WO1996029423A1 - Compositions and methods for inducing infection by retroviral vectors outside of their host range - Google Patents

Abstract

Methods and compositions for increasing the host range of a retrovirus or retroviral vector administered to a cell or an organism by co-administering a formulation comprising, for example, an adenovirus, are set forth. The host range of ecotropic, xenotropic and amphotropic retroviruses may be thereby expanded.

Description

DESCRIPTION

Compositions and Methods for Inducing Infection by Retroviral Vectors Outside of Their Host Range

Background of the Invention

This invention relates to compositions and methods for increasing the range of cells susceptible to infection by retroviral vectors. Infection of cells by retroviral vectors is normally limited to cells which express a membrane receptor that is specifically recognized by the protein or glycoprotein product of the retroviral env (envelope glycoprotein) gene. Such cells comprise what is known as the host range. Kozak, C. A. 1985. Susceptibility of wild mouse cells to exogenous infection with xenotropic leukemia viruses: Control by a single dominant locus on chromosome I. J. Virol. 55:690-695; Rein, A. and Schultz, A. 1984. For example, different murine leukemia viruses use different cell surface receptors. Virology 136:144-152; Weiss, R., Teich, N. , Varmus, H. and Coffin, J. 1984. RNA Tumor Viruses. Cold Spring Harbor Laboratory, New York, NY. The high-affinity interaction of the env gene product on the surface of the retrovirus with its cognate receptor on the target cell is thought to be directly involved in the process by which the retrovirus particle interacts with, and is internalized into, the cell. DeLarco, J. and Todaro, G. J. 1976. Membrane receptors for murine leukemia viruses: Characterization using the purified viral envelope glycoprotein, gp71. Cell 8:365-371. While the mechanistic function of the env-receptor interaction is not completely known, several processes in the early stages of infection may require a specific env-receptor interaction including the association of the retrovirus with the target cell, endocytosis into the cell, and entry of the retrovirus across the cellular membrane into the cytoplasm. The host range of a retrovirus is normally limited to those species that express a functional cognate receptor. Weiss, R., Teich, N. , Varmus, H. and Coffin, J. 1984. RNA Tumor Viruses. Cold Spring Harbor Laboratory, New York, NY. For example, ecotropic retrovirus are capable of transducing cells from many rodent species but not human cells; amphotropic retrovirus are capable of transducing cells from a large number of mammalian species including both rodents and humans; and xenotropic retrovirus are capable of transducing human and rat cells, but are not capable of transducing murine cells. Id.

The murine receptor for ecotropic retrovirus (MCAT) has been most extensively cloned and characterized. The MCAT protein has been shown to be an amino acid trans- porter that also serves as the receptor for the ecotropic env gp70 protein. Albritton, L. M., Tseng, L. , Scadden, D. and Cunningham, J. M. 1989. A putative murine eco¬ tropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57:659-666; Kozak, C. A., Albritton, L. M. and Cunningham, J. 1990. Genetic mapping of a cloned sequence responsible for susceptibility to eco¬ tropic murine leukemia virus. J. Virol. 64:3119-3121. The MCAT gene is congruent with the Rec-1 locus, a locus defined by classical genetic mechanisms which determines susceptibility to ecotropic MLV infection. Gazdar, A. F., Oie, H., Lalley, P., Moss, W. W., Minna, J. D. and Francke, U. 1977. Identification of mouse chromosomes required for murine leukemia virus replication. Cell 11:949-956; Kozak, C. A., Albritton, L. M. and Cunningham, J. 1990. Genetic mapping of a cloned sequence responsible for susceptibility to ecotropic murine leukemia virus. J. Virol. 64:3119-3121. Various studies with MCAT demonstrate that expression of a functional cognate receptor is necessary for ecotropic retroviral infection. For example, the MCAT gene has been shown to exhibit tissue specific expression and is not expressed on murine hepatocytes in vivo, Closs, E. L., Inne, H. M. , Rinkes, R. , A, B., and Cunningham, J. M. 1993. Retroviral infection and expression of cationic amino acid trans¬ porters in rodent hepatocytes. J. Virol. 67:2097-2102, a tissue known to be resistant to ecotropic retroviral infection in vivo. Jaenisch, R. 1976. Germ line integra¬ tion and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci . USA 73:1260-1264. The specificity of the interaction between the retrovirus and its surface receptor is evident in the fact that the human homologue of MCAT (HCAT) is not able to mediate ecotropic infection of human cells despite the fact that it exhibits considerable similarity to its murine homo¬ logue and is capable of binding gp70, though the affinity of the gp70-HCAT interaction is 7.6-fold lower than that between gp70 and MCAT. The amphotropic receptor has recently been cloned which should enable similar studies with this receptor system. Miller, D. G., Edwards, R. H. and Miller, A. D. 1994. Cloning of the cellular receptor for the amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus. Proc. Natl. Acad. Sci. USA 91:78-82; van Zeijl, M. , Johann, S. V., Closs, E., Cunningham, J., Eddy, R., Shows, J. B. and OHara, B. 1994. A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family. Proc. Natl. Acad. Sci. USA 91:1168-1172.

Transfection of human cells with the MCAT gene renders these cells susceptible to infection with eco¬ tropic retrovirus. This activity is dependent upon certain determinants within the MCAT gene. Site-directed mutagenesis has shown that replacement of two amino acids in the HCAT sequence with their murine counterparts (YGE_1)S „,) produces a receptor that is capable of mediating eco¬ tropic infection. Significantly, these replacements did not alter the affinity of the interaction between gp70 and the receptor. Albritton, L. M. , Kim, J. W. , Tseng, L. and Cunningham, J. M. 1993. Envelope-binding domain in the cationic amino acids transporter determines the host range of ecotropic murine retroviruses. J. Virol. 67:2091-2096. In contrast, the replacement of the same two amino acids in MCAT with their human counterparts (PGV,.,.,,,) reduces the affinity of the interaction between gp70 and MCAT and eliminates the function of the receptor. These studies demonstrate that binding of gp70 is necessary, but not sufficient, for ecotropic infection. The role of MCAT in retroviral infection beyond binding gp70 is not known. It is also known that structural modifications of the env gene or the surface of the retrovirus particle can alter the tropism of the viral vector and allow retrovirus to infect cells outside of their normal host range. For example, the addition of carbohydrates with a terminal galactose to the surface of ecotropic retroviral particles enhances the ability of these vectors to infect human cells (hepG2) expressing the asialoglycoprotein receptor. Neda, H., Wu, C. H. and Wu, G. Y. 1991. Chemical modifi¬ cation of an ecotropic murine leukemia virus results in redirection of its target specificity. J. Biol . Chem. 266:14143-14146. Crosslinking antibodies to MHC Class I and II antigens, or anti-human transferrin receptor to the outside of the virus particle extends the host range of ecotropic vectors by targeting to alternative receptors. Roux, P., Jeanteur, P. and Piechaczyk, M. 1989. A versa¬ tile and potentially general approach to the targeting of specific cell types by retroviruses: Application to the infection of human cells by means of major histocompati- bility complex murine leukemia virus-derived vectors. Proc. Natl. Acad. Sci. USA 86:9079-9083; Goud, B., LeGrain, P. and Buttin, G. 1988. Antibody-mediated binding of a murine ecotropic Moloney retroviral vector to human cells allows internalization but not the establishment of the proviral state. Virology 163:251-254. In addition, cloning of the VSV G protein (an env homolog from VSV) into an env(-) MoMLV vector was found to increase the efficiency of infection in a variety of cell lines norm- ally resistant to ecotropic virus, including primary human cells. Emi, N. , Friedmann, T. and Yee, J.-K. 1991. Psuedotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus. J. Virol. 65:1202-1207. It is also known that the susceptibility of a target cell to retroviral infection can be modified by the introduction of a gene expressing a functional receptor. For example, transfection of human cells with the gene encoding the murine ecotropic receptor MCAT or certain mutant forms of the MCAT gene renders these cells susceptible to infection with ecotropic retroviral vectors. Albritton, L. M. , Kim, J. W. , Tseng, L. and Cunningham, J. M. 1993. Envelope-binding domain in the cationic amino acids transporter determines the host range of ecotropic murine retroviruses. J. Virol. 67:2091-2096.

Retroviral vectors are important vehicles for somatic gene therapy. Adams, R, M., Soriano, H. E., Wang, M.,

Darlington, G., Steffen, D. and Ledley, F. D. 1992.

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors. Proc. Natl. Acad. Sci. USA 89:8981-8985; Miller, A. D. 1990. Retrovirus packaging cells. Hum. Gene Ther. 1:5-14. A variety of different retroviral vectors have been constructed for studies in animal or human cells using different env gene sequences. Most studies have focused on the use of vectors with the amphotropic env gene 4070A, Miller, A. D. 1990. Retrovirus packaging cells. Hum. Gene Ther. 1:5-14; Miller, A. D. and Rosman, G. J. 1989. Improved retroviral vectors for gene transfer and expression. BioTechniques 7:980-990, since such vectors can be used effectively in both rodent models and humans.

It would be useful to develop methods for enhancing the ability of retroviruses to infect certain cells, particularly human cells, which are relatively resistant to amphotropic retroviral infection. Summary of the Invention

The present invention is useful for gene therapy. The present invention features compositions and methods for efficiently integrating recombinant genes into human cells using viral particles that are incapable of infect¬ ing human cells by natural pathways without genetic modification of the target cell or covalent modification of the viral particle. One of the factors which threatens to limit the applicability of retroviral vectors for clinical gene therapy is concern about the possibility of infectious spread of replication-competent retrovirus

(RCR) arising by natural recombination between the defective retroviral vector, elements of the packaging cells, including the env genes, and even endogenous (murine) retroviral sequences. Emergence of replication competent retrovirus with amphotropic tropism (i.e., capable of infecting human cells) has been observed in virus lots produced for clinical trials. FDA. 1993. Vaccine Advisory Committee, Public Meeting on Gene Therapy, October 25, 1993.

The surprising finding that in the presence of, for example, adenovirus or adenoviral components ecotropic vectors which do not normally infect human cells do infect human cells has great utility. For example, the use of ecotropic vectors for gene therapy, in conjunction with formulations which enhance the entry of these particles into human cells, would reduce the risk of such recombi¬ nations, since such vectors could be constructed and used in the absence of any genetic elements capable of recognizing receptors on human cells.

In one embodiment the invention comprises a mixture of a substantially pure adenovirus and a substantially pure retrovirus .

In another embodiment the invention comprises a mixture of a substantially pure adenovirus and a substantially pure ecotropic retrovirus. In another embodiment the invention comprises a mixture of a substantially pure adenovirus and a substantially pure xenotropic retrovirus.

In another embodiment the invention comprises a mixture of a substantially pure adenovirus capsid protein and a substantially pure retrovirus.

In another embodiment the invention comprises a mixture of a substantially pure adenovirus capsid protein and a substantially pure retrovirus. In another embodiment the invention the substantially pure adenovirus capsid protein is a penton base protein. The retrovirus of the present invention contains a nucleic acid encoding for the expression of another nucleic acid, a protein, polypeptide or peptide. It was demonstrated that adenovirus enhances the efficiency of gene transfer using retroviral vectors. It was also demonstrated that co-infection of cells with retrovirus and replication- defective adenovirus allowed retroviral vectors to transduce cells outside of their normal host range. The efficient infection of human cells by ecotropic retrovirus and murine cells by xenotropic retrovirus was demon¬ strated. The ability of retroviral vectors to infect cells outside of their normal host range in the presence of virus such as adenovirus has significant implications for the use of retroviral vectors in gene therapy as well as the pathogenesis of retrovirus induced diseases.

The process of adenoviral infection begins with binding of the adenovirus particle to the host cell via cell-surface receptors followed by clustering of these receptors and invagination of the host cell membrane into a coated pit. The coated pit is internalized as a clathrin-coated vesicle. Following disassociation of the clathrin coat, an endocytotic vesicle is formed and a proton pump present in the endosomal membrane causes the internal pH to drop. The drop in pH causes conformational changes in the adenoviral capsid which disrupts the endosomal membrane leading to release of adenoviral particles into the host cytoplasm. Blumenthal, R., Seth, P., Willingham, M. C. and Pastan, I. 1986. pH-Dependent lysis of liposomes by adenovirus. Biochemistry 25:2231-2237. Defer, C, Belin, M.-T., Caillet-Boudin, M.-L. and Boulanger, P. 1990. Human adenovirus-host cell interactions: comparative study with members of subgroups B and C. J. Virol. 64:3661-3673. This ability of adenovirus to induce release of endosomal contents into the cytoplasm of infected cells may be responsible for the increased efficiency of DNA-mediated gene transfer in the presence of adenovirus. Curiel, D. T., Agarwal, S., Wagner, E. and Cotten, M. 1991. Adenovirus enhancement of transferrin-polylysine- mediated gene delivery. Proc. Natl. Acad. Sci. USA 88:8850-8854. By expand the host range of a retrovirus or retrovirus vector is meant that an organism not normally infected by a retrovirus or retroviral vector may be infected by that retrovirus or retroviral vector. Additionally, a cell type of an organism not normally infected by a retrovirus or retroviral vector may be infected by that retrovirus or retroviral vector. For example, an ecotropic vector which normally infects only many rodent species but not human cells will infect at least some human cells. A xenotropic vector which normally infects only human and rat cells, but is not otherwise capable of infecting murine cells will infect murine cells. An amphotropic retrovirus normally capable of transducing cells from a large number of mammalian species including both rodents and humans, however, not all cell types of a particular species may be normally infectable by an amphotropic retrovirus, may infect cell types, for example human primary hepatocytes otherwise not infectable.

By adenovirus is meant, for example, type 5, or other adenoviruses which have been described as effective viral vectors as is known by those of ordinary skill in the art, and which contain various deletions for safety as defective viral particles as also are known by those of ordinary skill in the art.

By partially expressed adenovirus is meant an adenovirus in which less than the complete viral genome is expressed, however, those elements whose expression increases the host range of a retrovirus co-administered with the partially expressed adenovirus are expressed. Such necessary elements of the viral genome may be determined by routine screening. By retroviruses are meant RNA viruses whose life cycle includes their replication by a DNA intermediate that integrates into the host genome, including all known trophisms and modifica¬ tions of retroviruses for use as recombinant vectors. By a retroviral vector is meant a particle constructed by recombinant means containing genes derived from the core proteins, enzymes, or envelope proteins of a retrovirus; genes comprising the long terminal repeat and packaging sequences or homologous genes capable of performing these functions and a non-homologous (to the retrovirus) gene that the practitioner wishes to introduce into a target cell . A variety of proteins can be encoded by the non- homologous sequences in the retrovirus. These proteins can be post-translationally modified to be proteins, glycoproteins, lipoproteins, phosophoproteins, etc. Those proteins which can be expressed may function as intra- cellular or extracellular structural elements, ligands, hormones, neurotransmitters, growth regulating factors, enzymes, serum proteins, receptors, carriers for small molecular weight compounds, drugs, immunomodulators, onco- genes, tumor suppressors, toxins, tumor antigens^', anti¬ gens. These proteins may have a natural sequence or a mutated sequence to enhance, inhibit, regulate, or eliminate their biological activity.

Specific examples of these compounds include extracellular matrix proteins, collagens, cytoskeletal proteins, cytokines including IL-1, IL-4, IL-6, IL-8, IL- 10, IL-1 receptor antagonist, a soluble IL-1 receptor, complement proteins, growth factors including insulin, p53, SDJ, RL growth inhibitors, IGF-1, IGF-2, EGF, NGF, PDGF, ciliary nerve growth factor, FGF, TGF-α, TGF-/3, interferons { , β , γ) , p53, Rb, or SDI, transplantation antigens, histocompatibility antigens (including class I and II transplantation antigen) , allogeneic transplan¬ tation antigen, xenogeneic transplantation antigens, bacterial antigens, parasitic antigens, viral antigens, cell adhesion antigen, tumor specific antigens, receptors for natural ligands, receptors for drugs, genetically modified receptors for natural ligands, steroid receptors, genetically modified steroid receptors, receptors for angiostatic steroids, enzymes of prostaglandin metabolism, enzymes for collagen and extracellular matrix degradation including metalloproteinases, enzymes for synthesis and secretion of synovial fluid, vitreous fluid, or fluid of the inner ear, enzymes for thyroid hormone synthesis, receptors for peptide backbones for peptidoglycan, receptors for platelet Factor 4, receptors for angio- genesis modulator factors, hormones including somato- tropin, thyrotropin, prolactin, endorphin, thyroglobulin, and thyroid peroxidase, and serum proteins such as clotting factors (VIII, IX, VII) .

By ligand is meant an element within a formulation that interacts with a specific receptor on a cell with high affinity. In specific embodiments, the interaction of the ligand with the receptor induces internalization of the complex into the endosome.

By co-infection is meant co-administration of a mixture of a substantially pure retrovirus and substan¬ tially pure adenovirus for the purpose of introducing a retroviral vector into a target cell. (The term transducing is equivalent to the term infecting.)

By mixture of a retrovirus and adenovirus is meant a formulation comprising the retrovirus and adenovirus which is administered to an organism or cell simultaneously or the administration of a retrovirus and adenovirus separately, but in close temporal order such that the organism or cell will be exposed to both the retrovirus and adenovirus at substantially the same time.

By a substantially pure retrovirus and a substan- tially pure adenovirus is meant a formulation comprising the retrovirus and adenovirus which is controlled as to its composition and does not contain contaminants such as other viruses, bacteria, or chemicals except those that are specifically and purposefully present. By formulation is meant the use of retrovirus in a mixture incorporating adenovirus or synthetic materials capable of the same membrane binding and endosomal release functions as the adenovirus. Further, the term formula¬ tion used herein refers to retroviral vectors combined with elements such as viruses, proteins, carbohydrates, synthetic organic compounds, or in-organic compounds, capable of enhancing the entry of retroviral particles into non-permissive cells. Examples of such elements include particulates comprising attenuated or defective viruses, liposomes, polymers, proteins, lipoproteins; ligands comprising peptides, carbohydrates, or lipids; endosomal release elements comprising peptides or lipids.

By endosomal release is meant translocation of the retroviral vector from the membrane or endosomal compart- ment into the cytoplasm of the cell. The retroviral particle may remain intact or endosomal release may involve only the genetic material of the retroviral vector in association with elements of the retroviral particle, formulation, or target cell. By target cell is meant a cell in vivo or ex vivo desired to be transformed with a gene present within a retroviral vector, including cells of hepatic, hemato- poietic, endothelial, epithelial and mesothelial origin.

By transformation is meant the introduction of genetic material into a target cell for therapeutic, diagnostic, or research purposes. By ecotropic retrovirus is meant a retrovirus capable of transducing cells from many rodent species but not human cells.

By amphotropic retrovirus is meant a retrovirus capable of transducing cells from a large number of mammalian species including both rodents and humans, however, not all cell types of a particular species may be normally infectable by an amphotropic retrovirus.

By xenotropic retrovirus is meant a retrovirus capable of transducing human and rat cells, but not capable of transducing murine cells.

By nucleic acid is meant both RNA and DNA including: cDNA, genomic DNA, plasmid DNA or condensed nucleic acid, nucleic acid formulated with cationic lipids, nucleic acid formulated with peptides, cationic polymers, RNA or mRNA. In a preferred embodiment, the nucleic acid administered is plasmid DNA which comprises a vector, phosphoproteins.

Other and further objects, features and advantages will be apparent from the following description of the presently preferred embodiments in the invention.

Detailed Description of the Invention

The drawings will first briefly be described.

Drawings : Figure 1. Infection of human HeLa cells with ecotropic retrovirus in the presence of adenovirus. HeLa cells plated in 35 mm tissue culture plates were infected with the ecotropic retrovirus zenβgal alone (A) or in the presence of 10⁸ pfu/ml adenovirus (B) and stained with X- Gal 48 hours after infection.

Figure 2. Titer of ecotropic virus on human cells in the presence of adenovirus. Human HeLa and PLC/PRF cells were plated in 35mm culture plates and transduced with ecotropic retrovirus zenβgal in the presence of differing concentrations of adenovirus from 10⁶ to 10⁸ pfu/ml and the number of X-gal staining cells were counted. Control titer was determined by infection of murine NIH3T3 cells. Figure 3. Stable infection of human cells with ecotropic retrovirus and murine cells with xenotropic retrovirus in the presence of adenovirus. Cells were infected with NEO-R containing retrovirus produced in an ecotropic packaging cell line (GP+E) or xenotropic packaging cell line (GP+X(N2)) alone or in the presence of 10⁸ pfu/ml dl312 adenovirus. Cells were selected after 18 hours with 0.5 mg G418/ml and maintained under this selec¬ tion for 10 days. The resulting colonies were stained with methylene blue and photographed. Legend: A. Human HeLa cells infected with ecotropic N2 alone in the presence of adenovirus; B. Human HeLa cells infected with ecotropic N2 alone; C. NIH3T3 cells infected with xenotropic N2 in the presence of adenovirus; D. NIH3T3 cells infected with xenotropic N2 alone.

Figure 4. Identification of provirus in infected cells by PCR. Panel A. Human (HeLa) cells were infected with GP+E(N2) retrovirus in the presence of varying amounts of dl312 adenovirus. Cells were harvested 48 hours after transduction and proviral DNA identified by PCR for a 790 bp NEO-R fragment and Southern blotting as described below. Lane M: Molecular weight markers, Lanes 1-5: quantitative control reactions containing 10^_1(1) to 10^"5(5) dilutions of a sample having a single copy of NEO-R per genome. Lane A: HeLa, uninfected. Lane B: HeLa + GP+E(N2) , 8 μg/ml polybrene. Lane C: HeLa + GP+E(N2) + 10⁸ pfu/ml DL312 adenovirus + 0 μg/ml polybrene. Lane D: HeLa + GP+E(N2) + 10⁸ pfu/ml DL312 adenovirus + 8 μg/ml poly¬ brene. Lane W: water PCR blank. For quantitation, the blot was examined using a Betagen Blot Analyzer, and the resulting counts from the control curve were plotted against the number of proviral copies per genome, demon- strating a log:linear response (panel B) . Regression analysis allowed the determination of the number of proviral copies/genome as a function of cpm in amplified product. The same virus was titered by classical selection methods, and the number of colonies formed compared to the number of proviral copies/plate giving similar results (panel C) . Figure 5. Effects of retroviral and adenoviral concentration on infection of murine NIH3T3 cells with xenotropic retrovirus. NIH3T3 cells were transduced with varying dilutions of GP+X(N2) in the presence of varying amounts of dl312 adenovirus and the number of proviral copies/plate determined by PCR. Data are expressed as the apparent titer of the xenotropic retrovirus on the NIH3T3 cells (y-axis) as a function of the concentration of adenovirus (x-axis) , and dilution of the retrovirus stock

(z-axis) . Figure 6. Preincubation of cells with adenovirus does not make cells permissive to retroviral infection. Human HeLa cells were infected with ecotropic virus in the presence of adenovirus or after pre-incubation of cells with adenovirus and subsequent washing of unbound virus from the plate. The apparent titer of the xenotropic vector is shown for simultaneous infection, preincuba- tions, and infection without exposure to adenovirus.

Figure 7. The effect of adenovirus co-administered with an amphotropic retrovirus is illustrated in panels A and B.

Administration

The nucleic acid sequence contained in the retroviral vector and the adenovirus or adenovirus protein can be administered to patients having a disease or condition for which the nucleic acid sequence encodes a therapeutic protein, polypeptide or peptide. Routes of administration may include intramuscular, intravenous, aerosol, oral (tablet or pill form) , topical, systemic, ocular, as a suppository, intraperitoneal and/or intrathecal. The specific delivery route of a retroviral vector and the adenovirus or adenovirus protein will depend on their uses.

Methods of delivery that may be used include in vivo where the viral particles are administered directly to the patient and ex vivo with subsequent rei plantation or administration of the transfected cells.

The nucleic acid sequence contained in the retroviral vector and the adenovirus, partial adenovirus or adeno- virus protein may also be systemically administered. Systemic absorption refers to the accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include: intravenous, intramuscular, sub- cutaneous, intraperitoneal, intranasal, intrathecal and ophthalmic. Descriptions of useful systems are provided in the art cited above, all of which is hereby incor¬ porated by reference.

A nucleic acid sequence contained in the retroviral vector and the adenovirus, partial adenovirus or adeno¬ virus protein may be administered utilizing an ex vivo approach whereby cells are removed from an animal, infected with the viruses and reimplanted into the animal. The liver can be accessed by an ex vivo approach by removing hepatocytes from an animal, transducing the hepatocytes in vi tro with the viruses and reimplanting them into the animal ( e . g. , as described for rabbits by Chowdhury et al, Science 254: 1802-1805, 1991, or in humans by Wilson, Hum. Gene Ther. 3: 179-222, 1992) incorporated herein by reference.

The viruses may be administered utilizing an in vivo approach whereby the gene will be administered directly to an animal by intravenous injection, intramuscular injec¬ tion, or by catheterization and direct delivery of the gene via the blood vessels supplying the target organ. Expression may be achieved using a skeletal muscle- specific promoter for the nucleic acid sequence encoding a protein, polypeptide or peptide.

Example 1

Retrovirus and adenovirus The N2 retroviral vector, which carries the Tn7 neomycin resistance gene (NEO-R) , has been described elsewhere. Armentano, D., Yu, S. F., Kantoff, P. W., von-Ruden, T., Anderson, W. F. and Gilboa, E. 1987. Effect of internal viral sequences on the utility of retroviral vectors. J. Virol. 61:1647-1650, incorporated herein by reference. Ecotropic N2 virus was generated using the GP+E86 packaging line. Markowitz, D., Goff, S. and Bank, A. 1988. A safe packaging line for gene transfer: Separating viral genes on two different plasmids. J. Virol. 62:1120-1124, incorporated herein by reference. An ecotropic vector containing the E. coli /3-galactosidase gene (Zenβgal) was provided by Dr. Philippe Soriano (Fred Hutchinson Cancer Research Center) and virus was similarly produced using the GP+E86 cell line. The GP+E86 cell line produces virus with the gp70 ecotropic env gene, the sequence of which has been published. Shinnick, T. M. , Lerner, R. A. and Sutcliffe, J. G. 1981. Nucleotide sequence of Moloney murine leukaemia virus . Nature 293:543-548. The LNL6/PA317/c8 cell line which produces an amphotropic derivative of N2 was constructed by Dusty Miller, Bender, M. A., Palmer, T. D., Gelinas, R. E. and Miller, A. D. 1987. Evidence that the packaging signal of the Moloney murine leukemia virus extends into the gag region. J. Virol. 61:1639-1646, in the PA317 cell line. Miller, A. D. and Buttimore, C. 1986. Redesign of retro¬ virus packaging cell lines to avoid recombination leading to helper virus production. Mol . Cell. Biol . 6:2895-2902. This packaging cell line produces a vector particle with the 4070A amphotropic ENV gene product, the sequence of which has been published. Ott, D. E., Friedrich, R. and Rein, A. 1990. Sequence analysis of amphotropic and 10A1 murine leukemia viruses: Close relationship to mink cell focus-inducing viruses. J. Virol. 64:757-766. The N2/GP+X/2 cell line produces a vector with a xenotropic env gene, the complete sequence of which has been published. O'Neill, R. R., Buckler, C. E., Theodore, T. S., Martin, M. A. and Repaske, R. 1985. Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus. J. Virol. 53:100-106. Xenotropic virus was produced using the plasmid pXenv, constructed by Dr. David Steffen as described in Adams, R. M., Soriano, H. E., Wang, M. , Darlington, G. , Steffen, D. and Ledley, F. D. 1992. Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors. Proc. Natl. Acad. Sci. USA 89:8981-8985, and the strategy described by Markowitz D., Goff, S. and Bank, A. 1988. A safe packaging line for gene transfer: Separating viral genes on two different plas ids . J. Virol. 62:1120-1124.

For retroviral harvest, producer cells were grown to near-confluence in Dulbecco's modified minimum essential medium (GIBCO) supplemented with 10% fetal bovine serum

(JRH) . Virus were harvested in serum-containing media overnight and filtered through a 0.45 μm syringe filter

(Gelman) . Virus was used immediately or frozen for up to 3 months at -80°C in 15 ml aliquots. Replication defective adenovirus (ElA-deleted) dl312, Jones, N. and Shenk, T. 1979. Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo. Cell 17:683-689, was provided by Dr. Tom Shenk (Princeton University) . An adenoviral stock was prepared as follows. The 293 cells were inoculated with dl312 virus in serum- containing DMEM media at an MOI of approximately 10:1, then allowed to recover in fresh serum-containing media for approximately 48 hours until cytopathic effect was observed and >90% of the cells were floating. Cellular material was recovered and centrifuged at 250 x g for 5 minutes at room temperature. The pellet was frozen and thawed three times in PBS to lyse the cells and insoluble material was removed with a 250 x g spin for 5 minutes at 10°C. The supernatant was layered onto a CsCl step- gradient comprising 1.5 g/ml, 1.35 g/ml, and 1.25 g/ml in 14 x 89 mm centrifuge tubes (Beckman) and centrifuged for 1 hour at 35,000 rpm (150,000 x g) at 10°C in an SW40ti rotor. The band was visually identified, collected, placed on a CsCl equilibrium gradient of 1.35 g/ml, and spun for 18 hours at 150,000 x g at 10°C. The visible band containing the virus was again collected and was dialyzed with Spectro/Por 7: (28 mm, 50,000 MW) against 3 changes of 10 mM Tris-HCl (pH 7.5) , 1 mM MgCl₂, and 10% Glycerol over a 2 -hour period. After dialysis, the virus was aliquotted in 500 μl, single-use aliquots and stored in 10% Glycerol at -80°C. The adenoviral titer in estimated pfu/ml was determined by spectrophotometry.

Cell lines

Murine NIH3T3 cells are classically infectable with ecotropic and amphotropic retrovirus but are uninfectable by xenotropic virus. Rat 1 cells are classically infect¬ able with ecotropic, xenotropic, and amphotropic retro¬ virus. Human HeLa cells and 293 cells were obtained from the ATCC. PLC/PRF, a human hepatoma cell line, was pro¬ vided by Dr. Gretchen Darlington. Human cells are classically infectable with xenotropic and amphotropic virus but not ecotropic virus. Cells were grown on Falcon tissue culture plates. NIH3T3, HeLa, 293, and rat-1 cells were grown in DMEM, supplemented with 10% HyClone. PLC/PRF cells were grown in 3:1 ME :Weymouth, supplemented with 10% Fetal bovine serum.

Infection procedures

For infection, target cells including NIH3T3, rat-1, PLC/PRF, and HeLa were grown to 70% confluence and placed in fresh media. Adenoviral or retroviral supernatant were serially diluted in PBS and added simultaneously. Polybrene was added to all infections at a concentration of 8 μg/ml. After an 18 hour incubation, the cells were washed three times with PBS and fed with serum-containing media. For the sequential viral addition experiments, HeLa cells were grown to 70% confluence, 10⁸ adenoviral particles/ml were added with 8 μg/ml final concentration of polybrene, cells were incubated for 10 minutes at 37°C, washed three times with several volumes of PBS, and then fed with media containing 1 ml of 10⁵ cfu/ml titer zen/3gal retrovirus and 8 μg/ml polybrene.

Analytical procedures

Three different methods were used for determining titer. 1) Titer was determined for all NEO-R containing strains by selection with 500 μg/ml G418 for 10 days and counting colonies after staining with methylene blue. 2) Titer was determined for _/S-galactosidase containing strains by histochemical staining for S-galactosidase expression in infected cells was performed with 5-bromo-4- chloro-3-indolyl beta-galactopyranoside (X-gal) . MacGregor, G. R., Mogg, A. E., Burke, J. F. and Caskey, C. T. 1987. Somatic Cell Molecular Genetics 13:253-265. Cells staining blue and unstained cells were counted in a 1 mm-wide band across the plate in both the horizontal and the vertical direction. The number of cells staining with X-gal in the area counted was normalized to the total surface area of the dish and dilution of the administered virus to determine the apparent titer. 3) Semi- quantitative PCR was employed to determine the number of proviral copies of viral DNA per genome equivalent in infected cells. Cellular DNA was harvested by SDS/ proteinase-K lysis and phenol extraction. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D. , Seidman, J. G., Smith, J. A. and Struhl, K. 1987. Current Protocols in Molecular Biology. John Wiley and Sons, New York, NY. PCR was performed on 1 μg total DNA from each sample with the simultaneous amplification of a control curve using oligonucleotides and conditions described previously. (Adams, R. M. , Soriano, H. E., Wang, M. , Darlington, G., Steffen, D. and Ledley, F. D. 1992. Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors. Proc. Natl. Acad. Sci. USA 89:8981-8985; Morgan, R. A., Cornetta, K. and Anderson, W. F. 1990. Applications of the poly erase chain reaction in retroviral- ediated gene transfer and the analysis of gene-marked human TIL cells. Hum. Gene Ther. 1:135-149.) Amplification of the proviral NEO-R gene produced a 790 bp fragment . The control curve consisted of DNA from a murine cell line known to contain a single copy of the NEO-R provirus per genome diluted into normal DNA of the appropriate species at serial 1:10 dilutions so that the total amount of DNA in the PCR reaction remained constant. PCR products were visualized on a 1% agarose gel, trans¬ ferred via Southern transfer to a nylon (NYTRAN, S & S, NH) membrane, probed with the NEO-R fragment, labeled with t³²P] , and analyzed both by autoradiography and quanti- tation of the fragments by Betagen blot analyzer.

All titer data is presented as the mean of triplicate samples. Standard deviations of titers determined using G418 selection and histochemistry for β-galactosidase were <5% of the mean. Standard deviations for titers deter- mined by PCR were uniformly <10% of the mean. Regression analysis of the control dilution was used to obtain a numerical estimate of the number of proviral copies/genome equivalent .

The entry of retroviral particles into target cells was assessed using the fluorescence dequenching methods described originally for studies with HIV. Sinangil, F., Loyter, A. and Volsky, D. J. 1988. Quantitative measure¬ ment of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching. FEBS Lett. 239:88-92. For assay of viral entry, a cell- free supernatant of GP+E(N2) containing 2xl0⁸ cfu of virus was harvested and centrifuged for 2 hours at 9,000 rpm in a Beckman SS-34 rotor (17,500 x g) at 4°C. The virus sediment was resuspended in a solution of 20 mM Tris-HCl (pH 7.4) , 100 mM NaCl, 1 mM EDTA and repurified by centri- fugation over a 20%-60% sucrose pad at for 18 hours at 25,000 rp in a SW40 rotor (160,000 x g) at 4°C. The visible virus band was recovered in 1 ml total volume, aliquotted in 50 μl aliquots, and stored at -80°C. When the virus was prepared for use, a vial was defrosted and 8.5 μl of 1 mg/ml ethanolic solution of octadecylrhod- amine-B chloride (R-18, Molecular Probes, OR) was added. The mixture was allowed to sit at room temperature for 15 minutes in the dark, after which the solution was diluted to 1.5 ml with PBS and centrifuged for 15 minutes at 14,000 rpm in an Eppendorf centrifuge (10,000 x g) at 4°C to remove the free dye. The virus particles were resus¬ pended in 100 μl PBS and stored at 4°C until ready for use.

Viral Entry Assay

Viral entry was assayed using 5xl0⁶ cfu of R-18 labeled GP+E(N2) retrovirus added to HeLa cells at 70% confluence on Primaria 6-well plates in the presence of 8 μg/ml polybrene. Cells were incubated with the virus for 30 minutes at 4°C then washed three times with PBS to remove unbound virus. The cells were then fed with fresh media and moved to 37°C for 30 minutes. After the final incubation, the cells were removed from the plate by trypsinization, and resuspended in 3 ml PBS. Fluorescence

(560 nm excitation, 608 nm emission) was measured before and after addition of 100 μl of a 5% stock solution of

Triton X-100 (Sigma) . Background fluorescence was esti- mated by mixture of the cells with labeled virus particles immediately before fluorescence measurement, so that no intermixing of R-18 labeled virus and cell membrane could occur. Specific dequenching with R-18 was calculated as described. Sinangil, F., Loyter, A. and Volsky, D. J. 1988. Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching. FEBS Lett. 239:88-92. The addition of Triton X-100 causes complete dequenching of R-18 labeled virus. The amount of dequenching caused by internalization of the R-18 labeled virus and dilution in cellular membranes is expressed as a fraction of observed fluorescence divided by total dequenching in the presence of Triton X-100.

Example 2

Infection of human cells with ecotropic retrovirus in the presence of adenovirus

Human PLC/PRF (hepatoma) or HeLa cells were infected with the zenβgal ecotropic retrovirus in the presence of different concentrations of adenovirus, and cells trans¬ duced with the retrovirus were identified by staining with X-gal. Transduced HeLa cells were identified in cultures co-infected with dl312 adenovirus (figure 1, panel A) but not in cultures infected with retrovirus alone (figure 1, panel B) . The number of transduced cells increased as a function of the concentration of adenovirus from 10⁶ to 10⁸ pfu/ml (figure 2) . Considerable cytopathicity was observed at higher concentrations of adenovirus, especi¬ ally in HeLa cells which are known to be permissive to replication of E1A deficient adenovirus such as dl312. Shenk, T., Jones, N. , Colby, W. and Fowlkes, D. 1980. Functional analysis of adenovirus-5 host-range deletion mutants defective for transformation of rat embryo cells. Cold Spring Harbor Symposia on Quantitative Biology 44:367-375. In control experiments, the same ecotropic virus was used to infect murine cells in the presence of different concentrations of adenovirus. There was no significant effect of adenovirus on the efficiency of infection of murine cells with ecotropic vectors.

To confirm that X-gal staining reflected authentic, stable infection, HeLa cells were transduced with an ecotropic retrovirus N2 containing the selectable NEO-R gene and titered using classical selection methods (figure 3) . Colony formation was observed in the presence of adenovirus (figure 3A) , but not in cultures infected with retrovirus alone (figure 3B) . In control experiments, the presence of adenovirus did not significantly increase the efficiency of infection of HeLa cells by amphotropic or xenotropic vectors.

Example 3

Infection of murine cells (NIH3T3) with xenotropic retrovirus in the presence of adenovirus To determine whether the infection of human cells with ecotropic virus in the presence of adenovirus reflected a specific interaction between adenovirus and the ecotropic eπv/receptor system, identical experiments were performed using a xenotropic vector GP+X(N2) to infect murine cells. Murine cells are normally outside the host range of the xenotropic vector, and no colonies were observed after infection with GP+X(N2) and G418 selection (figure 3D) . In contrast, colonies were observed in cultures infected with GP+X(N2) in the presence of adenovirus (figure 3C) . In control experi¬ ments, the presence of adenovirus did not proportionally enhance the efficiency of infection of murine cells with ecotropic or amphotropic retrovirus . It should be noted that an absolute increase in apparent titer on permissive cells equivalent to that observed outside of the host range would not be significant.

Example 4

Infection of primary human hepatocytes with amphotropic retrovirus in the presence of adenovirus Human hepatocytes were collected utilizing the following procedure: collagenase/EDTA disassociation of the tissue, plating in serum-containing medium, with a change to SUM/Chow defined medium after six hours. At 24 hours post-plating, plates were infected under standard conditions (including 8mg/μl f.c. polybrene) with:

5xl0⁶ cfu LNL6 5xl0⁶ cfu LNL6 + 10⁸ pfu/ml Adenovirus 5xl0⁶ cfu LNL6 + 10⁷ pfu/ml Adenovirus 5x10^s cfu LNL6 + 10⁶ pfu/ml Adenovirus The cells were infected in this combination over¬ night, after which the media was removed, and the cells were allowed to grow for another 48 hours, after which DNA was harvested by phenol/chloroform extraction. PCR for the integrated LNL6 provirus was performed by a standard protocol utilizing primers amplifying a 790 bp fragment of the viral NEO-R gene. Amplification was performed via both 20-cycle PCR and 25-cycle PRC, in order to highlight apparent differences in the infection efficiency between samples. PCR samples were electrophoresed in EtBr- containing agarose, and the Southern blot performed was transferred for hybridization with a radiolabeled NEO-R probe. (See figure 7 where the numbers represent adenoviral concentrations in the infection media, 10ⁿ ) . The increase in infection efficiency is most evident between the 0 adenovirus band and that of 10^B in the 20- cycle experiment. For quantitative analysis, the samples were again subjected to the NEO-PCR, this time with the standard quantitative controls: serial dilution of single-NEO-R- copy/cell DNA in tenfold steps with control DNA from the same source PCR, yielding a measure of infection effi- ciency. After Southern blot hybridization of the samples with radiolabeled NEO-R DNA, the blot was analyzed on a Betagen blot analyzer, and the resulting infection efficiency quantified. The infection estimates from the experiment were as follows: +0 AdV +10⁶AdV +10⁷ AdV -H0⁸AdV

4% 7% 4%(*) 18% *This band was hampered by a blot artifact, and probably should have been higher.

These experiments, although performed in a single subject, are evidence of an enhancement phenomena in human hepatocytes which correlates with observations in other systems where infection is hampered by lack of receptor expression. This evidence is highlighted by observations that the amphotropic receptor is not efficiently expressed by human hepatocytes under the culture conditions that were used. Northern hybridization of human hepatocytes after 48 hours in SUM/Chow media do not show appreciable expression of the amphotropic receptor gene, compared to the high level present in rat cells in culture, or the level observed in human hepatoma cell lines (which are shown to be quite infectable by amphotropic vectors) .

Example 5

Ouantitation of apparent retroviral titer on cells outside the normal host range

Quantitative PCR was used to assess the efficiency of infection using various vectors, cells, and infection conditions. The quantitative PCR assay was validated in two experiments. First, increasing amounts of DNA from cells containing a single copy of integrated provirus was mixed with DNA from non-transduced cells to establish a standard, control curve for the relationship between the amount of provirus and PCR product (figure 4, panel A, lanes 1-5) . There was a high log linear correlation (>0.90) between the number of proviral copies in the reac¬ tion template and the amount of PCR product (figure 4B) . This control was run concurrently with all experiments and the apparent titer is interpolated from the amount of PCR product in the same relative to the standard curve. Second, cells infected with GP+X(N2) or GP+E(N2) were titered by both G418 selection and PCR analysis to compare the results obtained with these two methods. Both methods gave equivalent results (figure 4 panel C) . The efficiency of infection of murine NIH3T3 cells with GP+X(N2) was shown as a function of both retroviral dilution and adenoviral dilution. The fraction of cells transduced with the retrovirus increased both as a func- tion of increasing retroviral concentration and increasing adenoviral concentration in a dose-dependent manner (figure 5) .

To further assess the generality of this phenomenon, the apparent titer of different xenotropic, amphotropic, and ecotropic vectors was determined by quantitative PCR on several different cell lines using optimal infection conditions (table 1) . There was no demonstration of sig¬ nificant infection of human cells by ecotropic virus or murine cells by xenotropic virus. This control confirms the expected host range of each of the vectors used in these experiments and the target cells. In each instance, the addition of adenovirus was associated with at least a two order of magnitude increase in the number of cells transduced outside of the host range. The apparent titers of vectors on cells outside of their host range in certain experiments was up to 1-10% of the apparent titer on nominally permissive cells.

Example 6

Mechanism of enhanced retroviral infection outside of the normal host range

To further understand the mechanism by which the presence of adenovirus enhances retroviral infection of cells outside their normal host range, we assayed the entry of ecotropic retrovirus into HeLa Cells using R18 stained virus particles. The principle of this experiment is that retrovirus stained with R18 will not exhibit fluorescence due to quenching, but the entry of virus into a target cell, and subsequent dispersion of the R18 throughout the infected cell membranes, leads to dequench- ing and an increase in the measurable florescence. Triton X-100 treatment of the virus with triton also causes dequenching and serves as a quantitative control.

In control experiments, incubation of R-18 labeled GP+E(N2) with murine cells resulted in significant fluorescence dequenching, while incubation of the labeled virus to human HeLa cells showed little fluorescence dequenching (DQ) . Sinangil, F., Loyter, A. and Volsky, D. J. 1988. Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching. FEBS Lett. 239:88-92. When R18/GP+E(N2) was added to HeLa cells in the presence of adenovirus, there was a marked increase in fluorescence dequenching. Thus, infection of NIH3T3 cells with eco¬ tropic GP+E(N2) resulted in DQ=17.5%; infection of HeLa cells with ecotropic GP+E(N2) resulted in background activity DQ=1.7%. In contrast, infection of HeLa cells with ecotropic GP+E(N2) in the presence of 10⁸ Adv/ml resulted in DQ=19.8%, a value equivalent to that seen for infection of permissive NIH3T3 cells. This result suggests that the effect of adenovirus is to enhance entry of the retroviral particles into cells outside of their normal host range.

To assess whether the adenoviral effect required both virus to be present at the time of infection, or whether adenovirus infection acted on the cells to render then susceptible to retroviral infection, experiments were performed in which cells were pre-incubated with adeno¬ virus and the adenovirus was removed before infection of cells with retroviral vectors. Studies on adenovirus infection (using Ad2) have demonstrated internalization of as much as 80-85% of adenoviral particles into infected cells within 10 minutes of initial exposure. Greber, U. F., Willetta, M. , Webster, P. and Helenius, A. 1993. Stepwise dismantling of adenovirus 2 during entry into cells. Cell 75:477-486. Accordingly, human HeLa cells were exposed to adenovirus for 10 minutes at 37°C and subsequently washed three times with 10X volumes of PBS to remove residual virus. Cells were then infected with zenβgal retrovirus for 18 hours using standard procedures and the efficiency of retroviral infection was assessed by X-gal staining. The efficiency of infection in cultures pre-infected with adenovirus was almost unchanged from the levels observed in controls performed in the absence of adenovirus (figure 6) . This result suggests that the enhanced retroviral infection of cells outside their normal host range requires the coincident presence of adenovirus and retrovirus in the media.

These experiments demonstrate stable retroviral infection of cells outside of host range barriers when the infection is performed in the presence of a replication- defective adenovirus. This appears to be a general phenomenon which is not related to a specific host range, target cell, selectable marker, or assay method. The efficient infection of several human cell lines with several different ecotropic retroviral vectors using classical (G418) selection assays, quantitative PCR, and histochemical staining for jβ-galactosidase was demon¬ strated. Efficient infection of murine cells with xenotropic retrovirus using G418 selection and PCR assays was also demonstrated. In each instance, the tropism of the virus was confirmed by their inability to infect cells outside of their normal host range in the absence of adenovirus and_. the characteristics of the cells were confirmed by their susceptibility to infection with various retrovirus under conventional conditions. In the presence of adenovirus, however, both ecotropic and xenotropic vectors were able to infect cells outside the host range with an apparent titer as high as 1-10% of the titer determined on cells within the host range. This effect is shown to be quantitatively dependent upon both the concentration of the retroviral vector and the replication-defective adenovirus in the infection media. Extensive previous experience with various types of retrovirus has uniformly demonstrated that infection requires the presence of an appropriate cognate receptor for the env gene on the surface of the target cell. The presence or absence of the cognate receptor determines the host range of a particular retrovirus as well as its tropism or interference group. While previous works demonstrate that tropism can be altered by changing the specific interaction between the virus particle and cognate receptors on the target cell, they also confirm the need for such a specific interaction for infection to occur. In marked contrast, the present invention describe a distinctly different phenomenon, namely the efficient infection of cells which lack a cognate receptor for the retrovirus. Significantly, this is observed without covalent or genetic modification of either the retroviral vector or the target cell.

Several observations suggest that in the present invention, adenovirus acts by enhancing the entry of retrovirus into cells which lack a cognate receptor, and that it is facilitated retroviral entry, rather than any general effect on the infectivity of the target cell, that accounts for the ability of retrovirus to infect cells outside of their normal host range. First, co-infection with adenovirus does not increase the infection of cells within the normal host range. Thus, adenovirus does not appear to be enhancing any common step of infection. Second, studies, with virus fluorescently labeled with R18 demonstrate that the entry of retrovirus into cells lacking the cognate receptor is enhanced in the presence of adenovirus. This experiment demonstrates that adeno- virus enhances the earliest steps of retroviral entry into the cell. Third, pre-incubation of cells with adenovirus does not produce enhanced infection outside of the host range. This suggests that the adenovirus particles must be present simultaneously with the retroviral particles for enhanced infection, and that infection is not enhanced by expression of adenoviral gene products or any persisting effect of adenovirus on the target cell. Table 1

Apparent Titer of Virus with Various Tropism on Murine, Human, and Rat Cells in the Presence and Absence of Co- infection with Adenovirus

Retrovirus Ecotropic Ecotropic Amphotropic Amphotropic Xenotropic Xenotropic

Adenovirus - - - - - -

(+/-)

NIH3T3 1.4xl0⁷ 4.3xl0⁶ 2.4x10⁶ 6.3x10" < 0.1 1.2x 10"

RAT-1 l . lxlO⁷ 1.8xl0⁷ 2.6x10⁶ 3.3x10" 5.9x10' 2.6x10"

HeLa < 0.1 6.0x10' 3.2x10" 6.0x10" 7.1xl0⁵ 1.6x10"

PLC/PRF < 0.1 4.5x10" ND ND 6.0xl0⁵ 8.4x10^s

Example 7

Enhanced retroviral transduction using semisynthetic formulations The general effectiveness of adenovirus on different cell types and with different retroviral vectors indicates that a similar effect may be achieved using synthetic formulations that mimic the dual effects of adenovirus on receptor binding to stimulate endocytosis and enhanced endosomal release. Lipoproteins resemble adenovirus in size and have high affinity receptors on certain cell types. Other particulates may be constructed that exhibit similar size and receptor affinities comprised of poly¬ mers, peptides, carbohydrates, and or lipids coupled to appropriate ligands. The ligands may themselves be comprised of peptides, carbohydrates or lipids and may be selected for affinities for specific receptors on the surface of target cells.

These particles may be modified by addition of peptides known to enhance endosomal release in the same manner as adenovirus. These peptides may be derived from the penton protein of adenovirus, the influenza virus hemagglutinin, or other synthetic peptides developed with similar biological activity, for example, GALA.

Particles comprised of synthetic materials thus resembling adenovirus may be mixed with ecotropic retro- virus and applied to human cells. The interaction of the synthetic particulate material with the membrane will induce endocytosis resulting in internalization of the retroviral particle. Once within the endosome, acidifi¬ cation with result in release of the retroviral material into the body of the cell.

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

Claims

What is claimed is:

1. A composition comprising a mixture of a substantially pure retrovirus, said retrovirus including a non-homologous gene, and a formulation which expands the host range of said retrovirus .

2. The composition of claim 1 wherein said formulation is a substantially pure adenovirus.

3. The composition of claim 1 wherein said formulation is a substantially pure partially expressed adenovirus.

4. The composition of claim 1 wherein said formulation is an adenovirus capsid protein.

5. The composition of claim 4 wherein said adenovirus capsid protein is a penton base protein.

6. The composition of claim 1 wherein said formulation is a lipoprotein.

7. The composition of claim 1 wherein said formulation is influenza virus hemagglutinin.

8. The composition of claim 1 wherein said formulation is a synthetic peptide.

9. The composition of claim 8 wherein said synthetic peptide is GALA.

10. The composition of claim 1 wherein said substantially pure retrovirus is ecotropic.

11. The composition of claim 10 wherein said substantially pure retrovirus is zenβgal .

12. The composition of claim 10 wherein said substantially pure retrovirus is N2.

13. The composition of claim 1 wherein said substantially pure retrovirus is xenotropic.

14. The composition of claim 1 wherein said substantially pure retrovirus is amphotropic.

15. A method for expanding the host range of a retrovirus comprising the step of administering a substan- tially pure retrovirus, said retrovirus including a non- homologous gene, and a formulation which expands the host range of said retrovirus .

16. The method of claim 15 wherein said formulation which expands the host range of said retrovirus is selected from the group consisting of a substantially pure adenovirus, a substantially pure partially expressed adenovirus, an adenovirus capsid protein, a penton base protein, a lipoprotein, influenza virus hemagglutinin or a synthetic peptide.

17. The method of claim 15 wherein said substantially pure retrovirus is ecotropic.

18. The method of claim 15 wherein said substantially pure retrovirus is xenotropic.

19. The method of claim 15 wherein said substantially pure retrovirus is amphotropic.