PEPTIDES FOR EFFICIENT GENE TRANSFER

Abstract: The present invention relates to nucleic acid condensates comprising a nucleic acid bound to a polycationic peptide, and in particular a CWK (cysteine, tryptophan, lysine) polycationic peptide, and to methods of making and using such condensates. The invention further relates to a novel pharmaceutical compositions comprising condensed DNA incorporated into matrices (gene-activated matrices) that may be utilized for delivery of nucleic acids into targeted cells. The invention further relates to methods for producing gene-activated matrices involving the addition of polycationic peptides, and in particular a CWK polycationic peptide, to negatively charged DNA prior to incorporation into a matrix. The invention further relates to the linkage of the polycationic peptides to ligand molecules, thus permitting targeting of the DNA to specific targeted cell types. The present invention provides pharmaceutical formulations and methods that are applicable to wound healing and a wide variety of genetic or acquired diseases.

Description: PEPTIDES FOR EFFICIENT GENE TRANSFER This invention was made with government support awarded by the National Institutes of Health (Grant Numbers GM48049 and DE13004). The United States government has certain rights in the invention. 1. INTRODUCTION The present invention relation to the introduction of nucleic acids into cells. In particular, the present invention relates to compositions and methods of nucleic acid formulation for nucleic acid delivery. The invention further relates to methods for producing condensed nucleic acids involving the addition of polycationic peptides, and in particular a CWK (cysteine, tryptophan, lysine) polycationic peptide, to negatively charged nucleic acid. The addition of a polycationic peptide to the nucleic acid results in partial neutralization of the nucleic acid charge and extensive compaction of the nucleic acid particles. The invention further relates to the linkage of the polycationic peptides to ligand molecules, thus permitting targeting of the nucleic acid to specific targeted cell types and/or the attenuation of non-specific transfer to non-targeted cells. The use of ligand/nucleic acid complexes has the advantage of providing a natural mechanism for delivery of nucleic acid directly to cells and even to the nucleus, thus avoiding endosomal destruction. The condensed nucleic acids can be administered to a subject by various methods known to those of skill in the art, e.g., intravenous injection. In a preferred embodiment, the present invention relates to novel pharmaceutical compositions comprising condensed nucleic acid incorporated into matrices (gene-activated matrices) that may be utilized for delivery of nucleic acids into targeted cells. The invention further relates to methods for producing gene- activated matrices involving the addition of polycationic peptides, and in particular a CWK polycationic peptide, to negatively charged nucleic acid prior to incorporation into a matrix. The use of condensed nucleic acid in the gene- activated matrices results in stabilization of the nucleic acid during in vi tro formulation, increases the stability of nucleic acid in vivo and increases the efficiency of nucleic acid transfer in vivo, thereby providing a more efficient localized sustained gene delivery system. The present invention provides pharmaceutical formulations and methods that are applicable to wound healing and a wide variety of genetic or acquired diseases. 2. BACKGROUND OF THE INVENTION 2.1. GENE THERAPY Gene therapy was originally conceived of as a specific gene replacement therapy for correction of heritable defects to deliver functionally active therapeutic genes into targeted cells. Initial efforts toward somatic gene therapy have relied on indirect means of introducing genes into tissues, called ex vivo gene therapy, e.g., target cells are removed from the body, transfected or infected with vectors carrying recombinant genes, and re-implanted into the body ("autologous cell transfer"). A variety of transfection techniques are currently available and used to transfer nucleic acid in vi tro into cells; including calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, liposome mediated nucleic acid transfer or transduction with recombinant viral vectors. Such ex vivo treatment protocols have been proposed to transfer nucleic acid into a variety of different cell types including epithelial cells (U.S. Patent 4,868,116; Morgan and Mulligan WO87/00201; Morgan et al . , 1987, Science 237:1476-1479; Morgan and Mulligan, U.S. Patent No. 4,980,286), endothelial cells (WO89/05345) , hepatocytes (WO89/07136; Wolff et al . , 1987, Proc. Natl. Acad. Sci. USA 84:3344-3348; Ledley et al . , 1987 Proc. Natl. Acad. Sci. 84:5335-5339; Wilson and Mulligan, WO89/07136; Wilson et al . , 1990, Proc. Natl. Acad. Sci. 87:8437-8441) fibroblasts (Palmer et al . , 1987, Proc. Natl. Acad. Sci. USA 84:1055-1059; Anson et al . , 1987, Mol . Biol. Med. 4:11-20; Rosenberg et al . , 1988, Science 242:1575- 1578; Naughton & Naughton, U.S. Patent 4,963,489), lymphocytes (Anderson et al . , U.S. Patent No. 5,399,346; Blaese, R.M. et al . , 1995, Science 270:475-480) and 5 hematopoietic stem cells (Lim, B. et al. 1989, Proc. Natl. Acad. Sci. USA 86:8892-8896; Anderson et al . , U.S. Patent No. 5,399,346) . Direct in vivo gene transfer has recently been attempted with formulations of DNA trapped in liposomes (Ledley et al . , 10 1987, J. Pediatrics 110:1); or in proteoliposomes that contain viral envelope receptor proteins (Nicolau et al . , 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1068); and DNA coupled to a polylysine-glycoprotein carrier complex. In addition, "gene guns" have been used for gene delivery into cells 15 (Australian Patent No. 9068389) . It has been shown that naked DNA, or DNA associated with liposomes, can be formulated in liquid carrier solutions for injection into interstitial spaces for transfer of DNA into cells (Feigner, WO90/11092) . 20 One of the problems associated with gene therapy is the lack of methods for local delivery of nucleic acid to targeted host cells versus systemic delivery. Current gene therapy methods utilize delivery vehicles such as liposomes, or recombinant viral vectors which are capable of systemic 25 gene delivery but fail to selectively transfer DNA locally to the targeted host cell. In contrast, gene-activated matrices can be used to deliver DNA selectively to targeted host cells through transplantation of the matrices to the site of gene delivery. 30 Perhaps one of the greatest problems associated with currently devised gene therapies, whether ex vivo or in vivo, is the inability to transfer DNA efficiently into a targeted cell population and to achieve high level expression of the gene product in vivo . Viral vectors are regarded as the 35 most efficient system, and recombinant replication-defective viral vectors have been used to transduce (i.e. , infect) cells both ex vivo and in vivo . Such vectors have included retroviral, adenovirus and adeno-associated and herpes viral vectors. While highly efficient at gene transfer, the major disadvantages associated with the use of viral vectors include the inability of many viral vectors to infect non- dividing cells; problems associated with insertional mutagenesis; inflammatory reactions to the virus and potential helper virus production; inflammatory reactions to the recombinant proteins, and/or production and transmission of harmful virus to human patients. The efficiency of gene transfer into cells directly influences the resultant gene expression levels. In addition to the low efficiency of most cell types to take up and express foreign DNA, many targeted cell populations are found in such low numbers in the body that the efficiency of presentation of DNA to the specific targeted cell types is even further diminished. In approaches to increase the efficiency of gene transfer into cells the nucleic acid is typically complexed with carriers that facilitate the transfer of the DNA across the cell membrane for delivery to the nucleus. The carrier molecules bind and neutralize the charge of the condensed DNA thereby facilitating DNA transfer across the membrane. In addition, the carrier molecules act as scaffolding to which ligands may be attached in order to achieve site or cell specific targeting of DNA. The most commonly used DNA condensing agent for the development of nonviral gene delivery systems include polylysine in the size range of dp 90-450 (Misoux, P. et al., Nucleic Acid Res., 1993, 21:871-878) and low molecular weight glycopeptides (Wadhwa et al., 1995, Bioconjugate Chemistry 6:283-291). Polylysine amino groups have been derivatized with transferrin, glycoconjugates, folate, lectins, antibodies or other proteins to provide specificity in cell recognition, without comprising its binding affinity for DNA. However, the high molecular weight and polydispersity of polylysine also contribute to toxicity and a lack of chemical control in coupling macromolecular ligands which leads to heterogeneity in polylysine-based carrier molecules. This can complicate the formulation of DNA carrier complexes and limits the ability to systematically optimize carrier design to achieve maximal efficiency. Clearly, there is a need for improved methods of gene delivery. Such methods should be amenable to use with virtually any gene of interest and permit the introduction of genetic material into a variety of cells and tissues. 2.2. PHARMACEUTICAL FORMULATIONS Pharmaceutical formulations containing DNA have been generated to deliver DNA intracellularly for use in gene therapy. Such formulations include, for example, liposomes, microspheres and/or nanospheres. One advantage associated with the use of such formulations includes their ability to provide sustained or controlled release of pharmaceutical agents such as DNA. Efforts to formulate supercoiled DNA within liposomes, nanospheres and/or microspheres has been hampered by the fragmentation of DNA associated with the mechanical forces normally required for the emulsification of sustained DNA delivery vehicles. Such forces include mixing, sonication, vortexing and shaking. For example, DNA nanosphere particles are formed using an established water-oil-water double emulsion method. Unfortunately, each emulsifying step involves a mixing (e.g., sonication) step that results in fragmentation of DNA. Accordingly, it would be extremely advantageous to have available methods for ensuring the stability of supercoiled DNA formulated within gene delivery vehicles (i.e., without fragmentation of the DNA). These devices would be particularly advantageous for delivering DNA for use in gene therapy. 2.3. RECEPTOR-MEDIATED GENE DELIVERY Receptor-mediated gene delivery has emerged as a potentially useful approach for introduction of DNA into cells in vivo . An advantage of this gene delivery method is the ability to target DNA to specific tissue or cell types based on the recognition of ligands by unique receptors expressed on the cell surface (Wu, G.Y. et al., 1988, J. Biol. Chem. 263:14621-14624; Christiano et al., 1993, Proc. Natl. Acad. Sci., U.S.A. 90:2122-2126; Huckett et al., 1990, Biochem. Pharmacol. 40:253-263; Perales, J.C. et al . , 1994, Eur. J. Biochem. 226:255-266). In addition, this particular delivery system is not limited by the size of the DNA and the system does not involve the use of infectious agents. Receptor-mediated gene transfer has considerable potential for use in human gene therapy if the method can be developed to a point where it is both a reliable and efficient approach for delivery in targeted host cells. The advantage of receptor-mediated gene delivery is that DNA molecules may be targeted to specific cell types (in a mixed population of cells) utilizing receptor-ligand instructions. The major shortcomings of currently available techniques are transient, variable and low level expression of the transferred DNA and toxicity. Any method designed to increase the efficiency of transfer of DNA into the cell will facilitate the successful development of receptor-mediated gene delivery protocols. 3. SUMMARY OF THE INVENTION The present invention relates to compositions and methods for the introduction of nucleic acids into cells (both in vivo and in vitro) at improved efficiencies. In particular, the present invention relates to condensed nucleic acids and methods for preparation of such condensed nucleic acids for use in nucleic acid delivery, e.g., gene delivery. The invention contemplates the use of any polycationic peptide that results in condensation of nucleic acid, and in particular, a CWK (cysteine, tryptophan, lysine) polycationic peptide. The nucleic acid-peptide condensates of the present invention are particularly suited for specific delivery of nucleic acid into targeted host cells. The condensed nucleic acids can be administered to a subject by various methods known to those of skill in the art, e.g., intravenous injection. The nucleic acid-peptide condensates of the present invention are found to possess enhanced stability in vivo, thus increasing the efficiency of sustained gene transfer in the host. In addition, the CWK- polycationic peptide nucleic acid complex may be attached to ligands, yielding low molecular weight carriers useful for site-specific gene delivery. In one embodiment of the invention, the nucleic acid- peptide condensates are derivatized with polyethylene glycol (PEG), preferably by covalent attachment of PEG to the CWK peptide, to form PEG-peptide-nucleic acid condensates. Such condensates display an improved ability to avoid non-specific gene transfer to non-targeted cells. The PEG moiety also provides a suitable site for the attachment of ligands and other molecules capable of, for example, targeting the condensates to specific cells or promoting DNA uptake by the cell nucleus. In a preferred embodiment, the invention relates to gene-activated matrices comprising the nucleic acid-peptide condensates of the present invention incorporated into said matrices, as well as methods for preparation of such matrices for use in nucleic acid delivery. The nucleic acid-peptide condensates are incorporated into matrices for use in local delivery of nucleic acids into targeted host cells. It is believed that the mechanism of peptide-mediated gene transfer is related to the efficiency of condensing nucleic acid into small particles. While not limited to any particular -theory, it is believed that the CWK polycationic peptide plays a specific role in neutralizing the charge of the nucleic acid and organizing the nucleic acid into small condensates that exhibit enhanced gene transfer efficiency. Thus, the use of such peptide-mediated gene transfer is especially well suited when large fragments of nucleic acid, or multiple plasmids, are to be formulated into matrices. Additionally, the methods of the invention provide a means for stabilizing nucleic acid during in vitro formulation of the gene-activated matrices containing the condensed nucleic acid. Condensation of the nucleic acid prior to incorporation into matrices is found to protect the nucleic acid against the fragmentation associated with the mechanical forces required for formation of the nucleic acid incorporated matrices. The gene-activated matrices comprising condensed nucleic acid are also found to be more stable in vivo, thus increasing the efficiency of sustained local gene transfer in the host. In addition, the CWK- polycationic peptide DNA complex may be attached to ligands yielding low molecular weight carriers useful for site specific gene delivery. In one embodiment, the method comprises the formation of a gene-activated matrix comprising a CWK polycationic peptide associated with DNA. The result is a matrix incorporating condensed DNA that can be introduced into a host under conditions such that said DNA is delivered across the cell membrane. The gene-activated matrices may be derived from any biocompatible material. The condensed DNA may encode any of a variety of therapeutic proteins and may be used to treat a wide variety of genetic or acquired diseases. Such proteins may include growth factors, cytokines, hormones or any other proteins capable of regulating the growth, differentiation or physiological function of cells. In a specific embodiment of the present invention condensed DNA preparations may be incorporated into polylactic-polyglycolic (PLGA) microspheres and/or nanospheres. Currently, methods for incorporating DNA into microspheres and/or nanospheres use an established water-oil- water double emulsion method which involves at least one sonication step and which results in fragmentation of the DNA. The use of polycationic peptides provides a method for protecting DNA from sonication induced fragmentation that normally occurs during incorporation of DNA into microspheres and/or nanospheres. The present invention also contemplates the use of the peptides of the present invention in receptor-mediated gene transfer (both in vi tro and in vivo) . In one embodiment, the method comprises linking the DNA to a CWK polycationic peptide having a covalently attached ligand, which is selected to target a specific receptor on the surface of the cell of interest. The DNA is taken up by the cell, transported to the nucleus and expressed. The present invention is particularly well suited for wound healing based on the discovery that repair cells involved in the wound healing process will naturally proliferate and migrate to the site of injury and infiltrate a gene-activated matrix where they will take up and express DNA. In an embodiment of the invention, the condensed DNA compositions may be used to transfer DNA into mammalian repair cells at the site of a wound. The present invention also provides pharmaceutical formulations and methods that may also be used as a method of gene therapy and may be applicable to a wide variety of genetic or acquired diseases. The condensed DNA may comprise genes encoding therapeutically useful proteins such as growth factors, cytokines, hormones, etc. The present invention provides compositions and methods for increasing the efficiency of gene transfer through condensation of DNA prior to incorporation into a matrix. Condensation of DNA increases the efficiency of gene transfer through stabilization of the DNA during in vi tro preparation of the condensed DNA. It is not intended that the present invention be limited by the nature of the nucleic acid. The target nucleic acid may be native or synthesized nucleic acid. The nucleic acid may be from a viral, bacterial, animal or plant source. 4. BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Schematic figure showing plasmid DNA condensed with the Alk-CWK 18 peptide. FIG. 2. Gel Electrophoresis of Supercoiled, Circular, and Linear DNA. The result of converting supercoiled pCMVL (lane 1) to open circular (lane 2) and linear (lane 3) DNA is shown. Each lane was loaded with 0.5 μg of DNA and electrophoresed. FIG. 3. Gene Transfer Efficiency of Supercoiled, Circular, and Linear DNA. The transfection efficiency of the three types of DNA shown in FIG. 2 were compared. Each transfection utilized 10 μl of DNA combined with 3 nmol of Alk-CWK 18 μg in 200 μl of 5 mM Hepes pH 7.4. The results established a slight (10%) reduction in gene transfer when using open circular DNA and a 90% reduction when the DNA is linear relative to supercoiled DNA. FIG. 4. Stability of Peptide/DNA Condensates to Ultrasonication. Gel electrophoresis with ethidium staining was used to demonstrate the fragmentation of plasmid DNA on sonication for 15, 30 and 60 sec (lanes 2, 3, & 4) relative to standard DNA (lane 1) . After complexation with Alk-CWK 18 and sonication for 60 seconds, plasmid DNA failed to migrate on gel electrophoresis (lane 5) . Treatment of the sonicated condensed DNA with trypsin restored migration but led to the formation of some linear DNA (lane 6) . Omission of sonication (lane 7) or both sonication and condensation with Alk-CWK 18 (lane 8) resulted in identical banding patterns indicating that linear DNA was an artifact of contaminated trypsin. FIG. 5. Gene Transfer Efficiency Of Sonicated Peptide/DNA Condensates. The expression of luciferase following in vi tro transfection of HepG2 cells is compared for Alk-CWK 18 /DNA condensates subjected to 0, 15, 30 and 60 sec of sonication, relative to plasmid DNA sonicated for 60 sec then condensed with Alk-CWK 18 (open bar) . FIG. 6. Sodium Chloride Induced Dissociation of Peptide/DNA Condensates. The stability of peptide/DNA condensates to sonication in the presence of 0-1 M sodium chloride is demonstrated using gel electrophoresis. Alk-CWK 8 (panel A) , Alk-CWK 18 (panel B) and dimeric-CWK 18 (panel C) DNA condensates were treated with 0.1, 0.2, 0.3, 0.4, 0.6, 0.8 and 1 M sodium chloride prior to 60 sec sonication and trypsin digestion. The dissociation of the peptide/DNA condensate was observed at 0.1 M for Alk-CWK 8 (panel A), 0.4 M Alk-CWK 18 (panel B) , and above 0.8 M for dimeric-CWK 18 (panel C) DNA condensates. FIG. 7. Serum Stability of Peptide/DNA Condensates. Uncondensed plasmid DNA (panel A) , Alk-CWK 18 /DNA condensates (panel B) , Alk-CWK 8 /DNA condensates (panel C) , and LipofectAce/DNA complexes (panel D) were incubated with mouse serum at 37°C and analyzed by gel electrophoresis. Time points were analyzed at 0, 5, 15, 30, 45, 60, 120, and 180 min in lanes 1-8 respectively. The rapid conversion of supercoiled DNA to linear DNA is evident in panels A and C and the formation of fragmented DNA is nearly complete at 2 hrs indicating a lack of protection afforded linear DNA is evident in panels A and C and the formation of fragmented DNA is nearly complete at 2 hrs indicating a lack of protection afforded by Alk-CWK 8 . LipofectAce/DNA complex showed a similar profile but the DNA fragmentation was slower than for uncondensed DNA. Panel B illustrates the protective effects of Alk-CWK 18 during the same three hour incubation. FIG. 8. In Vitro Study, Cell Culture Model. Following transfection of cultured cells significant amounts of heat stable alkaline phosphatase was found to be reproducibly expressed by 293T cells following gene transfer in vi tro . FIG. 9. In Vitro Study, Canine Model. The surgical procedure involved the placement of 8 mm x 8 mm cylindrical osteotomy defects in the metaphysis of the R and L femur and tibia (each dog) . Three weeks after implantation, gap tissues were harvested, snap frozen, powdered, and processed for heat-stable alkaline phosphatase activity (Tropix) . SC- mg refers to a GAM implant that contains 8.0 mg supercoiled plasmid DNA. C-μg refers to an independent GAM implant that contains lOOμg of condensed plasmid DNA. FIG. 10. QELS Particle Size and Zeta Potential Analysis of Peptide DNA Co-Condensates. Particle size analysis was used to characterize peptide DNA co-condensates prepared at 50 μg/ml of DNA and varying mol% of Alk-CWK 18 and PEG-CWK 18 as shown in panel A. The zeta potential of DNA co-condensates is shown in panel B. The mean particle size changes from 65 to 80 nm whereas the zeta potential of DNA co-condensates decreases from +35 to +10 mV with increasing mol% of PEG- CWK 18 . FIG. 11. RP-HPLC Analysis of Peptide DNA Co- Condensates. The time course of dialysis of free Alk-CWK 18 (•), free PEG-CWK 18 (■), Alk-CWK 18 DNA condensates (T) , PEG- CWK 18 DNA (A), and co-condensates of 25:75 (♦) , 50:50 (0), 75:25 (0) mol% of Alk-CWK 18 : PEG-CWK 18 bound to DNA was determined by tryptophan fluorescence in the retentate (panel A) . After 75 hrs of dialysis, peptide DNA condensates in the retentate were dissociated with sodium chloride and directly chromatographed by RP-HPLC. Panels B-F illustrate chromatograms resulting from 100 mol% PEG-CWK 18 DNA condensates (panel B) , DNA co-condensates prepared with 75:25 (panel C) , 50:50 (panel D) , 25:75 (panel E) PEG-CWK 18 :Alk- CWK 18 , and 100 mol% Alk-CWK 18 DNA condensates (panel F) . FIG. 12. Solubility of Peptide DNA Condensates. Particle size analysis was performed as a function of DNA concentration using 100 mol% Alk-CWK 18 (•) and 100 mol% PEG- CWK^ (A) DNA condensates and using Alk-CWK 18 : PEG-CWK 18 DNA co- condensates prepared with 50 (♦) and 90 (■) mol% PEG-CWK 18 . The particle size increased to >400 nm above 500 μg/ml for Alk-CWK 18 DNA condensates but remained at <100 nm for PEG-CWK 18 DNA condensates throughout concentrations up to 2 mg/ml. 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to compositions and methods for the introduction of nucleic acids into cells (both in vivo and in vi tro) at improved efficiencies. In particular, the present invention relates to condensed nucleic acids and methods for preparation of such condensed nucleic acids for use in nucleic acid delivery, e.g., gene delivery. The invention contemplates the use of any polycationic peptide that results in condensation of nucleic acid, and in particular, a CWK (cysteine, tryptophan, lysine) polycationic peptide. The nucleic acid-peptide condensates of the present invention are particularly suited for specific delivery of nucleic acid into targeted host cells. The condensed nucleic acids can be administered to a subject by various methods known to those of skill in the art, e.g., intravenous injection. The nucleic acid-peptide condensates of the present invention are found to possess enhanced stability in vivo , thus increasing the efficiency of sustained gene transfer in the host. In addition, the CWK- polycationic peptide nucleic acid complex may be attached to ligands yielding low molecular weight carriers useful for cell and/or tissue specific gene delivery. In a preferred embodiment, the present invention relates to compositions comprising condensed nucleic acid incorporated into matrices that may be utilized for efficient delivery of nucleic acids into targeted cells and methods for formulating such compositions. The invention further relates to methods for producing gene-activated matrices. The composition and methods of the invention can be used to increase gene transfection efficiencies through condensation of nucleic acid and incorporation into matrices prior to contact with the targeted cell. The method involves the ionic interaction between a polycationic peptide, and in particular a CWK polycationic peptide, to negatively charged nucleic acid resulting in partial neutralization of the charge and condensation of nucleic acid. The invention is based, in part, on the discovery that the addition of a CWK polycationic peptide to nucleic acid is capable of increasing gene transfer efficiency through condensation of nucleic acid. The increased efficiency of gene transfer may result from stabilization of the nucleic acid during in vi tro formulation of the gene-activated matrix. Condensation of the nucleic acid protects the nucleic acid from the fragmentation associated with the mechanical forces, i.e., mixing, vortexing and sonication, utilized during formation of certain types of matrices. For example, it was discovered that particles of condensed nucleic acid are strongly protected from fragmentation induced by mechanical forces such as sonication. Such protection is important for successful encapsulation of nucleic acid into certain types of matrices, such as nanospheres and microspheres that require sonication during their production. In addition, condensation of nucleic acid increases the efficiency of gene transfer through stabilization of nucleic acid in vivo resulting in an increased ability of the compositions of the invention to provide a local, sustained nucleic acid delivery system. In an embodiment of the invention the condensed nucleic acid is incorporated into any biocompatible matrix material. Such materials may include, but are not limited to, biodegradable or non-biodegradable materials that support all attachment and growth, powders or gels. Materials may be derived from synthetic polymers or naturally occurring proteins such as collagen, other extracellular matrix proteins, or other structural macromolecules . In a further embodiment of the invention condensed nucleic acid may be incorporated into polylactic-polyglycolin (PLGA) microspheres and/or nanospheres and used for delivery of nucleic acid into targeted host cells. Such microspheres and nanospheres comprise a biodegradable polymeric core having a nucleic acid incorporated therein. In yet another embodiment of the invention, the polycationic peptides of the present invention may be used in receptor-mediated gene transfer protocols. In such an embodiment, nucleic acid is covalently linked to the CWK polycationic peptide which is attached to a ligand, wherein the ligand is selected to target a specific receptor on the surface of the tissue of interest. In preferred embodiment, a DNA-peptide-ligand is incorporated into a matrix for delivery of DNA to specific targeted host cells. The DNA is taken up by the targeted cell, transported to the nucleus and expressed for varying amounts of time. In certain cases, the use of ligand/DNA complexes has the advantage of providing a natural mechanism for delivery of DNA directly to the nucleus, thus avoiding endosomal destruction. In an embodiment of the invention, the methods of the invention may be used as a drug delivery system for transfer of DNA into targeted host cells. The DNA to be used in the practice of the invention may include DNA encoding translational products (i.e., proteins) or transcriptional products ( i . e . , antisense or ribozymes) that act as therapeutic agents for treatment of wounds and diseases. For example, the DNA may comprise genes encoding therapeutically useful proteins such as growth factors, cytokines, hormones, etc. Additionally, the DNA may encode antisense or ribozyme molecules that may inhibit the translation of mRNAs encoding proteins that, for example, inhibit wound healing, induce inflammation or cause disease. Furthermore, the DNA may encode a nucleic acid capable of forming a DNA triple helix, thereby attenuating transcription. The invention is demonstrated, by way of examples, wherein the efficiency of both in vitro and in vivo transfer of DNA into cells is increased by addition of polycationic CWK peptide to the DNA. 5.1. THE CONDENSED DNA COMPOSITIONS The present invention relates to compositions comprising nucleic acid molecules ionically bound to polycationic peptides that result in condensation of the DNA, and in particular, to a CWK polycationic peptide. The invention also relates to methods for increasing gene-transfer efficiencies through condensation of DNA prior to contact with the target cell. The particularly preferred compositions of the invention can be used to transfer a wide variety of therapeutic nucleic acids into targeted cells. The compositions comprise nucleic acid molecules bound to a polycationic peptide, and in particular, a CWK polycationic peptide. Although exemplified herein by specific CWK polycationic peptides, e.g., CWK 8 , CWK 18 , Alk- CWK 18 , PEG-CWK 18 , and 18 KWC-CWK 18 , one of skill in the art will recognize that the present invention provides a wide array of related CWK polycationic peptides capable of condensing a nucleic acid. In particular, the size of the peptides, the ordering and number of particular amino acids, as well as the nature of the specific amino acids themselves can be altered to provide various alternate embodiments of the CWK polycationic peptide. All such embodiments fall within the scope of the instant invention. In a preferred embodiment of the invention, the CWK polycationic peptide comprises the peptide element C;ι . -W m -K n , where C is cysteine, W is tryptophan, K is lysine, and 1, m and n are integers greater than or equal to one. The value of 1 is preferably one. The value of m is preferably less than ten, more preferably less than six, still more preferably less than four, and most preferably one. The value of n is preferably between 6 and 50, more preferably between 8 and 40, still more preferably between 10 and 30, even more preferably between 13 and 20, and most preferably 18. The specific ordering of the amino acid residues comprising the CWK polycationic peptide can be varied to achieve alternate embodiments of the invention. Thus, in some embodiments of the invention the C residues or residues will be located at the C-terminus or at an interior location of the peptide. Likewise, the K and W residues can be located throughout the peptide, either contiguous to one another or interspersed with one another. Furthermore, certain amino acid residues in the CWK polycationic peptide structure can be replaced with other amino acid residues without significantly deleteriously affecting the activity of the peptides, and in some instances the replacement can even enhance the activity of the peptides. Thus, also contemplated by the present invention are altered, or mutated, forms of the CWK polycationic peptide wherein at least one defined amino acid residue in the structure is substituted with another amino acid residue. One critical feature affecting the activity of the polycationic peptide of the invention is believed to be its ability to condense DNA; it will be recognized that in preferred embodiments of the invention, the amino acid substitutions are conservative, i.e., the replacing amino acid residue has physical and chemical properties that are similar to the amino acid residue being replaced. In particular, the K residue or residues can be replaced by any other amino acid, whether naturally occurring or non- natural, that exists as a cation under the contemplated conditions of use. Such conservative substitutions are described more fully below. The W residue or residues can be replaced with other hydrophobic residues capable of interacting with a nucleic acid, preferably aromatic amino acid residues such as tyrosine and phenylalanine, or non- coded analogs of these naturally occurring amino acids. The C residue or residues can likewise be replaced, preferably with another amino acid capable of cross-linking or otherwise forming a covalent linkage with another molecule. For purposes of determining conservative amino acid substitutions, the amino acids can be conveniently classified into two main categories — hydrophilic and hydrophobic— depending primarily on the physical-chemical characteristics of the amino acid side chain. These two main categories can be further classified into subcategories that more distinctly define the characteristics of the amino acid side chains. For example, the class of hydrophilic amino acids can be further subdivided into acidic, basic and polar amino acids. The class of hydrophobic amino acids can be further subdivided into apolar and aromatic amino acids. The definitions of the various categories of amino acids that define structure are summarized in TABLE I, below. TABLE I CLASSIFICATIONS OF COMMONLY ENCOUNTERED AMINO ACIDS Classification Genetically Non-Genetically Encoded Encoded Hydrophobic Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4- Cl), Phe(2-F), Phe(3-F), Phe(4-F), hPhe Apolar L, V, I, M, G, A, P t-BuA, t-BuG, Melle, Nle, MeVal, Cha, McGly, Aib Aliphatic A, V, L, I b-Ala, Dpr, Aib, Aha, MeGly, t-BuA, t-BuG, Melle, Cha, Nle, MeVal Hydrophilic Acidic D, E Basic H, K, R Dpr, Orn, hArg, Phe(p-NH 2 ), Dbu, Dab Polar c, Q, N, S, T Cit, AcLys, MSO, bAla, hSer Helix-Breaking P, G D-Pro and other D-amino acids (in L-peptides) While the above-defined categories have been exemplified in terms of the genetically encoded amino acids, the amino acid substitutions need not be, and in certain embodiments preferably are not, restricted to the genetically encoded amino acids. Indeed, the polycationic peptides may contain genetically non-encoded amino acids. Thus, in addition to the naturally occurring genetically encoded amino acids, amino acid residues in the polycationic peptides may be substituted with naturally occurring non-encoded amino acids and synthetic amino acids such as those summarized in TABLE I. Certain commonly encountered amino acids which provide useful substitutions for the polycationic peptides include, but are not limited to, β-alanine (β-Ala) and other omega- amino acids such as 3-aminopropionic acid, 2,3- diaminopropionic acid (Dpr) , 4-aminobutyric acid and so forth; -aminoisobutyric acid (Aib) ; e-aminohexanoic acid (Aha) ; δ-aminovaleric acid (Ava) ; N-methylglycine or sarcosine (MeGly) ; ornithine (Orn) ; citrulline (Cit) ; t-butylalanine (t-BuA) ; t-butylglycine (t-BuG) ; N-methylisoleucine (Melle) ; phenylglycine (Phg) ; cyclohexylalanine (Cha) ; norleucine (Nle) ; naphthylalanine (Nal) ; 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1, 2, 3, -tetrahydroisoquinoline-3-carboxylic acid (Tic) ; β-2-thienylalanine (Thi) ; methionine sulfoxide (MSO) ; homoarginine (hArg) ; N-acetyl lysine (AcLys) ; 2,4- diaminobutyric acid (Dbu); 2, 3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH 2 )); N-methyl valine (MeVal); homocysteine (hCys) , homophenylalanme (hPhe) and homoserine (hSer) ; hydroxyproline (Hyp) , homoproline (hPro) , N- methylated amino acids and peptoids (N-substituted glycines) . It is to be understood that TABLE I is for illustrative purposes only and does not purport to be an exhaustive list of amino acid residues that can be used to substitute the core peptides described herein. Other amino acid residues not specifically mentioned herein can be readily categorized based on their observed physical and chemical properties in light of the definitions provided herein. While in most instances, the amino acids of the polycationic peptide will be substituted with L-enantiomeric amino acids, the substitutions are not limited to L-enantiomeric amino acids. Thus, also included in the definition of "mutated" or "altered" forms are those situations where an L-amino acid is replaced with an identical D-amino acid (e.g., L-Arg - D-Arg) or with a D- amino acid of the same category or subcategory (e.g. , L-Arg → D-Lys), and vice versa. The polycationic peptides can also be extended at one or both termini or internally with additional amino acid residues that do not substantially interfere with, and in some embodiments even enhance, the structural and/or functional properties of the polycationic peptide. Preferably, such extended peptides will substantially retain the net properties of the polycationic peptides, i.e., condensation of DNA. It is to be understood that the present invention also contemplates peptide analogues wherein one or more amide linkage is optionally replaced with a linkage other than amide, preferably a substituted amide or an isostere of amide. Thus, while the amino acid residues within polycationic peptides are generally described in terms of amino acids, and preferred embodiments of the invention are exemplified by way of peptides, one having skill in the art will recognize that in embodiments having non-amide linkages, the term "amino acid" or "residue" as used herein refers to other bifunctional moieties bearing groups similar in structure to the side chains of the amino acids. Additionally, one or more amide linkages can be replaced with peptidomimetic or amide mimetic moieties which do not significantly interfere with the structure or activity of the peptides. Suitable amide mimetic moieties are described, for example, in Olson et al., 1993, J. Med. Chem. 36:3039-3049. In a preferred embodiment of the present invention, the sulfhydryl group of a CWK polycationic peptide serves to covalently bridge the peptide to another molecule. The sulfhydryl group can be blocked, for example by alkylation with an alkylating group such as iodoacetic acid. Alternatively, the sulfhydryl can be used to link a CWK polycationic peptide to another molecule by means of a disulfide bridge. In a preferred embodiment of the invention the disulfide bridge will link the CWK polycationic peptide to another peptide, preferably another CWK polycationic peptide, forming dimeric-CWK. In another preferred embodiment, CWK polycationic peptide is covalently linked to PEG, typically through attachment at the sulfhydryl group, to form a PEG-CWK. PEG can be attached to the sulfhydryl by means of, for example, a vinyl sulfone on the PEG. As discussed below, the PEG moiety is useful for providing a site of attachment for other molecules, particularly proteins, peptides or other ligands capable of, for example, targeting the nucleic acid condensates to a particular cell type, or promoting DNA uptake by the nucleus. In addition, as discussed below, the PEG moiety has been shown to increase DNA condensate solubility and inhibit non-specific DNA transfer to non-targeted cells. Methods for alkylating, forming disulfide bridges, and attaching PEG and other molecular moieties are well known to those of skill in the art . By "nucleic acid" is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sultone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil) . It is not intended that the present invention be limited by the nature of the nucleic acid employed. The target nucleic acid may be native or synthesized nucleic acid. The nucleic acid may be from a viral, bacterial, animal or plant source. The nucleic acid may be DNA or RNA and may exist in a double-stranded, single-stranded or partially double- stranded form. Nucleic acids useful in the present invention include, by way of example and not limitation, oligonucleotides such as antisense DNAs and/or RNAs; ribozymes; DNA for gene therapy; viral fragments including viral DNA and/or RNA; DNA and/or RNA chimeras; various structural forms of DNA including single-stranded DNA, double-stranded DNA, supercoiled DNA and/or triple-helix DNA; Z-DNA; and the like. The nucleic acids may be prepared by any conventional means typically used to prepare nucleic acids in large quantity. For example, DNAs and RNAs may be chemically synthesized using commercially available reagents and synthesizers by methods that are well-known in the art ( see, e . g. , Gait, 1985, Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, England) . RNAs may be produce in high yield via in vitro transcription using plasmids such as SP65 (Promega Corporation, Madison, WI) . In some circumstances, as where increased nuclease stability is desired, nucleic acids having modified internucleoside linkages may be preferred. Nucleic acids containing modified internucleoside linkages may also be synthesized using reagents and methods that are well known in the art. For example, methods for synthesizing nucleic acids containing phosphonate phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate, formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate, dimethylene-sulfide (-CH 2 -S-CH 2 ) , dimethylene-sulfoxide (-CH 2 - SO-CH 2 ), dimethylene-sulfone (-CH 2 -S0 2 -CH 2 ) , 2'-0-alkyl, and 2 ' -deoxy-2 ' -fluoro phosphorothioate internucleoside linkages are well known in the art (see Uhlmann et al . , 1990, Chem. Rev. 90:543-584; Schneider et al . , 1990, Tetrahedron Lett. 3JL:335 and references cited therein). The nucleic acids may be purified by any suitable means, as are well known in the art. For example, the nucleic acids can be purified by reverse phase or ion exchange HPLC, size exclusion chromatography or gel electrophoresis. Of course, the skilled artisan will recognize that the method of purification will depend in part on the size of the DNA to be purified. The nucleic acid itself may act as a therapeutic agent, such as for example an antisense DNA that inhibits mRNA translation, or the nucleic acid may encode a variety of therapeutic transcription or translation products that will be expressed by the repair cells. Useful transcription products include antisense RNAs, ribozymes, viral fragments and the like. Useful translation products include proteins such as, for example, membrane proteins, transcription factors, intracellular proteins, cytokine binding proteins, and the like. In a preferred embodiment of the invention, the nucleic acid is a DNA molecule that encodes gene products that stimulate or promote healing of wounded or damaged tissues in vivo or alleviate the symptoms of disease. The DNA molecules may include genomic or cDNAs that code for a variety of factors that stimulate or promote healing, including extracellular, cell surface and intracellular RNAs and proteins. Alternatively, the DNA molecules may encode functional proteins which complement absent or defective gene products arising from genetic defects. Examples of extracellular proteins include growth factors, cytokines, therapeutic proteins, hormones and peptide fragments of hormones, inhibitors of cytokines, peptide growth and differentiation factors, interleukins, chemokines, interferons, colony stimulating factors, angiogenic factors and extracellular matrix proteins such as collagen, laminin and fibronectin. Specific examples of such proteins include, but are not limited to, the superfamily of TGF-β molecules including the five TGF-β isoforms and bone morphogenetic factors (BMP) , latent TGF-β binding proteins (LTBP) , keratinocyte growth factor (KGF) , hepatocyte growth factor (HGF) , platelet derived growth factor (PDGF) , insulin-like growth factor (IGF), the basic fibroblast growth factors (FGF-1, FGF-2), vascular endothelial growth factor (VEGF) , Factor VIII and Factor IX, erythropoietin (EPO) , tissue plasminogen activator (TPA) , activins and inhibins. Examples of hormones that stimulate wound healing that may be used in the practice of the invention include growth hormone (GH) and parathyroid hormone (PTH) . Examples of cell surface proteins include the family of cell adhesion molecules ( e . g. , the integrins, selections, Ig family members such as N-CAM and Ll, and cadherins) , cytokine signaling receptors such as the type I and type II TGF-β receptors and the high-affinity FGF receptor and non- signaling co-receptors such as betaglycan and syndecan. Examples of intracellular RNAs and proteins include the family of signal transducing kinases, cytoskeletal proteins such as talin and vinculin, cytokine binding proteins such as the family of latent TGF-β binding proteins and nuclear trans acting proteins such as transcription factors and enhancing factors. The DNA molecules may also encode proteins that block pathological processes, thereby allowing the natural wound healing process to occur unimpeded. Examples of blocking factors include ribozymes that destroy RNA function and DNAs that, for example, code for tissue inhibitors of enzymes that destroy tissue integrity, e.g., inhibitors of metalloproteinases associated with osteoarthritis . One may obtain the DNA segment encoding the protein of interest using a variety of molecular biological techniques, generally known to those skilled in the art. For example, cDNA or genomic libraries may be screened using primers or probes with sequences based on the known nucleotide sequences. Polymerase chain reaction (PCR) may be used to generate the DNA fragment encoding the protein of interest. Alternatively, the DNA fragment may be obtained from a commercial source. Modified gene sequences, i.e. genes having sequences that differ from the gene sequences encoding the native proteins, are also encompassed by the invention, so long as the modified gene still encodes a protein that functions to stimulate healing in any direct or indirect manner. These modified gene sequences include modifications caused by point mutations, modifications due to the degeneracies of the genetic code or naturally occurring allelic variants, and further modifications that have been introduced by genetic engineering, i.e., by the hand of man. Techniques for introducing changes in nucleotide sequences that are designed to alter the functional properties of the encoded proteins or polypeptides are well known in the art. Such modifications include the deletion, insertion or substitution of bases, and thus, changes in the amino acid sequence. Changes may be made to increase the activity of a protein, to increase its biological stability or half-life, to change its glycosylation pattern, and the like. All such modifications to the nucleotide sequences encoding such proteins are encompassed by this invention. The DNA encoding the transcription or translation products of interest may be recombinantly engineered into variety of host vector systems that also provide for replication of the DNA in large scale. These vectors can be designed to contain the necessary elements for directing the transcription and/or translation of the DNA sequence taken up by the repair cells at the wound in vivo . Vectors that may be used include, but are not limited to, those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. For example, plasmid vectors such as pcDNA3, pBR322, pUC 19/18, pUC 118, 119 and the M13 mp series of vectors may be used. Bacteriophage vectors may include λgtlO, λgtll, λgt18-23, λZAP/R and the EMBL series of bacteriophage vectors. Cosmid vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors. Alternatively, recombinant virus vectors including, but not limited to, those derived from viruses such as herpes virus, retroviruses, vaccinia viruses, adenoviruses, deno- associated viruses or bovine papilloma viruses may be engineered. While integrating vectors may be used, non- integrating systems, which do not transmit the gene product to daughter cells for many generations are preferred for wound healing. In this way, the gene product is expressed during the wound healing process, and as the gene is diluted out in progeny generations, the amount of expressed gene product is diminished. In order to express a biologically active protein, the nucleotide sequence coding for the protein may be inserted into an appropriate expression vector, i . e . , a vector which contains the necessary elements for the transcription and translation of the inserted coding sequences. Methods which are well known to those skilled in the art can be used to construct expression vectors having the protein coding sequence operatively associated with appropriate transcriptional/translational control signals. These methods include in vi tro recombinant DNA techniques and synthetic techniques. See, for example, the techniques described in Sambrook, et al . , 1992, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al . , 1989, Current Protocolsin Molecular Biology, Greene Publishing Associates & Wiley Interscience, N.Y. The genes encoding the proteins of interest may be operatively associated with a variety of different promoter/enhancer elements. The promoter/enhancer elements may be selected to optimize for the expression of therapeutic amounts of protein. The expression elements of these vectors may vary in their strength and specificities. Depending on the host/vector system utilized, any one of a number of suitable transcription and translation elements may be used. The promoter may be in the form of the promoter which is naturally associated with the gene of interest. Alternatively, the DNA may be positioned under the control of a recombinant or heterologous promoter, i . e . , a promoter that is not normally associated with that gene. For example, tissue specific promoter/enhancer elements may be used to regulate the expression of the transferred DNA in specific cell types. Examples of transcriptional control regions that exhibit tissue specificity which have been described and could be used include, but are not limited to, elastase I gene control region which is active in pancreatic acinar cells (Swift et al . , 1984, Cell 38:639-646; Ornitz et al . , 1986, Cold Soring Harbor Sv o. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 2:42S-51S); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315 : 115-122) ; immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al . , 1984, Cell 38:647-658; Adams et al . , 1985, Nature 318:533-538; Alexander et al . , 1987, Mol. Cell. Biol. 7:1436-1444): albumin gene control region which is active in liver (Pinkert et al . , 1987, Genes and Devel. 1:268-276) alpha-fetoprotein gene control region which is active in liver (Krumlauf et al . , 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al . , 1987, Science 235:53-58); alpha-1-antitrypsin gene control region which is active in liver (Kelsey et al . , 1987, Genes and Devel. 1 : 161-171); beta-globin gene control region which is active in myeloid cells (Magram et al . , 1985, Nature 315: 338- 340; Kollias et al . , 1986, Cell 46:89-94) ; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al . , 1987, Cell 4.8: 703-712) ; myosin light chain-2 gene control region which is active in skeletal muscle (Shani, 1985, Nature 314 :283-286) ; and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al . , 1986, Science 234:1372-1378) . Promoters isolated from the genome of viruses that grow in mammalian cells, (e.g., vaccinia virus 7.5K, SV40, HSV, adenoviruses MLP, MMTV, LTR and CMV promoters) may be used, as well as promoters produced by recombinant DNA or synthetic techniques. In some instances, the promoter elements may be constitutive or inducible promoters and can be used under the appropriate conditions to direct high level or regulated expression of the gene of interest. Expression of genes under the control of constitutive promoters does not require the presence of a specific substrate to induce gene expression and will occur under all conditions of cell growth. In contrast, expression of genes controlled by inducible promoters is responsive to the presence or absence of an inducing agent. Specific initiation signals are also required for sufficient translation of inserted protein coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire coding sequence, including the initiation codon and adjacent sequences are inserted into the appropriate expression vectors, no additional translational control signals may be needed. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the protein coding sequences to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of transcription attenuation sequences, enhancer elements, etc. In addition to DNA sequences encoding therapeutic proteins of interest, the scope of the present invention includes the use of ribozymes or antisense DNA molecules that may be transferred into or expressed by the mammalian repair cells. Such ribozymes and antisense molecules may be used to inhibit the transcription of DNA or translation of RNA encoding proteins that inhibit the healing process. Transferred or expressed anti-sense RNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. Transferred or expressed ribozymes, which are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA may also be used to block protein translation. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences. RNA molecules may be generated by transcription of DNA sequences encoding the RNA molecule. It is also within the scope of the invention that multiple genes, combined on a single genetic construct under control of one or more promoters, or prepared as separate constructs of the same or different types, may be used. Thus, an almost endless combination of different genes and genetic constructs may be employed. Certain gene combinations may be designed to achieve synergistic effects on cell stimulation and regeneration for healing. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic gene combinations, or even gene- protein combinations. 5.2. PREPARATION OF POLYCATIONIC PEPTIDE BOUND NUCLEIC ACIDS The nucleic acid molecules to be bound to the polycationic peptide may be prepared using methods that are well known to those skilled in the art (see, e.g., techniques described in Sambrook et al., 1992, Molecular Cloning, A Laboratory Manual, Cold spring Harbor Laboratory, N.Y.). The polycationic peptides, and in particular the CWK polycationic peptides, can be synthesized. Core peptides may be prepared using conventional step-wise solution or solid phase synthesis (see, e.g., Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al . , Eds., 1997, CRC Press, Boca Raton Florida, and references cited therein; Solid Phase Peptide Synthesis: A Practical Approach, Atherton & Sheppard, Eds., 1989, IRL Press, Oxford, England, and references cited therein) . The polycationic peptides of the invention can be purified by art-known techniques such as reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like. The actual conditions used to purify a particular polycationic peptide will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art. The preparation and use of polycationic peptides is taught in PCT Appl . No. 98/19711, which is herein incorporated by reference in its entirety. CWK polycationic peptide/DNA complexes are prepared with DNA concentrations ranging from 10-100 μg/ml and CWK polycationic peptide/DNA ratios (nanomole carrier/μg DNA) varying from 0.2-1.0. The optimized complex was prepared at a DNA concentration of 50 μg/ml and a carrier-DNA ratio of 0.3 by adding CWK peptide to DNA while vortexing and allowing the mixture to incubate at room temperature for 30 minutes. The amounts and volumes of carrier and DNA were linearly scaled for preparing different amounts of complex. Solvents to be used included 5mM Hepes, pH 7.4. The relative binding affinity of peptides for DNA can be determined using a fluorescence displacement assay. Titration of peptide with DNA in the presence of intercalator dye leads to a decrease in fluorescence intensity, resulting in titration curves that have minima at the peptide/DNA stoichiometry of maximal binding. Simultaneously, condensation of DNA can be monitored by light scattering, which produces curves that indicate the stoichiometry of maximal condensation. A quantitative measure of the size of the condensed DNA particles can be determined using quasielectric laser scattering (QELS) . QELS analysis provides a mean diameter and the distribution of particles. Nucleic acid condensates with increased solubility and enhanced selectivity against non-specific incorporation into non-targeted cells can be achieved by using PEGylated CWK polycationic peptides. Nucleic acid condensates comprising such peptides can exhibit at least three orders of magnitude greater discrimination against non-specific nucleic acid transfer compared to condensates comprising alkylated CWK polycationic peptides. The ability to form DNA co- condensates that incorporate both PEG and targeting ligands attached to CWK polycationic peptides provides the means for the skilled practitioner to systematically optimize gene delivery formulations for maximum efficacy. 5.3. PREPARATION OF DNA/RECEPTOR-TARGETING LIGAND Receptor mediated gene transfer takes advantage of the ability of receptors located on the cell surface to efficiently bind and internalize a ligand. The components for use in receptor-mediated gene delivery methods of the present invention include (i) a nucleic acid encoding one or more gene products; (ii) a ligand moiety capable of binding to a receptor expressed on the surface of a cell and responsible for the initial interaction of the DNA-ligand complex with the cell membrane; and (iii) a linking CWK polycationic peptide that binds to both the DNA and the ligand and is responsible for the formation of the DNA-ligand complex. The ligand moieties to be used in the practice of the invention, include but are not limited to, naturally occurring proteins or macromolecules that bind to cell surface receptors, structural motifs taken from natural ligands with high affinity for cell surface receptors, or antibodies that recognize epitopes in the extracellular portion of the cell surface receptor and that bind to the cell surface receptor and induce internalization of the DNA- ligand complex. In a specific embodiment of the invention, cytokine fibroblast growth factor 2 (FGF-2) may be conjugated to a CWK polycationic peptide complexed to a DNA molecule. The FGF2 signaling receptor is well known to be highly expressed at sites of tissue repair, and, in particular, on the surface of granulation tissue fibroblasts . Thus, in this setting, repair cells which are localized to the wound site and which express high levels of FGF2 receptor would become transfected and eventually produce DNA-encoded agents that may, for example, enhance wound healing. Additionally, FGF2 is normally shuttled with its receptor from the cell surface to the nucleus, providing a natural mechanism for delivery of the ligand/DNA complex to the nucleus thus avoiding lysosomal destruction. The FGF-2 can be covalently attached to a DNA condensate by means of a PEGylated peptide. In this embodiment of the present invention, the DNA condensate can be formed from CWK polycationic peptides that have been derivatized with PEG- vinyl sulfone. Free thiol groups can be introduced to FGF-2 with dithiobis (succinimidyl propionate) followed by reduction with DTT. These thiol groups can then be reacted with the vinyl sulfone on the end of PEG to create a covalent thiol ether linkage, resulting in the attachment of FGF-2 to the vinyl sulfonate on the surface of the condensed DNA. The expression level of transferred genes is strongly influenced by the degree to which they traffic to lysosomes (relative to nuclear trafficking) . In yet another embodiment of the invention, a fusogenic peptide can be synthesized that undergoes a pH sensitive conformational change in a pre- lysosomal compartment to circumvent cellular trafficking to lysosomes, which leads to rapid DNA turnover. The fusogenic peptide is a 24 mer that possesses a sequence closely analogous to the heme agglutinin viral coat protein that is responsible for viral escape from endosomes (Plank, C. et al., 1994, J. Biol. Chem. 269:12918-12924). During endosome acidification, the peptide undergoes a conformational change from a random coil to an extended rod that inserts into the endosome, resulting in disruption and escape of the condensed DNA into the cytosol. Therefore, the fusogenic peptide strategy represents an additional targeting strategy designed to efficiently deliver DNA from the cell surface to the nucleus. Such fusogenic peptides can be attached to the surface of condensed DNA by reaction with vinyl sulfone on PEG as described above. This will position the fusogenic peptide on the surface of the particle where it can interact with the endosomal membrane as it does in the viral system. The transfection efficiency of the condensed DNA can be compared with, and without, derivatization with fusogenic peptide and optimal levels of covalently attached fusogenic peptide will be determined by comparing the expression levels while changing the loading density of peptide. In yet another embodiment of the invention, a nuclear localizing peptide can be conjugated to the surface of condensed DNA using a single cysteine residue located at the C-terminus to react with a PEG vinyl sulfone. A nuclear localizing peptide can be utilized to enhance the expression levels of the transferred DNA by facilitating the transport of the DNA into the nucleus. For example, a peptide previously shown to enhance the nuclear transport of BSA can be attached to the DNA-peptide condensate. The peptide is a 13 er derived from the conserved region of nuclear proteins known to interact with the nuclear transporter. 5.4. PREPARATION OF GENE-ACTIVATED MATRICES Any biocompatible matrix material containing CWK peptide-condensed DNA encoding a therapeutic agent of interest, such as a translational product, i.e. therapeutic proteins, or transcriptional products, i.e. antisense or ribozymes, can be formulated and used in accordance with the invention. Specific examples and methods of using gene- activated matrices are described in U.S. Patent Application Ser. No. 08/631,338, herein incorporated by reference in its entirety. The gene-activated matrices of the invention may be derived from any biocompatible material. Such materials may include, but are not limited to, biodegradable or non- biodegradable materials formulated into scaffolds that support cell attachment and growth, powders or gels. Matrices may be derived from synthetic polymers or naturally occurring proteins such as collagen, other extracellular matrix proteins, or other structural macromolecules . The polycationic peptide, and in particular, the CWK peptide linked condensed DNA incorporated into the matrix may encode any of a variety of therapeutic proteins depending on the envisioned therapeutic use. Such proteins may include growth factors, cytokines, hormones or any other proteins capable of regulating the growth, differentiation or physiological function of cells. The CWK peptide linked condensed DNA may also encode antisense or ribozyme molecules which inhibit the translation of proteins that inhibit wound repair and/or induce inflammation. In one aspect of the invention, compositions are prepared in which the condensed CWK peptide linked DNA encoding the therapeutic agent of interest is associated with or impregnated within a matrix to form a gene-activated matrix. The matrix compositions function (i) to facilitate ingrowth of cells (targeting) ; and (ii) to harbor DNA until cells arrive (delivery) . Once the gene-activated matrix is prepared it is stored for future use or placed immediately in the host. The type of matrix that may be used in the compositions, devices and methods of the invention is virtually limitless and may include both biological and synthetic matrices. The matrix will have all the features commonly associated with being "biocompatible", in that it is in a form that does not produce an adverse, allergic or other untoward reaction when administered to a mammalian host. Such matrices may be formed from both natural or synthetic materials. The matrices may be non-biodegradable in instances where it is desirable to leave permanent structures in the body; or biodegradable where the expression of the therapeutic protein is required only for a short duration of time. The matrices may take the form of sponges, implants, tubes, telfa pads, band-aids, bandages, pads, lyophilized components, gels, patches, powders or nanoparticles . In addition, matrices can be designed to allow for sustained release of the condensed DNA over prolonged periods of time. The choice of matrix material will differ according to the particular circumstances and the site of the wound that is to be treated. Matrices such as those described in U.S. Patent 5,270,300, incorporated herein by reference, may be employed. Physical and chemical characteristics, such as, e.g., biocompatibility, biodegradability, strength, rigidity, interface properties and even cosmetic appearance may be considered in choosing a matrix, as is well known to those of skill in the art. Appropriate matrices will both deliver the CWK linked condensed DNA molecule and also act as an in situ scaffolding through which mammalian cells may migrate. Where the matrices are to be maintained for extended periods of time, non-biodegradable matrices may be employed, such as sintered hydroxyapatite, bioglass, aluminates, other bioceramic materials and metal materials, particularly titanium. A suitable ceramic delivery system is that described in U.S. Patent 4,596,574, incorporated herein by reference. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate; and they may be processed to modify particular physical and chemical characteristics, such as pore size, particle size, particle shape, and biodegradability. Polymeric matrices may also be employed, including acrylic ester polymers and lactic acid polymers, as disclosed in U.S. Patents 4,521,909, and 4,563,489, respectively, each incorporated herein by reference. Particular examples of useful polymers are those of orthoesters, anhydrides, propylene-cofumarates, or a polymer of one or more γ-hydroxy carboxylic acid monomers, e.g. , γ-hydroxy auric acid (glycolic acid) and/or γ-hydroxy propionic acid (lactic acid) . In preferred embodiments, it is contemplated that a biodegradable matrix will likely be most useful. A biodegradable matrix is generally defined as one that is capable of being reabsorbed into the body. Potential biodegradable matrices for use in connection with the compositions, devices and methods of this invention include, for example, biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polyanhidrides, matrices of purified proteins, and semi-purified extracellular matrix compositions. Other biocompatible biodegradable polymers that may be used are well known in the art and include, by way of example and not limitation, polyesters such as polyglycolides, polylactides and polylactic polyglycolic acid copolymers ("PLGA") (Langer and Folkman, 1976, Nature 263:797-800); polyethers such as polycaprolactone ("PCL"); polyanhydrides; polyalkyl cyanoacrylates such as n-butyl cyanoacrylate and isopropyl cyanoacrylate; polyacrylamides; poly (orthoesters) ; polyphosphazenes; polypeptides; polyurethanes; and mixtures of such polymers. It is to be understood that virtually any polymer that is now known or that will be later developed suitable for the sustained or controlled release of nucleic acids may be employed in the present invention. In preferred embodiments, the biocompatible biodegradable polymer is a copolymer of glycolic acid and lactic acid ("PLGA") having a proportion between the lactic acid/glycolic acid units ranging from about 100/0 to about 25/75. The average molecular weight ("MW") of the polymer will typically range from about 6,000 to 700,000 and preferably from about 30,000 to 120,000, as determined by gel-permeation chromatography using commercially available polystyrene of standard molecular weight, and have an intrinsic viscosity ranging from 0.5 to 10.5. The length of the period of continuous sustained or controlled release of condensed DNA from the matrix according to the invention will depend in large part on the MW of the polymer and the composition ratio of lactic acid/glycolic acid. Generally, a higher ratio of lactic acid/glycolic acid, such as for example 75/25, will provide for a longer period of controlled of sustained release of the nucleic acids, whereas a lower ratio of lactic acid/glycolic acid will provide for more rapid release of the nucleic acids. Preferably, the lactic acid/glycolic acid ratio is 50/50. The length of period of sustained or controlled release is also dependent on the MW of the polymer. Generally, a higher MW polymer will provide for a longer period of controlled or sustained release. In the case of preparing, for example, matrices providing controlled or sustained release for about three months, when the composition ratio of lactic acid/glycolic acid is 100/0, the preferable average MW of polymer ranges from about 7,000 to 25,000; when 90/10, from about 6,000 to 30,000; and when 80/20, from about 12,000 to 30,000. Another type of biomaterial that may be used is small intestinal submucosa (SIS) . The SIS graft material may be prepared from a segment of jejunum of adult pigs. Isolation of tissue samples may be carried out using routine tissue culture techniques such as those described in Badylak et al., 1989, J. Surg. Res. 47:74-80. SIS material is prepared by removal of mesenteric tissue, inversion of the segment, followed by removal of the mucosa and superficial submucosa by a mechanical abrasion technique. After returning the segment to its original orientation, the serosa and muscle layers are rinsed and stored for further use. Another particular example of a suitable material is fibrous collagen, which may be lyophilized following extraction and partial purification from tissue and then sterilized. Matrices may also be prepared from tendon or dermal collagen, as may be obtained from a variety of commercial sources, such as, e.g. , Sigma and Collagen Corporation. Collagen matrices may also be prepared as described in U.S. Patents 4,394,370 and 4,975,527, each incorporated herein by reference. In addition, lattices made of collagen and glycosaminoglycan (GAG) such as that described in Yannas & Burke, U.S. Patent 4,505,266, may be used in the practice of the invention. The collagen/GAG matrix may effectively serve as a support or "scaffolding" structure into which mammalian cells may migrate. Collagen matrix, such as those disclosed in Bell, U.S. Patent No. 4,485,097, may also be used as a matrix material. The various collagenous materials may also be in the form of mineralized collagen. For example, the fibrous collagen implant material termed UltraFiber™, as may be obtained from Norian Corp., (1025 Terra Bella Ave., Mountain View, CA, 94043) may be used for formation of matrices. U.S. Patent 5,231,169, incorporated herein by reference, describes the preparation of mineralized collagen through the formation of calcium phosphate mineral under mild agitation in si tu in the presence of dispersed collagen fibrils. Such a formulation may be employed in the context of delivering a nucleic acid segment to a bone tissue site. Mineralized collagen may be employed, for example, as part of gene- activated matrix therapeutic kit for fracture repair. At least 20 different forms of collagen have been identified and each of these collagens may be used in the practice of the invention. For example, collagen may be purified from hyaline cartilage, as isolated from diarthrodial joints or growth plates. Type II collagen purified from hyaline cartilage is commercially available and may be purchased from, e.g., Sigma Chemical Company, St. Louis. Type I collagen from rat tail tendon may be purchased from, e.g., Collagen Corporation. Any form of recombinant collagen may also be employed, as may be obtained from a collagen-expressing recombinant host cell, including bacterial yeast, mammalian, and insect cells. When using collagen as a matrix material it may be advantageous to remove what is referred to as the "telopeptide" which is located at the end of the collagen molecule and known to induce an inflammatory response. The collagen used in the invention may, if desired be supplemented with additional minerals, such as calcium, e.g., in the form of calcium phosphate. Both native and recombinant type collagen may be supplemented by admixing, absorbing, or otherwise associating with, additional minerals in this manner. The CWK linked condensed DNA matrices of the invention can be transferred to the patient using a variety of techniques. Procedures for transfer of the matrices into a patient include injection into a patient at the site of the wound. Alternatively, the matrices may be surgically placed at the site of the wound either as a therapeutic implant or as a coated device. Therapeutic kits containing a biocompatible matrix and DNA form another aspect of the invention. In some instances the kits will contain preformed gene-activated matrices, thereby allowing the physician to directly administer the matrix within the body. Alternatively, the kits may contain the components necessary for formation of a gene-activated matrix. In such cases the physician may combine the components to form the gene-activated matrices which may then be used therapeutically by placement within the body. In an embodiment of the invention the matrices may be used to coat surgical devices such as suture materials or implants. In yet another embodiment of the invention, gene-activated matrices may include ready to use sponges, tubes, band-aids, lyophilized components, gels, patches or powders and telfa pads. 5.5. PREPARATION OF MICROSPHERES AND/OR NANOSPHERES Microspheres and/or nanospheres are comprised of a biocompatible, biodegradable polymeric core and have at least one pharmaceutical agent, such as condensed DNA of the present invention entrapped, entrained, embedded or otherwise incorporated therein. Typically, the microspheres and/or nanospheres comprise about 0.001% to 30% (w/v) pharmaceutical agent, preferably about 1% to 15% (w/v) pharmaceutical agent. Pre-formed microspheres and/or nanospheres may be prepared using methods commonly employed in the art, such as the methods described in U.S. Patent No. 5,478,564 to Wantier et al . ; European Patent Application EP 190,833 to Yamamoto et al . ; U.S. Patent No. 5,480,656 to Okada et. al . ; and Alle ann et al . , 1992, Intl. J. Pharmaceutics 87:247-253. Alternatively, microspheres and/or nanospheres may be obtained by stirring a coating emulsion of the invention for about 18 hours at room temperature to evaporate organic solvents. The spheres are recovered by ultracentrifugation, washed several times with water and dried in a lyophilizer. Pre-formed or partially-formed microspheres and/or nanospheres encapsulating the condensed DNA may also be suspended in a suitable solvent. Methods for producing such formulations are described in detail in U.S. Patent Application 09/065,892, herein incorporated by reference in its entirety. Such compositions are particularly suited for coating medical devices with microsphere and/or nanosphere coatings. Optionally, the spheres or coating suspensions may further include pharmaceutical agents that facilitate particulate intracellular DNA and/or RNA processing. Such agents include, by way of example and not limitation, compounds that block or disrupt lysosomal action such as chloroquine, cytochalasin B, colchicine, polyvinylpyrrolidone, sucrose, polylysine, and the like. Such compounds will facilitate gene transfer and entry into the cell nucleus. Of course, it is to be understood that in many instances it may be desirable to modify the surface of the microspheres and/or nanospheres or to incorporate additional agents into the microspheres and/or nanospheres. For example, it may be desirable to impart the microspheres and/or nanospheres with the ability to target and bind specific tissues or cells, to be retained at the administration site, to protect incorporated pharmaceutical agents, to exhibit antithrombogenic effects and/or to prevent aggregation. As a specific example, it may be desirable to 5 incorporate receptor-specific molecules, such as for example antibodies, into the microspheres and/or nanospheres to mediate receptor-speci ic particle uptake. Agents and methods for imparting microspheres and nanospheres with these and additional desirable properties are well known in the art 0 ( see, e . g. , Troster et al . , 1990, Intl. J. Pharmaceutics 61:85-100; Davis et al . , 1993, J. Controlled Release 24:157- 163; Muller et al . , 1993, Intl. J. Pharmaceutics 89:25-31; Maruyama et al . , 1994, Biomaterials 15 . : 1035-1042; Leroux et al . , 1994, J. Biomed. Materials Res. 28:471-481). Any of 15 these methods may be used in conjunction with the present invention. The condensed DNA formulations of the invention can be transferred to the patient using various techniques. For example, micro-and/or nanospheres containing the condensed 0 DNA can be transferred directly to the host by the hand of the physician, either as a therapeutic implant or as a coated device (e.g., suture, stent, coated implant, etc.). 5.6. USES OF THE POLYCATIONIC PEPTIDE BOUND 25 CONDENSED DNA The present invention provides pharmaceutical formulations and efficient methods for transferring nucleic acid molecules into a host target cell. The invention is applicable to a wide variety of genetic or acquired diseases. ™ Such genetic diseases include but are not limited to enzyme defect diseases such as adenosine deaminase deficiency. The correct gene for the defective enzyme can be transferred into host cells using the DNA-peptide condensates of the present invention. In addition, nucleic acids encoding for „ polypeptides such as immunoregulatable factors, clotting factors or polypeptide hormones may also be transferred into a host. For example, in hosts whose insulin production or utilization is impaired, DNA encoding insulin may be transferred using DNA-peptide condensates. The expression of other specific proteins, including but not limited to Factor VIII, luteinizing releasing hormone growth factors and the interleukins . In addition, the invention is applicable to wound healing situations including, but are not limited to, bone repair, tendon repair, ligament, repair, blood vessel repair, skeletal muscle repair, and skin repair. For example, using the condensed DNA technology, cytokines, growth factors, systemic hormones, extracellular matrix proteins, and other proteins that regulate growth and differentiation produced by transfected cells can influence other cells in the wound, i . e . , through binding of cell surface signaling receptors, thereby stimulating and amplifying the cascade of physiological events normally associated with the process of wound healing. The end result is the augmentation of tissue repair and regeneration. The present invention is particularly well suited for wound healing, based on the discovery that repair cells involved in the wound healing process will naturally proliferate and migrate to the site of tissue injury and infiltrate a gene-activated matrix. Surprisingly, these repair cells, which are normally difficult to efficiently transfect, either in vivo or in vi tro, are extremely efficient at taking up and expressing DNA when activated to proliferate by the wound healing process. The DNA transfer methods and compositions of the present invention will have a wide range of applications as a drug delivery method for stimulating tissue repair and regeneration in a variety of different types of tissues. These include but are not limited to bone repair, skin repair, connective tissue repair, organ regeneration, or regulation of vasculogenesis and/or angiogenesis . The use of condensed DNA compositions may also be used to treat patients with impaired healing capacity resulting from, for example, the effects of aging or diabetes. The condensed DNA compositions may also be used for treatment of wounds that heal slowly due to natural reasons, e.g., in the elderly, and those who do not respond to existing therapies, such as in those individuals with chronic skin wounds. The methods of the present invention include the grafting or transplantation of the condensed DNA compositions containing the DNA of interest into the host. Procedures for transplanting the compositions may include surgical placement, or injection into the host. In instances where the compositions, including, for example, nano-and microspheres containing condensed DNA are to be injected, the condensed DNA compositions are drawn up into a syringe and injected into a patient at the site of the wound. Multiple injections may be made in the area of the wound. Alternatively, the condensed DNA compositions may be surgically placed at the site of the wound either as a therapeutic implant or as a coated device. The amount of DNA needed to achieve the purpose of the present invention i.e. stimulation of wound repair and regeneration, is variable depending on the size, age and weight of the host. 6. EXAMPLE: CWK PEPTIDE MEDIATED DNA CONDENSATION The example described below demonstrates CWK peptide mediated DNA condensation. In addition, as described below, DNA condensation increased the efficiency of gene transfer. 6.1. MATERIALS AND METHODS 6.1.1. PREPARATION OF CWK PEPTIDES Alkylated Cys-Trp-Lys 8 (Alk-CWK 8 ) , alkylated Cys-Trp-Lys 18 (Alk-CWK 18 ), and dimeric CWK 18 (K 18 WC-CWK 18 ) were synthesized and characterized as described previously (in Wadhwa, M.S. et al., 1997, Bioconj . Chem. 8:81-88)). pCMVL was produced in E. coli and purified using a Qiagen Ultrapure-100 kit (Santa Clarita, CA) . TPCK treated Trypsin was obtained from Worthington Biochemicals (Freehold, NJ) Micro BCA™ protein assay reagent kit was obtained from Pierce (Rockford, IL) . MEM, bovine calf serum, electrophoresis grade agarose, and LipofectAce™ (1:2.5 w/w dimethyldioctadecyammonium bromide and dioleolylphosphatidylethanolamine) were obtained from Gibco BRL (Gaithersburg, MD) . Nrul restriction enzyme was purchased from Boehringer Mannheim (Indianapolis, IN) . 6.1.2. GENERATION OF LINEAR AND CIRCULAR pCMVL Supercoiled plasmid pCMVL (Wadhwa, M.S. et al., 1997, Bioconj . Chem. 8:81-88) was linearized with 2\7rul which recognizes the sequence TCG/CGA at bp 206 of the pRC/CMV cloning vector from Invitrogen (San Diego, CA) . One hundred units of rul was used to cleave lOOμg of pCMVL in 200 μl of SURE/Cut buffer at 37°C for 1 hour. The linear DNA was purified by precipitation with 150 μl of ethanol at -20°C followed by centrifugation at 13,000 g for 5 min. at 4°C and then analyzed by 1% agarose gel electrophoresis. Open circular pCMVL was prepared by creating single stranded nicks in the supercoiled pCMVL. The DNA (100 μg) was heated to 70°C in lOOμl of TAE buffer, pH 8.0 for 1 hour and then purified using ethanol precipitation and analyzed by gel electrophoresis. 6.1.3. PREPARATION AND CHARACTERIZATION OF PEPTIDE/DNA CONDENSATES Peptide/DNA condensates were prepared at a DNA concentration of 50 μl/ml in 5 mM Hepes pH 7.4 using a stoichiometry of 0.3 nmol of peptide per μg of DNA. DNA (150 μl of 0.1 μg/μl) was added dropwise to a microfuge tube containing 4.5 nmols of peptide in 150 μl of buffer. Peptide/DNA condensates formed instantly, although physical measurements were carried out after 30 min. to allow the particle size to stabilize. Particle size analysis was performed on 350 μl of the undiluted DNA/peptide complex using a Nicomp 370 Autodilute Particle Sizer (Nicomp, Santa Barbara, CA) . 6.1.4. SONICATION OF DNA CONDENSATES A 100 W Microson XL-2000 ultrasonic probe homogenizer (Kontes, Vineland, NJ) with a vibrational amplitude of 5 was used throughout the study. The probe tip was placed at 3/4 depth into a 1.5 ml microfuge tube containing 300 μl of sample. The sonication time was varied from 15-60 sec and the probe tip was washed between samples using deionized water. Sonicated DNA samples were analyzed by gel electrophoresis on a 1% agarose gel prepared in TAE buffer (pH 8.0, 40 mM Tris acetate, 2 mM EDTA) containing 0.5 mg/ml ethidium bromide. Prior to electrophoresis, peptide/DNA condensates (2.5 μg DNA) were digested for 30 min. with 5 μg of trypsin (0.2 U) prepared in a final volume of 50 μg of 5 mM Hepes pH 7.4 in order to release plasmid DNA from the condensate. The DNA (0.75 μg/15 μl) was combined with 3 μl of loading buffer (0.25 wt% bromophenol blue, 0.25 wt xylene cyanol FF, and 30 wt glycerol in water) and then loaded onto the gel and electrop oresed for 1.5 hr at 70 V. DNA bands were visualized following detaining of the ethidium bromide on a transilluminator and photographed on Polaroid 667 black and white film. DNA condensates were prepared using either Alk-CWK 8 , Alk-CWK 18 or dimeric-CWK 18 , then adjusted to 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1 M sodium chloride and sonicated for 60 sec. to fragment uncondensed DNA. The samples where then digested with trypsin and electrophoresed on an agarose gel. 6.1.5. SERUM STABILITY OF PEPTIDE/DNA CONDENSATES The DNAse activity in freshly prepared mouse serum was determined to be 4.2 U/μl according to the method of Kunitz. CWK 18 /DNA condensates (5 μg/100 μl) prepared in 5 mM Hepes pH 7.4 and 75 mM sodium chloride were combined with 100 μl of mouse serum and allowed to incubate at 37°C for 3 hours, while rapidly freezing lOμl aliquots at intermediate time points. Prior to electrophoresis, 10 μl of 10 mg/ml SDS was added to each aliquot along with 5 μl of 68 mM EDTA and 3 μl of loading buffer, and samples were applied to an agarose gel containing 0.05 w/v% SDS and electrophoresed for 1 hour at 70 V. The gel was destined in water for 24 hours to remove SDS and increase the detection of the bands by ethidium bromide. The stability of a LipofectAce/DNA complex was also studied by combining 30 μl of LipofectAce with 20 μl of 5 mM Hepes containing 75 mM sodium chloride and 50 μl of DNA (10 μg/100 μl of 5 mM Hepes pH 7.4) . The complexes were then combined with 100 μl of mouse serum, incubated at 37°C for 3 hours while removing time points, and then electrophoresed as described above for peptide/DNA condensates. 6.1.6. IN VITRO GENE TRANSFER Gene transfer experiments were performed on HepG2 cells grown to 30% confluence in MEM supplemented with 10% fetal bovine serum as reported previously. The DNA condensate (10 μg of DNA) was applied to the cells in 2% fetal bovine serum with 100 μM chloroquine and allowed to incubated for 5 hours after which time the media was replaced with MEM containing 10% fetal bovine serum. After 24 hours, cells were harvested and analyzed for the presence of luciferase. The expression level of luciferase was normalized for protein in each well and the relative light units were converted to fmol of luciferase/mg of protein using a standard curve as reported previously. 6.1.7. PREPARATION OF COLLAGEN STRUCTURAL MATRIX CARRIER Plasmid DNA was prepared by standard methods. The Alk- CWK 18 peptide was synthesized 50μg as described above. To prepare condensed plasmid DNA, DNA and peptide (in Hepes- buffered mannitol: 5 mM Hepes, pH 7.4, 5% mannitol) were slowly mixed together by gentle vortexing. The mixture was incubated at room temperature for at least 30 min. and then frozen and lyophilized. To prepare collagen matrices containing condensed DNA, 0.5 ml of sterile bovine type I collagen paste was added to individual wells of a 24-well tissue culture plate. Lyophilized condensed DNA was added to the paste, and mixed well by stirring. 3 x 10 5 293T cells were mixed into the DNA-collagen paste, and the wells were filled with tissue culture medium. After 48 h incubation in a tissue culture hood, and the contents of each well were transferred to a 15 ml tube, pelleted (10,000 rpm, 10 min., 4C) . Cells within the collagen paste were thoroughly lysed in TMNC buffer and the lysate was clarified. Supernatant (50 μl) was transferred to a clean microfuge tube, and alkaline phosphatase activity was assayed (scintillation counting) using kit reagents and the manufacturer's protocols. The control group, which was run in parallel, consisted of cells and collagen but no plasmid DNA of any kind. 6.1.8. PREPARATION OF DNA-PLGA MICROSPHERES AND/OR NANOSPHERES A condensed DNA composition is prepared as described in Example 6.1.3. The DNA-polymer emulsion is further emulsified with an aqueous solution of polyvinyl alcohol (2.5 w/w, 15 mL, 30,00-70,00 ave. MW PVA) by sonication at 65 Watts for 10 min. at 0°C to yield a W/O/W emulsion. The W/O/W emulsion is stirred with a magnetic stirring bar at room temperature for 18 hours to evaporate organic solvent. The spheres are recovered by ultracentrifugation, washed three times with water, resuspended in water by sonication for 30 seconds, and the resultant suspension lyophilized. 6.1.9. IN VIVO CANINE MODEL The surgical procedure involved the placement of 8 mm x 8 mm cylindrical osteotomy defects in the metaphysis of the R and L femur and tibia of each dog. Fixation was not required to stabilize defect sites. Each defect site is marked by the placement of a stainless steel ring at the opening and a bebe at the base. Rings were sterilized with ethylene oxide gas prior to surgery. Sterile GAM implants (pure sterile plasmid DNA + bovine type I collagen, lyophilized) were placed and held in the osteotomy gap until surrounded by clotted blood. After surgery, animal ambulation was unlimited for the time course of the experiment. Three weeks after implantation, gap tissues were harvested, snap frozen, powdered, and processed for heat-stable alkaline phosphatase activity (Tropix) . SC-mg refers to a GAM implant that contains 8.0 mg supercoiled plasmid DNA. C-μg refers to an independent GAM implant that contains lOOμg of condensed plasmid DNA. Condensed plasmid DNA were prepared as described above. 6.2. RESULTS Peptide/DNA condensates were prepared at a concentration of 20 μg/ml of DNA and at stoichiometry of 0.3 nmol of Alk- CWK 18 per μg of DNA. Sonicated DNA was sonicated for 60 seconds prior to condensation with Alk-CWK 18 . The peptide size analysis of peptide/DNA condensates is presented in TABLE II. The particle size diameter represents the mean diameter of the particles. σ represents the standard deviation of the population. TABLE II. PARTICLE SIZE ANALYSIS OF PEPTIDE/DNA CONDENSATES DNA Particle Size Population Morphology Diameter (nm) σ (nm) Supercoiled 46.9 32.4 Circular 61.0 36.0 Linear 72.6 37.4 Sonicated 44.8 28.8 Circular DNA was prepared by base hydrolysis of supercoiled DNA. In its native form, plasmid DNA exists as a mixture of both supercoiled and open circular DNA forms that resolve on gel electrophoresis (FIG. 2, lane 1). Treatment of plasmid DNA at pH 8.0 with elevated temperature (70°C) accelerated the hydrolysis of supercoiled DNA to form predominantly circular DNA within 2 hours (FIG. 2A, lane 2) . Linear DNA was prepared by restriction digestion with Nru I, which cleaved the plasmid prior to the CMV promoter leaving the essential coding region for luciferase and the CMV promoter intact (FIG. 2, lane 3) . Condensation of DNA with peptides possessing lysine repeats of either 18 or 36 residues resulted in the formation of fully condensed 50-70 nm diameter particles. Linear, supercoiled, and circular DNA condensates could not be distinguished through particle size on QELS analysis as indicated in Table 1. However, their transfection efficiency was significantly different as indicated in FIG. 3. The gene transfer efficiency of circular DNA was only reduced 10% compared to supercoiled DNA whereas linear DNA condensates were nearly 90% less efficient at transfection cells. The influence of shear stress and endonuclease attack on the stability of supercoiled plasmid DNA was analyzed. Condensed and uncondensed DNA were challenged with 100 W sonication for up to 60 sec. Sonication of uncondensed plasmid DNA for as little as 15 sec. resulted in extensive fragmentation as indicated in FIG. 4, lane 2, and prolonged sonication for 30-60 sec. further fragments DNA to form a finite size distribution (FIG. 4, lanes 3 and 4). In contrast, electrophoretic analysis of peptide condensed DNA sonicated for 60 sec. resulted in an empty lane (FIG. 4, lane 5) . This results from the failure of peptide/DNA condensates to dissociate and to stain with ethidium bromide. Trypsin was used to hydrolyze Alk-CWK 18 , allowing the plasmid DNA to migrate and stain normally in the gel (FIG. 4, lane 6) . The formation of 10% linear DNA was the result of endonuclease contamination of trypsin. This was deduced from control experiments in which trypsin digestion of peptide/DNA condensates as well as uncondensed plasmid DNA both produced a linear DNA band (FIG. 4, lane 7 and 8) . Peptide/DNA condensates were sonicated for up to 60 sec and used to transfect HepG2 cells to establish that sonication also does not alter gene transfer efficiency. The results showed no change in gene transfer efficiency for sonicated DNA relative to unsonicated condensates (FIG. 5) . Fragmented DNA also formed small (45 nm) peptide/DNA condensates, but these showed negligible gene expression activity (FIG. 5) . Since the stability of peptide/DNA condensates are also be influenced by the solution ionic strength the dissociation of peptide/DNA condensates in the presence of increasing sodium chloride concentration was analyzed in an attempt to disrupt the peptide/DNA binding. However, the failure to detect any DNA bands by gel electrophoresis after incubating the condensates with up to 5 M sodium chloride suggested that either the condensates failed to dissociate or they reformed during gel electrophoresis. To distinguish between these alternatives peptide/DNA condensates were treated with sodium chloride at concentrations ranging from 0-1 M and then sonicated for 60 sec. to fragment any uncondensed DNA. Trypsin was then added to hydrolyze Alk-CWK 18 and allow the DNA to migrate into the gel during electrophoresis. The resulting gel established that peptide/DNA condensates dissociate at sodium chloride concentrations below 1 M as revealed by the presence of fragmented DNA as indicated in FIG. 6. The validity of the method was established by comparing the sodium chloride concentration required to dissociate three different peptides. Previously, it was established that Alk-CWK 8 binds weakly to DNA but was able to fully condense DNA at a stoichiometry of 0.8 nmol per μg of DNA. As observed in FIG. 6A, the DNA remains protected by Alk-CWK 8 at 0.1 M sodium chloride (lane 2) but was degraded by sonication at all higher sodium chloride concentrations due to dissociation of the peptide/DNA complex. Alk-CWK 18 possesses a significantly greater affinity for DNA resulting in complete condensation at 0.3 nmol per μg of DNA. The results shown in FIG. 6B support this by demonstrating protection of the DNA by Alk-CWK 18 at concentrations up to 0.4 M sodium chloride. Likewise, dimeric-CWK 18 was previously found to have a slightly higher affinity for DNA but produced condensates that were the same size as Alk-CWK 18 . In agreement with these results, dimeric-CWK 18 /DNA condensates were more stable as demonstrated by their ability to resist sonicative induced fragmentation up to 0.6 M sodium chloride. To study the serum stability of peptide/DNA condensates a modification of the gel electrophoresis approach was required. In the presence of serum, trypsin was incapable of completely hydrolyzing the peptide to release DNA resulting in bands that migrated slower than open circular DNA on gel electrophoresis. Consequently, SDS was found to be an effective agent to dissociate the peptide/DNA complexes present in serum. The optimal result was obtained by adding 0.5% w/v% of SDS in the agarose gel and running buffer along with 0.3 w/v% SDS in the loading lane. Incubation of either DNA, peptide/DNA condensates, or LipofectAce/DNA complexes with freshly prepared mouse serum adjusted to 0.15 M sodium chloride was used to establish the in vitro stability of DNA. Direct analysis of the incubation time points by SDS-agarose electrophoresis established the uncondensed DNA rapidly converted from supercoiled to circular DNA and then began to form linear DNA within a 5 min incubation period (FIG. 7A, lane 2) . Further incubation resulted in progressive formation of linear DNA which then degraded to smaller oligonucleotides within 1 hour and resulted in complete fragmentation of DNA within 3 hours (FIG. 7A, lanes 6-8) . Conversely, Alk-CWK 18 condensed DNA was stable when exposed to serum during a three hour incubation period (FIG. 7B) , demonstrating the complete preservation of supercoiled DNA. This result suggested that the dissociation of Alk-CWK 8 /DNA condensates in normal saline (0.15 M) could leave DNA exposed to serum endonucleases . Incubation of Alk- CWK 8 /DNA condensates with serum resulted in a degradation profile that was identical to that observed for uncondensed DNA (FIG. 7C) . This adequately explains the three-order of magnitude difference in the transfection efficiency previously observed when transfecting cells with Alk-CWK 18 and Alk-CWK 8 /DNA condensates in the presence of fetal calf serum. This same rationale may also explain the ineffectiveness of LipofectAce/DNA transfection in the presence of serum. To establish this point, LipofectAce/DNA complexes were incubated in serum and time points ranging from 0-3 hours were analyzed by gel electrophoresis (FIG. 7C) . Although some protective effect was conferred by the presence of the cationic lipid formulation, the DNA still underwent a complete conversion from supercoiled to circular and linear DNA within 1 hour and was 50% depolymerized during the 3 hour incubation. To determine if condensed plasmid DNA could be formulated in a collagen structural matrix carrier for gene transfer into mammalian cells condensed DNA was mixed with a collagen paste. The condensed DNA/collagen matrix was mixed with 293T cells followed by analysis of the 293T cells for expression of the reporter gene product alkaline phosphatase. As indicated in FIG. 8 significant amounts of heat stable alkaline phosphatase are reproducibly expressed by the 293T cells. The data presented in FIG. 8 is the first direct evidence that condensed plasmid DNA can be formulated into a collagen structural matrix and successfully used for gene transfer. To test the efficiency of gene transfer in vivo condensed DNA mixed with bovine type I collagen was placed and held in an osteotomy gap in the metaphysis of the R and L femur and tibea of a canine. Three weeks following implantation, gap tissues were harvested, snap frozen and processed for detection of helper gene alkaline phosphatase activity. As indicated in FIG. 9, local gene transfer efficiency is increased in vivo . 7. EXAMPLE: PEG-MEDIATED INHIBITION OF NON-SPECIFIC NUCLEIC ACID TRANSFER The example described below demonstrates the preparation of PEG-CWK polycationic peptides and their use in forming DNA condensates. The resulting DNA condensates are characterized and shown to have increased solubility and reduced levels of non-specific gene transfer. 7.1. MATERIALS AND METHODS 7.1.1. SYNTHESIS OF PEG-CWK 10 PEG-vinylsulfone (PEG-VS, 5kDa) was purchased from Fluka (Ronkonkoma, NY) . Alk-CWK 18 and K 18 WC-CWK 18 were synthesized and characterized as described above. The synthesis of PEG- CWK 18 utilized K 18 WC-CWK 18 (0.5 μmol) , which was reduced to form 1 μmol of CWK 18 by reaction with 25 μmol of Tris-(2- carboxyethyl) phosphine (TCEP) in 0.5 ml of 0.1 M sodium phosphate pH 7 for 4 hrs at room temperature. PEG-CWK 18 was formed by reacting 1 μmol of reduced CWK 18 with 30 μmol of PEG-VS in a total volume of 1.2 ml of 0.1 M sodium phosphate pH 7 at room temperature for 12 hrs. The progress of the reaction was monitored by analytical RP-HPLC eluted at 1 ml/min with 0.1% TFA and a gradient of acetonitrile (5-65% over 30 min) while detecting by Abs 280nm . The reaction mixture was applied to a CM Sephadex C50 cation-exchange column (0.7 x 15 cm) eluted with 60 ml of water to remove free PEG-VS as the unbound fraction, then with 15 ml of 1.5 M sodium chloride while collecting 5 ml fractions. PEG-CWK 18 and CWK 18 were detected by Abs 280nm and were pooled and desalted by 5 hr dialysis against 4 L of water in 1000 MWCO tubing, then freeze dried. PEG-CWK 18 was resolved from CWK 18 by injecting 0.5 μmol onto a semi- preparative C-18 RP-HPLC column (2 x 25 cm) eluted at 10 ml/min with 0.1% TFA and a gradient of acetonitrile (5 to 65% over 30 min) while detecting by Abs 280nm . The peak eluting at 25 min yielded 0.8 μmol of PEG-CWK 18 (80%) based on tryptophan absorbance (e 280nm = 5600 M "1 cm "1 ) . PEG-CWK 18 (1 μmol) was prepared for X H-NMR by D 2 0 exchange followed by dissolvingthe sample in 0.5 ml of D 2 0 (99.96%) containing acetone as an internal standard. X H-NMR spectra were generated on a Bruker 500 MHz spectrometer operated at 23°C. 7.1.2. FORMULATION OF PEPTIDE-DNA CONDENSATES Peptide DNA condensates were formed by adding 75 μg of DNA (pCMVL in 750 μl of 5 mM Hepes pH 7. ) to varying amounts of peptide (7.5 to 90 nmol in 750 μl of Hepes) while vortexing, followed by equilibration at RT for 1 h. Peptide binding to DNA was monitored by a fluorescent dye displacement assay. A 1 μg aliquot of the peptide DNA condensate was diluted to 1 ml in HEPES containing 0.1 μM thiazole orange. The fluorescence of the intercalated dye was measured on an LS50B fluorimeter (Perkin Elmer, UK) in a microcuvette by exciting at 500 nm while monitoring emission at 530 nm. The particle sizes of peptide DNA condensates were analyzed at a DNA concentration of 50 μg/ml in HEPES by quasielastic light scattering (QELS) . The particle surface charge was determined by zeta potential analysis using a Brookhaven ZetaPlus (Brookhaven Instruments) . The solubility of peptide DNA condensates were determined by measuring particle size as a function of DNA concentration (50 μg/ml to 2 mg/ml) at a constant peptide: DNA stoichiometry of 0.4 nmol of peptide per μg of DNA corresponding to a charge ratio (NH 4 + :P0 4 ) of 2.3:1. DNA co-condensates were prepared by admixing Alk-CWK 18 and PEG-CWK 18 in ratios ranging from 0 to 100 mol%, and condensing DNA at a charge ratio of 2.3:1 as described above. To establish the mol ratio of peptides bound to DNA, condensates were dialyzed in a fixed volume (0.5 ml) dialyzer for 75 hrs against water using a 100,000 MWCO membrane. Peptide DNA condensates in the retentate (0.5 ml) were dissociated by adding 50 μl of 5M sodium chloride in 0.1% TFA. Alk-CWK 18 and PEG-CWK 18 were quantified by injecting 1 nmol of peptide (100 μl) onto analytical RP-HPLC eluted with 0.1% TFA and a gradient of acetonitrile (5 to 65% over 30 min) while detecting tryptophan by fluorescence (λ ex 280 nm , λ em 350 nra ) . The peak integration areas were used to quantify Alk- CWK 18 and PEG-CWK 18 with reference to standard curves developed for each peptide. 7.1.3. IN VITRO GENE EXPRESSION HepG2 cells were plated at 1.5 x 10 5 cells per 35 mm well and grown to 40-70% confluence in MEM supplemented with 10% fetal calf serum (FCS) . Peptide DNA condensates (10 μg of DNA) were added dropwise to triplicate sets of cells in 2% FCS containing 80 μM chloroquine. After 5 h incubation at 37°C, the media was replaced with MEM supplemented with 10% FCS, and luciferase expression was determined at 24 h. Cells were washed twice with ice-cold phosphate buffered saline (calcium and magnesium free) and then treated with 0.5 ml of ice-cold lysis buffer (25 mM Tris hydrochloride pH 7.8, 1 mM EDTA, 8 mM magnesium chloride, 1% Triton X-100, 1 mM DTT) for 10 min. The cell lysate was scraped, transferred to 1.5 ml microcentrifuge tubes, and centrifuged for 7 min at 13,000 g at 4°C to pellet debris. Lysis buffer (300 μl) , sodium-ATP (4 μl of a 180 mM solution, pH 7, 4°C) and cell lysate (100 μl, 4°C) were combined in a test tube, briefly mixed and immediately placed in a luminometer. Luciferase relative light units (RLU) were recorded on a Lumat LB 9501 (Berthold Systems, Germany) with 10 sec integration after automatic injection of 100 μl of 0.5 mM D-luciferin (prepared fresh in lysis buffer without DTT) . The expression level of luciferase was normalized for protein using the Bradford assay, and the relative light units were converted to fmol of luciferase/mg of protein using a standard curve developed by adding luciferase to cell supernatant. Each experimental result represents the mean and standard deviation derived from a triplicate set of transfections . LipofectAce (Gibco BRL, 1:2.5 w/w dimethyl dioctadecylammonium bromide and dioleoylphosphatidylethanolamine) was used to mediate gene transfection according to the manufacturer's instructions. DNA/LipofectAce complexes were prepared by combining 10 μg of DNA in 100 μl of serum free media (SFM) with 60 μl of LipofectAce prepared in 150 μl of SFM. The LipofectAce DNA complex was then diluted with 1.7 ml of SFM and used to transfect HepG2 cells for 5 h followed by replacement of the transfecting media with MEM supplemented with 10% FBS . The cells were incubated for a total of 24 h, then harvested, and analyzed for luciferase as described above. 7.2. RESULTS PEG was covalently attached to the Cys of CWK 18 to prepare a PEG-peptide possessing an irreversible covalent linkage (PEG-CWK 18 ) . The reaction was optimized by systematically changing the pH and the stoichiometry of peptide to PEG while monitoring product formation by analytical RP-HPLC. Since the reaction of CWK 18 with PEG-VS at pH 7 was slow (12 hours), it was most efficient to add TCEP to reduce 18 KWC-CWK 18 and allow it to remain in the reaction to block its re-formation during conjugation with PEG-VS. A mol ratio of PEG-VS :CWK 18 of 30:1 at pH 7 resulted in optimal conjugation to form PEG-CWK 18 . At sub-optimal stoichiometries or lower pH the reaction was incomplete, whereas at higher pH dimeric-CWK 18 re-formed as the major product. RP-HPLC analysis of the crude reaction product of PEG- CWK 18 demonstrated a nearly complete disappearance of CWK 18 with the formation of a new peak eluting at 25 min. Despite the apparent complete resolution of the desired product, careful examination revealed that PEG-VS co-eluted with PEG- CWK 18 . This was evident from NMR analysis which determined a 10-fold excess of PEG relative to CWK 18 in the HPLC purified product. Consequently, PEG-CWK 18 was purified using cation exchange to remove excess PEG-VS, and then by RP-HPLC to remove unreacted CWK 18 , resulting in a product that rechromatographed as a single peak on RP-HPLC. Proton NMR analysis identified resonances assigned to the , β, γ, δ, and e protons of the Lys residues as well as the Trp aromatic resonances. Integration of protons at δ 3.67 ppm (PEG) relative to the signal at 2.97 ppm (Lys e) produced a peak ratio of 13.5:1, corresponding to a 1:1 conjugate of PEG 122 and CWK 18 . The DNA binding affinity of Alk-CWK 18 and PEG-CWK 18 were compared using a fluorescent dye displacement assay. A coincident titration curve for each peptide with a asymptote at 0.3 nmol per μg of DNA corresponding to a charge ratio of 1.8:1 suggested that both peptides bind to DNA with equivalent affinity. The particle size and zeta potential of DNA condensates prepared with Alk-CWK 18 and PEG-CWK 18 were examined as a function of peptide: DNA stoichiometry. The mean diameter for PEG-CWK 18 DNA condensates was 90 nm at a charge ratio of 1.8:1 or higher whereas the mean diameter for Alk-CWK 18 DNA condensates was 60 nm. In contrast to the negligible differences in particle size, a large drop in zeta potential to +10 mV was identified for PEG-CWK 18 DNA condensates at a charge ratio of 1.8:1 compared to +35 mV for Alk-CWK 18 DNA condensates . Since PEG-CWK 18 and Alk-CWK 18 possess equivalent DNA binding affinity, admixtures of the two peptides were used to prepare DNA co-condensates. The average particle size increased from 65 to 80 nm using admixtures of Alk-CWK 18 and PEG-CWK 18 varying from 0 to 100 mol% while keeping the charge ratio constant at 2.3:1, as indicated in FIG. 10A. Likewise, as indicated in FIG. 10B, the zeta potential decreased from +35 mV to +10 mV as the stoichiometry of PEG-CWK 18 increased, suggesting the formation of DNA co-condensates composed of Alk-CWK 18 and PEG-CWK 18 . To further confirm the formation of DNA co-condensates, unbound peptides were removed by microdialysis and the ratio of peptides bound to DNA was determined by HPLC. Control experiments established the nearly complete removal (>90%) of free Alk-CWK 18 or PEG-CWK 18 from the retentate after 75 hrs of dialysis (FIG. 11A) . However, the dialysis of DNA co- condensates prepared at charge ratios of 2.3:1 resulted in the removal of unbound peptide and retention of >35% of the tryptophan fluorescence. As indicated in FIG. 11B-F, dissociation and RP-HPLC analysis of the retained peptide allowed recovery of Alk-CWK 18 and PEG-CWK 18 at ratios that agreed to within 15% of the input ratio for each DNA co- condensate in which loss of PEG-CWK 18 was greater than that of Alk-CWK 18 . DNA condensate solubility was evaluated by examining the particle size of concentrated solutions. Alk-CWK 18 DNA condensates increased in particle size from 60 to 400 nm when increasing DNA concentration from 50 to 500 μg/ml, then formed visible flocculates at higher concentrations. Alternatively, PEG-CWK 18 DNA condensates maintained a mean diameter of <100 nm throughout concentrations ranging from 0.05-2 mg/ml and showed no sign of increasing in size (FIG. 12) . DNA co-condensates containing 50 mol% PEG-CWK 18 and Alk- CWK 18 possessed similar poor solubility properties to that of 100 mol% Alk-CWK 18 DNA condensates. However, co-condensates composed of 90 mol% PEG-CWK 18 and 10 mol% Alk-CWK 18 were nearly as soluble as 100 mol% PEG-CWK 18 DNA condensates, revealing only slightly larger mean particle diameter at DNA concentrations of 1 mg/ml or higher (FIG. 12) . The in vitro gene expression of DNA condensates prepared with Alk-CWK 18 , PEG-CWK 18 and admixtures of Alk-CWK 18 and PEG- CWK 18 were compared by measuring luciferase expression in HepG2 cells 24 hrs post-transfection. PEG-CWK 18 DNA condensates blocked gene transfer by three orders of magnitude compared to Alk-CWK 18 DNA condensates. The inhibition was only ten-fold when transfecting with DNA co- condensates prepared with 50 mol% PEG-CWK 18 and only two-fold using co-condensates composed of 25 mol% PEG-CWK 18 . It is apparent that many modifications of this invention as set forth here may be made without departing from the spirit and scope thereof. The specific embodiments described hereinabove are given by way of example only and not by way of limitation. The invention, therefore, is limited only by terms of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method for transferring a nucleic acid into a mammalian cell, comprising contacting a mammalian cell with a nucleic acid condensate comprising a CWK polycationic peptide bound to a nucleic acid, under conditions wherein the nucleic acid condensate is delivered across the cell membrane. 2. A method for transferring a nucleic acid into a mammalian cell, comprising binding a CWK polycationic peptide to a nucleic acid to form a peptide-nucleic acid complex, incorporating the peptide-nucleic acid complex into a matrix and introducing said complex into a mammalian cell under conditions wherein the peptide- nucleic acid complex is delivered across the cell membrane . 3. A method for transferring a nucleic acid into a mammalian cell, comprising applying a matrix containing a CWK polycationic peptide bound to a nucleic acid into the body of a host so that the cells of the host infiltrate the matrix, and acquire the DNA molecule. 4. The method of Claim 2 or 4, wherein the matrix is biocompatible. 5. The method of Claim 4, wherein the biocompatible matrix is collagenous, metal, hydroxyapatite, bioglass, aluminate, bioceramic materials, purified proteins or extracellular matrix compositions. 6. The method of Claim 5, wherein the biocompatible matrix is collagen. 7. The method of Claim 6, wherein the collagen is type II collagen. 8. A method for producing a nucleic acid condensate, comprising contacting a nucleic acid with a CWK polycationic peptide under conditions wherein the nucleic acid binds to the CWK polycationic peptide to 5 form a condensate. 9. The method of any one of Claims 1-8, wherein the nucleic acid is DNA. 10 10. The method of Claim 9, wherein the DNA encodes a therapeutic protein. 11. The method of Claim 9, wherein the DNA encodes a growth factor. 15 12. The method of Claim 9, wherein the DNA is more than one DNA molecule. 13. The method of Claim 11, wherein the growth factor is 20 transforming growth factor-βeta (TGF-β) , fibroblast growth factor (FGF) , platelet derived growth factor (PDGF), insulin like growth factor (IGF), or bone morphogenic factor (BMP) . 25 14. The method of Claim 10, wherein the therapeutic protein is a hormone. 15. The method of Claim 14, wherein the hormone is growth hormone (GH) . 30 16. The method of Claim 14, wherein the hormone is human parathyroid hormone (PTH) . 17. The method of any one of Claims 1-16, wherein the CWK 35 polycationic peptide comprises CWK 18 . 18. The method of any one of Claims 1-17, wherein the CWK polycationic peptide comprises PEG. 19. The method of any one of Claims 1-17, wherein the CWK polycationic peptide is alkylated. 20. The method of any one of Claims 1-19, wherein the CWK polycationic peptide comprises a receptor targeting ligand. 21. The method of Claim 20, wherein the receptor targeting ligand is fibroblast growth factor-2 (FGF-2), a fusogenic peptide, or a nuclear localizing peptide. 22. A pharmaceutical composition comprising a nucleic acid condensate containing a nucleic acid bound to a CWK polycationic peptide. 23. A pharmaceutical composition comprising a biocompatible matrix containing a nucleic acid bound to a CWK polycationic peptide. 24. The pharmaceutical composition of Claim 22 or 23, wherein the nucleic acid is a DNA molecule having a promoter operably linked to a sequence encoding a therapeutic protein. 25. The pharmaceutical composition of Claim 22 or 23, wherein the nucleic acid is a DNA molecule having a promoter operably linked to a sequence encoding a growth factor. 26. The pharmaceutical composition of Claim 25, wherein the growth factor is TGFβ, FGF, PDGF, IGF, or BMP. 27. The pharmaceutical composition of Claim 24, wherein the therapeutic protein is a hormone. 28. The pharmaceutical composition of Claim 27, wherein the hormone is GH. 28. The pharmaceutical composition of Claim 27, wherein the hormone is PTH. 29. The pharmaceutical composition of any one of Claims 22- 28, wherein the composition contains more than one DNA molecule . 30. The pharmaceutical composition of any one of Claims 23- 29, wherein the biocompatible matrix is collagenous, metal, hydroxyapatite, bioglass, aluminate, bioceramic materials, metal materials, purified proteins or extracellular matrix compositions. 31. The pharmaceutical composition of Claim 30, wherein the biocompatible matrix is collagen. 32. The pharmaceutical composition of Claim 31, wherein the collagen is type II collagen. 33. The pharmaceutical composition of any one of Claims 22- 32, wherein the CWK polycationic peptide comprises CWK 18 . 34. The method of any one of Claims 22-33, wherein the CWK polycationic peptide comprises PEG. 35. The method of any one of Claims 22-33, wherein the CWK polycationic peptide is alkylated. 36. The method of any one of Claims 22-35, wherein the CWK polycationic peptide comprises a receptor targeting ligand. 7. The method of Claim 36, wherein the receptor targeting ligand is FGF-2, a fusogenic peptide, or a nuclear localizing peptide.

Inventors: BONADIO JEFFREY F, LABHASETWAR VINOD D, LEVY ROBERT J, RICE KEVIN G, BONADIO, JEFFREY, F., LABHASETWAR, VINOD, D., LEVY, ROBERT, J., RICE, KEVIN, G.
Applicants: BONADIO JEFFREY F, LABHASETWAR VINOD D, LEVY ROBERT J, RICE KEVIN G, UNIV MICHIGAN, BONADIO, JEFFREY, F., LABHASETWAR, VINOD, D., LEVY, ROBERT, J., RICE, KEVIN, G., THE REGENTS OF THE UNIVERSITY OF MICHIGAN
Application year: 1999
Main classification: A61K48
IPC codes: A61K38, A61K48, A61P17, C07K14, C12N15