WO2002094318A1 - Vector for targeted delivery to cells - Google Patents

Abstract

A non-viral single fusion protein vector for targeted cellular delivery which comprises a cell targeting moiety, such as herugulin; a cell penetration penton moiety; and optionally a polynucleotide binding moiety, such as a polylysine sequence. The vector may further comprise an active agent, such as a therapeutic agent. Compositions comprising the vector and methods of utilizing the compositions are also provided.

Description

VECTOR FOR TARGETED DELIVERY TO CELLS

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of provisional application serial number 60/292,192 filed May 18, 2001 the disclosure of which are hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION The invention is related to the field of vectors for targeted delivery of agents to cells.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH Some of the work described in this application was supported by grant number DAMD179919378 from the Department of Defense (Aπny) and SP50H6550055-03 from the National Heart, Lung and Blood Institute.

BACKGROUND OF THE INVENTION Growing concerns about gene therapy vector safety have spurred the ongoing development of physically targeted gene delivery vehicles. Among the advantages to targeting gene therapy is the lack of undesirable delivery to tissues other than the target tissue. Additionally, physical targeting should require lower doses of vector in comparison to an untargeted vector, and presumably, reduce the immunogenicity and toxicity of the vector.

Despite the potent immune response elicited by adenovirus vectors (Hian, Y. et at Journal of Clinical Investigation 1997; 99: 1098-1106, Yang, Y., et al Immunity 1994; 1: 433-442), the highly efficient cell entry mechanism of the virus remains a desirable feature for gene delivery to both dividing and non-dividing cells. Particularly, the cell binding and entry functions imparted by the fiber and penton capsid proteins of adenovirus serotype 5 (Ad5) are a major reason why adeno viruses continue to be widely used as vehicles for gene transfer (Kozarsky, K.F. & Wilson, J.M. Current Opinion in Genetics & Development 1993; 3: 499-503, Greber, U.F. et al Cell 1993; 75: 477-486). The antenna-like fiber proteins that protrude from each vertex of the icosahedral-shaped viral capsid bind with high affinity to ubiquitous cell surface Coxsackievirus Adenovirus Receptor (CAR) proteins (Bergelson, J.M. et al. Science 1997; 275: 1320-1323). This interaction initiates the infection mechanism of the virus, and is followed by the binding of the homopentameric penton base proteins, which lie at the base of each fiber, to cell surface integrins (Wickham, TJ. et al Cell 1993; 73:309-319). h tegrin receptor-mediated endocytosis provides the cellular entry of the virus, but traps it in cellular endosomes. Endosome escape appears to be mediated by the penton, thus allowing entry of the virus to the cytosol (Seth, P. et al Molecular & Cellular Biology 1984; 4:1528-1533, Karayan, L. et al. Journal of Virology 1997; 71: 8678-8689).

While improvements have been made to vectors there are disadvantages to existing vector systems. For example, the adenovirus enhanced transfection (A VET) system developed by Curiel et al. AVET uses targeting ligands chemically conjugated to a whole virus, which is bulky and complicated. In addition the AVET technology requires the production of adenovirus. Other vector technologies consist of several separable parts thereby increasing the difficulty of preparing pharmaceutical compositions with sufficient homogeneity to be useful as a pharmaceutical composition. What is needed is a technology that provides targeted delivery of nucleic acids, proteins, or drugs to a specific-cell type using a single fusion protein delivery vehicle which would allow the production of the vector in pharmaceutical quantities. A single fusion protein drug delivery vehicle may be more tangible than one containing several separable parts, like the penton-fiber complexes. Therefore, the delivery system can be In addition a non-viral vector is perceived safer than viral delivery systems and therefore more desirable.

SUMMARY OF THE INVENTION

h general this invention relates to a non-viral protein vector for targeted cellular delivery of agents to cells, compositions comprising the vector and applications utilizing the compositions. More specifically, this invention relates to a non-viral protein vector comprising a cell targeting moiety and a cell penetration moiety and optionally a polynucleotide binding moiety (e.g., a DNA binding moiety such as a polylysine sequence) for targeted delivery of an agent to a cell. It is an object of this invention to provide a non- viral single fusion protein vector for targeted cellular delivery which comprises: a cell targeting moiety (e.g., heregulin moiety); and a cell penetration moiety (e.g., all or part of a penton protein).

It is a further object of this invention to provide a non- viral single fusion protein vector for targeted cellular delivery which comprises: a cell targeting moiety; a cell penetration moiety; and further comprises a polynucleotide binding moiety (e.g., polylysine sequence).

It is an object of this invention to provide a non- viral a non- viral single fusion protein vector comprising a herugulin moiety for cell targeting and a penton moiety (e.g., HerPBKlO) for cell penetration, and optionally a polylysine sequence for a polynucleotide binding moiety for targeted delivery to a breast cell.

It is another object of this invention to provide a non- viral single fusion protein vector linked with an active agent, such as a therapeutic agent.

It is a further object of this invention to provide nucleic acid sequences and amino acid sequences for all or part of the fusion protein and active agent.

It is yet another object of this invention to provide compositions, such as pharmaceutical compositions, comprising the fusion protein vector or the fusion protein vector linked to an active agent.

It is an object of this invention to provide methods of utilizing the protein vectors of the invention (e.g., cell sorting, gene therapy vectors etc).

It is another object of this invention to provide a method of targeted cell delivery comprising contacting a cell with the non-viral single fusion protein vector or the non- viral single fusion protein vector linked to an active agent.

It is a further object of this invention to provide a method of targeted cell delivery in a subject comprising delivering to a subject the non- viral single fusion protein vector or the non- viral single fusion protein vector linked to an active agent. Yet another object of this invention is to provide kits comprising the compositions of the inventions and for use in the methods described herein.

DESCRIPTION OF THE FIGURES Figures 1A-1B. Construction and production of recombinant proteins. (A) Schematic representation of proteins. Each bar represents the N to C terminal orientation of each protein (not drawn to scale). (B) Immunodetection of recombinant proteins. All proteins were electrophoresed under denaturing conditions. HerPBKlO, PBK10, and PB were detected by a polyclonal antiserum directed against Ad5 capsid proteins (penton, hexon and fiber). Her and HerKlO were detected by an anti-histidine tag monoclonal antiserum.

Figures 2A-2D. DNA mobility shift analyses of lysine-tagged proteins. (A & B) HerPBKlO binds to linear and plasmid DNA. HerPBKlO was pre-incubated with (A) 200 ng of a 1 kb ladder whose sizes range from 75 base pairs to 12 kilobases, or (B) 200 ng of as kb plasmid (pGFPemd-cmv) that is used in subsequent gene delivery assays. (C) HerKlO binds to DNA. HerKlO was preincubated with 500 ng of pGFPemd-cmv. (D) DNA binding occurs through the polylysine domain. The plasmid, pGFPemd-cmv (350 ng) was electrophoresed alone (Lane 1) or after preincubation with 1 micromolar concentrations of HerPBKlO (Lane 2), HerKlO (Lane 3), Her (Lane 4), and PB (Lane 5).

Figures 3A-3H. Cell binding activity of recombinant proteins. MDA-MB-453 human breast cancer cells were incubated with GFP-Her (1 μM) alone (A) or with the indicated competitors (B-F and H), then quantified by FACS. The molar ratio of competitor to GFP- Her are shown in parentheses. Untreated and treated cell populations are shown by white and shaded histograms, respectively. (G) Summary of multiple FACS analyses. MDA- MB-453 cells were incubated with GPF-Her (0.1 μM) and increasing concentrations of either a non-specific competitor (Knob, gray line), Her (black line), HerPBKlO (dotted line), or Her K10 (dashed line).

Figure 4. DNA protection analysis. Plasmid DNA (pGFPemd-cmv, 350 ng) was mixed with protamine and/or HerPBKlO at the indicated ratios, incubated in 20% active (non-heat inactivated) fetal bovine serum, and electrophoresed at 5Ov. The origin of electrophoresis, supercoiled DNA, and relaxed (nicked) plasmids are indicated. Figures 5A-5C. Gene delivery to human breast cancer cells in culture. (A) HerPBKlO mediates gene delivery to MDA-MB-453 cells. Plasmid DNA (pGFPemd-cmv) was mixed with protamine and/or HerPBKlO at the indicated ratios and cells were assayed by detection of green fluorescence. Values are plotted as the percent of GFP positive cells. (B) Gene delivery to MDA-MB-453 cells in the presence of serum. Protamine was pre-incubated with the same plasmid used in H2PO complexes at a protamine to DNA (w/w) ratio of 7. Protamine/DNA complexes or H2PO were incubated with cells in either the absence or the presence of serum, (B) and (C), transduction was determined by counting the percentage of GFP positive cells of treated cell populations over untreated ones, and plotting the values as a percentage of H2PO-treated cells in the absence of serum. Error bars represent standard deviation. Experiments were performed in triplicate (C) Specificity of gene delivery by H2PO. All complexes contain a protamine to DNA (w/w) ratio of 7. H2PO, PBK10, and HerKlO complexes contain 1 μM HerPBKlO, PBK10, and HerKlO, respectively. The peptides GRGDTP and Her were incubated at a 100-fold and 8-fold molar excess, respectively, over HerPBKl 0.

Figures 6A-6H. Receptor binding activity of HerPBKlO. Micrographs were captured at lOx magnification under UV light (A, C, F, & G) or regular light (B, D, F, & H). MDA- MB-453 cells were incubated with GFP-Her (0.1 μM) in the absence (A & B), or presence of HerPBKlO at a 10-fold (C & D), or 82-fold (E & F) molar excess. (G & H), untreated cells.

Figures 7A-7F. H2PO-mediated gene delivery to MDA-ML3-453 cells in culture. Micrographs were captured under UV light (A, C, & F) or regular light (B, D, & F). (A & B), 4x magnification of cells treated with protamine + DNA. Cells treated with H2PO were visualized at 4x (C & D) and lOx (E & F) magnification.

Figure 8. H2PO-mediated delivery of a luciferase reporter gene to MDA-MB-453 cells in culture. Cells were seeded at a density of 5x10⁴ cells/well of a 96-well dish and grown overnight. Cells were treated with H2PO complexes containing 0.1 μg pGL3 per well and the indicated concentrations of HerPBKlO and protamine, then assayed for luciferase activity 30 hours later. Luciferase activity is expressed as relative light units (RLU)/mg of total protein.

Figure 9. Effect of chloroquine on gene delivery. MDA-MB-453 cells were treated either in the absence (filled bars) or presence (open bars) of a lOOμM final concentration of chloroquine. Relative transduction is expressed as a percent of GFP positive cells relative to that mediated by H2PO in the absence of chloroquine.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g.,

Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2^nd edition (1989);

CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.); "PCR: A PRACTICAL APPROACH"

(M. MacPherson, et al., LRL Press at Oxford University Press (1991)); PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)); ANTIBODIES, A

LABORATORY MANUAL (Harlow and Lane, eds. (1988)); and ANIMAL CELL CULTURE (R.I.

Freshney, ed. (1987)).

Definitions

As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

The terms "polynucleotide sequence" refers to a stretch of nucleotide residues. The polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, ohgonucleotides and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc .), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

As used herein, "polypeptide," "peptide" and "protein" are used interchangeably and include reference to a polymer of amino acid residues and/or amino acid analogs. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid, e.g., a peptidomimetic, as well as to naturally occurring amino acid polymers. The terms also apply to polymers containing conservative amino acid substitutions such that the polypeptide remains functional. The term polypeptide also includes concatemer units of a motif, or a contiguous amino acid sequence within a larger amino acid sequence, or polypeptides comprising the motif.

The term "cancer" includes a myriad of diseases, characterized by inappropriate cellular proliferation of a variety of cell types. Examples include, but are not limited to, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, melanoma, Kaposi's sarcoma, lung cancer, colon cancer, kidney cancer, prostate cancer, brain cancer, bone cancer, hemopoietic cancers, sarcomas, cervical cancer, heart cancer, head and neck cancers, brain tumors, such as gliablastoma, or any highly vascularized malignant tumor or an epithelial cell derived tumor.

The term "subject" refers to any animal, preferably a vertebrate such as a mammal. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Veterinary uses are also intended to be encompassed by this invention.

A "pharmaceutical composition" is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM. SCI., 20th Ed. (Mack Publ. Co., Easton (2000)).

An "effective amount" refers to an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Examples of beneficial or desired results include, but are not limited to, decrease or amelioration or inhibition of cancer cell growth or metastasis, in for example breast cancer.

The term "therapeutic agent" includes any number of compounds which will be apparent to one of skill upon review of this disclosure that act as anti-neoplasties, anti- angiogenics, antinflamatories or other agents administered to induce a desired therapeutic effect in a subject.

Non- Viral Fusion Protein Vector

The invention provides a non-viral fusion protein vector for targeted cellular delivery of active agents to a cell. The non-viral single fusion protein vector for targeted cellular delivery comprise a cell targeting moiety and a cell penetration moiety. The non- viral fusion protein vector can further comprise a polynucleotide binding moiety. The moieties of the non-viral fusion protein may be arranged in any order in the vector, for example, the cell penetration moiety may be flanked by the cell targeting moiety and the polynucleotide binding moiety. By way of example, the vector can comprise herugulin as the cell targeting moiety, the Ad5 penton protein as the cell penetration moiety and polylysine as the DNA binding (e.g., the HerPBKlO fusion protein vector in Examples).

Cell Targeting Moiety

A "cell targeting moiety," as used herein, refers generally to compounds capable of specifically delivering a molecule, reacting with or otherwise recognizing or binding to a target cell. Examples of cell targeting moieties include, but are not limited to, immunoglobulins or binding fragments thereof, receptor ligands such as lymphokines (e.g., Medical Immunology (2001) 10^th Edition, Edited by Parslow et al, Lange Medical Books/McGraw-Hill medical publishing Division, NYC, NY), cytokines (e.g., Medical Immunology, supra) or growth factors, cell surface antigens, solubilized receptor proteins, hormones and viral envelope proteins. The cell targeting moiety may be any type of molecule. Nonlimiting examples include proteins, polypeptides or peptides and can also include carbohydrates, drugs, lipids, polynucleotides or ohgonucleotides (e.g., sense or antisense; oligopeptides from library screening or biopanning) or any other molecule which selectively binds to a target cell.

The cell targeting moiety is selected on the basis of the cell to be targeted. By way of example, for targeting a breast cancer cell, herugulin (Holmes et al. (1992) Science 256:1205-1210) can be used as the targeting moiety; for targeting cells expressing CD4 receptors (e.g., T lymphocytes, monocytes, macrophages, EBV transformed B cells, see Medical hnmunology (2001) 10^th Edition, Edited by Parslow et al, Lange Medical Books/McGraw-Hill medical publishing Division, NYC, NY), the gpl20 envelope protein (Lasky et al. (1998) Cell 50:975-985) for cells expressing the transferrin receptor (e.g., T lymphocytes erthyroblasts, cervical carcinoma), transferrin (Wagner et al. (1990) PNAS (USA) 87:3410-3414) can be used as the cell targeting moiety, or for cells expressing Epidermal Growth Factor (EGF) receptors (e.g., gliabalstoma, lung, breast, head, neck, bladder and ovarian concers), the EGF ligand can be used as the cell targeting moiety (Fominaya et al. (1998) Gene Therapy 5:521-530. Cell Penetration Moiety

The cell penetration moiety may be any moiety which facilitates entry of the vector into the cell. Examples of cell penetration moieties include, but are not limited to, a penton protein from any serotype of adenovirus (see Field Virology (4^th Edition) Publishers Williams and Wilkins) or a GALA protein from a rhino virus (Nicol et al. (1996) Biophysical Journal 71:3288-301). By way of example, the cell penetration can be the penton protein is from the adenovirus serotype 5 (Ad5) (Medina-Kauwe (2001) Gene Therapy 8:795-803 and Medina-Kauwe (2000) Gene Therapy 8:1753-1761). All or part of the penton protein may be used as the cell penetration moiety. Also included in this invention are fusion proteins comprising the penton protein or GALA protein with conservative substitutions. A "conservative substitution," refers to a change in the amino acid composition of the protein or polypeptide that does not substantially alter the protein or polypeptide' s activity. "Conservatively modified variations" of a particular amino acid sequence refers to amino acid substitutions of those amino acids that are not critical for functional activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids do not substantially alter activity. Conservative substitution tables providing functionally similar amino acids are well known in the art. The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue.

"Chemical derivative" refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Examples of such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those proteins or peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine.

Modified penton proteins may also be used in the fusion protein vector. Mutations may be introduced to alter or enhance the activity of the targeted penton. For example, a mutation to the RGD motif (Karayan et al. (1997) Journal of Virology 71:8678-89) of the penton moiety removes the specific binding of the penton to v integrins. In the context of HerPBKlO (see Examples), the mutation enhances the binding of the mutant protein, HerPBrgdKlO, to breast cancer cells. Additional mutations may also improve the activity of the fusion protein. For example, a mutation may be introduced in the LDV motif of the penton, which directs binding to α4 integrins. Despite the ability of the heregulin moiety to direct high affinity binding of HerPBKlO to heregulin-specifc receptors, the introduction of such mutations contributes to the specificity of binding that is to be directed solely by the targeting ligand. Other ligands that may be used in place of heregulin may bind to their respective targets with less affinity than that of heregulin, thus requiring the removal of other lower affinity binding sites, such as the RGD and LDV, from the penton component, hi addition to introducing mutations at sites that are responsible for binding to specific receptors, mutations may be introduced that improve the infracellular trafficking of the complex after cell-specific binding. Such mutations may be used to enhance the internalization rate of the complexes into cells, the endosomal penetration activity, the translocation of the complex from the endosome to the cytosol, or the translocation from the cytosol to the nucleus.

Polynucleotide Binding Moiety

The non-viral fusion protein vector can optionally comprise a variety of polynucleotide binding moieties. Examples of polynucleotide binding moieties that may be used include but are not limited polycationic polypeptides such as polylysine or polyarginine, all or part of a histone protein (Gao et al. (1996) Biochemistry 35:1227-1036, a protamine protein (Gao et al., supra) or the DNA binding motifs of transcription factors such as GAL 4 DNA binding domain (Fominaya et al. (1998) Gene Therapy 5:521-530).

Generation of the Non- Viral Fusion Protein Vector

The vector described herein can be generated by a variety of methods. The vector is a chimeric molecule formed by the joining or linking two or more moieties. In a a preferred embodiment, the cell targeting moiety, the cell penetration moiety and the polynucleoside binding domain are a single polypeptides. If one or more moieties is a polypeptide, the bond between the polypeptide and the other moiety may be covalent or noncovalent. An example of a covalent bond is a peptide bond between two proteins or polypeptides. Examples of non-covalent bond include, but are not limited to , hydrogen bonds, electrostatic interactions and van der Waal's forces. By way of example, the two or more moieties and/or agent can also be linked via chemical conjugation. Chemical modification before chemical conjugation may be effected. Chemical modifications before chemical conjugation can be effected. These modifications include, for example, derivitazation for the purpose of linking the moieties polypeptide to the functional moiety, either directly or through a linking compound, by methods that are well known in the art of protein chemistry. In one preferred chemical conjugation embodiment, the means of linking the polypeptide and the functional moiety comprises a heterobifunctional coupling reagent which ultimately contributes to formation of an intermolecular disulfide bond between the two moieties. Other types of coupling reagents that are useful in this capacity for the present invention are described, for example, in U.S. Patent 4,545,985. Alternatively, an intermolecular disulfide may conveniently be formed between cysteines in each moiety which occur naturally or are inserted by genetic engineering (see below). The means of linking moieties may also use thioether linkages between heterobifunctional crosslinking reagents or specific low pH cleavable crosslinkers or specific protease cleavable linkers or other cleavable or noncleavable chemical linkages. The means of linking protein moieties of the vectors may also comprise a peptidyl bond formed between moieties which are separately synthesized by standard peptide synthesis chemistry or recombinant means.

In the case of chemical conjugation between a polypeptide and a non- proteinaceous molecule (e.g. an active agent such as a polynucleotide), a covalent bond between the two is preferred. Examples of active sites on the polypeptide or on the functional moiety for covalent bonds include sulfhydryl-reactive groups (e.g., methanethiosulfonyl groups, dithiopyridyl groups, other reactive disulfides, and cystine), alkylating agents (e.g., α-halo ketones, α-diazo ketones), and acylating agents (e.g., activated esters such as 2,4-dinitrophenyl esters and pentafluorophenyl esters, and certain anhydrides). Other suitable active sites are known to those of skill in the art.

Covalent bonding of the polypeptide and the functional moiety of this invention is not required for the compounds of the present invention. Non-covalent bonding can take place via suitable electrostatic interactions with, for example, ammonium ion and carboxylic acid groups present in the polypeptide or in the functional moiety. Alternatively, the moieties can be linked in a non-continuous manner. For example, a linking group between the polypeptide and the functional moiety may comprise of two parts, which are selected to be complimentary binding groups, for example, two complimentary ohgonucleotides or an avidin-biotin pair. Other complementary binding groups will be apparent to those of skill upon review of this disclosure. In addition to the chemical modifications made to the moieties prior to linking are are also envisioned. Such modifications include but are not limited to, derivitization with polyethylene glycol (PEG) to extend time of residence in the circulatory system and reduce immunogenicity, according to well known methods (see for example, Lisi, et al, Applied Biochem. 4:19 (1982); Beauchamp, et al., Anal. Biochem. 131:25 (1982); and Goodson, et al., Bio/Technology 8:343 (1990)).

If the moieties of the vector are all proteins or polypeptides, the vector (e.g., fusion protein) may be expressed as a single polypeptide from a polynucleotide sequence encoding a single contiguous fusion protein. Byway of example the nucleic acid encoding the two or three moieties of the vector and or agent may be inserted into an expression vector and introduced into a cell. The fusion protein may then be isolated by methods known in the art. Methods of producing recombinant fusion proteins are well known to those of skill in the art (Ausubel et. al., (1987) in "Current Protocols in Molecular Biology" John Wiley and Sons, New York, N.Y); see also Chaudhary, et al, Nature 339:394 (1989); Bafra, et al, J. Biol. Chem. 265:15198 (1990); Bafra, et al, Proc. Nat'lAcad. Sci. USA 86:8545 (1989); Chaudhary, et al, Proc. Nat'lAcad. Sci. USA 87:1066 (1990), describe the preparation of various single chain fusion proteins). If the active agent linked to the vector is also a protein or polypeptide, a polynucleotide sequence encoding the protein or polypeptide is also attached to the polynucleotide encoding the vector.

h another embodiment, the polypeptides and the fusion proteins of this invention are synthesized recombinantly. Recombinant techniques are well known to those of skill and are described, in brief, below. The nucleic acids which encode the polypeptides and the functional moieties, whether RNA, cDNA, genomic DNA, or a hybrid of the various combinations, are isolated from biological sources or synthesized in vitro.

The nucleic acids which encode the two or more polypeptide moieties of this invention can be synthetically produced or isolated from biological sources. By way of example, the polynucleotides may be isolated from genomic or cDNA libraries. Methods for generating these libraries from source organisms, e.g., animals or bacteria, are known to those of skill and can be found in many practice guides, including Berger & Kimmel, GUIDE To MOLECULAR CLONING TECHNIQUES, METHODS IN ENZYMOLOGY VOL. 152, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. MOLECULAR CLONING - A LABORATORY MANUAL (2ND ED.) VOL. 1-3, Cold Springs Harbor

The proteins or fusion protein vector of this invention may also be chemically synthesized by methods known in the art.( e.g., Merrifield, J. Am. Chem. Soc. 85:2149 (1963); Barany & Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in THE PEPTEDES: ANALYSIS, SYNTHESIS, BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDE SYNTHESIS, PART A.,; Merrifield, et al, J. Am. Chem. Soc. 85: 2149-2156 (1963); and Stewart, et al, SOLID PHASE PEPTIDE SYNTHESIS, 2ND ED. Pierce Chem. Co., Rockford, 111. (1984).. Various automatic synthesizers and sequencers are commercially available and can be used in accordance with known protocols.

Agent

The vector may further comprise an agent, preferably an active agent. The agent may be any type of molecule, from, for example, chemical, nutritional or biological sources. The agent may be a naturally occurring or synthetically produced. For example, the agent may encompass numerous chemical classes (e.g. are organic molecule, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons). Such molecules may comprise functional groups necessary for structural interaction with proteins or nucleic acids. By way of example, chemical agents may be novel, untested chemicals, agonists, antagonists, or modifications of known therapeutic agents.

The agent may also be a biomolecule including, but not limited to, peptides, saccharides, fatty acids, antibodies, steroids, purines, pryimidines, toxins conjugated cytokmes, derivatives or structural analogs thereof or a molecule manufactured to mimic the effect of a biological response modifier. Examples of agents from nutritional sources include, but is not limited to, extracts from plant or animal sources or extracts thereof. Preferred agents include antisense ohgonucleotides or antibodies.

The agent may be obtained from a wide variety of sources including libraries of synthetic or natural compounds or alternatively the agent may be commercially available. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced, natural or synthetically produced libraries or compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to random or directed chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. The choice of agent is governed by the intended us of the subject vector. By way of example if the fusion protein is to be used in cell sorting or imaging the agent may be a radioactive isotope (e.g., I , P , S ), a fluorescent molecule or a gene encoding a fluorescent protein (e.g., fluorescien, rhodamine, luciferase GMP) a pigment or dye. By way of example, the non- viral vector of the subj ect invention may be linked to a protein substrate which fluoresces upon cleavage by a protease (e.g., see U.S. Patent No.6,174,673). The agent maybe a therapeutic agent: Examples of therapeutic include, but are not limited to, a radioactive molecule (e.g., 1¹²⁵, P ³², S ³⁵, see for e.g., U.S.Patent Nos.: 6,287,537, 6, 090,365), drug (e.g. adriamycin, tamoxifen, taxol), antibiotic, antibody (see herein below), cytotoxic molecule, hormones (GMCSF, angiostatin, endostatin) or a ribozyme. Cytotoxic moiety includes, but is not limited to, abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof. For example, PE and DT are bacterial toxins that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use in a fusion protein by removing the native targeting component of the toxin (e.g., domain la of PE and the B chain of DT) and replacing it with a different moiety, such as a polypeptide which specifically binds to a cell to be killed. "PE38" and "PE40" refer to a 38 kD and a 40 kD, respectively, cytotoxic moiety derived from PE. See, for example, U.S. Patents 5,082,927 and 5,696,237 as well as Chaudhary, et al, Nature 339:394 (1989) for descriptions of and methods of making and using PE40 and Chaudhary, et al. , Proc. Nat 7 Acad. Sci. USA 87:308 (1990) and Benhar, et al, Bioconjug. Chem. 5:321 (1994) for descriptions of PE 38 as well as methods for making and using PE38.

The agent may be a nucleic acid sequence such as ,a plasmid containing a gene that encodes a therapeutic gene product, linearized double-stranded DNA, artificial chromosomes, chromosomal DNA, viral DNA, ohgonucleotides, RNA, and antisense RNA may also be used. By way of example, for a breast cancer cell, the vector maybe linked to a gene encoding herpes simplex viruses thymidine kinase (HSV-TK). Any of these nucleic acid moieties may also be conjugated, covalently (e.g., chemical conjugation, derivatization, linkers etc) or non-covalently, to a peptide, drug, fluorescent or radioactive molecule. The agent (e.g., peptides, drugs, nucleic acids etc) may be linked to the fusion protein by methods known in the art (e.g., covalent or non-covalent ligation or binding). The assembly of the targeted penton with drugs or peptides may or may not necessitate the use of protamine for the formation of a therapeutic complex.

The agent may be also be a polyclonal and/or monoclonal antibody, including fragments and immunologic binding equivalents thereof, which are capable of specifically binding to the a desired target, h general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (Campbell, 1984; Kohler and Milstein, 1975). These include, e.g., the trioma technique and the human B-cell hybridoma technique (Kozbor, 1983; Cole, 1985). The antibodies or antigen binding fragments may also be produced by genetic engineering. The technology for expression of both heavy and light chain genes in E. coli is the subject the PCT patent applications; publication number WO 901443, WO901443, and WO 9014424 and in Huse et al, 1989 Science 246:1275-1281. Techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies to be used as an agent of the present invention. Alternatively, the antibody may be used in a labeled form to permit detection (e.g., imaging, cell contrast). Antibodies can be labeled, e.g., through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as fluorescein or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labeling are well-known in the art, e.g., see Sternberger, 1970; Bayer, 1979; Engval, 1972; Goding, 1976.

The antibodies utilized in practicing this invention also can be modified to create chirneric antibodies. Chimeric antibodies are those in which the various domains of the antibodies' heavy and light chains are coded for by DNA from more than one species making the antibody compositions more compatible with a host system by minimizing potential adverse immune system responses. This may be accomplished in a variety of ways, including modifying the antibodies to create chimeric antibodies (e.g., antibodies in which the various domains of the antibodies' heavy and light chains are coded for by DNA from more than one species), such as humanized antibodies (Oi, et al. (1986) BioTechniques 4(3):214). Humanized antibodies (i.e., non immunogenic in a human) may be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding, but non immunogenic portion (i.e., chimeric antibodies, see for e.g., Robinson et al, International Patent Application 184,187; Taniguchi M., European Patent Application 171,496; Morrison et al., European Patent Application 173, 494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al, 1987 Proc. Natl. Acad. Sci. USA 84:3439; Nishimura et al., 1987 Cane. Res. 47:999; Wood et al., 1985 Nature 314:446; Shaw et al., 1988 J. Natl. Cancer ist. 80: 15553, all incorporated herein by reference). General reviews of "humanized" chimeric antibodies are provided by Morrison S., 1985 Science 229:1202 and by Oi et al., 1986 BioTechniques 4:214. Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (Jones et al., 1986 Nature 321:552; Verhoeyan et al., 1988 Science 239:1534; Biedleret al. 1988 J. Immunol. 141 :4053, all incorporated herein by reference). Anti idiotype antibodies may also be used. The antibody described herein above, may also be used as a cell targeting moiety.

Methods

The non-viral vector of this invention have a variety of uses. For example, the vector can be used for cell sorting (e.g., FACS), metabolic studies or , imaging of targeted diseased tissues (e.g., magnetic resonance imaging, U.S. Patent No.: 6,232,295 or cell specific contrast U.S. Patent No.: 6,232,295), generate antibodies or to determine the parameters of a malignant lesion. In addition the vector may be coupled with a therapeutic agent and utilized as vector for delivery of a therapy to a subject, such as gene therapy or targeted drug delivery. In one embodiment, the invention provides a method of targeted cell delivery comprising contacting a cell with the non- viral single fusion protein vector or the non- viral single fusion protein vector linked to an active agent. A variety of cells may be used in the methods of the subject application. By way of example, the cells to be used in the disclosed methods exhibit inappropriate cellular proliferation, such as, cancer cells. Nonlimiting examples of cancer cells that may be used include, but are not limited to, breast, cervix, bone, heart, lymphoma, melanoma, sarcoma, leukemia, retinoblastoma, hepatoma, myeloma, glioma, mesothelioma or carcinoma cells. The cells used in the methods may be primary cultures (e.g., developed from biopsy or necropsy specimens) or cultured cell lines. Methods of maintaining primary cell cultures or cultured cell lines are well known to those of skill in the art. If cultured cell lines are used, preferably the cell lines are mammalian cancer cells, most preferably human cancer cells, such as MDA-MB-453 human breast cancer cells. Alternatively, the cells may be non- cancer cells which comprise an expression library or gene library or primary cultures isolated from inormal tissues. In yet another embodiment, the cells used in the method may be intended for use in ex vivo gene therapy (Jurecic and Belmont (1995) "Gene Transfer into Human Hematopoletic Cells in Somatic Gene Therapy" CRC Press p. 15, Spyridonides et al. Blood 91:1820-1827). Examples of such cells include T-lymphocytes, dendritic cells, hematopoietic cells.

Also provided is a method of targeted cell delivery in a subject comprising delivering to a subject the non- viral single fusion protein vector or the non- viral single fusion protein vector linked to an agent, such a therapeutic agent, imaging agent etc. A variety of diseases or conditions may be freated or prevented utilizing the fusion protein vector of the invention linked with a therapeutic agent. By way of example, such diseases include , but are not limited to, cancer, such as breast cancer, autoimmune diseases, such as rheumatoid arthritis (e.g., the therapeutic agent may be an antiinflammatory agent). By way of example, in a subject with breast cancer, a fusion protein vector comprising hergulin as the the cell targeting moiety, the penton protein as the cell penetration moiety, polylysine as the polynucleotide binding moiety and linked with a therapeutic agent such as a gene encoding HS V-TK or the drug adriamycin.

Alternatively, the non-viral single fusion protein vector may be linked with an immunogen and delivered to a subject for use as a vaccine. In yet another embodiment, the non-viral single fusion protein vector may be linked with an imaging agent (e.g., radioactivity etc) and delivered to a subject for use as a diagnostic.

Dosages for the therapeutic agent may be based on established ranges utilized by physicians for therapeutic purposes. One of skill in the art will appreciate that individualization of dosage may be required to achieve the maximum response for a given subject It is further understood by one skilled in the art that the dosage administered to a individual being freated may vary depending on the individuals age, severity or stage of the infection and response to the course of treatment. One skilled in the art will know the clinical parameters to evaluate to determine proper dosage for the subject being freated by the methods described herein. Such dosages may be administered as often as necessary and for the period of time judged necessary by the physician. Administration of the vector linked to a therapeutic agent serves to ameliorate, attenuate or abolish the abnormal proliferation of cancer cells, such as breast cancer cells in the subject. Thus, for example, in a subject afflicted with cancer, the therapeutic administration of one or more of the vectors, either alone or in conjunction with other therapeutic agents serves to attenuate or alleviate the cancer or facilitate regression of cancer in the subject. Also contemplated is administration of the vector linked to a therapeutic agent to a subject prior to any clinical signs of disease. Examples of such individuals includes, but is not limited to, subjects with a family history of a disease such as breast cancer, subjects carrying a deleterious genetic mutation or subjects at risk of disease reoccurrence.

Animal Model System

The vector may be evaluated first in animal models. The safety of the compositions and methods of treatment is determined by looking for the effect of treatment on the general health of the treated animal (weight change, fever, appetite behavior etc.) monitoring of generalized toxicity, electrolyte renal and hepatic function, hematological parameters and function measurements. Pathological changes may be detected on autopsies. By way of example, nude mice carrying breast carcinoma xenografts can be used as a breast cancer model (Jeschke et al. (1995) Int. J. Cancer 60:730-739).

Any animal based (e.g., recombinant and non-recombinant) model systems may be used to assess the in vivo efficacy of the vector linked to a therapeutic agent and to provide effective dosage ranges. Efficacy in cell culture can also be used to determine effective amounts.

Pharmaceutical Compositions

While it is possible for the fusion protein vectors or the fusion protein vectors linked with an active agent to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation. The formulations of the present invention, are for both veterinary and human use, comprises one or more of the fusion protein vectors linked with one or more active agents together with one or more pharmaceutically acceptable carriers and, optionally, other active agents (e.g., chemotherapeutic agents, antibiotics, etc) or therapeutic ingredients. The formulations may optionally comprise protamines. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The characteristics of the carrier will depend on the route of administration. Such a composition may additionally contain carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The formulations may be prepared by any method well-known in the pharmaceutical art (see, e.g., see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975).

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in a effective amount and a variety of dosage forms. For example, an effective concentration of the compositions of the invention may be administered orally, topically, intraocularly, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, transdermally or by any other effective means. Aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intraperitoneal, oral, intercranial, cerebrospinal fluid, pleural cavity, occular, or topical (lotion on the skin) administration. By way of example, for systemic cancers such as breat cancer administration via intravenous, subcutaneously, or parenterally is preferred.

EFFECTIVE AMOUNTS

An effective amount or therapeutically effective of the antisense ohgonucleotides or functional equivalents thereof to be administered to a subject in need of treatment may be determined in a variety of ways. By way of example, the effective amount for the vector coupled with a therapeutic agent be administered may be chosen based on effectiveness in inhibiting the growth of cultured cancer cells or efficacy in an animal model. For known thereapeutic agents, dosages currently in use may be administered.

By way of example, a general range of suitable effective dosage that may be established first with an in vitro dose curve then by an in vivo dose curve. Concentration curves bracketing from about one nanomolar to about 1 millimolar can be tested first with the in vitro dose curve than an in vivo dose curve. The daily dose may be administered in a single dose or in portions at various hours of the day. Initially, a higher dosage may be required and may be reduced over time when the optimal initial response is obtained. By way of example, treatment may be continuous for days, weeks, or years, or may be at intervals with intervening rest periods. The dosage may be modified in accordance with other treatments the individual may be receiving. However, the method of treatment is in no way limited to a particular concentration or range and may be varied for each individual being treated and for each derivative used.

One of skill in the art will appreciate that individualization of dosage may be required to achieve the maximum effect for a given individual. It is further understood by one skilled in the art that the dosage administered to a individual being treated may vary depending on the individuals age, severity or stage of the disease and response to the course of treatment. One skilled in the art will know the clinical parameters to evaluate to determine proper dosage for the individual being treated by the methods described herein. Clinical parameters that may be assessed for determining dosage include, but are not limited to, tumor size, alteration in the level of tumor markers used in clinical testing for particular malignancies. Based on such parameters the treating physician will determine the therapeutically effective amount of antisense oligo nucleotides or functional equivalents thereof to be used for a given individual. Such therapies may be administered as often as necessary and for the period of time judged necessary by the treating physician.

Kits

Also provided are kits comprising the fusion protein vector either alone or linked with an active agent. Such kits may be incorporated into a multiwell configuration or any other configuration. The kits may further comprise one or more additional regents for utilizing or administering the fusion protein or the fusion protein linked with an active agent, such as for example, buffers, primers, enzymes, labels and the like. The kits may further comprise, or be packaged with, an instrument for assisting with the performance of an assay or administration of the compositions. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle. The kits of the invention may also include an instruction sheet defining administration of the compositions. The kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. Other instrumentation includes devices that permit the reading or monitoring of reactions.

All books, articles, and patents referenced herein are incorporated by reference. The following examples illustrate various aspects of the invention and in no way intended to limit the scope thereof.

EXAMPLES

A recombinant penton protein containing a decalysine sequence (PBK10) can bind DNA and mediate gene delivery into cells in culture in the absence of whole virus (Medina- Kauwe et al Gene Therapy 2001; 8:795-803). This delivery is mediated mainly through integrin receptor binding. When mixed at optimized ratios with plasmid DNA and the DNA condensing agent, protamine, effective gene delivery complexes were formed (PBKlO/protamine/plasmid DNA) and have been designated 3PO.

The ligand protein used herein, is the receptor-binding domain of heregulin-α. This ligand binds with high affinity to heterodimers of HER2/3 or HER2/4 receptor subunits, which are overexpressed on certain mammary tumor cell lines (Bacus, S.S. et al American Journal of Clinical Pathology 1994; 102: 513-24, Carraway, K.L., 3rd & Cantley, L.C. Cell 1994; 78: 5-8, Carraway, K.L., 3rd et al. Journal of Biological Chemistry 1994; 269: 14303-14306, Caraway, K.L., 3rd et al Current Opinion in Neurobiology 1995; 5: 606-6 12, Goldman, R et al Biochemistry 1990; 29: 11024-11028, Hung, M.C. et al Gene 1995; 159: 65-71, Press, M.F. et al Progress in Clinical & Biological Research 1990; 354A: 209- 221, Slamon, DJ. et. al. Science 1987; 235:177-182, Slamon, D.J. & Clark, G.M. Science 1988; 240:- 1795-1798, Yarden, Y. & Weinberg, R.A. Proceedings of the National Academy of Sciences of the United Slates of America 1989; 86: 3179-3183.). The binding kinetics between heregulin isomers and their target receptors have been well characterized using classical receptor binding techniques, and the intemalization activities of the receptor subunits have been studied (Holmes, W.E. et al. Science 1992; 256: 1205-1210, Lenferink, A.E. et al. EMBO Journal 1998; 17: 3385-3397, Li, W. et al Oncogene 1996; 12: 2473- 2477, Sliwkowski, M.X. et al Journal of Biological Chemistry 1994; 269: 14661-14665, Tzahar, E. et al Journal of Biological Chemistry 1994; 269: 25226-25233, Waterman, H. et al Journal of Biological Chemistry 1998; 273: 13819-13827). The heregulin ligand is rapidly internalized after binding to MDA-MB-453 human breast cancer cells (Medina-Kauwe, L.K. et al BioTechniques 2000; 29: 602-609). as demonstrated in th examples below, appending the heregulin ligand to the recombinant penton protein, results in direct the delivery of conjugated plasmid DNA specifically to these breast cancer cells by way of heregulin receptor binding and intemalization, while preserving the endosomolytic function of the penton which enhances transduction efficiency.

MATERIALS AND METHODS

Cells, plasmids, and peptides 293 cells, HeLa cells, and MDA-MB-453 human breast cancer cells were maintained in DMEM, 10% fetal bovine serum, at 37°C, 5% CO2. The reporter plasmid, pGFPemd-cmv [R] control vector (Packard Instrument Company, Meriden, CT, USA), was used for gene delivery assays. The RGD-containing peptide (GRGDTP) was obtained from

Sigma (St. Louis, MO, USA).

DNA constructs

A common 5' oligonucleotide primer containing the sequence 5'- ATCGAAGGATCCATGCGG CGCGCGGCGATGTAT3' was used to amplify both wild- type and lysine-tagged penton sequences from a pJM17 adenoviral genome template. The sequences of the 3' primers are 5'- GCATCAGAATTCTCAAAAAGTGCGGCTCGATAG- 3' (PB) and 5'- CATGAATTCA(TTT)₁₀AAAAGTGCGGCTCGATAGGA-3'

(PBK10). A BamHI restriction site was introduced in the 5' primer and an EcoRI restriction site was introduced in the 3' primers for in-frame insertion of both the wild-type and lysine- tagged pentons into the pRSET-A bacterial expression plasmid (Invitrogen, Carlsbad, CA, USA). This plasmid expresses the recombinant protein as an N-terminally histidine-tagged fusion for affinity purification by nickel chelate affinity chromatography. Polymerase chain reaction (PCR) amplification was used to add a sequence encoding a short polyglycine linker to the amino (N) -terminus of PBK10. The sequence encoding the linker contains a SacLI restriction site for additional cloning. The heregulin targeting ligand was produced by PCR amplification of the epidermal growth factor (EGF)-like domain of the heregulin gene 29 using a 5' oligonucleotide primer containing a BamHI site and a 3' primer containing a SacIJ site for cloning in frame with PBK10. The targeting ligand was added to the lysine- tagged construct to create HerPBKlO by ligating the PCR product just N-terminal to PBK10. Construction of Her and GFP-Her are described elsewhere (Medina-Kauwe, L.K. et al BioTechniques 2000; 29: 602-609). HerKlO was created by PCR amplification of the

90 Her construct using the existing 5' Her primer and a 3' oligonucleotide primer containing the sequence 5'- ATGAATTCA(TTT)₁₀AGATCTACTTCCACCACTTCCACC-3'.

Protein expression and purification from bacteria

Overnight cultures of BL21(DE3)pLysS (Novagen, Madison, WI, USA) bacterial transformants were inoculated 1:50 in LB containing O.lmg/ml ampicillin and 0.034 mg/ml chloramphenicol. At OD600 -0.6, cultures were induced with 1 mM IPTG and grown 4 more hours at 37 C with shaking. Cultures were harvested and pelleted. Cell pellets were resuspended in lysis buffer (50mM Na-phosphate, pH 8.0; 500 mM NaCl; 5-10 mM imidazole; lmM phenylmethylsulfonyl fluoride) and lysed by addition of 01% Triton X-100 and one cycle of freeze-thawing. Supematants were recovered, added to Ni-NTA resin

(Qiagen, Valencia, CA, USA) pre-equilibrated in lysis buffer, and incubated for 1 hour on ice. The resin containing bound protein was washed with 10 ml of lysis buffer, then 6 ml of a solution of 50mM Na-phosphate, pH 8.0; 500mM NaCl; 60mM imidazole, and protein was eluted with 2 ml of a solution of 50mM Na-phosphate, pH 8.0; 500mM NaCl; 400 mM imidazole. Proteins were desalted on YM-10 nominal molecular weight limit spin columns

(Millipore, Bedford, MA, USA) and their concentrations measured using the BioRad protein quantitation assay (Bio-Rad Laboratories, Hercules, CA, USA).

Protein Detection Denaturing polyacrylamide gel electrophoresis was performed in a discontinuous gel buffer system as previously described. Proteins were electrically transferred onto nitrocellulose using 39 mM Glycine, 48 mM Tris-HCl, 0.0375% SDS, 20% methanol in a Bio-Rad semi-dry transfer cell set at constant voltage (15v) for 30 minutes. Blots were blocked with 3% milk in PBS. Anti-His Tag antisera (Sigma, St. Louis, MO, USA) was used at a 1:3000 dilution in blocking buffer. Anti-Ad5 antisera (Access Bio Medical, San Diego, CA, USA) was used at a 1:8000 dilution in blocking buffer. Antibody-antigen complexes were detected by incubation with horeseradish peroxidase (HRP) conjugated secondary antibodies (Sigma, St. Louis, MO, USA), reaction with chemiluminescence detection reagents (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA), and exposure to film (Hyperfϊlm ECL; Amersham Pharmacia Biotech hie, Piscataway, NJ, USA).

Cell Binding Assay

Cells were treated with (GFP-tagged proteins as described elsewhere (Medina- Kauwe, L.K. et al BioTechniques 2000; 29: 602-609). Briefly, 293 cells or MDA-MB-453 cells grown to 75% confluency were lifted with phosphate-buffered saline (PBS) supplemented with 2mM EDTA, centrifuged, washed, then added to PBS containing GFP- tagged proteins and 3% milk to reduce non-specific binding. After a one hour incubation on ice, cells were centrifuged, washed thoroughly, resuspended in 0.5ml PBS and measured by FACS scan.

DNA Mobility Shift Assays DNA was mixed with proteins at the indicated ratios in HEPES-buffered saline

(HBS; 150 mM NaCl, 20 mM HEPES, pH 7.3) for 30 minutes at room temperature in a total volume of 10 microliters. After incubation, 2 microliters of sample dye was added to the mixtures, mixes were loaded on a 0.8% agarose gel, and electrophoresed at 50 volts. Gels were stained with ethidium bromide after electrophoresis to visualize DNA bands. The 1 kb ladder was obtained from Life Technologies Inc., Rockville, MD. USA.

DNA Protection Assay

Plasmid and proteins were added together at various ratios in a total volume of 20 microliters and incubated at room temperature for 30 minutes. Four microliters of active (non-heat inactivated) fetal bovine serum were added to each mix and mixtures were incubated at 37°C. SDS at a 1% final concentration was added to each mix to release the proteins from the DNA. Four microliters of sample dye was added to each mix, and mixtures were electrophoresed on agarose gels as described earlier.

Cell Delivery Assays

Cells were grown on 24-well dishes to approximately 75% confluency. Proteins and plasmids (3.5 μg) were incubated together at various ratios at room temperature for 30 minutes in 0.3ml of Adhesion Buffer (DMEM, 2mM MgCl₂, 20 mM HEPES}, then added to cell monolayers. Cells were exposed to mixes for 3-5 hours at 37°C, 5% C02, before the addition of complete serum. Cells were trypsinized and counted using a hemocytometer under UV light microscopy to determine the percentage and absolute numbers of GFP positive cells.

UV Microscopy

An Olympus EVIT-2 inverted microscope fined with an FITC filter was used to visualize GFP-positive cells. Photomicrographs were taken at 4x and lOx magnification.

FACS analysis

Where indicated, 5000-10,000 events are counted on a Becton Dickinson FAC- Scan™ analyzer (Becton Dickinson, Franklin Lakes, NJ, USA) using a 15 mW air-cooled argon laser set at 488 nm and recorded with a 530 nm emission filter in the FL1 emission channel. Cell populations are represented on a FACS histogram plotting green fluorescence intensity on a logarithmic scale against cell number. Fluorescence intensity of cell populations is indicated by a shift to the right of the histographical plots of treated cells. Fluorescence enhancement was determined by obtaining the number of gated fluorescent events for untreated and treated cells.

Example 1 Description and bacterial production of recombinant proteins

Recombinant genes encoding several specific proteins were constructed (Fig. 1A). One of the constructs encoded the wild-type Ad5 penton (PB). A second construct encoded the Ad5 penton protein containing a carboxy (C) -terminal fusion of 10 lysines for binding DNA (PBK10). A third construct added an amino (N) -terminal cell specific ligand to PBK10 to produce the targeted fusion protein, HerPBKlO. The ligand alone was also made (Her; also known as eHRG) (Medina-Kauwe, L.K. et al BioTechniques 2000; 29: 602-609) and as a C-terminal decalysine fusion protein (HerKlO). Additionally, the ligand was produced as an N-terminal fusion to green fluorescent protein (GFP-Her; also known as GFP-eHRG) (Medina-Kauwe, L.K. et al BioTechniques 2000; 29: 602-609).

The PB, PBK10, and Her can be produced in bacteria as soluble proteins (Medina-

Kauwe et al Gene Therapy 2001; 8:795-803, Medina-Kauwe, L.K. et al BioTechniques 2000; 29: 602-609). Using a similar bacterial expression system, HerPBKlO and HerKlO were produced. The ligand peptide used here is derived from the receptor binding domain of heregulin and contains an EGF-like motif that confers binding specificity to HER2- containing receptors (Holmes, W.E. et al. Science 1992; 256: 1205-1210, Harris, A. et al. Biochemical & Biophysical Research Communications 1998; 251: 220-224). The version of ligand presented here has successfully directed the cell-specific binding of refroviral vectors when genetically engineered into the viral envelope (Han, X. et al Proceedings of the National Academy of Sciences of the United States of America 1995; 92: 9747-9751). The heregulin receptor binding by this peptide is not impaired when fused to foreign sequences, such as GFP (Medina-Kauwe, L.K. et al BioTechniques 2000; 29: 602-609).

Polyclonal antiserum specific to Ad5 capsid proteins recognizes all three penton proteins (HerPBKlO, PBK10, and PB), thus confirming their identities (Fig. IB). Additional control proteins, Her, HerKlO, and GFP-Her (not shown) were also highly expressed in bacteria and could be recognized by antibodies directed against the recombinant proteins. All proteins migrate at their expected molecular weights under denaturing conditions (Fig. IB).

Example 2 HerPBKlO binds DNA

The polylysine tracts encoded by the recombinant penton constructs, PBK10 and HerPBKlO, should impart DNA binding function to the fusion proteins by interacting with the negatively charged phosphate backbone of nucleic acids. To determine DNA binding ability, increasing concentrations of protein were incubated with constant amounts of a 5 kilobase (kb) plasmid DNA (pGFPemd-cmv), and the resulting effect on DNA mobility analyzed on an agarose gel. At concentrations where the amount of polylysine completely binds, and thus neutralizes, all of the DNA, the plasmid appears immobilized on the gel. PBK10 immobilizes the plasmid at the predicted protein-to-DNA (w/w) ratio of 22 (Medina-Kauwe et al Gene Therapy 2001; 8:795-803). The predicted ratio of HerPBKlO to plasmid to produce the same effect is 29, and our results agree with this prediction (Fig. 2B). HerKlO exhibits a similar DNA mobility retardation pattern (Fig. 2C). Interestingly, the same amount of HerPBKlO to neutralize a 5 kb plasmid also neutralizes a DNA ladder (Fig. 2A). These results show that HerPBKlO can bind both double-sfranded circular and linear DNA. To confirm that DNA binding occurs specifically through the polylysine sequence, non-polylysine tagged proteins, PB and Her, were incubated with the same 5 kb plasmid. On an agarose gel, plasmid incubated with HerPBKlO (Fig. 2D, lane 2) or HerKlO (Fig. 2D, lane 3) is retarded in mobility whereas equivalent concentrations of Her (Fig. 2D, lane 4) or PB (Fig. 2D, lane 5) produce no such shift in mobility, thus establishing that DNA binding occurs predominantly through the polylysine domain (Fig. 2D).

Example 3

HerPBKlO binds heregulin receptors

The human breast cancer cell line, MDA-MB-453, which over-expresses HER2-containing receptors, was used to test receptor binding of HerPBKlO. Receptor binding activity was determined by an established assay that uses fluorescence activated cell sorting (FACS) to measure the amount of heregulin ligand required to displace the fluorescently-tagged heregulin ligand, GFP-Her (Medina-Kauwe et al. (200) Biotechniques 29:602-609 and Medina-Kauwe et al. (2001) 29:602-609 and Medina-Kauwe et al. (2001) Gene Therapy 8:1753-1761. After MDA-MB-453 cells are incubated with GFP-Her and washed thoroughly to remove unbound proteins, the cells exhibit bright, punctate foci under ultraviolet (UV) light microscopy (Fig. 6A). A concenfration of HerPBKlO equal to that of GFP-Her produces slightly less bright foci, whereas ten-fold and higher amounts of HerPBKlO substantially reduce the visible foci (Fig. 6, C & E), suggesting that HerPBKlO competes for the same binding sites as the fluorescently tagged heregulin ligand. FACS analysis of MDA-MB-453 cells bound by GFP-Her are two orders of magnitude brighter than untreated cells (Fig. 3A). A ten-fold higher concentration of non GFP-tagged Her reduces this fluorescence by nearly 95% (Fig. 3, C & G). The Ad5 knob protein, which we produced and purified from E. coli similar to Our other recombinant proteins, is used here as a non-specific competitor. A ten-fold higher concentration of the knob protein has no effect on cell fluorescence, as previously established (Medina-Kauwe et al. (2001) supra) (Fig. 3, B & G). Increasing concentrations of HerPBKlO (Fig. 3, D & G) and HerKlO (Fig. 3, F & G) proteins, produce reductions in cell fluorescence similar to that produced by Her, as measured by FACS assay, suggesting that the receptor binding of HerPBKlO is unchanged from that of free heregulin ligand. In addition, the incubation of pGFPemd-cmv plasmid with HerPBKlO has no effect on the ability of HerPBKlO to compete away cell fluorescence (Fig. 3E), indicating that DNA binding does not interfere with receptor binding activity, furthermore, a mutation in the RGD integrin binding motif further enliances binding to herugulin receptors, as shown by the sharper shift in fluorescence produced by HerPBgrdKlO competitor (Figure 3H).

Example 4 Protamine protects HerPBKl 0/DNA complexes from serum protease degradation

The addition of protamine to PBK10/DNA complexes protects the plasmid from serum nuclease activity and enhances gene delivery to cells in serum-containing culture medium without overriding the receptor-specific binding of the complexes (Medina-Kauwe et al. (2001) Gene Therapy 8:1753-1761). To confirm the ability of protamine to protect HerPBKl 0-bound DNA, we incubated HerPBKl 0/DNA complexes formed at a standardized protein to DNA (w/w) ratio of 3 with increasing concentrations of protamine in the presence of 20% active serum. Complexes that are sensitive to nuclease activity are detected by the conversion of the plasmid from the supercoiled form to the open circle form, as described elsewhere (Gao, X. & Huang, L. Biochemistry 1996; 35: 1027-1036). In the presence of serum, nearly all of the plasmid DNA, whether in the presence or absence of HerPBKlO, is convened to nicked forms by 30 minutes at 37°C (Fig. 4). However, adding increasing concentrations of protamine preserves supercoiled DNA in the presence of active serum for up to 45 minutes (Fig. 4). A protamine to DNA ratio (w/w) of 7 assures complete protection of the plasmid from nuclease-induced degradation. Example 5

HerPBKlO- mediated gene delivery to breast cancer cells in culture

To test the gene delivery capacity of complexes formed between HerPBKlO, DNA, and protamine, the pGFPemd-cmv reporter plasmid was used. The experiments were first performed in the absence of serum to determine the ratios of DNA to proteins required for optimal gene delivery. HerPBKl 0/DNA complexes formed at a protein to DNA (w/w) ratio of 3 were incubated with increasing concentrations of protamine as described earlier. MDA-MB-453 human breast cancer cells were exposed to these complexes and assayed three days post-treatment for the expression of GFP. h the absence of HerPBKlO, fransduction is nearly undetectable at all of the protamine concentrations that were tested (Fig. 5A, open bars). In the presence of HerPBKlO, increasing the concenfration of protamine from 0 to 7 μg of protamine per μg of DNA enhances the green fluorescence from less than 50-fold to greater than 270-fold over control treated cells (Fig. 5A, filled bars). A protamine to DNA w/w ratio greater than 7 reduces fransduction to 150-fold over control treated cells, presumably by competing with HerPBKlO for DNA binding. In subsequent assays, complexes were formed at a HerPBKlO to DNA (w/w) ratio of 3 and a protamine to DNA (w/w) ratio of 7, and will be referred to hereinafter as H2PO. Fluorescence microscopy of MDA-MB-453 monolayers detects numerous GFP-positive cells after treatment with H2PO (Fig. 7, C & E) but not with DNA complexed to protamine alone (Fig. 7A), thus the HerPBKlO protein appears to be responsible for enhanced fransduction.

H2PO can also mediate the delivery and expression of a luciferase gene from the plasmid, pGL3 (Fig. 8). Increasing the concentration of HerPBKlO from 0 to 5 ng in H2PO complexes containing pGL3 enhances the luminescence detected in transduced MDA-MB- 453 cells by up to 34-fold over protamine alone, hi the absence of HerPBKlO, delivery of pGL3 by protamine alone produces negligible luciferase activity at all of the protamine concentrations that were tested. Cells treated with lipofectin produced almost 8 times the luminescence of cells treated with H2PO (not shown).

The ability of H2PO to mediate similar levels of gene delivery in the presence of 1% and 10% serum, respectively was examined. DNA complexed with protamine alone produces nearly undetectable gene delivery both in the absence and presence of serum (Fig. 5B). H2PO enhances this delivery by nearly 20-fold in the absence of serum (Fig. 5B). Importantly, similar levels of gene delivery by H2PO are observed in the presence of either 1% or 10% serum (Fig. 5B). Thus, whereas the addition of protamine to the complex appears to mediate a protective effect, HerPBKlO is still required for enhanced levels of gene delivery.

Example 6

HerPBKl 0-mediated gene delivery is specific and requires the penton moiety

To test the specificity of H2PO-mediated gene delivery, free heregulin protein (Her) was used as a competitor for heregulin receptor binding on MDA-MB-453 human breast cancer cells. In the presence of eight-fold higher concentrations of Her compared to HerPBKlO, relative fransduction (as measured by GFP fluorescence) is reduced by nearly 90% (Fig. 5C). Also tested was H2PO fransduction of cells that do not express high levels of heregulin receptors. H2PO-mediated fransduction of MDA-MB-231 cells, which express low to undetectable levels of HER-2 and -4 (Han et al. (1995) PNAS 92:9747-9751), produces only 12% of the GFP-fluorescence of MDA-MB-453 cells (Fig. 5C). Together, these findings suggest that gene delivery by H2PO is mediated predominantly by specific binding to the heregulin receptor.

To determine whether integrins, the natural receptors for the penton protein, are also involved in mediating H2PO fransduction, an RGD-containing peptide (GRGDTP) was used as a competitor for α_v integrin receptor binding. We have shown previously that similar complexes formed between PBK10, DNA, and protamine (3PO) mediate gene delivery by integrin binding that is competitively inhibited by GRGDTP (Medina-Kauwe et al. (2001) Gene Therapy 8:975-803). Interestingly, in this case, even at a concentration up to 100-fold higher than HerPBKlO, GRGDTP produces no significant effect on H2PO- mediated gene delivery (Fig. 5C). Taken together, these findings indicate that cell binding by H2PO is not mediated by the integrin binding motif found on the penton moiety of HerPBKlO, but rather by the heregulin receptor binding domain, Her. This findings also indicate that the requirement for integrin receptor-mediated endocytosis may be substituted by intemalization via the heregulin receptor. The requirement for the heregulin domain to enhance targeted delivery is further demonsfrated by comparison with the use of PBK10 as a targeting moiety in 3PO complexes exposed to MDA-MB-453 cells. The delivery of the same concentration of a reporter plasmid to MDA-MB-453 cells by HerPBKlO exceeds that of PBK10 by more than ten-fold (Fig. 5C).

The importance of the penton moiety is underscored when it is removed from the HerPBKlO construct to create the deletion mutant, HerKlO. Whereas this protein binds DNA and heregulin receptors in a similar fashion to HerPBKlO (Fig. 2C and Fig. 3, F & G), complexes made between HerKlO, pGFPemd-cmv, and protamine produced 97% less green fluorescence than H2PO, and did not enhance fransduction over protamine and DNA alone (Fig. 5C). As competition for integrin binding by the RGD peptide did not appear to significantly affect overall fransduction levels, the penton domain in HerPBKlO may be required for the lysis of the endosomal vesicle after endocytosis.

To confirm whether H2PO is capable of endosome escape, H2PO was added to cells in the presence or absence of the acidofropic reagent, chloroquine. Although chloroquine inhibits endosome acidification, it also accumulates in infracellular vesicles, thereby inducing osmotic swelling and release of endosomal contents (Fominaya et al. (1998) Gene Therapy 5:521-532 and Uhereh et al. (1998) J. Biol. Chem. 273:8830-8841. The presence of chloroquine has a notable effect on DNA delivery mediated by protamine alone, enhancing gene transfer nearly 4-fold (Fig. 9). In contrast, no effect is observed on H2PO- mediated gene transfer, which consistently maintained a nearly 5 -fold higher gene delivery over protamine alone.

The data demonstrate that the DNA/protein conjugate complex, H2PO, can be successfully targeted to breast cancer cells by means of the novel fusion protein, HerPBKlO, which consists of a targeting ligand, an endosomolytic component and a DNA binding domain. The receptor-binding motif of heregulin is used to mediate specific binding to breast cancer cells, and this ligand is rapidly endocytosed after cell binding. The penton protein used here does not participate in binding to an integrin receptor, but is likely essential for lysis of the cellular endosome after intemalization. Protamine protects the complexes from degradation by serum proteins. At the optimal ratios of protein to DNA, these complexes can transduce MDA-MB-453 human breast cancer cells in vitro by way of receptor-specific binding and intemalization that is inhibited by free heregulin ligand. Accordingly, this method effectively targets gene delivery to cells expressing the receptor for heregulin.

Provided herein is the first demonstration of targeting the adenoviral penton protein for gene delivery by way of recombinant fusion to a receptor-specific ligand. The ability of the penton protein to mediate gene transfer across the endosome membrane has been established previously, suggesting that the penton is sufficient for receptor-mediated endocytosis and vesicle lysis (Medina-Kauwe et al Gene Therapy 2001; 8:795-803, Fender, P. et al Nature Biotechnology 1997; 15: 52-56). Studies of rhinovirus-mediated gene transfer suggest that viral fusogenic peptides form pores in the endosomal membrane in response to vesicle acidification, thus allowing the passage of endosomal contents into the cytosol (Prchla, E. et al Journal of Cell Biology 1995; 131 : 111-123). Pore formation is the most plausible explanation for the membrane penetration activity of the DT translocation domain as well (O'Keefe, D.O. et al Proceedings of the National Academy of Sciences of the United States of America 1992; 89: 6202-6206). Lysis of the endosome by adenovirus, however, is likely due to a different mechanism since molecules of differing size are similarly released from endosomes in vitro by adenovirus, whereas the upper size exclusion by rhinovi s fusogenic peptides suggest the formation of pores of limiting size. This presents an advantage for gene transfer by penton proteins since it overrides any size limitations imposed by channel forming peptides.

A previous study has reported that the interaction of the penton protein with α_vβ₅ integrins is required for the promotion of endosomal penefration (Wickham, TJ. et al Journal of Cell Biology 1994; 127: 251-264). In the examples, it was demonstrated that integrin binding is not a determinant for penton-mediated gene transfer when the complexes are directed to a different receptor. The α_v integrins do not significantle contribute to the gene transfer process by H2PO based on the observation that integrin-specific blocking peptides have no effect on gene delivery. In contrast, the nearly complete abolishment of delivery by free heregulin suggests that heregulin receptor binding and intemalization is the predominant mode of cell entry by H2PO. In comparison to 3PO complexes, which are directed to integrin receptors, the incorporation of the heregulin ligand markedly improves delivery to MDA-MB-453 cells. This may be due to the higher affinity of heregulin for its receptor in comparison to the affinity of the penton protein to α_v integrins in addition to the amplification of the heregulin receptor subunits on MDA-MB-453 cells.

These examples, demonstrate the feasibility of producing a chimeric penton protein for non-viral targeted gene delivery. As a protein-based gene delivery system, the HerPBKlO fusion protein presented here avoids the complications associated with producing targeted viral vectors. As each domain of the fusion protein can be conceived as a separate entity providing a distinct function to the overall molecular complex, modular replacement of the targeting ligand is possible, thus producing novel proteins targeted to other cell types.

It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Claims

What is claimed is:

1. A non- viral single fusion protein vector for targeted cellular delivery, wherein said vector comprises a cell targeting moiety and a cell penetration moiety.

2. The vector of claim 1, wherein the cell targeting moiety is an immuno globulins or binding fragments thereof, a receptor ligand, a growth factor, a cell surface antigen, a solubilized receptor protein, a hormone or a viral envelope protein.

3. The vector of claim 1, wherein the cell targeting moiety is a herugulin protein, a gpl20 envelope protein, a transferrin protein, or an EGF protein.

4. The vector of claim 3, wherein the cell targeting moiety is herugulin.

5. The vector of claim 1, wherein the cell penetration moiety is an adenovirus penton protein or a rhinovirus GALA protein.

6. The vector of claim 5, wherein the cell penefration moiety is said adenovims penton protein.

7. The vector of claim 6, wherein the cell penetration moiety is a serotype 5 adenovims penton protein.

8. The vector of claim 5, wherein the cell penetration moiety is a mutant penton protein.

9. The vector of claim 1, wherein said vector further comprises a polynucleotide binding moiety.

10. The vector of claim 9, wherein said polynucleotide binding moiety is a polycationic polypeptide, a histone protein, a protamine protein or a DNA biding motif of a transcription factor.

11. The vector of claim 10, wherein said polycationic polypetide is polylysine or polyarginine.

12. The vector of claim 9, wherein said polynucleotide binding moiety is a protamine protein.

13. The vector of claim 1, further comprising an active agent.

14. The vector of claim 13, wherein the active agent is further comprising an imaging agent or a cell contrast agent.

15. The vector of claim 14, wherein the agent is radioactive isotope, a fluorescent molecule a pigment, or dye.

16. The vector of claim 15, wherein said radioactive isotope is the agent is 1 ¹²⁵, P ³², or S ³⁵.

17. The vector of claim 15, wherein said fluorescent molecule is fluorescien, rhodamine, or luciferase.

18. The vector of claim 14, wherein the agent is a therapeutic agent.

19. The vector of claim 18, wherein the thereapeutic agent is a drug, an antibiotic, an antibody, a cytotoxic molecule, a hormones or a polynucleotide encoding a therapeutic gene product.

20. The vector of claim 19, wherein the cytotoxic molecule is abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof.

21. The vector of claim 19, wherein said polynucleotide encodes HSV-TK.

22. A method of targeted cell delivery, comprising contacting a cell with the non-viral single fusion protein vector or the non-viral smgle fusion protein vector linked to an active agent of claims 1-21.

23. The method of claim 22, wherein the cell is a cancer cell.

24. The method of claim 23, wherein the cancer cell is a breast cancer cell.

25. A method of targeted cell delivery in a subject, comprising delivering to a subject the non- viral single fusion protein vector or the non- viral single fusion protein vector linked to an active agent of claims 1-21.

26. The method of claim 25, wherein the subject has breast cancer.

27. A non- viral single fusion protein vector for targeted cellular delivery, wherein said vector comprises a hergulin protein as a cell targeting moiety and an adenovims penton protein as a cell penefration moiety.

28. A non- viral single fusion protein vector for targeted cellular delivery, wherein said vector comprises a hergulin protein as a cell targeting moiety, an adenovims penton protein as a cell penetration moiety and a protamine protein as a polynucleotide binding moiety.

29. The vector of claim 27, further comprising an active agent.

30. The vector of claim 28, further comprising an active agent.