl fatty acid covalently linked to an amino moiety of a peptide, or alternatively an amino moiety of a spacer, forming an amide linkage. "Nα-palmitoyl" refers to a palmitoyl moiety linked to an α amino moiety of an amino acid. "Nε-palmitoyl" refers to a palmitoyl moiety linked to an ε-amino moiety of a lysine residue. The term "N,N- dipalmityl" as used herein refers to a molecule which contains at least one, if not both, palmityl moieties attached to a glycine linker. The term "palmityl" refers to an alkyl moiety comprising sixteen carbon atoms. The palmityl moiety of moieties can be attached to the D-amino moiety of glycine. In another preferred embodiment, the invention relates to the molecular complex, where the lipophilic binding peptide has the following structure:

In preferred embodiments, the invention relates to the molecular complex, where the peptide moiety of the lipophilic lytic peptide consists of a sequence of amino acids selected from the group consisting of GLFEALLELLESLWELLLEA; GLFEALEELWEA; GLFLLEEWLE; GLFLLEEWLEK; GLFEALEELWEAK; GLFEALLELLESLWELLLEAK; Suc-GLFKLLEEWLE; and Suc-GLFKLLEEWLEK, where Sue is a succinyl moiety. In another preferred embodiment, the lipophilic lytic peptide consists essentially of a sequence of amino acids selected from the group consisting of GLFEALLELLESLWELLLEA; GLFEALEELWEA; GLFLLEEWLE; GLFLLEEWLEK; GLFEALEELWEAK; GLFEALLELLESLWELLLEAK; Suc-GLFKLLEEWLE; and Suc- GLFKLLEEWLEK, where Sue is a succinyl moiety. Peptides of the invention can exist in forms in which the carboxyl termini can be free (-COOH) , or blocked with a variety of functional groups . An example of a functional group commonly used to block the carboxyl termini of peptides is an amide moiety (-CONH₂) .

In another preferred embodiment, the invention relates to the molecular complex, where the hydrophobic moiety or moieties of the lipophilic lytic peptide are selected from the group consisting of N-palmitoyl; Nα-palmitoyl; Nε- palmitoyl; Nα, Nε-dipalmitoyl; Nα, Nα-dipalmityl; Nε,Nε- dipalmityl; and N, N-dipalmityl-glycyl .

In yet another preferred embodiment, the invention relates to molecular complexes, where the lipophilic lytic peptide has the following structure:

In yet another preferred embodiment, the invention relates to the molecular complex, where the molecular complex has a -/+/- charge ratio of 1/3/1, 1/2/1, 1/1/1, 4/0/1, 2/1/0, 4/2/1, 4/3/1, and 4/4/1. These ratios are not meant to be limiting as one skilled in the art could readily practice the methods of the invention using any ratio of the components. The -/+/- ratio refers to the phosphate moieties of the nucleic acid molecule / the basic amino acids of the lipophilic binding peptide / the acidic amino acids of the lipophilic lytic peptide ratio. The charge ratio can be determined prior to formulating the nucleic acid molecule, lipophilic binding peptide, and lipophilic lytic peptide. The charge ratio can be calculated theoretically at pH 7.0. Thus, the peptide moiety of a lipophilic binding peptide, which consists of the amino acid sequence KKKKKKKKKWK, will have a charge at pH 7.0 of +9 by virtue of the ten positive charges on each D-amino group of lysine which are neutralized by one negatively charged carboxyl terminus. Fatty acid moieties attached to peptides of the invention will not confer any charge to these peptides .

In yet another preferred embodiment, the invention relates to the molecular complex, where the molecular complex has a -/- charge ratio of 1/4, 1/3, 1/2, and 1/1. The -/- ratio is the phosphate moieties of the nucleic acid molecule / acidic amino acids of the lipophilic lytic peptide ratio. These ratios are not meant to be limiting as one skilled in the art could readily practice the methods of the invention using any ratio of the components disclosed herein.

In yet another preferred embodiment, the invention relates to the molecular complex, where the molecular complex has a -/+ charge ratio of 1/4, 1/3, 1/2, and 1/1. The -/+ ratio is the phosphate moieties of the nucleic acid molecule / basic amino acids of the lipophilic binding peptide ratio. These ratios are not meant to be limiting as one skilled in the art could readily practice the methods of the invention using any ratio of the components disclosed herein. In another aspect, the invention features a method of delivering a macromolecule into the cells of an organism. The method comprises the step of administering to the organism a molecular complex which comprises (a) a lipophilic lytic peptide comprising a peptide moiety and one or more hydrophobic moieties; (b) a lipophilic binding peptide comprising a peptide moiety and one or more hydrophobic moieties; (c) a multifunctional peptide comprising a peptide moiety and optionally comprising one or more hydrophobic moieties; and (d) the nucleic acid molecule .

The term "organism" relates to any living entity comprising at least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal.

The term "mammalian" or "mammal" as used herein refers to any organism that is warm blooded. Mammalian organisms relate to mice, rats, rabbits, goats and sheep, more preferably monkeys and apes, and most preferably humans. The term "administration" or "administering" refers to a procedure for introducing a macromolecule, preferably a nucleic acid molecule, into the body of an organism. The molecular complex can be administered directly to a target tissue or administered by systemic delivery. In particular, administration may be accomplished by direct injection into the tissue. In another embodiment, a molecular complex may be administered intravenously, by hypospray, or in conjunction with polyvinyl pyrrolidone (PVP) . Routes of administration include intramuscular, aerosol, oral, topical, systemic, nasal, ocular, intraperitoneal and/or intratracheal . Molecular complexes of the invention can be administered to an organism by direct injection into the organism or by removing cells from an organism and transforming these cells with the nucleic acid molecules delivered by the molecular complexes of the invention.

As used herein "transformation" or "transformed" is a mechanism of gene transfer which involves the uptake of nucleic acid molecules by a cell or organism. It is a process or mechanism of inducing transient or permanent changes in the characteristics (expressed phenotype) of a cell. Such changes are by a mechanism of gene transfer whereby nucleic acid molecules are introduced into a cell in a form where they can express a specific gene product or alters the expression or effect of endogenous gene products.

In yet another aspect, the invention relates to a method of delivering a macromolecule into the cells of an organism, which comprises the step of administering to the organism a molecular complex comprising, (a) a lipophilic lytic peptide comprising a peptide moiety and one or more hydrophobic moieties; and (b) the macromolecule. This type of molecular complex delivers the macromolecule to cells in a non-condensed state, but in a state that is active in vivo . Examples of this type of non-condensed delivery of macromolecules to cells are provided herein by example.

In another aspect, the invention relates to a method of delivering a macromolecule into the cells of an organism, which comprises the step of administering to the organism a molecular complex comprising: (a) a lipophilic binding peptide comprising a peptide moiety and one or more hydrophobic moieties; and (b) the macromolecule. This type of molecular complex delivers the macromolecule to cells in a condensed state, a state that is active in vivo . Examples of this type of condensed delivery of macromolecules to cells are provided herein by example. The condensed molecular complexes of the invention are stable in vivo as they retain their condensed properties in the presence of physiological concentrations of salt. The condensed macromolecules can exist as colloidal suspensions. Colloidal dispersions consist of at least two discrete phases, namely, one or more dispersed internal phases and a continuous external phase called a dispersion medium or vehicle. The diameter of the colloidal particles in the internal phases can be determined by light scattering experiments. The diameter of the colloidal particles are preferably less than 200 nm in diameter and more preferably less than 100 nm in diameter for the methods described herein. The properties of colloidal particles are well known to those skilled in the art and are reviewed in basic pharmaceutical sciences textbooks.

In a preferred embodiment, the invention relates to the method of delivering a macromolecule to cells where the macromolecule is a nucleic acid molecule.

In a preferred embodiment, the invention relates to the method of delivering a macromolecule to cells where the step of administering the molecular complex consists of contacting the cells of the organism with the molecular complex. When cells exist within an organism, the cells can be contacted by piercing the surface of the organism with a hypodermic needle and by directly releasing the molecular complex through the needle into the cells of the organism. When the cells exist outside of the organism, the cells can be contacted by simply adding a formulation comprising the molecular complexes of the invention to the medium bathing the cells.

In another preferred embodiment, the invention relates to the method of delivering a macromolecule to cells, where the step of administering the molecular complex consists of techniques selected from the group consisting of parenteral injection, intramuscular injection, intravenous injection, intraarticular, and administration by inhalation. Parent- eral administration includes intravenous, subcutaneous, intraperitoneal, intramuscular, and intramedullary injection. These modes of administration are well-known to those skilled in the medical profession and are described in readily obtained manuals for performing such modes of administration .

In yet another preferred embodiment, the invention relates to the method of delivering a macromolecule to cells, where the nucleic acid molecule comprises a gene.

The term "gene" as used herein refers to a recombinant nucleic acid molecule that encodes a polypeptide in cells.

In another preferred embodiment the invention relates to the method of delivering a macromolecule to cells, where the gene is expressed in the cells.

The term "expressed" as used herein refers to a process in which DNA is transcribed into RNA and/or a process in which RNA is translated into a polypeptide in a cell. DNA is transcribed into RNA in a cell by the operation of RNA polymerase on the DNA molecule. RNA is translated into a polypeptide in the cell when a ribosome binds to the RNA and recruits tRNA molecules to compile amino acids into a polypeptide. Examples are provided herein which demonstrate the method of expressing a gene in cells . Examples set forth herein describe the methods of contacting the cells with one of the molecular complexes of the invention, the type of surface ligand necessary for targeting the molecular complex to specific cells, and the subsequent expression of the gene from the nucleic acid molecule. The gene can be expressed when the complex comprises the nucleic acid molecule and a lipophilic lytic peptide as well as a lipophilic binding peptide. Expression of a gene can also be achieved with a molecular complex containing only the nucleic acid and a lipophilic lytic peptide. Furthermore, expression of a gene can be achieved by contacting cells with a molecular complex of the invention containing only the nucleic acid molecule and a lipophilic binding peptide. In a preferred embodiment, the invention relates to the method of delivering a macromolecule to cells, where the organism is a mammal.

In yet another preferred embodiment, the invention relates to the method of delivering a macromolecule to cells, where the macromolecule is delivered to a specific tissue in the organism.

The term "specific tissue" as used herein refers to delivering the nucleic acid molecule using methods of the invention to one set of tissues over another set of tissues. Hence the specific delivery of a nucleic acid molecule to a tissue refers to the delivery of a nucleic acid to one tissue over another in a ratio greater than one to one. An example of the delivery of a nucleic acid molecule to a specific tissue is set forth herein by example, where the nucleic acid molecules expressed in liver at higher expression levels than the nucleic acid molecules expressed in lung.

In other preferred embodiments, the invention relates to the method of delivering macromolecules to cells, where the specific tissue is selected from liver, lung, spleen, kidney, heart, muscle, and blood.

In preferred embodiments, the invention relates to methods of delivering nucleic acid molecules to cells where the nucleic acid molecule comprises a gene, and where the gene is expressed in a specific tissue.

In another preferred embodiment, the invention relates to the method of delivering macromolecules to cells, where the macromolecule is delivered to a specific cell type in the organism.

The term "specific cells" as used herein refers to delivering the nucleic acid molecule using methods of the invention to one set of tissues over another set of tissues. Hence the specific delivery of a nucleic acid molecule to a tissue refers to the delivery of a nucleic acid to one tissue over another in a ratio greater than one to one. An example of the delivery of a nucleic acid molecule to a specific tissue is set forth herein by example, where the nucleic acid molecules in liver at higher expression levels than the nucleic acid molecules express in lung.

In another preferred embodiment, the invention relates to the method of delivering nucleic acid molecules to cells, where the specific cell type is selected from the group consisting of Kupffer cells, adipocyte cells, red blood cells, white blood cells, macrophages, synoviolcytes, and bone forming cells.

As used herein the term "bone-forming cell" refers to those cells which promote bone growth. Non-limiting examples include osteoblasts, stromal cells, inducible osteoprogenitor cells, determined osteoprogenitor cells, chondrocytes, as well as other cells capable of aiding bone formation . In preferred embodiments, the invention relates to methods of delivering nucleic acid molecules to cells, where the nucleic acid molecule comprises a gene and where the gene is expressed in a specific cell type.

In other preferred embodiments, the invention relates to the method of delivering nucleic acid molecules to cells, where the lipophilic binding peptide further comprises one or more saccharide moieties.

In a preferred embodiment, the invention relates to a method of delivering nucleic acid molecules to cells, where the saccharide moiety or moieties of the lipophilic binding peptide are selected from the group consisting of galactose, glucose, and sucrose. In yet another preferred embodiment, the invention relates to methods of delivering macromolecules to cells, where the saccharide moiety or moieties of the lipophilic binding peptide are connected to the peptide by N-, 0-, or S- linkages.

In another preferred embodiment, the invention relates to the method of delivering macromolecules to cells, where the saccharide moiety or moieties are connected to the lipophilic binding peptide by one or more spacer moieties. In other preferred embodiments, the invention relates to the method of delivering macromolecules to cells, where the spacer moiety or moieties are selected from the group consisting of:

and

In a preferred embodiment, the invention relates to the method of delivering macromolecules to cells, where the peptide moiety of the lipophilic binding peptide consists of the amino acid sequences selected from the group consisting of KKKKKKKKKWK, (K)_nW, and YKA(K)_nWK, where n = 0 through 40. Preferably n is equal to 4, 5, 6, 7, 8, 10, 12, and 40. In another preferred embodiment, the peptide moiety of the lipophilic binding peptide consists essentially of the amino acid sequences selected from the group consisting of KKKKKKKKKWK, (K)_nW, and YKA(K)_nWK, where n = 0 through 40. In yet another preferred embodiment, the invention relates to the method of delivering macromolecules to cells, where the hydrophobic moiety or moieties of the lipophilic binding peptide are selected from the group consisting of N- palmitoyl; Nα-palmitoyl; Nε-palmitoyl; Nα, Nε-dipalmitoyl; Nα,Nα-dipalmityl; Nε,Nε-dipalmityl; and N, N-dipalmityl- glycyl.

In another preferred embodiment, the invention relates to the method of delivering macromolecules to cells, where the lipophilic binding peptide has the following structure:

In yet another preferred embodiment, the invention relates to the method of delivering nucleic acid molecules to cells, where the peptide moiety of the lipophilic lytic peptide consists of a sequence of amino acids selected from the group consisting of GLFEALLELLESLWELLLEA; GLFEALEELWEA; GLFLLEEWLE; GLFLLEEWLEK; GLFEALEELWEAK; GLFEALLELLESLWELLLEAK; Suc-GLFKLLEEWLE; and Suc-GLFKLLEEWLEK, where Sue is a succinyl moiety. In another preferred embodiment, the peptide moiety of the lipophilic lytic peptide consists essentially of a sequence of amino acids selected from the group consisting of GLFEALLELLESLWELLLEA; GLFEALEELWEA; GLFLLEEWLE; GLFLLEEWLEK; GLFEALEELWEAK; GLFEALLELLESLWELLLEAK; Suc-GLFKLLEEWLE; and Suc-GLFKLLEEWLEK, where Sue is a succinyl moiety.

In other preferred embodiments, the invention relates to methods of delivering macromolecules to cells, where the hydrophobic moiety of the lytic peptide is selected from the group consisting of N-palmitoyl; Nα-palmitoyl; Nε-palmitoyl; Nα, Nε-dipalmitoyl; Nα,Nα-dipalmityl; Nε, Nε-dipalmityl; and N, N-dipalmityl-glycyl .

In another preferred embodiment, the invention relates to the method of delivering macromolecules to cells, where the lipophilic lytic peptide has the following structure:

.

In yet another preferred embodiment, the invention relates to the method of delivering macromolecules to cells where the molecular complex has a -/+/- charge ratio of 1/3/1, 1/2/1, 1/1/1, 4/0/1, 2/1/0, 4/2/1, 4/3/1, and 4/4/1, where the -/+/- ratio is phosphate moieties of the nucleic acid molecules / basic amino acids of the lipophilic binding peptides / acidic amino acids of the lipophilic lytic peptide ratio. These ratios are not meant to be limiting as one skilled in the art could readily practice the methods of the invention using any ratio of the components disclosed herein.

In preferred embodiments, the invention relates to the method of delivering macromolecules to cells, where the molecular complex has a -/- charge ratio of 1/4, 1/3, 1/2, and 1/1 where the -/- ratio is the phosphate moieties of the nucleic acid molecule / acidic moieties of the lipophilic lytic peptide ratio. These ratios are not meant to be limiting as one skilled in the art could readily practice the methods of the invention using any ratio of the components disclosed herein.

In preferred embodiments, the invention relates to the method of delivering macromolecules to cells, where the -/+ charge ratio is 1/3, 1/2, and 1/1, where the -/+ ratio is the phosphate moieties of the nucleic acid molecule / basic amino acids of the lipophilic binding peptide ratio. These ratios are not meant to be limiting as one skilled in the art could readily practice the methods of the invention using any ratio of the components disclosed herein. In preferred embodiments, the invention relates to the method of delivering macromolecules to cells, where the organisms is fasted before administering the molecular complex.

The term "fast" or "fasted" as used herein refers to the diet of an organism which contains less nutrients than it would normally intake. Thus, an animal being delivered a molecular complex of the invention may be restricted to a diet of only water for a period of time. Alternatively, a patient administered a molecular complex of the invention may be given instructions to limit itself to a diet of only water for a period of time. The organism may also be administered or given instructions to intake only a limited number of restricted nutrients during the fasting period. The period of time for fasting may be as short as six hours and could extend to more than 48 hours. A preferred amount of time for fasting is a 24 hour period.

Preferred embodiments related to fasting capitalize upon the physiological changes that occur upon fasting to facilitate in vivo delivery of a macromolecule. In blood, the fasting state is characterized by a decrease in the concentration of serum lipoprotein particles. It has been recently shown in rats that fasting is also accompanied by a significant decrease in Kupffer cell phagocytic activity and cytokine production in the liver. Sankary et al . , 1995, Hepa tology 22 (4) : 1236-1242. Furthermore, it has been demonstrated that intra-lysosomal protease activity is diminished in the livers of fasting rats. Harikumar et al . , 1985, Biochem-Int . 11 (3) : 311-318. The effects of fasting are not limited to the blood and liver, as it has been shown that fasting can reduce protease content in rat lung mast cells with a subsequent decrease in histamine synthesis. Rouleau et al . , 1994, Agen ts-Actions 42 (1 -2) : 7-12. In light of these known responses to fasting, it is logical to assume that delivery of macromolecules, especially those to the liver and lungs, can be improved by fasting subjects prior to the administration of molecular complexes.

The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention and from the claims.

Detailed Description Of The Preferred Embodiments

The invention relates in part to molecular complexes for delivering macromolecules (e.g., nucleic acid molecules) into cells. These molecular complexes can comprise lipophilic peptides that condense and stabilize the macromolecule. These lipophilic binding peptides can also comprise moieties that target the macromolecule to specific cells in an organism. In addition, the molecular complexes may comprise lipophilic peptides that can lyse endosomal structures within cells and thereby enhance delivery of the macromolecule into cells.

The invention also relates in part to methods of delivering macromolecules to cells using the molecular complexes described herein. Thus, the present invention provides for efficient and specific delivery of nucleic acid molecules into cells.

The molecular complexes described herein can contain multiple components. The molecular complexes may comprise, consist of, or consist essentially of: (1) a macromolecule of a known chemical composition, specifically a nucleic acid molecule; (2) a lipophilic peptide capable of stabilizing and condensing the macromolecule, preferably a nucleic acid molecule; (3) a lipophilic lytic peptide that facilitates the delivery of macromolecules into a cell by lysing endosomal bodies in which the molecular complex is trapped in cells; (4) a targeting moiety that recognizes and binds to a cell surface receptor or antigen or is capable of entering a cell through cytotic mechanisms; (5) a moiety that is capable of delivering the molecular complex to cell nuclei or initiating delivery of the molecular complex to cell nuclei; and/or (6) a macromolecule or nucleic acid molecule capable of covalently binding or reversibly binding to the moieties of (2), (3), (4), and/or (5). Additionally,

(2), (3), (4), and/or (5) may be covalently or no - covalently bonded or associated with each other. I . Macromolecules Of The Invention

A variety of macromolecules can be delivered to a cell using the molecular complexes of the invention. These macromolecules include proteins, peptides, lipids, carbohydrates, peptidomimetics, organic molecules, and preferably nucleic acid molecules. A preferred type of polypeptide delivered to cells using the molecular complexes of the invention are toxins, such as ricin and other cytotoxic agents. The specific delivery of toxins to cells can potentially eradicate harmful cells in an organism. This application is particularly useful in the treatment of certain cancers .

Nucleic acid molecules can refer to DNA and RNA molecules. The nucleic acid molecules can exist in a naked state, in which the DNA or RNA are prepared such that they do not contain a significant ratio of associated molecules or salts. Alternatively, the nucleic acid molecules can exist in a state in which they are complexed to other molecules, such as radio-labels or dyes. These molecules can be associated with the nucleic acid molecules in a covalent or non-covalent reversibly associated fashion.

The nucleic acid molecules may exist as recombinant vectors containing a variety of nucleic acid elements . These elements can include promoter elements, ribosome binding elements, drug resistance elements, replication binding elements, and genes.

A variety of proteins and polypeptides can be encoded by a gene harbored within a nucleic acid molecule of the invention. Those proteins or polypeptides include hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, cytokines, viral antigens, parasitic antigens, bacterial antigens and chemically synthesized polymers and polymers biosynthesized and/or modified by chemical, cellular and/or enzymatic processes. Specific examples of these compounds include proinsulin, insulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin- like growth factor II, insulin growth factor binding proteins, epidermal growth factor, TGF-α, TGF-β, dermal growth factor (PDGF) , angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin) , matrix proteins (Type IV collagen, Type VII collagen, laminin) , oncogenes ( ras, fos, myc, erb, src, sis, j un) , E6 or E7 transforming sequence, p53 protein, cytokine receptor, IL-1, IL-6, IL-8, IL-2, α, β, or γlFN, GMCSF, GCSF, viral capsid protein, and proteins from viral, bacterial and parasitic organisms. Other specific proteins or polypeptides which can be expressed include: phenylalanine hydroxylase, α-1-antitrypsin, cholesterol-7α-hydroxylase, truncated apolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoprotein Al, LDL receptor, scavenger receptor for oxidized lipoproteins, molecular variants of each, VEGF, and combinations thereof. Other examples are clotting factors, apolipoproteins, drugs, tumor antigens, viral antigens, parasitic antigens, and bacterial antigens. Other examples can be found above in the discussion of nucleic acid. One skilled in the art readily appreciates that these proteins belong to a wide variety of classes of proteins, and that other proteins within these classes can also be used. These are only examples and are not meant to be limiting in any way.

It should also be noted that the genetic material which is incorporated into the cells from the molecular complex includes (1) nucleic acid molecules not normally found in the cells; (2) nucleic acid molecules which are normally found in the cells but not expressed at physiological significant levels; (3) nucleic acid molecules normally found in the cells and normally expressed at physiological desired levels; (4) other nucleic acid molecules which can be modified for expression in cells; and (5) any combination of the above .

In addition, the macromolecules of the invention may relate to nucleic acid molecules that can cleave RNA molecules in specific regions. The nucleic acid molecules which can cleave RNA molecules are referred to in art as ribozymes, which are RNA molecules themselves. Ribozymes can bind to discrete regions on a RNA molecule, and then specifically cleave a region within that binding region or adjacent to the binding region. Ribozyme techniques can thereby decrease the amount of polypeptide translated from formerly intact message RNA molecules. The methods of the invention can enhance the delivery of ribozyme nucleic acid molecules to cells using the molecular complexes described herein.

Furthermore, the macromolecules of the invention may relate to nucleic acid molecules which can bind to specific RNA sequences or DNA sequences. Nucleic acid molecules which are designed to specifically bind to regions on RNA molecules or DNA molecules are utilized in antisense techniques. Antisense techniques include the delivery of RNA or DNA to cells that are homologous to message RNA sequences in the cell or two specific sequences in genomic DNA. The antisense nucleic acid molecules bind to the message RNA molecules and block the translation of these message RNA molecules. Antisense techniques can thereby block or partially block the synthesis of particular polypeptides in cells. The methods of the invention can enhance the delivery of antisense nucleic acid molecules to cells using the molecular complexes described herein. II . Lipophilic Binding Peptides

Lipophilic binding peptides can form non-covalent complexes with macromolecules, preferably nucleic acid molecules, and thereby condense these molecules into small particles. The small diameter particles are then capable of diffusing into the cells. In addition, these smaller particles can render nucleic acid molecules more stable with respect to degradation within the cells. Furthermore, the particles are colloidally stable, meaning that the particles remain as an aqueous suspension comprising physiological concentrations of salt and do not aggregate. These properties enable lipophilic binding peptides to facilitate the delivery of nucleic acid molecules from the outside of cells, through the plasma membrane, and, if desired, to the nucleus of cells.

Lipophilic binding peptides contain at least one hydrophobic moiety, preferably a fatty acid moiety, that enhances the hydrophobicity of the peptide and resultant molecular complex, and thereby increases the affinity of the molecular complex for lipid membranes.

In general, parameters that are important for lipophilic binding peptides include the following. First, the peptide must contain sufficient basic residues, i.e., lysine, arginine, ornithine, and γ-aminobutyric acid residues, to permit ionic interaction with anionic macromolecules, such as nucleic acid molecules. Second, the peptide must have sufficient length to form a stable helix, eleven or twelve residues, and condense the DNA to small particles. Third, the peptide helix that forms upon interaction with DNA can be stabilized by mechanisms similar to leucine zipper formation which renders a condensing agent less susceptible to decreasing condensation in response to changes in ionic strength. Finally, the lysine or arginine sequence of the binding peptide may serve as a nuclear localization sequence.

Ill . Lipophilic Lytic Peptides

The present invention also relates in part to lipophilic lytic peptides to avoid the problems of endosomal/lysosomal degradation in the delivery of macromolecules, preferably nucleic acid molecules, to cells. Like the lipophilic binding peptides, the lipophilic lytic peptides also harbor a peptide moiety and one or more hydrophobic moieties, preferably fatty acids. The peptide moiety of the lipophilic lytic peptides confer a endosomal lysis property to nucleic acid complexes such that they are not degraded by lysosomes in the cell.

The lytic moiety of the lipophilic peptide can function by: (1) a membrane fusion mechanism, i.e., fusogenic, whereby the lipophilic lytic peptide associates or fuses with the endosomal membrane to allow the endosomal contents to leak into the cytoplasm; (2) a membrane destabilization mechanism whereby the lipophilic lytic peptide disrupts the structural organization of the endosomal membrane thereby causing leakage through the endosome into the cytoplasm; or (3) other known or unknown mechanisms which cause endosomal lysis. This term includes, but is not limited to, synthetic compounds such as a GLFEALLELLESLWELLLEA peptide, lytic peptides, or derivatives thereof, and naturally occurring lytic agents such as viruses and virus components. For example, lytic peptides may also function by fusing or destabilizing the cell membrane, thereby allowing macromolecules into the cytoplasm without entering endosomes .

In general, parameters that are important for amphiphilic peptide mediated endosomal lysis include the following. First, Hydrophobicity : The peptide must have a high enough hydrophobicity for the hydrophobic face of the peptide to interact with and penetrate phospholipid- cholesterol membranes, i.e., lipid binding per se is not sufficient. Red cell hemolysis assays give indications of which peptides will have useful activity. Second, Peptide aggregation: The ability to aggregate plays an important role in lysis and transfection. Third, pH sensitivity: The amphiphilic peptide is preferably pH sensitive. Lysis activity can be controlled by the introduction of lysine, arginine and histidine residues into the hydrophilic face of the lytic peptide. Fourth, Lipid membrane interaction: The peptide must have a hydrophobic carboxyl terminal to permit interaction with lipid membranes, e.g., tyrosine substitution for tryptophan greatly reduces activity. Finally, Peptide chain length: The length is preferably greater than ten residues in order to obtain stable helix formation and lipid membrane penetration and rupture.

A preferred lytic peptide is the JTS-1 peptide or derivatives thereof. The amino acid sequence of JTS-1 lytic peptide is GLFEALLELLESLWELLLEA. One skilled in the art will readily appreciate and understand that such nomenclature is the standard notation accepted in the art for designating amino acids. The JTS-1 lytic peptide and derivatives are designed as an α-helix, which contains a sequence of amino acids such that the side chains are distributed to yield a peptide with hydrophobic and hydrophilic sides. Such α-helixes are termed amphipathic or amphiphilic. The hydrophobic side contains highly apolar amino acid side chains, both neutral and non-neutral. The hydrophilic portion of the peptide contains an extensive number of glutamic acids but could also contain aspartic acid, as well as polar or basic amino acids. The JTS-1 peptide would include any derivatives or modifications of the backbone thereof. The lytic peptide undergoes secondary structure changes at acidic pH resulting in the formation of oligomeric aggregates which possess selective lytic properties. Following entry into the cell, the transforming nucleic acid may recombine with that of the host. Such transformation is considered stable transformation in that the introduction of gene(s) into the chromosome of the targeted cell where it integrates and becomes a permanent component of the genetic material in that cell. Gene expression after stable transformation can permanently alter the characteristics of the cell leading to stable transformation. In addition, the transforming nucleic acid may exist independently as a plasmid or a temperate phage, or by episomes. An episomal transformation is a variant of stable transformation in which the introduced gene is not incorporated in the host cell chromosomes but rather remains in a transcriptionally active state as an extrachromosomal element .

IV. Cell Targeting Moieties

In addition to the lipophilic lytic peptides and lipophilic binding peptides, the present invention relates in part to molecular complexes containing one or more molecules which target the molecular complexes to specific tissues. Examples of these targeting molecules are surface ligands as well as nuclear ligands. The surface ligands are capable of binding to a cell surface receptor and entering a cell via cytotic mechanisms (e.g., endocytosis, potocytosis, pinocytosis) . By using surface ligands specific to certain cells, nucleic acid molecules can be delivered directly to the desired tissue using the molecular complexes of the invention. The nuclear ligands are also capable of recognizing and transporting molecular complexes, preferably nucleic acid molecules, through the nuclear membrane to the nucleus of a cell. Such nuclear ligands help enhance the targeting nucleic acid molecules to the nucleus.

A receptor is a molecule to which a ligand binds specifically and with relatively high affinity. It is usually a protein or a glycoprotein, but may also be a glycolipid, a lipid polysaccharide, a glycosaminoglycan or a glycocalyx. For purposes of this invention, epitopes to which an antibody or its fragments binds is construed as a receptor since the antigen/antibody complex undergoes endocytosis. Furthermore, surface ligand includes anything which is capable of entering the cell through cytosis (e.g., endocytosis, potocytosis, pinocytosis).

A cell surface receptor includes, but is not limited to, a folate receptor, biotin receptor, lipoic acid receptor, low-density lipoprotein receptor, asialoglyco- protein receptor, insulin-like growth factor type II/cation- independent mannose-6-phosphate receptor, calcitonin gene- related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilage induction factor receptor or glycosylphosphatidylinositol (GPI) -anchored proteins (e.g., β- adrenergic receptor, T- cell activating protein, Thy-1 protein, GPI-anchored 5' nucleotidase) . These are nonlimiting examples.

Ligands are chemical compounds which can bind to receptors. Ligands of the invention include naturally occurring ligands such as asialoorosomucoid, asialoglyco- protein, folate, lipoic acid, biotin, as well as those compounds listed in PCT publication WO 93/18759, hereby incorporated by reference. Ligands of the invention also relate to synthetic molecules which can bind to the extracellular or intracellular portions of receptors with high affinity. High affinity represents an equilibrium dissociation constant between 10 μM to 0.1 pM.

One skilled in the art will readily recognize that the ligand chosen will depend on which receptor is being bound. Since different types of cells have different receptors, this provides a method of targeting nucleic acid molecules to specific cell types, depending on which cell surface ligand is used. Thus, the preferred cell surface ligand may depend on the targeted cell type.

The spacers described herein refer to a chemical structures which link two molecules to one another. The spacer normally binds each molecule on a different part of the spacer molecule. The spacer can be a hydrophilic molecule comprising about 6 to 30 carbon atoms. The spacer can also contain between 6 to 16 carbon atoms. In addition to the spacer molecules set forth herein by example, the spacer moieties can include, but are not limited to, a hydrophilic polymer of [ ( gly) { ser) ₃] _k wherein i ranges from 1 to 6, j ranges from 1 to 6, and k ranges from 3 to 20. In addition, the spacer and binding molecule compounds include, but are not limited to, those compounds disclosed in PCT publication WO 93/18759, WO 96/40958, and PCT US 97/00454

(U.S. Application Serial No. 08/584,043), hereby incorporated by reference herein in their entirety including all figures, tables, and drawings. Furthermore, the spacer may include, but is not limited to, repeating omega-amino acid of the structure [NH- (CH₂CH₂) _n-CO-]_m, where n = 1-3 and m = 1-20, a disulfide structure (CH₂CH₂-S-S-CH₂CH₂-) _n, where n = 1-20, or an acid sensitive multifunctional molecule with the structure -CO-CH₂-C = CH-CO-NH-CH₂-CH₂-S - .

COOH

V. Therapeutic Applications of the Invention The methods of the invention can relate to cultured cells, which can utilized in basic research and therapeutic applications. The methods of the invention can also relate to a transgenic animal whose cells contain the nucleic acid molecules delivered via the molecular complexes of the invention. These cells include germ or somatic cells. Transgenic animal models can be used for dissection of molecular carcinogenesis and disease, assessing potential chemical and physical carcinogens and tumor promoters, exploring model therapeutic avenues and livestock agricultural purposes.

The methods of use also include a method of treating humans, which is another aspect of the present invention. The method of treatment includes the steps of administering the nucleic acid transporters as described above so as to deliver a desired nucleic acid molecule to a cell or tissue for the purposes of expression of the nucleic acid molecular by the cells or tissues. Cell or tissue types of interest can include, but are not limited to, liver, muscle, lung, endothelium, joints, skin, bone, and blood. The methods of the invention include methods for delivering macromolecules into specific cells using the molecular complexes of the invention. In particular, macromolecules can be delivered into hepatocyte cells by contacting these cells with a molecular complex containing a targeting moiety that specifically binds to receptors expressed on the outside of cells. For this purpose, the asialoorosomucoid protein can be used as a cell surface ligand. Thus, a combination of the asialoorosomucoid protein attached to a lipophilic binding peptide of the invention, a lipophilic lytic peptide of the invention, and the macromolecule to be delivered to cells can comprise a molecular complex formulation suitable for macromolecule delivery to hepatocyte cells.

Preferred modes of delivery of macromolecules are to hepatocyte cells within the liver, described herein by example . A method for delivering macromolecules to bone-forming cells, for example, comprises the step of contacting the bone-forming cell with a molecular complex of the invention. In particular, the surface ligands used for this purpose can include, but are not limited to, bone morphogenetic protein or cartilage induction factor.

A method for delivering macromolecules to synoviolcytes or macrophages, for example, can utilize the molecular complexes and methods set forth herein. The molecular complexes can utilize ligands recognized by synoviolcytes and/or macrophages.

VI . Administration

Administration as used herein refers to the route of introducing the molecular complexes of the invention into the body of cells or organisms. Administration includes parenteral, intravenous, intramuscular, topical, or oral methods of delivery. The molecular complexes of the invention can also be administered directly to a target tissue or through systemic delivery.

Prior to administration, the molecular complexes of the invention can be formulated with at least one other type of molecule. For example, the molecular complexes can be dialyzed into a solution containing a saccharide, such as glucose, and then dialyzed again in saline solution. The molecular complexes can also be formulated with other molecules such as PVP as described herein. Formulation techniques are provided herein by example. In particular, the present invention can be used for administering nucleic acid molecules to cells. Routes of administration include intramuscular, aerosol, nasal, oral, topical, systemic, ocular, intraperitoneal and/or intratracheal . A preferred method of administering molecular complexes is by intravenous delivery. Another preferred method of administration is by direct injection into the cells.

In addition, another means to administer the nucleic acid transporters of the present invention is by using a dry powder form for inhalation. One compound which can be used is polyvinylpyrrolidone ("PVP"), an amorphous powder. PVP is a polyamide that forms complexes with a wide variety of substances and is chemically and physiologically inert. Specific examples of suitable PVP ' s are Plasdone-C®15, MW 10,000 and Plasdone-C®30, MW 50,000. Furthermore, molecular complexes of the invention can be administered through an aerosol composition or liquid form into a nebulizer mist and thereby inhaled.

The special delivery route of any selected molecular complex will depend on the particular use for the nucleic acid associated with the nucleic acid transporter. In general, a specific delivery program for each nucleic acid transporter used will focus on uptake with regard to the particular targeted tissue, followed by demonstration of efficacy. Uptake studies will include uptake assays to evaluate cellular uptake of the nucleic acid and expression of the specific nucleic acid of choice. Such assays will also determine the localization of the target nucleic acid after uptake, and establishing the requirements for maintenance of steady-state concentrations of expressed protein. Efficacy and cytotoxicity is then tested. Toxicity will not only include cell viability but also cell function.

The chosen method of delivery should result in cytoplasmic accumulation and optimal dosing. The dosage will depend upon the disease and the route of administration but should be between 1-1000 mg/kg of body weight/day. This level is readily determined by standard methods. It could be more or less depending on the optimal dosing. The duration of treatment extends through the course of the disease symptoms, possibly continuously. The number of doses will depend upon disease delivery vehicle and efficacy data from clinical trials. In vivo delivery may also be enhanced by adding other volumes such as 1 mL injection volume per 25 grams of body weight.

Methods of establishing the levels of nucleic acid molecules delivered to cells by the molecular complexes of the invention are provided herein.

A. Administration of Macromolecules to Cultured Cells

The methods of the invention can be utilized to deliver macromolecules to cultured cells. This application is useful for basic research studies, pharmacological studies, and medical treatments, for example. In particular, medical treatments can be conducted by removing dysfunctional cells from a patient, culturing the cells, modifying the cells by methods of the invention, and replacing the modified cells into the patient. The methods of the invention can be utilized to deliver a gene therapeutic into cultured cells or deliver other compounds into cells. Microinjection and transfection techniques required for this application are well known to persons of ordinary skill in the art.

B . Administration of Macromolecules to Muscle

The muscular dystrophies are a group of diseases that result in abnormal muscle development, due to many different reasons. These diseases can be treated by using the direct delivery of genes with the nucleic acid transporters of the present invention resulting in the production of normal gene product. The methods of the invention can be utilized to deliver nucleic acid molecules that produce various antigens for vaccines against a multitude of infections of both viral and parasitic origin. The detrimental effects caused by aging can also be treated using the methods of the invention. Since the injection of growth hormone protein promotes growth and proliferation of muscle tissue, a growth hormone gene can be delivered to muscle, resulting in both muscle growth and development, which is decreased during the later portions of the aging process. Genes expressing other growth related factors can be delivered, such as Insulin Like Growth Factor-I (IGF-I) . Furthermore, any number of different genes may be delivered by this method to the muscle tissue.

IGF-I can be utilized as a ligand that is linked to the molecular complex of the invention for the delivery of nucleic acid molecules to muscle. IGF-I is transported into cells by receptor-mediated endocytosis. This polypeptide is 70 amino acids in length and is a member of the growth promoting polypeptides structurally related to insulin. It is involved in the regulation of tissue growth and cellular differentiation affecting the proliferation and metabolic activities of a wide variety of cell types, since the polypeptide has receptors on many types of tissue. The advantage of an IGF-I/ molecular complex is that the specificity and the efficiency of the delivery is greatly increased due to a great number of cells coming into contact with the ligand/molecular complex with uptake through receptor-mediated endocytosis. Thus, using the molecular complexes of the invention coupled to IGF-I provides a technique for the delivery of nucleic acid molecules to muscle cells. This technique provides a treatment of diseases and abnormalities that affect muscle tissues. In addition to the above, Factor IX can also be delivered to the muscle cells. DNA encoding Factor IX can be delivered to cells using the molecular complexes of the invention. As a result, the molecular complexes of the invention can incorporate nucleic acid molecules encoding Factor IX for the treatment of cells that are Factor IX deficient and are susceptible to disease and abnormalities due to such a deficiency. DNA encoding Factor IX can be coupled or associated with lipophilic binding peptides and/or lipophilic lytic peptides as described herein. The complex can then be delivered directly to muscle cells for expression. A preferred ratio of DNA to binding peptide to lytic peptide is 1:3:1. Direct injection of these molecular complexes to the muscle tissue is preferred. Use of the molecular complexes of the invention for the delivery of nucleic acid molecules expressing Factor IX to muscle cells provides treatment of diseases and abnormalities that affect muscle tissues.

C. Administration of Macromolecules to Osteogenic Cells

There are many other problems that occur during the aging process, but one major problem is osteoporosis, which is the decrease in overall bone mass and strength. The methods of the present invention can be used to deliver genes to cells that promote bone growth. Osteoblasts are the main bone forming cells in skeletal organisms, but there are other cells that are capable of aiding in bone formation. For example, stromal cells found in bone marrow are the source of stem cells for osteoblasts. Stromal cells differentiate into a population of cells known as Inducible Osteoprogenitor Cells (IOPC) , which under induction of growth factors, differentiate into Determined Osteoprogenitor Cells (DOPC) . It is this population of cells that mature directly into bone producing cells. The IOPCs are also found in muscle and soft connective tissues. Another cell involved in the bone formation process is the cartilage-producing cell known as the chondrocyte.

A factor that stimulates the differentiation of IOPCs is Bone Morphogenetic Protein (BMP). This 19,000 MW protein was first identified from demineralized bone. Another factor similar to BMP is Cartilage Induction Factor (CIF) , which functions to stimulate IOPCs to differentiate also, starting the pathway of cartilage formation, cartilage calcification, vascular invasion, resorption of calcified cartilage, and finally induction of new bone formation. Cartilage Induction Factor has been identified as being homologous to Transforming Growth Factor β.

Since osteoblasts are involved in bone production, genes that enhance osteoblast activity can be delivered directly to these cells. Genes can also be delivered to the IOPCs and the chondrocytes, which can differentiate into osteoblasts, leading to bone formation. BMP and CIF are the ligands that can be used to deliver genes to these cells. Genes delivered to these cells promote bone formation or the proliferation of osteoblasts. The polypeptide, IGF-I stimulates growth in hypophysectomized rats which could be due to specific uptake of the polypeptide by osteoblasts or by the interaction of the polypeptide with chondrocytes, which result in the formation of osteoblasts. Other specific bone cell and growth factors can be used through the interaction with various cells involved in bone formation to promote osteogenesis .

Nonlimiting examples of genes expressing the following growth factors which can be delivered to these cell types are Insulin, Insulin-Like Growth Factor-1, Insulin-Like

Growth Factor-2, Epidermal Growth Factor, Transforming Growth Factor-α, Transforming Growth Factor-β, Platelet Derived Growth Factor, Acidic Fibroblast Growth Factor, Basic Fibroblast Growth Factor, Bone Derived Growth Factors, Bone Morphogenetic Protein, Cartilage Induction Factor, Estradiol, and Growth Hormone. All of these factors have a positive effect on the proliferation of osteoblasts, the related stem cells, and chondrocytes. As a result, BMP or CIF can be used as conjugates to deliver genes that express these growth factors to the target cells by the intravenous injection of the molecular complexes of the invention. Using the molecular complexes described herein with the use of specific ligands for the delivery of nucleic acid to bone cells provides treatment of diseases and abnormalities that affect bone tissues.

D. Administration of Macromolecules to Synoviolcytes The inflammatory attack on joints in animal models and human diseases may be mediated, in part, by secretion of cytokines such as IL-1 and IL-6 which stimulate the local inflammatory response. The inflammatory reaction may be modified by local secretion of soluble fragments of the receptors for these ligands. The complex between the ligand and the soluble receptor prevents the ligand from binding to the receptor which is normally resident on the surface of cells, thus preventing the stimulation of the inflammatory effect. Therapy consists of the construction of a vector containing the soluble form of receptors for appropriate cytokines (for example, IL-1), together with promoters capable of inducing high level expression in structures of the joint and a formulation which enables efficient uptake of this vector. This DNA can then used in conjunction with the macromolecular complexes of the present invention. Macromolecules can be injected into affected joints where the secretion of an inhibitor for IL-1, such as a soluble IL-1 receptor or natural IL-I inhibitor, modifies the local inflammatory response and resulting arthritis .

This method is useful in treating episodes of arthritis which characterize many "autoimmune" or "collagen vascular" diseases. This method can also prevent disabling injury of large joints by inflammatory arthritis.

In addition, current therapy for severe arthritis involves the administration of pharmacological agents including steroids to depress the inflammatory response. Steroids can be administered systemically or locally by direct injection into the joint space.

Steroids normally function by binding to receptors within the cytoplasm of cells. Formation of the steroid- receptor complex changes the structure of the receptor so that it becomes capable of translocating to the nucleus and binding to specific sequences within the genome of the cell and altering the expression of specific genes. Genetic modifications of the steroid receptor can enable the receptor to bind naturally occurring steroids with higher affinity, or bind non-natural, synthetic steroids, such as RU486. Other modifications can be made to create steroid receptors which are "constitutively active", meaning that they are capable of binding to DNA and regulating gene expression in the absence of steroid in the same way that the natural steroid receptor regulates gene expression after treatment with natural or synthetic steroids.

Of particular importance is the effect of glucocorticoid steroids such as cortisone, hydrocortisone, prednisone, or dexamethasone which are the most important drugs available for the treatment of arthritis . One approach to treating arthritis is to introduce a vector in which the nucleic acid cassette expresses a genetically modified steroid receptor into cells of the joint, e.g., a genetically modified steroid receptor which mimics the effect of glucocorticoids but does not require the presence of glucocorticoids for effect. This is termed the glucocortico-mimetic receptor. This is achieved by expression of a constitutively active steroid receptor within cells of the joint which contains the DNA binding domain of a glucocorticoid receptor. This induces the therapeutic effects of steroids without the systemic toxicity of these drugs. Alternatively, steroid receptors which have a higher affinity for natural or synthetic glucocorticoids, such as RU486, can be introduced into the joint. These receptors exert an increased anti-inflammatory effect when stimulated by non-toxic concentrations of steroids or lower doses of pharmacologically administered steroids. Alternatively, constitution of a steroid receptor which is activated by a novel, normally-inert steroid enables the use of drugs which would affect only cells taking up this receptor. These strategies obtain a therapeutic effect from steroids on arthritis without the profound systemic complications associated with these drugs. Of particular importance is the ability to target these genes differentially to specific cell types (for example synoviol cells versus lymphocytes) to affect the activity of these cells.

As described in U.S. Patent No. 5,364,791 to Vegeto, et al . , entitled "Progesterone Receptor Having C Terminal Hormone Binding Domain Truncations," and U.S. Application, Serial No. 07/939,246, entitled "Mutated Steroid Hormone Receptors, Methods for Their Use and Molecular Switch for Gene Therapy," Vegeto, et al., filed September 2, 1992, both hereby incorporated by reference (including drawings), genetically modified receptors, such as the glucocortico- mimetic receptor, can be used to create novel steroid receptors including those with glucocortico-mimetic activity. The steroid receptor family of gene regulatory proteins is an ideal set of such molecules . These proteins are ligand activated transcription factors whose ligands can range from steroids to retinoids, fatty acids, vitamins, thyroid hormones and other presently unidentified small molecules. These compounds bind to receptors and either up- regulate or down-regulate transcription. A preferred receptor of the present invention is modification of the gluco-corticoid receptor, i.e., the glucocorticoid-mimetic receptor. These receptors can be modified to allow them to bind various ligands whose structure differs from naturally occurring ligands, e.g., RU486. For example, small C-terminal alterations in amino acid sequence, including truncation, result in altered affinity and altered function of the ligand. By screening receptor mutants, receptors can be customized to respond to ligands which do not activate the host cells own receptors. A person having ordinary skill in the art will recognize, however, that various mutations, for example, a shorter deletion of carboxy terminal amino acids, will be necessary to create useful mutants of certain steroid hormone receptor proteins . Steroid hormone receptors which may be mutated are any of those receptors which comprise the steroid hormone receptor super family, such as receptors including the estrogen, progesterone, glucocorticoid-α, glucocorticoid-β, mineral corticoid, androgen, thyroid hormone, retinoic acid, and Vitamin B3 receptors. Furthermore, DNA encoding for other mutated steroids such as those which are capable of only transrepression or of only transactivation are also within the scope of the above embodiment. Such steroids could be capable of responding to RU486 in order to activate transrepression.

In addition to the above, the present invention can also be used with the following method. Drugs which inhibit the enzyme prostaglandin synthase are important agents in the treatment of arthritis. This is due, in part, to the important role of certain prostaglandin in stimulating the local immune response. Salicylates are widely used drugs but can be administered in limited doses which are often inadequate for severe forms of arthritis. Gene transfer using the present invention is used to inhibit the action of prostaglandin synthase specifically in affected joints by the expression of an antisense RNA for prostaglandin synthase. The complex formed between the antisense RNA and mRNA for prostaglandin synthase interferes with the proper processing and translation of this mRNA and lowers the levels of this enzyme in treated cells. Alternatively RNA molecules are used for forming a triple helix in regulatory regions of genes expressing enzymes required for prostaglandin synthesis. Alternatively, RNA molecules are identified which bind the active site of enzymes required for prostaglandin synthesis and inhibit this activity. Alternatively, genes encoding enzymes which alter prostaglandin metabolism can be transferred into the joint.

These have an important anti-inflammatory effect by altering the chemical composition or concentration of inflammatory prostaglandin.

Likewise, the present invention is useful for enhancing repair and regeneration of the joints. The regenerative capacity of the joint is limited by the fact that chondrocytes are not capable of remodeling and repairing cartilaginous tissues such as tendons and cartilage. Further, collagen which is produced in response to injury is of a different type lacking the tensile strength of normal collagen. Further, the injury collagen is not remodeled effectively by available collagenase. In addition, inappropriate expression of certain metalloproteinases is a component in the destruction of the joint.

Gene transfer using promoters specific to chondrocytes (i.e., collagen promoters) is used to express different collagens or appropriate collagenase for the purpose of improving the restoration of function in the joints and prevent scar formation.

The delivery of gene therapeutics to cells in the joint can provide a therapeutic effect. Gene delivery for these purposes is affected by direct introduction of DNA into the joint space where it comes into contact with chondrocytes and synovial cells. Further, the genes permeate into the environment of the joint where they are taken up by fibroblasts, myoblasts, and other constituents of periarticular tissue.

E . Administration of Macromolecules to the Lungs

Macromolecular complexes of the present invention can also be used in reversing or arresting the progression of disease involving the lungs, such as lung cancer. One embodiment involves use of intravenous methods of administration to delivery nucleic acid encoding for a necessary molecule to treat disease in the lung. Molecular complexes comprising nucleic acid molecules that express a necessary protein or RNA can be directly injected into the lungs or blood supply so as to travel directly to the lungs. Furthermore, the use of an aerosol or a liquid in a nebulizer mist can also be used to administer the desired nucleic acid to the lungs. Finally, a dry powder form, such as PVP discussed above or a powder resulting from lyophilization of the peptide/nucleic acid molecule complexes of the invention, can be used to treat disease in the lung. The dry powder form is delivered by inhalation. These treatments can be used to control or suppress lung cancer or other lung diseases by expression of a particular protein encoded by the nucleic acid chosen.

Examples

The examples below are not limiting and are merely representative of various aspects and features of the present invention. Chemical compounds and solvents utilized for the synthesis of components of the invention can be substituted with compounds and solvents with similar chemical properties. In addition, amounts and volumes described in the following procedures are dependent upon the scale of the technique. A person of ordinary skill in the art could scale the amounts and volumes of reagents appropriately.

Example 1: Preparation Of Lipophilic Lytic Peptide The following lipophilic lytic peptide was synthesized:

Two palmityl moieties were attached to a glycine, and the resulting dipalmityl-glycine moiety was covalently linked to the epsilon amino moiety of a lysine residue placed at the C-terminus of GLFEALEELWEA.

The amino acid sequence of the lipophilic lytic peptide is meant to provide endosome lytic activity at acid pHs, and the hydrophobic chains are meant to promote hydrophobic association of the lytic peptide to a lipophilic binding peptide, described herein by example.

Preparation of the lipophilic lytic peptide involved (a) the synthesis of dipalmityl glycine; (b) attachment of the dipalmityl glycine to the epsilon amine of the C- terminal lysine (on a solid phase resin) ; (c) the synthesis of the rest of the peptide; and (d) purification of the lipopeptide by HPLC. These procedures are described below.

Preparation of Hexadecylamido Hexadecanoic Acid

1.1 Two hundred mL of toluene was heated to about 60°C. 1.2 Hexadecylamine (24.15 g, 0.1 mol) was dissolved in the warm toluene.

1.3 Diisopropylamine (17.4 mL, 0.1 mol) was added to the solution. Stirring and heating was continued for one hour . 1.4 A solution of 30.34 mL (0.1 mol) of palmitoyl fluoride in 100 mL toluene was then added in a dropwise fashion.

1.5 The mixture was allowed to cool to room temperature after overnight stirring at 60°C. The white precipitate was collected then recrystallized with hot ethyl acetate .

Preparation of N, -Dihexadecylamine

2.1 Hexadecylamido hexadecanoic acid (9.6 g, 0.02 mol) was dissolved in 200 mL warm, dry THF.

2.2 Lithium aluminum hydride (IM in ether, 50 mL) was added dropwise to the stirring warm solution.

2.3 The solution was stirred and heated until no starting amide was observable by TLC. 2.4 Wet THF was added dropwise to destroy excess

LiAlH₄.

2.5 The solvents were evaporated. The resulting residue was treated with IM NaOH then extracted with ether and ethyl acetate. 2.6 The combined organic extracts were washed with water, dried over K₂C0₃, then evaporated to give about 9.4 g amine .

Preparation of t-Butyl N, N-Dihexadecylglycinate

3.1 N, N-dihexadecylamine (9.4 g) was dissolved in 50 mL warm toluene.

3.2 t-Butylbromoacetate (3 mL) , 18-crown-6 (0.1 g) , and K₂C0₃ (2.75 g) were added to the solution.

3.3 The solution was heated overnight until no amine was present by TLC. 3.4 The solution was extracted with water. The organic phase was dried over MgSO. and evaporated. 3.5 The residue was purified on silica gel using hexanes : ethyl acetate (5:2) to give a yellow oil.

Preparation of N, N-Dihexadecylglycinate (Dipalmityl Glycine)

4.1 Fifty mL of ether was cooled in an ice bath. A stream of HCl gas was bubbled into the solution for about one hour. Periodic measurements of the weight of ether were taken. When the weight became constant, the ether was assumed to be saturated with gas.

4.2 The oily t-butyl N, N-dihexadecylglycinate was dissolved in the ether. The solution was allowed to stir on ice for about 30 minutes.

4.3 The solution flask was stoppered and placed in the refrigerator for 48 hours. The precipitate was filtered from the solution and stored in a vacuum desiccator until used.

Attachment of C-terminals Lysine Residue to Resin

5.1 Dde-Lys (Fmoc) -OH was attached to MBHA Rink Amide resin using standard coupling procedures. The 0.25 mmol scale coupling was performed on the ABI peptide synthesizer. 5.2 After coupling, the resin was tested using the Kaiser ninhydrin test. The resin tested positive for free amine at this step.

Attachment of Dipalmityl Glycine to Lysine Side Chain

6.1 The following solution was prepared with stirring: 20 mL NMP

135 mg HOBt 520 mg PyBOP 50 uL diisopropylamine

522 mg dipalmityl glycine (prepared in Step 4 above) The solution was warmed slightly in order to fully dissolve all components in NMP.

6.2 The resin prepared in Step 5 was added to the solution and stirred for 3 hours at 50°C. 6.3 The resin was then tested again using the Kaiser ninhydrin test. The resin tested negative indicating successful coupling.

6.4 The resin was filtered from the solution and rinsed twice with 10 mL NMP.

Removal of the Dde Protecting Group

7.1 The resin was deprotected by two 3 minute treatments with a solution of 2% hydrazine hydrate in NMP.

7.2 The resin was washed with NMP then dichloromethane . 7.3 A final Kaiser ninhydrin test showed the presence of free amine sites (positive test) after this deprotection step.

Extension of Peptide Sequence

8.1 The deprotected resin was loaded onto the automated peptide synthesizer.

8.2 The remaining amino acid sequence (GLFEALEELWEA) was attached using standard procedures.

8.3 After completion of the synthesis, the peptide was cleaved from the resin using a TFA: water : anisole : - ethanedithiol (95:3:1:1) solution.

8.4 The cleaved peptide was lyophilized to a dry, white powder.

Purification Procedure

9.1 The crude material was dissolved in 0.1% TFA in water. 9.2 The peptide was purified on the preparative HPLC under the following conditions:

Column: C₈ pH stable reverse phase column

Flow rate: 5 mL/min

Buffer A: 0.1% TFA in water

Buffer B: 10% buffer A; 90% acetonitrile

Method: initial %B = 50%

at 25 min = 100%

at 30 min = 50%

Wavelength: 215 nm

The peptide peak was collected at 24.5 minutes on this gradient.

9.3 The purified peptide was lyophilized to a dry, white powder. The sample was analyzed by analytical HPLC and mass spectroscopy .

Chemicals for Lipophilic Lytic Peptide Synthesis TABLE 1

Name Physical Properties Amount Needed

Hexadecylamine MW = 241.46 24.15 g (0.1 mol)

Diiisopropylamine MW = 129.25; d=0.742 12.92 g, 17.4 mL(p.l mol)

Palmityl chloride MW = 274.88; d=0.906 27.49 g, 30.34 mL (0.1 mol)

Lithium aluminum IM in ether 30 mL hydride

tButylbromoacetate MW = 195.06; d=1.321 3.9 g, 2.95 mL (0.02 mol)

Potassium carbonate MW - 138.21 2.76 g (0.02 mol)

18-Crown-6 MW=264.32 0.1 g (catalytic amount)

HCl ether 50 mL

HOBt MW = 135.1 135 mg 0.001 mol)

ByBOP MW - 520.3 520 mg 0.001 mol)

Dde-Lys(Fmoc)-OH MW=532.58 533 mg 1 mmol)

MBHA Rink Amide resin

Fmoc-Gly-OH MW=297.31 297 mg (1 mmol)

Fmoc-Leu-OH MW=353.42 353 mg (1 mmol)

Fmoc-Phe-OH MW=387.44 387 mg (1 mmol)

Fmoc-Glu(OtBu)-OH MW=425.48 425 mg (1 mmol)

Fmoc-Ala-OH MW=311.3 311 mg (lmmol)

Fmoc-Trp(Boc)-OH MW=526.6 527 mg (1 mmol) Instruments for Synthesis and Purification of Lipophilic Lytic Peptide :

Peptide synthesizer

Preparative HPLC

Analytical HPLC

Example 2 : Determination Of Lipophilic Lytic Peptide

Activity

The lytic activity of the lipophilic lytic peptide was assessed by monitoring the lysis of red blood cells. The protocol for the assay comprises the following steps:

1. Washing of Red Blood Cells (RBCs)

1.1 Obtain fresh whole blood preserved in 0.38% sodium citrate. The blood should be stored in a glass Vacutainer. Plastic containers should be avoided for storing and handling of blood due to adsorption of RBCs.

1.2 Spin whole blood in a clinical centrifuge at approximately 2,500 rpm for 10 minutes. Remove resulting supernatant (plasma) and wash with an equal volume of IX phosphate buffered saline. 1.3 Repeat step 1.2 for a total of three washes.

1.4 Store blood at 4°C. Blood can be stored in this manner for at least one week, possibly longer if blood is oxygenated by inverting the tube several times daily.

2. Counting of RBCs Using a Two-chamber Hemocytometer 2.1 Make a 1:10000 dilution of RBCs in IX PBS using disposable glass test tubes.

2.2 Place 15 mL of the RBC dilution between the cover slip and one chamber of the hemocytometer. Count the RBCs in the four corner squares and the center square. 2.3 Repeat step 2.2 with the second hemocytometer chamber.

2.4 Average the counts from the ten chambers and determine the number of RBCs/mL: Cells per mL = Ave . count per square x dilution factor x 10"

3. Hemolysis Assay

3.1 Prepare the following solutions:

A) 0.2 g Triton X-100 in 10 mL of 150 mM NaCl/20 mM Sodium citrate buffer

(pH 5.0)

B) 0.2 g Triton X-100 in 10 mL of 150 mM NaCl/20 mM Sodium citrate buffer

(pH 7.4) C) 1.0 mL stock solution of the peptide in the appropriate buffer (pH 5.0 and pH 7.4)

3.2 Using a multichannel pipette, fill the wells of a 96-well microtiter plate with 100 μl of buffer (pH 5.0 or 7.4) . 3.3 Next deliver 100 μL of 2% Triton X-100 in pH 5.0 buffer to wells A:l - C:l and 100 μL of the peptide stock solution (pH 5.0) to wells D:l - F:l.

3.4 To wells A:7 - C:7 add 100 μL of 2% Triton X-100 in pH 7.4 buffer and 100 μL of the peptide solution (pH 7.4) to wells D:7 - F:7.

3.5 Perform 30μl serial dilutions.

3.6 Dilute recently counted RBCs to a final cell density of lxlO⁸ cells per ml with the appropriate pH buffer. Pipette 100 μL of this RBC solution into the appropriate wells. RBCs at pH 5.0 and pH 7.4 should be pipetted into Rows G and H to determine background as well as basal RBC lysis .

3.7 Incubate plates at 37°C for 1 hour.

3.8 Following incubation, centrifuge the microtiter plate in a clinical centrifuge at 3,000 rpm for 5 minutes.

3.9 Following centrifugation, transfer 100 μL of supernatant to another microtiter plate for analysis. Read the absorbance of the samples at 540 nm using a microtiter plate reader. The activity of lytic peptides was sensitive to modification or derivatization of amino acid residues, or attachment of other sequences, including hydrophobic groups. The lipophilic lytic peptide was designed such that the hydrophobic chains were aligned with the hydrophobic side of the peptide. Furthermore, the use of glycine instead of lysine for attachment of the dipalmityl group was meant to promote tighter packing of the hydrophobic chains.

It was important to confirm that attaching hydrophobic chains to the lytic peptide did not abolish its endosomolytic activity. Therefore, the lipophilic lytic peptide was tested in a hemolytic assay. The results demonstrated that the lytic activity was retained and further, the activity was still pH selective (Figure 2). The hemolytic values for the lipophilic lytic peptide synthesized in the preceding example, designated GM227.3, are compared to its parent sequence GM226.14 and to another potent lytic peptide GM225.1 in Table 2. Table 2

ID# Sequence pH Selectivity mg ml % of Triton Activity

GM225.1 GLFEALLELLESLWELLLEA* Selective 0.0065 102

GM226.14 GLFEALEELWEA Selective 0.012 132

GM227.3 GLFEALEELWEAK Selective 0.0083 90.9 (-G(Pam)₂)

GM227.5 Pam-GLFEALEELWEA Non-Selective 0.0024 83.9

GM227.6 GLFEALEELWEAK(-Pam) Selective 0.018 52.4

* Peptide portion contains a free carboxy terminus while the rest of the peptides contain amidated carboxy termini.

In addition to the peptides of Table I, the peptide GM226.18, which has the amino acid sequence LLFKLLEWLE, was also shown to be active in the hemolytic assay. Table I also displays hemolytic activities of two another lipopeptide analogs : GM227.5 and GM227.6. The former has a single palmityl group attached at the N-terminus, and the latter has a single palmityl group attached at the epsilon amine of lysine placed C-terminal to the parent lytic sequence. The GM227.5 lost its pH selectivity following N- terminal modification. The maximum lytic activity was much reduced compared to the parent sequence GM226.14 in the case for the GM227.6 peptide, although pH selectivity was retained. In contrast, GM227.3 retained lytic activity along with pH selectivity. It appears that there was a benefit in attaching the hydrophobic chain through a glycine rather than directly to the epsilon amine of lysine.

Example 3: Preparation Of A Lipophilic Condensing Peptide

The following is a protocol for the synthesis and purification of lipophilic binding peptide, designated GM 246.3:

1. Preparation of (Fmoc) ₂-K- (k_boC) ₈-W_{b0C de}~Resin

1.1 A 0.5g (0.25 mmole scale) portion of MBHA Rink Amide resin (Novabiochem 01-64-0037) was weighed into the reaction vessel.

1.2 The following protected amino acids were sequentially coupled to the resin using standard Fmoc procedures on the ABI 433A Peptide Synthesizer: Fmoc-Lys_Dde- OH, Fmoc-Trp_Boc~OH, Fmoc-Lys_Boc-OH (8 cycles), and Fmoc-Lys_FmOC- OH.

2. Preparation of (PAM) ₂-K- (K_BOc) 8- _BocK_Dde-Re5in

2.1 The (Fmoc) ₂-K- (K_Boc) ₈-W_BocK_Dde- esin was treated with piperidine (5.5 ml, 15 min) to remove the Fmoc protecting groups. The presence of free amino groups was verified with the Kaiser ninhydrin test before coupling.

2.2 The resin was treated with 2.0M DIEA in 20 ml NMP.

2.3 Then 600 ml (8X excess) of palmityl chloride [CH₃- (CH)ι_.-COCl] was added and the reaction mixture was magnetically stirred overnight at room temperature (RT, 25°C) .

2.4 After the reaction, the resin was washed with NMP and CH₃OH. 3. Preparation of (Pam) ₂-K- (K_BOc) ₈-W_BocK (Fmoc-AEEA) (Resin) 3.1 The (Pam) ₂-K- (K_Boc) ₈-W_BocK_Dde-Resin (0.3 mmol) was treated with 2% hydrazine (2 x 8 ml x 3 min) to remove the Dde protecting group. 3.2 Then, 362 mg (0.94 mmol) of Fmoc-AEEA-OH (from Perseptive Biosystems) was coupled to the peptide resin on the ABI 433A peptide synthesizer by standard FastMoc chemistry using HOBT/PyBOP activation.

4. Preparation of (Pam) ₂-K- (K_BoC) ₈-W_BocK (AEEA) -K_Dde-K_(Fmoc)2 (Resin)

4.1 The (Pam) ₂-K- (K_BOC) ₈-W_BocK( Fmoc-AEEA) (Resin) was treated with piperidine (5.5 ml, 15 min) to remove the Fmoc protecting group.

4.2 This was followed by coupling with Fmoc-Lys-Dde on the ABI synthesizer using standard FastMoc chemistry.

4.3 The Fmoc protecting group was then removed by treatment with piperidine (5.5 ml, 15 min) .

4.4 This was followed by another coupling with Fmoc- Lys-Fmoc on the ABI synthesizer using standard FastMoc chemistry.

5. Preparation of (Pam) ₂-K- (K_Boc) ₈-W_BocK-AEEA-K (Fmoc-AEEA) - K (Fmoc-AEEA) ₂ (Resin)

5.1 The (Pam) ₂-K-(K_Boc)₈-W_BocK (AEEA) -K_Dde-K_(Fmoc)2 (Resin) was treated with 2% hydrazine (2 x 8 ml x 3 min) to remove both the two Fmoc and the Dde protecting groups.

5.2 This was followed by coupling with 0.5 mmol Fmoc- AEEA-OH for two hours on the ABI synthesizer using HOBT/PyBop activation. 6. Preparation of (Pam) ₂-K- (K_Boc) ₈-W_BocK-AEEA-K (AEEA-AcGal) - K(AEEA-AcGal)₂ (Resin)

6.1 The resin was treated with piperidine (5.5 ml, 15 min) to remove the Fmoc protecting groups. 6.2 This was followed by coupling with AcGalTP (10X molar excess) on the ABI synthesizer using HOBT/PyBop activation .

7. Cleavage of Peptide from Resin to Produce (Pam) ₂-K-K₉WK- AEEA-K (AEEA-AcGal ) -K (AEEA-AcGal ) ₂ 7.1 The ( Pam) ₂-K- (K_Boc)₈-W_BocK-AEEA-K (AEEA-AcGal ) -K(AEEA- AcGal)₂ (Resin) was cleaved with TFA:EDT:H₂0 (95:2.5:2.5).

7.2 The crude peptide was purified by preparative RP- HPLC and the pure fractions were pooled and lyophilized.

8. Deacetylation of AcGal Peptide

8.1 The peptide was dissolved in 0. IN NaOH and incubated at RT for about 40 minutes. The solution was then neutralized with 0. IN HCl.

8.2 The crude peptide was then purified by preparative RP-HPLC and the pure fractions were pooled and lyophilized.

8.3 The authenticity of the pure peptide was verified by analytical RP-HPLC, mass spectroscopy, amino acid analysis, analytical HPLC.

Materials for the synthesis and purification of GM246.3;

Table 3 ITEM PHYSICAL AMOUNT REQUIRED PROPERTIES

MBHA Rink Amide resin 100-200 mesh 0.5 g @ 0.5mmole/gsubstn Fmoc-Lys_Dde-OH MW = 532.6 1065 mg (2 mmol)

Fmoc-Lys_Fmoc-OH MW = 590.7 1181 mg (2 mmol)

Fmoc-Lys_Boc-OH MW = 468.5 3748 mg (8 mmol)

Fmoc-T _Boc-OH MW = 526.6 526.6 mg (1 mmol)

Fmoc-OSu MW = 337.3 1012 (3 mmol)

HOBt MW = 135.1 1891 mg (14 mmol)

PyBOP MW = 520.3 7284 mg (14 mmol)

Palmityl chloride MW = 274.88, d - 0.906 0.6 ml (2 mmole)

Hydrazine MW = 32.05, d = 1.021 2 ml

Piperidine MW = 85.15, d = 0.973 110 ml

Diisopropylethylamine, DIEA MW = 129.25, d = 0.742 40

Acetonitrile MW = 41.05, d = 0.786 1 liter

TFA MW = 114.02, d = 1.480 50 ml

EDT MW = 94.20, d = 1.123 2.5 ml

AcGalTP MW = 436 1090 mg

Dichloromethane MW = 84.93, d - 1.325 500 ml

N-methylpyrrolidone, NMP M W = 85.15, d = 0.819 2000 ml

Fmoc-AEEA-Linker MW = 385.4 1156 mmol Example 4 : Determining The Agglutination Activity Of The

Lipophilic Binding Peptide

The following is a protocol for an agglutination assay for determining the presence of galactose in samples. The assay utilizes the lectin from Ri cnus communis (RCAι₂₀) .

Materials :

Lectin from Ricinus communis (RCAι₂₀) - Sigma Cat#L7886 lx PBS - Gibco/BRL Cat#14040-026

Polystyrene four sided cuvettes - VWR Scientific Cat#58017-882

Cuvet lids - Evergreen Scientific Cat# 300-3126-020

25% Lactose

Parafilm

Procedure : 1) Access the kinetic function of spectrophotometer that measures absorbance at visible wavelengths. A Beckman DU640 UV/Visible spectrophotometer is sufficient for this analysis .

2) Set the analytical wavelength to 450nm. 3) Set interval time to 5 seconds

4) Set the total time to 300 seconds

5) Blank the instrument with 600 μL of lx PBS, 13μL of RCAι₂₀.

6) Mix 100 μL of formulation (~100ug/ml DNA) with 600 μL of lx PBS and scan.

7) Place 13 μL of RCA__.2o on the inside of a cuvet lid, place on cuvet, seal with parafilm, rapidly invert several times, place in the Beckman DU640 and begin reading.

8) Next, add 100 μL of 25% lactose to the side of the cuvet used in step 7. Gently recap the cuvet, wrap in parafilm, invert several times and place in the Beckman DU640 and begin reading.

9) Repeat steps 7-8 as needed.

10) Data from the Beckman DU640 can be transfered to disc as an ascii file. See the operating manual for the

Beckman DU640 for more details.

Example 5 : Molecular Complex Formulation Comprising

Lipophilic Lytic Peptide, Lipophilic Binding Peptide, And Macromolecules Formulations of nucleic acid molecules were prepared in isotonic (5%) glucose. The lipophilic lytic peptide and the lipophilic binding peptide were mixed together first in aqueous solution, and then the peptide mixture was formulated with a plasmid in a glucose solution. Formulations can also be prepared by mixing the various components of the invention in different proportions. Formulations were prepared with various ratios of the three components. The following results were obtained from experiments performed using the preparations as samples: a) The complexation of DNA was observed by agarose gel electrophoresis b) DNA condensation and particle sizes were observed by laser light scattering. c) The stability of the particles in ionic media was analyzed after exposing the condensed DNA particles to isotonic saline.

These experiments demonstrated that certain charge ratios of the components provided small particles that were also stable in ionic media whereas large/unstable particles or no particles were found at other charge ratios. A prototype formulation was selected with the charge ratio of 4/2/1 (-/+/-) of DNA/lipophilic binding peptide/lipophilic lytic peptide. This formulation had a mean particle size of approximately 120 nm (at a DNA concentration of 100 μg/ml) and these particles remained less than 200 nm in size when exposed to isotonic saline. This formulation was fractionated on a sucrose density gradient, and the fractions were analyzed by both agarose gel electrophoresis and SDS-polyacrylamide gel electrophoresis. These experiments demonstrated that the DNA complex contained both the peptides. This formulation was also tested for gene delivery and expression in vivo after tail vein administration in mice.

Small particles can be generated by using initially low concentrations of nucleic acid molecules, i.e., about 10 μg/mL concentration or less, and then concentrating the suspension by techniques well known to those skilled in the art. Examples of these concentration techniques are sedimentation by centrifugation under large gravitational force conditions, i.e., ultracentrifugation, and concentration with a membrane filter containing apparatus, which is commercially available. The particles generated by this procedure are less than 200 nm in diameter and are preferably less than 100 nm in diameter for the delivery of macromolecules into cells.

Example 6: In Vivo Activity Of Molecular Complex Formulation

The formulation was injected into the tail vein of mice and compared to controls (120 μg DNA in 1.2 ml formulation) . In vivo delivery may also be enhanced by adding other volumes such as 1 mL injection volume per 25 grams of body weight. The mice were sacrificed at 48 hours, their livers harvested, and analyzed for chloramphenicol hydrophobic transferase (CAT) gene expression. From the results, it was clear that naked DNA injected into the tail vein did not provide any reporter gene expression, similar to a non- injected control. The formulation with only the lipophilic binding peptide provided a low level of expression, while the highest level of expression was observed for the formulation comprising plasmid formulated with both the lipophilic lytic peptide and the lipophilic binding peptide. Tissue samples were processed in the following manner: The mouse liver was fixed in 10% neutral buffered formalin (NBF) for 6-8 hours at room temperature (RT) . After fixation the tissues were dehydrated at RT for 1 hour each in (a) 70% ethanol, (b) 90% ethanol, (c) 95% ethanol, and (d) 100% ethanol, 2 changes. They were then cleared in xylene for 2 changes, 50 minutes each at RT and infiltrated with paraffin at 58° C for 3 changes, 1 hour each. The tissue was then embedded in paraffin. Paraffin sections, were cut at 4-um, collected on Plus slides, and dried at 60° C for 1 hour prior to immunostaining.

The gene expression was then determined using the following procedure. The tissue sections were deparaffinized in 3 changes of xylene (5 min each) , transferred to 100% ethanol for 2 changes of 5 minutes each and placed in 1% hydrogen peroxide in methanol for 15 minutes to quench the endogenous peroxidase activity. The sections were then rinsed briefly in two changes of distilled water, placed in 3 changes of phosphate buffered saline (PBS) for 5 minutes each, and incubated in avidin and biotin for 15 minutes each with PBS rinses in between the steps. The sections were placed in 20% normal rabbit serum for 30 minutes prior to an overnight incubation in the primary antibody, sheep anti -CAT (1:100,000 dilution). This was followed by 3 changes of PBS for 5 minutes each, and then incubated in the secondary antibody (biotinylated rabbit anti sheep IgG, 1:400) for 1 hour. The sections were then rinsed in PBS for 5 minutes (3 changes each), and incubated for 1 hour in Vector eli te ABC solution (1:80) and rinsed again in PBS as before. To visualize the site of CAT protein, the sections were reacted with a solution of diaminobenzadine (DAB) for 7 minutes. They were then rinsed well with distilled water, counterstained in Mayer' s hematoxylin, dehydraded in ethanol, and coverslipped.

Procedure: incubate the cells in the following solutions and then discard the solutions:

1. Xylene 3 X 5 min.

2. 100% EtOH 2 X 5 min.

3. Methanol/H₂0₂ 15 min.

4. d H₂0 rinse rinse well

5. PBS 3 X 5 min.

6. Avidin 15 minutes

7. PBS rinse well

8. Biotin 15 minutes

9. PBS rinse well

10. 20% NRS 30 min.

11. 1° antibody B-M sheep anti-CAT 1: 100K - overnight

12. PBS 3 X 5 min.

13. 2° antibody Vector biotinylated rabbit anti-sheep IgG 1:400 - 1 hr.

14. PBS 3 X 5 min .

15. Vector elite ABC 1 hr. (1:80)

16. PBS 3 X 5 min.

17. DAB 7 min

18. dH₂0 rinse well

Counterstain with hematoxylin, dehydrate the cells, mount the cells, and place a coverslip over the cells. Example 7 : Delivery Of Macromolecules To Cells Using

Molecular Complexes Comprising Plasmid And Lipophilic Lytic Peptide

The level of expression for the formulation comprising the plasmid with both peptides was higher than the expression for a formulation containing plasmid with only the lipophilic lytic peptide. Both (a) the formulation comprising lipophilic binding peptide, lipophilic lytic peptide, and DNA and (b) the formulation comprising only lipophilic lytic peptide and DNA provided a higher level of expression as compared to a formulation of DNA alone in 5% glucose. This result suggested that the lipophilic lytic peptide is capable of interacting with DNA by itself even though it is anionic. The formulation only comprising lipophilic lytic peptide and plasmid also does not condense DNA into particles. This result represents a counterintuitive finding that demonstrated the use of the lipophilic lytic peptide as a delivery agent that can be used in simple formulations comprising only the macromolecule and the lipophilic lytic peptide.

Example 8 : Selective Delivery And Expression Of

Macromolecules To Specific Tissues

The levels of expression resulting from injection of the formulation comprising plasmid, lipophilic lytic peptide, and lipophilic binding peptide were also compared in three different organs: liver, lung, and spleen. The expression level in the liver was approximately 10-fold higher than in the lung and about 60-fold higher than in the spleen. These results indicated that the formulation was targeted to the liver selectively.

The methods for manipulating tissue samples and detecting gene expression are set forth herein by example. Example 9 : Delivery Of Macromolecules To Specific Cell

Types

The molecular complex formulation specifically delivered plasmid to the parenchymal hepatocytes in the liver. The targeting of the plasmid to these cells was confirmed by immunohistochemical staining of CAT expressing cells (using a CAT specific antibody) . Liver sections were stained and the results confirmed that the cells expressing the CAT within the liver were parenchymal hepatocytes.

Example 10: Fasting Enhances The Delivery Of

Macromolecules To Cells

Male rats were fasted for 24 hours prior to the administration of formulations composed of plasmid DNA complexed with a lipophilic binding peptide of the invention. The formulations were administered by portal vein injection and the animals were sacrificed for two hours after injection. Livers were harvested from the sacrificed animals and the amount of the plasmid DNA delivered to the liver cells was determined by quantitative polymerase chain reaction (PCR) techniques. The quantitative PCR techniques are commonly known to those skilled in the art in the form of commercially available kits.

Depending on the type of lipophilic binding peptide of the invention was utilized, the fasting period enhanced the delivery of the plasmid DNA molecule by 118-fold and 17-fold with respect to non-fasted animals. In addition to increasing the amount of DNA plasmid delivered to the specific tissues of cells, fasting also appeared to reduce the variability within a test group. Both fasted groups demonstrated significantly less variability as compared to non-fasted groups. One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The molecular complexes and the methods, procedures, treatments, molecules, specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims .

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference .

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. Other embodiments are within the following claims.

Claims

1. A molecular complex for delivering a macromolecule into cells of an organism, comprising:

(a) a lipophilic lytic peptide comprising a peptide moiety and one or more hydrophobic moieties;

(b) a lipophilic binding peptide comprising a peptide moiety and one or more hydrophobic moieties; and

(c) said macromolecule.

2. A molecular complex for delivering a macromolecule into cells, comprising:

(a) a lipophilic lytic peptide comprising a peptide moiety and one or more hydrophobic moieties; and

(b) said macromolecule.

3. A molecular complex for delivering a macromolecule into cells, comprising:

(a) a lipophilic binding peptide comprising a peptide moiety and one or more hydrophobic moieties; and (b) said macromolecule.

4. The molecular complex of claims 1, 2, and 3, wherein said macromolecule is a nucleic acid molecule.

5. The molecular complex of claims 1 and 3, wherein said lipophilic binding peptide further comprises one or more saccharide moieties.

6. The molecular complex of claim 5, wherein said saccharide moiety or moieties of said lipophilic binding peptide are selected from the group consisting of galactose, glucose, and sucrose.

7. The molecular complex of claim 5, wherein said saccharide moiety or moieties of said lipophilic binding peptide are connected to said peptide by N-, S-, or 0- linkages .

8. The molecular complex of claim 5, wherein said saccharide moiety or moieties are connected to said lipophilic binding peptide by one or more spacer moieties.

9. The molecular complex of claim 8, wherein said spacer moiety or moieties are selected from the group consisting of:

and

where n = 0 through 20.

10. The molecular complex of claims 1 and 3, wherein said peptide moiety of said lipophilic binding peptide consists of the amino acid sequences selected from the group consisting of:

KKKKKKKKKWK;

(K)„W;

YKA ( K) _nWK; where n = 0 through 40 .

11. The molecular complex of claims 1 and 3, wherein said hydrophobic moiety or moieties of said lipophilic binding peptide are selected from the group consisting of N- palmitoyl; Nα-palmitoyl; Nε-palmitoyl; Nα, ε-dipalmitoyl; Nα, Nα-dipalmityl; Nε, Nε-dipalmityl; and N, N-dipalmityl- glycyl .

12. The molecular complex of claims 1 and 2, wherein said lipophilic binding peptide has the following structure:

13. The molecular complex of claims 1 and 2, wherein said peptide moiety of said lipophilic lytic peptide consists of a sequence of amino acids selected from the group consisting of:

GLFEALLELLESLWELLLEA;

GLFEALEELWEA; GLFLLEEWLE; GLFLLEEWLEK;

GLFEALEELWEAK; GLFEALLELLESLWELLLEAK;

Suc-GLFKLLEEWLE; and

Suc-GLFKLLEEWLEK; where Sue is a succinyl moiety.

14. The molecular complex of claims 1 and 2, wherein said hydrophobic moiety or moieties of said lipophilic lytic peptide are selected from the group consisting of N- palmitoyl; Nα-palmitoyl; Nε-palmitoyl; Nα, Nε-dipalmitoyl; Nα,Nα-dipalmityl; Nε,Nε-dipalmityl; and N,N-dipalmityl- glycyl .

15. The molecular complex of claims 1 and 2, wherein said lipophilic lytic peptide has the following structure:

16. The molecular complex of claim 4, wherein said molecular complex has a -/+/- charge ratio of 1/3/1, 1/2/1, 1/1/1, 4/0/1, 2/1/0, 4/2/1, 4/3/1, and 4/4/1, wherein said - /+/- ratio is nucleic acid molecule/lipophilic binding peptide/lipophilic lytic peptide.

17. The molecular complex of claim 4, wherein said molecular complex has a -/- charge ratio of 1/4, 1/3, 1/2, and 1/1, wherein said -/- ratio is nucleic acid molecule/lipophilic lytic peptide.

18. The molecular complex of claim 4, wherein said molecular complex has a -/+ charge ratio of 1/3, 1/2, and 1/1, wherein said -/+ ratio is nucleic acid molecule/lipophilic binding peptide.

19. A method of delivering a macromolecule into cells of an organism, comprising the step of administering to said organism a molecular complex comprising: (a) a lipophilic lytic peptide comprising a peptide moiety and one or more hydrophobic moieties;

(c) said macromolecule.

20. A method of delivering a macromolecule into cells of an organism, comprising the step of administering to said organism a molecular complex comprising:

(b) said macromolecule.

21. A method of delivering a macromolecule into cells of an organism, comprising the step of administering to said organism a molecular complex comprising:

(a) a lipophilic binding peptide comprising a peptide moiety and one or more hydrophobic moieties; and

(b) said macromolecule.

22. The method of claims 19, 20 and 21, wherein said step of administering said molecular complex consists of contacting said cells of said organism with said molecular complex.

23. The method of claims 19, 20 and 21, wherein said step of administering said molecular complex consists of techniques selected from the group consisting of parenteral injection, intramuscular injection, intravenous injection, and administration by inhalation.

24. The method of claims 19, 20 and 21, wherein said macromolecule is a nucleic acid molecule.

25. The method of claims 19, 20 and 21, wherein said nucleic acid molecule comprises a gene.

26. The method of claim 25, wherein said gene is expressed in said cells.

27. The method of claims 18, 19, and 20, wherein said organism is a mammal.

28. The method of claims 19, 20 and 21, wherein said macromolecule is delivered to a specific tissue in said organism.

29. The method of claim 28, wherein said specific tissue is selected from the group consisting of liver, lung, spleen, kidney, heart, muscle, and blood.

30. The method of claim 28, wherein said macromolecule is a nucleic acid molecule.

31. The method of claim 30, wherein said nucleic acid molecule comprises a gene.

32. The method of claim 31, wherein said gene is expressed in said specific tissue.

33. The method of claims 19, 20, and 21, wherein said macromolecule is delivered to a specific cell type in said organism.

34. The method of claim 33, wherein said specific cell type is selected from the group consisting of Kupffer cells, adipocyte cells, red blood cells, white blood cells, macrophages, synovialcytes, and bone forming cells.

35. The method of claim 33, wherein said macromolecule is a nucleic acid molecule.

36. The method of claim 35, wherein said nucleic acid molecule comprises a gene.

37. The method of claim 36, wherein said gene is expressed in said specific cell type.

38. The method of claims 19 and 21, wherein said lipophilic binding peptide further comprises one or more saccharide moieties.

39. The method of claims 19 and 21, wherein said saccharide moiety or moieties of said lipophilic binding peptide are selected from the group consisting of galactose, glucose, and sucrose.

40. The method of claims 19 and 21, wherein said saccharide moiety or moieties of said lipophilic binding peptide are connected to said peptide by N-, 0-, or S- linkages .

41. The method of claims 19 and 21, wherein said saccharide moiety or moieties are connected to said lipophilic binding peptide by one or more spacer moieties.

42. The method of claim 19 and 21, wherein said spacer moiety or moieties are selected from the group consisting of:

and

where n = 0 through 20.

43. The method of claims 19 and 21, wherein said peptide moiety of said lipophilic binding peptide consists of the amino acid sequences selected from the group consisting of:

KKKKKKKKKWK; (K)_nW; and YKA(K)_nWK; where n = 0 through 40.

44. The method of claims 19 and 21, wherein said hydrophobic moiety or moieties of said lipophilic binding peptide are selected from the group consisting of N- palmitoyl; Nα-palmitoyl; Nε-palmitoyl; Nα, ε-dipalmitoyl; Nα,Nα-dipalmityl; Nε, Nε-dipalmityl; and N, N-dipalmityl- glycyl.

45. The method of claims 19 and 21, wherein said lipophilic binding peptide has the following structure:

46. The method of claims 19 and 20, wherein said peptide moiety of said lipophilic lytic peptide consists of a sequence of amino acids selected from the group consisting of:

GLFEALLELLESLWELLLEA; GLFEALEELWEA; GLFLLEEWLE;

GLFLLEEWLEK; GLFEALEELWEAK; GLFEALLELLESLWELLLEAK; Suc-GLFKLLEEWLE; and Suc-GLFKLLEEWLEK; where Sue is a succinyl moiety.

47. The method of claims 19 and 20, wherein said hydrophobic moiety or moieties of said lytic peptide are selected from the group consisting of N-palmitoyl; Nα- palmitoyl; Nε-palmitoyl; Nα, Nε-dipalmitoyl; Nα, α-dipalmityl; Nε, Nε-dipalmityl; and N, N-dipalmityl-glycyl .

48. The method of claims 19 and 20, wherein said lipophilic lytic peptide has the following structure:

49. The method of claim 24, wherein said molecular complex has a -/+/- charge ratio of 1/3/1, 1/2/1, 1/1/1, 4/0/1, 2/1/0, 4/2/1, 4/3/1, and 4/4/1, wherein said -/+/- ratio is nucleic acid molecule/lipophilic binding peptide/lipophilic lytic peptide.

50. The method of claim 24, wherein said molecular complex has a -/- charge ratio of 1/4, 1/3, 1/2, and 1/1, wherein said -/- ratio is nucleic acid molecule/lipophilic lytic peptide.

51. The method of claim 24, wherein said molecular complex has a -/+ charge ratio of 1/3, 1/2, and 1/1, wherein said -/+ ratio is nucleic acid molecule/lipophilic binding peptide .

52. The method of claims 19, 20, and 21, wherein said organism is fasted before administering said molecular complex.