HIGH EFFICIENCY EX VIVO TRANSDUCTION OF HEMATOPOIETIC STEM CELLS BY RECOMBINANT RETROVIRAL PREPARATIONS

Abstract: Compositions and methods for the efficient ex vivo introduction of nucleic acid into hematopoietic stem cells mediated by recombinant retrovirus particles is described. The recombinant vector constructs carried by the recombinant retrovirus particles code for the production of a desired gene product having a therapeutic application from a gene of interest. Upon re-introduction into a patient, the transduced hematopoietic stem cells produce a desired gene product in an amount sufficient to treat a particular disease state.

Description: HIGH EFFICIENCY EX VIVO TRANSDUCΗON OF HEMATOPOIETIC STEM CELLS BY RECOMBINANT RETROVΓRAL PREPARATIONS Technical Field The present invention relates generally to recombinant retroviruses and gene therapy, and more specifically, to recombinant retroviral particle preparations suitable for a variety of somatic cell gene therapy applications. Background of the Invention Since the discovery of DNA in the 1 40s and continuing through the most recent era of recombinant DNA technology, substantial research has been undertaken in order to realize the possibility that the course of disease may be affected through interaction with the nucleic acids of living organisms. Most recently, a wide variety of methods have been described for altering or affecting genes of somatic tissue (a process sometimes referred to as "somatic gene therapy"), including for example, viral vectors derived from retroviruses, adenoviruses, vaccinia viruses, herpes viruses, and adeno-associated viruses (see Jolly, C cer Gene Thercφy 7(1):51-64, 1994), as well as direct transfer techniques such as lipofection (Feigner etal, Proc. Natl. Acad. Sci. USA 84:1413-14X1, 1989), direct DNA injection (Acsadi etal, Nature 52:815-818, 1991), microprojectile bombardment (Williams etal, PNAS 88:2126- 2730, 1991), liposomes of several types (see, e.g., Wang etal., PNAS 84:1%51-7855, 1987) and administration of nucleic acids alone (WO 90/11092). Of these techniques, recombinant retroviral gene delivery methods have been most extensively utilized, in part due to: (1) the efficient entry of genetic material (the vector genome) into replicating cells; (2) an active, efficient process of entry into the target cell nucleus; (3) relatively high levels of gene expression; (4) the potential to target particular cellular subtypes through control of the vector-target cell binding and the tissue-specific control of gene expression; (5) a general lack of pre-existing host immunity; and (6) substantial knowledge and clinical experience which has been gained with such vectors. Briefly, retroviruses are diploid positive-strand RNA viruses that replicate through an integrated DNA intermediate. In particular, upon infection by the RNA virus, the retroviral genome is reverse-transcribed into DNA by a virally encoded reverse transcriptase that is carried as a protein in each retrovirus. The viral DNA is then integrated pseudo-randomly into the host cell genome ofthe infecting cell, forming a "provirus" which is inherited by daughter cells. Wild-type retroviral genomes (and their proviral copies) contain three genes (the gag, ol and env genes), which are preceded by a packaging signal (ψ), and two long terminal repeat (LTR) sequences which flank both ends. Briefly, the gag gene encodes the internal structural (nucleocapsid) proteins. The pol gene codes for the RNA-dependent DNA polymerase which reverse transcribes the RNA genome, and the env gene encodes the retroviral envelope glycoproteins. The 5' and 3' LTRs contain c/s-acting elements necessary to promote transcription and polyadenylation of retroviral RNA. Adjacent to the 5' LTR are sequences necessary for reverse transcription ofthe genome (the tRNA primer binding site) and for efficient encapsidation of retroviral RNA into particles (the psi sequence). Removal ofthe packaging signal prevents encapsidation (packaging of retroviral RNA into infectious virions) of genomic RNA, although the resulting mutant can still direct synthesis of all proteins encoded in the viral genome. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann, et al. (Cell 33:153, 1983), Cane and Mulligan (Proc. Nat'l. Acad. Sci. USA *7:6349, 1984), Ivffller, etal., Human Gene Thercφy 1:5-14, 1990, U.S. Patent Nos. 4,405,712; 4,861,719; 4,980,289 and PCT Application Nos. WO 89/02,468; WO 89/05,349 and WO 90/02,806). Briefly, a foreign gene of interest may be incorporated into the retrovirus in place ofa portion ofthe normal retroviral RNA. When the retrovirus injects its RNA into a cell, the foreign gene is also introduced into the cell, and may then be integrated into the host's cellular DNA as if it were the retrovirus itself. Expression of this foreign gene within the host results in expression ofthe foreign protein by the host cell. One disadvantage, however, of recombinant retroviruses is that they principally infect only replicating cells, thereby making efficient direct gene transfer difficult or impossible for cells characterized as largely non-replicating, such as hematopoietic stem cells. Indeed, some scientists have suggested that other, more efficient methods of gene transfer, such as direct administration of pure plasmid DNA, be utilized (Davis et al., Human Gene Therapy 4:133- 740, 1993) to introduce nucleic acid molecules into such cells. In order to increase the efficacy of recombinant retroviruses, the methods which have been suggested have principally been aimed at inducing the desired target cells to replicate, thereby allowing the retroviruses to infect the cells. Such methods have included, for example chemical treatment with 10% carbon tetrachloride in mineral oil (Kaleko, et al, Human Gene Therapy 2:21-32, 1991). However, such techniques are not preferred for use SUBSTITUTE SHEET (RULE 261 in ex vivo techniques designed to introduce nucleic acid molecules encoding therapeutic gene products into mammalian hematopoietic cells. Mammalian hematopoietic cells provide a diverse range of physiological activities. These cells are divided into lymphoid, myeloid and erythroid lineages. The lymphoid lineages, comprising B-cells, T-cells, and NK-cells, provides for the production of antibodies, regulation ofthe cellular immune system, detection of foreign agents in the Mood, detection of cells foreign to the host, and the like. The myeloid lineage, which includes monocytes, granulocytes, megakaryocytes as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like. The erythroid lineage provides the red blood cells, which act as oxygen carriers. Despite the diversity ofthe nature, morphology, characteristics and function of hematopoietic cells, these cells are derived from a single hematopoietic progenitor cell population, termed "stem cells." Stem cells are capable of self-regeneration and may become lineage restricted progenitors, which further differentiate and expand into specific lineages. As used herein, the term "stem cells" or "hematopoietic stem cells" refers to hematopoietic cells and not stem cells of other cell types. Further, unless indicated otherwise, "stem cells" refers to human hematopoietic stem cells. U.S. Patent No. 5,061,620 describes a substantially homogeneous stem cell composition and the manner of obtaining such a composition. See also the references cited therein. Stem cells constitute only a small percentage ofthe total number of hematopoietic cells. Hematopoietic cells are identifiable by the presence ofa variety of cell surface "markers." Such markers may be either specific to a particular lineage or progenitor cell or be present on more than one cell type. CD34 is a marker found on stem cells and a significant number of more differentiated progenitor cells. U.S. Patent No. 4,714,680 describes a population of cells expressing the CD34 marker. Table 1 summarizes probable phenotypes of stem cells in fetal, adult, and mobilized peripheral blood. In Table 1, myelomonocytic stands for myelomonocytic associated markers, NK stands for natural killer cells, FBM and ABM refer to fetal and adult bone marrow, respectively, and AMPB stands for adult mobilized peripheral blood. As used herein both infra, supra and in Table 1, the negative sign or, uppercase negative sign, (-) means that the level ofthe specified marker is undetectable above Ig isotype controls by FACS analysis, and includes cells with very low expression ofthe specified marker. The ability of stem cells to undergo substantial self-renewal as well as the ability to proliferate and differentiate into all ofthe hematopoietic lineages makes stem cells the target of choice for a number of gene therapy applications. Successful gene transfer into stem cells should provide long-term repopulation of an individual with the modified cells and their progeny, which will express the desired gene product. By contrast, gene transfer into more mature hematopoietic cells, such as T-cells, at best, provides only transient therapeutic benefit. Thus, there have been world-wide efforts toward finding effective methods of genetically modifying stem cells. For reviews of genetic modification of stem cells see Brenner (1993) J. Hematother. 2:7-17; Miller (1992) Nature 357:455-460; and Nienhuis (1991) Cancer 67:2700-2704. Most efforts to genetically modify stem cells have involved the use of retroviral vectors. Other methods such as liposome-mediated gene transfer or adeno-associated viral vectors have also been used. As discussed previously, retroviral vectors have been the primary vehicle due to the generally high rate of gene transfer obtained in experiments with cell lines, and the ability to obtain stable integration ofthe genetic material, which ensures that the progeny ofthe modified cell will contain the transferred genetic material. Efficient gene transfer into human stem cells has proven difficult due to a variety of factors. Currently used methods of retroviral transduction into human stem cells have a number of practical limitations. One limitation is the extremely low numbers of stem cells present in any tissue. Therefore, in transductions performed with relatively impure SUBST TUTΕ SHEET (RULE 26 Most efforts to genetically modify stem cells have involved the use of retroviral vectors. Other methods such as liposome-mediated gene transfer or adeno-associated viral vectors have also been used. As discussed previously, retroviral vectors have been the primary vehicle due to the generally high rate of gene transfer obtained in experiments with cell lines, and the ability to obtain stable integration ofthe genetic material, which ensures that the progeny ofthe modified cell will contain the transferred genetic material. Efficient gene transfer into human stem cells has proven difficult due to a variety of factors. Currently used methods of retroviral transduction into human stem cells have a number of practical limitations. One limitation is the extremely low numbers of stem cells present in any tissue. Therefore, in transductions performed with relatively impure populations of cells, the ratio of virus particles to stem cells will be quite low. This limitation is compounded by the relatively low titers generally obtained with most retroviral vectors, typically in the range of 10 5 to 10 6 infectious virions per milliliter. Also, the effect of more differentiated cells in culture on the growth or division of stem cells is not well understood. In addition, primitive stem cells typically are quiescent in culture; retroviral vectors require target cells to be cycling for stable integration ofthe retroviral DNA. Cytokines have been used to cause stem cells to cycle, which improves gene transfer efficiency, but the effect of various cytokines in driving stem cells to differentiation remains in question. The range of host cells that may be infected by a retrovirus or retroviral vector is determined by the viral envelope protein. Therefore, a lack or deficiency ofthe receptor for the given envelope protein would limit transduction efficiency. In addition, a lack ofthe requisite cellular factors involved in viral binding, penetration, uncoating ofthe retroviral vectors, viral replication or integration would limit transduction efficiency. It is the object ofthe present invention to provide ex vivo methods for using compositions of recombinant retroviral particles to deliver vector constructs encoding genes of interest to hematopoietic stem cells ex vivo. The transduced stem cells may then be re- administered to the patient by standard techniques, e.g., intravenous infusion to achieve a desired therapeutic benefit. Summary ofthe Invention The present invention provides compositions and methods for transducing hematopoetic stem cells Within one aspect ofthe invention a method is provided for the production of transduced hematopoetic stem cells comprising obtaining a population of hematopoetic stem cells from a patient and transducing the population of hematopoetic stem cells with a recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest. Within one embodiment ofthe invention wherein the vector construct encodes a molecule selected from the group consisting ofa protein, an active portion ofa protein and a RNA molecule with intrinsic biological activity. The protein or active portion ofa protein is selected from the group consisting ofa cytokine, a colony stimulating factor, a clotting factor, and a hormone. Within another embodiment ofthe invention methods are provided for treating a genetic disease, cancer, infectious disease, degenerative disease, inflammatory disease, cardiovascular disease, and autoimmune disease by administering to a patient a composition or reintroduction of a therapeutically effective amount ofthe population of transduced hematopoetic stem cells. In another embodiment the transduced population of hematopoetic stem cells are characterized as CD34 + Thy-1 + Lin". In another embodiment the recombinant reroviral vectors used to the transduce the hematopoitic stem cells are xenotropic retroviral vectors. Preferably the rerotroviral vector preparations used to transduce the hematopoietic stem cells are high titer preparations. In yet another embodiment the hematopoetic stem cells are expanded in vitro prior to re¬ introduction ofthe cells into the patient. In another aspect ofthe invention an in vivo delivery vehicle comprising transplantable hematopoetic stem cells which express a therapeutically effective amount ofa gene product encoded by a gene wherein the gene does not occur in hematopoetic stem cells or where the gene occurs in hematopoetic stem cells but is not expressed in the cells and wherein the gene can be modified to be expressed in hematopoetic stem cells is provided. In other aspects ofthe invention the transduced hematopoetic stem cells and compositions of hematopoetic stem cells encoding a gene of interest, hematopoetic stem cells transduced with a recombinant xenotropic retroviral vector, and hematopoetic stem cells characterized as CD34 + Thy-1 + Lin" transduced with recombinant retroviral particles are provided. In one embodiment of this aspect ofthe invention compositions are provided substantially free from contamination with replication competent retrovirus. Definition of Terms The following terms are used throughout the specification Unless otherwise indicated, these terms are defined as follows: "Event-specific promoter" refers to transcriptional promoter/enhancer or locus defining elements, or other elements which control gene expression as discussed above, whose transcriptional activity is altered upon response to cellular stimuli. Representative examples of such event-specific promoters include thymidine kinase or thymidylate syntheses promoters, alpha or beta interferon promoters and promoters that respond to the presence of hormones (either natural, synthetic or from other non-host organisms). "Tissue-specific promoter" refers to transcriptional promoter/enhancer or locus defining elements, or other elements which control gene expression as discussed above, which are preferentially active in a limited number of hematopoietic tissue types. Representative examples of such hematopoietic tissue-specific promoters include, but are not limited to, the IgG promoter, α- or β-globin promoters, T-cell receptor promoter, Granzyme A, Granzyme B, CD8, and CDI lb. "Transduction" involves the association ofa replication defective, recombinant retroviral particle with a cellular receptor, followed by introduction ofthe nucleic acids carried by the particle into the cell. "Transfection" refers to a method of physical gene transfer wherein no retroviral particle is employed. "Vector construct", "retroviral vector", "recombinant vector", and "recombinant retroviral vector" refer to a nucleic acid construct capable of directing the expression ofa gene of interest. The retroviral vector must include at least one transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein. Such vector constructs must also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector). Optionally, the vector construct may also include a signal which directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way example, such vectors will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof In order to express a desired gene product from such a vector, a gene of interest encoding the desired gene product is also included. Numerous aspects and advantages ofthe invention will be apparent to those skilled in the art upon consideration ofthe following detailed description which provides illumination of the practice of the invention SUBSTITUTE SHEET (RULE 2S) Detailed Description ofthe Invention The present invention is based on the unexpected discovery that recombinant retroviral particles carrying a vector construct comprising a gene of interest can be used ex vivo to efficiently transduce hematopoietic stem cells. As a result, recombinant retroviral particles according to the invention can be used for purposes of somatic gene therapy. A more thorough description of such recombinant retroviral particles, their production and packaging, and uses therefore is provided below. Generation of Recombinant Retroviral Vectors As noted above, the present invention provides compositions and methods comprising recombinant retroviral particles, including recombinant xenotropic retroviral vector particles, for use in ex vivo somatic gene therapy. The construction of recombinant retroviral vectors and particles is described in greater detail in an application entitled "Recombinant Retroviruses". Production of transduction competent recombinant xenotropic retroviral particles is described in U.S.S.N. 08/156,789, and U.S.S.N. 07/965,084, which are hereby incorporated by reference in their entirety. In general, the recombinant vector constructs described herein are prepared by selecting a plasmid with a strong promoter, and appropriate restriction sites for insertion of DNA sequences of interest downstream from the promoter. According to the invention, the recombinant vector construct is carried by a recombinant retrovirus. Retroviruses are RNA viruses with a single positive strand genome which in general, are nonlytic. Upon infection, the retrovirus reverse transcribes its RNA into DNA, forming a provirus which is inserted into the host cell genome. The retroviral genome can be divided conceptually into two parts The "trans-acting" portion consists of the region coding for viral structural proteins, including the group specific antigen (gag) gene for synthesis ofthe core coat proteins; the pol gene for the synthesis ofthe reverse transcriptase and integrase enzymes; and the envelope (env) gene for the synthesis of envelope glycoproteins The "cis-acting" portion consists of regions ofthe genome that is finally packaged into the viral particle These regions include the packaging signal, long terminal repeats (LTR) with promoters and polyadenylation sites, and two start sites for DNA replication The internal or "trans-acting" part ofthe cloned provirus is replaced by the gene of interest to create a "vector construct" When the vector construct is placed into a cell where viral packaging proteins are present, the transcribed RNA will be packaged as a viral particle which , in turn, will bud off from the cell. These particles are used to transduce tissue cells, allowing the vector construct to integrate into the cell genome. Although the vector construct expresses its gene product, the virus carrying it is replication defective because the trans-acting portion ofthe viral genome is absent. Various assays may be utilized m order to detect the presence of any replication competent iiifcuiiuus retrovirus. One preferred assay is the extended S + L" assay described in Example 4. In the broadest terms, the retroviral vectors ofthe invention comprise a transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post- translational modification of messenger, or post-transcriptional modification of protein. Such vector constructs must also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector). Optionally, the vector construct may also include a signal which directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way example, such vectors will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof. Such vectors do not contain one or more ofa complete gag, pol, or env gene, thereby rendering them replication incompetent. In addition, nucleic acid molecules coding for a selectable marker are neither required nor preferred. Preferred retroviral vectors contain a portion ofthe gag coding sequence, preferably that portion which comprises a splice donor and splice acceptor site, the splice acceptor site being positioned such that it is located adjacent to and upstream from the gene of interest. In a particularly preferred embodiment, the gag transcriptional promoter is positioned such that an RNA transcript initiated therefrom contains the 5' gag LTR and the gene of interest. As an alternative to the gag promoter to control expression ofthe gene of interest, other suitable promoters, some of which are described below, may be employed. In addition, alternate enhancers may be employed in order to increase the level of expression ofthe gene of interest. In preferred embodiments ofthe invention, retroviral vectors are employed, particularly those based on Moloney murine leukemia virus (MoMLV) MoMLV is a murine retrovirus which has poor infectivity outside of mouse cells. The related amphotropic N2 retrovirus will infect cells from human, mouse and other organisms Other preferred retroviruses which may be used in the practice ofthe present invention include gibbon ape leukemia virus (GALV) (Todaro, et al, Virology, 67:335, 1975; Wilson, et al, J. Vir., 63:2374, 1989), feline immunodeficiency virus (FIV) (Talbatt, et al, Proc. Nat'l. Acad. Sci. USA, 86:5143, 1984), and feline leukemia virus (FeLV) (Leprevette, et al, J. Vir., 50:884, 1984; Elder, et al. . Vir., 46:871, 1983; Steward, et al., 1 Vir., 55:825, 1986; Riedel, et al, J. Vir., 60:242, 1986), although retroviral vectors according to the invention derived from other type C retroviruses (Weiss, RNA Tumor Viruses, vols. I and II, Cold Spring Harbor Laboratory Press, N.Y.) can also be generated. A variety of promoters can be used in the vector constructs ofthe invention, including but not necessarily limited to the cytomegalovirus major immediate early promoter (CMV MIE), the early and late SV40 promoters, the adenovirus major late promoter, thymidine kinase or thymidylate synthase promoters, alpha or beta interferon promoters, event or tissue specific promoters, etc. Promoters may be chosen so as to potently drive high levels of expression or to produce relatively weak expression, as desired. As those in the art will appreciate, numerous RNA polymerase II and RNA polymerase III dependent promoters can be utilized in practicing the invention. In one embodiment, recombinant retroviral vectors comprising a gene of interest are under the transcriptional control of an event-specific promoter, such that upon activation of the event-specific promoter the gene is expressed. Numerous event-specific promoters may be utilized within the context ofthe present invention, including for example, promoters which are activated by cellular proliferation (or are otherwise cell-cycle dependent) such as the thymidine kinase or thymidylate synthase promoters (Merrill, Proc. Nat I. Acad. Sci. USA, 56:4987, 1989; Deng, et al, Mol. Cell. Biol, 9:4079, 1989); or the transferrin receptor promoter, which will be transcriptionally active primarily in rapidly proliferating cells (such as hematopoietic cells) which contain factors capable of activating transcription from these promoters preferentially to express gene products from gene of interest; promoters such as the alpha or beta interferon promoters which are activated when a cell is infected by a virus (Fan and Maniatis, EMBOJ., 5:101, 1989; Goodbourn, et al, Cell, 45:601, 1986); and promoters which are activated by the presence of hormones, e.g., estrogen response promoters. See Toohey et al, Mol. Cell. Biol, 6:4526, 1986; and promoters that are activated in response to cellualr stress or insult, e.g., electrophilic response elements (Friling, et al, PNAS, 57:6258, 1990) In another embodiment, recombinant retroviral vectors are provided which comprise a gene of interest under the transcriptional control of a tissue-specific promoter, such that upon activation ofthe tissue-specific promoter the gene is expressed A wide variety of tissue-specific promoters may be utilized within the context ofthe present invention SUBSTTTUTE SHEET (RULE 26) Representative examples of such promoters include: B-cell specific promoters such as the IgG promoter; T-cell specific promoters such as the T-cell receptor promoter (Anderson, et al, Proc. Natl. Acad Sci. USA, 55:3551, 1988; Winoto and Baltimore, EMBOJ., 8:29, 1989); bone-specific promoters such as the osteocalcin promoter (Markose, et al, Proc. Natl Acad. Sci. USA, 57:1701, 1990; McDonnell, et al, Mol Cell Biol, 9:3517, 1989; Kemer, etal, Proc. Natl. Acad. Sci. USA, 56:4455, 1989), the E -2 promoter, EL-2 receptor promoter, and the MHC Class TJ promoter, and hematopoietic tissue specific promoters, for instance erythoid specific-transcription promoters which are active in erythroid cells, such as the porphobilinogen deaminase promoter (Mignotte, etal, Proc. Natl Acad. Sci. USA, 56:6458, 1990), α- or β-globin specific promoters (van Assendelft, et al, Cell, 56:969, 1989, Forrester, et al, Proc. Natl. Acad. Sci. USA, 56:5439, 1989), endothelial cell specific promoters such as the vWf promoter, megakaryocyte specific promoters such as β- thromboglobulin, and many other tissue-specific promoters. Examples of promoters that may be used to cause expression ofthe introduced sequence in specific cell types include Granzyme A and Granzyme B for expression in T-cells and NK cells, the CD34 promoter for expression in stem and progenitor cells, the CD8 promoter for expression in cytotoxic T- cells, and the CDI lb promoter for expression in myeloid cells. Retroviral vectors according to the invention may also contain a non-LTR enhancer or promoter, e.g., a CMV or SV40 enhancer operably associated with other elements employed to regulate expression ofthe gene of interest. Additionally, retroviral vectors from which the 3' LTR enhancer has been deleted, thereby inactivating the 5' LTR upon integration into a host cell genome, are also contemplated by the invention. A variety of other elements which control gene expression may also be utilized within the context ofthe present invention, including, for example, locus-defining elements such as those from the β-globin gene and CD2, a T-cell marker. In addition, elements which control expression at the level of splicing, nuclear export, and or translation may also be included in the retroviral vectors. Representative examples include the β-globin intron sequences, the rev and rre elements from HTV-l, the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), a 219 nucleotide sequence that allows rev-independent replication of rev- negative HIV proviral clones, and a Kozak sequence. Rev protein functions to allow nuclear export of unspliced and singly spliced HIV RNA molecules. The MPMV element allows nuclear export of intron-containing mRNA. The CTE element maps to MPMV nucleotides 8,022-8,240 (Bray, et al. Biochemistry, 97:1256, 1994). In another preferred embodiment, the retroviral vector contains a splice donor (SD) site and a splice acceptor (SA) site, wherein the SA is located upstream ofthe site where the gene of interest is inserted into the recombinant retroviral vector. In a preferred embodiment, the SD and SA sites will be separated by a short, i.e., less than 400 nucleotide, intron sequence. Such sequences may serve to stabilize RNA transcripts. Such stabilizing sequences typically comprise a SD-intron-SA configuration located 5' to the gene of interest. The recombinant retroviral vectors ofthe invention will also preferably contain transcriptional promoters derived from the gag region σperabh/ positioned such that a resultant transcript comprising the gene of interest further comprises a 5' gag LTR (untranslated region) upstream ofthe gene of interest. The present invention also provides for multivalent vector constructs, the construction of which may require two promoters when two proteins are being expressed, because one promoter may not ensure adequate levels of gene expression ofthe second gene. In particular, where the vector construct expresses an antisense message or ribozyme, a second promoter may not be necessary. Within certain embodiments, an internal ribosome binding site (IRBS) or herpes simplex virus thymidine kinase (HSVTK) promoter is placed in conjunction with the second gene of interest in order to boost the levels of gene expression ofthe second gene. Briefly, with respect to IRBS, the upstream untranslated region ofthe immunoglobulin heavy chain binding protein has been shown to support the internal engagement of a bicistronic message (Jacejak, et al, Nature 353:90, 1991). This sequence is small, approximately 300 base pairs, and may readily be incorporated into a vector in order to express multiple genes from a multi-cistronic message whose cistrons begin with this sequence. Retroviral vector constructs according to the invention will often be encoded on a plasmid, a nucleic acid molecule capable of propagation, segregation, and extra- chromosomal maintenance upon introduction into a host cell. As those in the art will understand, any ofa wide range of existing or new plasmids can be used in the practice of the invention. Such plasmids contain an origin of replication and typically are modified to contain a one or more multiple cloning sites to facilitate recombinant use. Preferably, plasmids used in accordance with the present invention will be capable of propagation in both eukaryotic and prokaryotic host cells. Generation of Packaging Cells Another aspect ofthe invention relates to methods of producing recombinant retroviral particles incorporating the retroviral vectors described herein. In one embodiment, vectors are packaged into infectious virions through the use ofa packaging cell. Briefly, a packaging cell is a cell comprising, in addition tσ its natural genetic cuuipleiπenr, aϋϋithjnal nucleic acids coding for those retroviral structural polypeptides required to package a retroviral genome, be it recombinant (i.e., a retroviral vector) or otherwise. The retroviral particles are made in packaging cells by combining the retroviral genome with a capsid and envelope to make a transduction competent, preferably replication defective, virion. Briefly, these and other packaging cells will contain one, and preferably two or more nucleic acid molecules coding for the various polypeptides, e.g., gag, pol, and ewv, required to package a retroviral vector into an infectious virion. Upon introduction ofa nucleic acid molecule coding for the retroviral vector, the packaging cells will produce infectious retroviral particles. Packaging cell lines transfected with a retroviral vector according to the invention which produce infectious virions are referred to as "producer" cell lines. A wide variety of animal cells may be utilized to prepare the packaging cells ofthe present invention, including without limitation, epithelial cells, fibroblasts, hepatocytes, endothelial cells, myoblasts, astrocytes, lymphocytes, etc.. Preferentially, cell lines are selected that lack genomic sequences which are homologous to the retroviral vector construct, gag/pol expression cassette and ewv expression cassette to be utilized. Methods for determining homology may be readily accomplished by, for example, hybridization analysis (Martin et al, Proc. Natl. Acad. Sci., USA, 75:4892, 1981; and U.S.S.N. 07/800,921, supra). The most common packaging cell lines (PCLs) used for MoMLV vector systems (psi2, PA12, PA317) are derived from murine cell lines. However, murine cell lines are typically not the preferred choice to produce retroviral vectors intended for human therapeutic use because such cell lines are known to: contain endogenous retroviruses, some of which are closely related in sequence and retroviral type to the MLV vector system preferred for use in practicing the present invention, contain non-retroviral or defective retroviral sequences that are known to package efficiently; and cause deleterious effects due to the presence of murine cell membrane components. An important consideration in developing packaging cell lines useful in the invention is the production therefrom of replication incompetent virions, or avoidance of generating replication-competent retrovirus (RCR) (Munchau, et al. Virology. J 76.262, 1991 ) This will ensure that infectious retroviral particles harboring the recombinant retroviral vectors of the invention will be incapable of independent repUcation in target cells, be they in vitro or in vivo. Independent replication, should it occur, may lead to the production of wild-type virus, which in turn could lead to multiple integrations into the chromosome(s) of a patient's cells, thereby increasing the possibility of insertional mutagenesis and its associated problems. R€R production can occur in at least two ways: (1) through homologous recombinatiσrr between the therapeutic proviral DNA and the DNA encoding the retroviral structural genes ("gag/po and "ewv") present in the packaging cell line; and (2) generation of replication- competent virus by homologous recombination ofthe proviral DNA with the very large number of defective endogenous proviruses found in murine packaging cell lines. To circumvent inherent safety problems associated with the use of murine based recombinant retroviruses, as are preferred in the practice of this invention, packaging cell lines may be derived from various non-murine cell lines. These include cell lines from various mammals, including humans, dogs, monkeys, mink, hamsters, and rats. As those in the art will appreciate, a multitude of packaging cell lines can be generated using techniques known in the art (for instance, .see U.S.S.N. 08/156,789 and U.S.S.N. 08/136,739). In preferred embodiments, cell lines are derived from canine or human cell lines, which are known to lack genomic sequences homologous to that of MoMLV by hybridization analysis (Martin et al, supra). A particularly preferred parent dog cell line is D17 (ATCC. CRL 8543). HT-1080 (ATCC. CCL 121; Graham et al, Vir., 52:456, 1973) and 293 cells (Feigner et al, Proc. Nat'l. Acad. Sci. USA 84:1413, 1987) represent particularly preferred parental human cell lines. Construction of packaging cell lines from these cell lines for use in conjunction with a MoMLV based recombinant retroviral vector is described in detail in U.S.S.N 08/156,789, supra. Thus, a desirable prerequisite for the use of retroviruses in gene therapy is the availability of retroviral packaging cell lines incapable of producing replication competent, or "wild-type," virus. As packaging cell lines contain one or more nucleic acid molecules coding for the structural proteins required to assemble the retroviral vector into infectious retroviral particles, recombination events between these various constructs might produce replication competent virus, i.e., infectious retroviral particles containing a genome encoding all ofthe structural genes and regulatory elements, including a packaging signal, required for independent replication. In the past several years, many different constructions have been developed in an attempt to obviate this concern. Such constructions include: deletions in the 3' LTR and portions ofthe 5' LTR (see, Miller and Butti ore, Mol Cell Biol, 6:2895, 1 86). where two recombination events are necessary to form RCR; use of complementary portions of helper virus, divided among two separate plasmids, one containing gag znάpol, and the other containing ewv (see, Markowitz et al, J. Virol, 62: 1120; and Markowitz et al, Virology, 167: 600, 1988), where three recombination events are required to generate RCR The ability to express gag/pol and env function separately allows for manipulation of these functions independently. A cell line that expresses ample amounts of gag/pol can be used, for example, to address questions of titer with regarτr tσ errr Ong'ii tWi'iesultiiig ΠT measured low titers is the density of appropriate receptor molecules on the target cell or tissue. A second factor is the affinity ofthe receptor for the retroviral envelope protein. One report suggests that xenotropic vector, in the presence of replication-complement virus, may more effectively infect human hematopoietic progenitor cells (Eglitis, et al. , Biochem. Biophys. Res. Comm. 757:201, 1988). vector-containing particles, in the presence of replication-competent xenotropic virus, also infect cells from other species which are not easily infectable by amphotropic virus such as bovine, porcine, and equine cells (Delouis, et al, Biochem. Biophys Res. Comm. 769:80, 1990). In a preferred embodiment ofthe invention, packaging cell lines which express a xenotropic ewv gene are provided. Significantly, recombinant xenotropic retroviral particles produced from such packaging cell lines are substantially free from association with replication competent retrovirus ("RCR"). More recently, further improved methods and compositions for inhibiting the production of replication incompetent retrovirus have been developed. See co-owned U.S.S.N. 09/028,126, filed September 7, 1994. Briefly, the spread of replication competent retrovirus generated through recombination events between the recombinant retroviral vector and one or more ofthe nucleic acid constructs coding for the retroviral structural proteins may be prevented by providing vectors which encode a non-biologically active inhibitory molecule, but which produce a nucleic acid molecule encoding a biologically active inhibitory molecule in the event of such recombination. The expression ofthe inhibitory molecule prevents production of RCR either by killing the producer cell(s) in which that event occurred or by suppressing production ofthe retroviral vectors therein. A variety of inhibitory molecules may be used, including ribozymes, which cleave the RNA transcript of the replication competent virus, or a toxin such as ricin A, tetanus, or diphtheria toxin, herpes thymidine kinase, etc. As those in the art will appreciate, the teachings therein may be readily adapted to the present invention. In addition to issues of safety, the choice of host cell line for the packaging cell line is of importance because many ofthe biological properties (such as titer) and physical properties (such as stability) of retroviral particles are dictated by the properties ofthe host cell For instance, the host cell must efficiently express (transcribe) the vector RNA genome. prime the vector for first strand synthesis with a cellular tRNA, tolerate and covalently modify the MLV structural proteins (proteolysis, glycosylation, myristylation, and phosphorylation), and enable virion budding from the cell membrane. For example, it has been found that vector made from the mouse packaging line PA317 is retained by a 0.3 micron filter, while that made from a CA line will pass through. Furthermore, sera from primates, including humans, but not that from a wide variety of lower mammals or birds, is known to inactivate retroviruses by an antibody independent complement lysis method. Such activity is non-selective for a variety of distantly related retroviruses. Retroviruses of avian, murine (including MoMLV), feline, and simian origin are inactivated and lysed by normal human serum. See Welsh, et al, Nature, 257:612, 1975; Welsh, et al, Virology, 74:432, 1976; Banapour, et al, Virology, 752:268, 1986; and Cooper, et al, (1986) Immunology of the Complement System, Pub. American Press, Inc., pp: 139-162. In addition, replication competent murine amphotropic retroviruses injected intravenously into primates in vivo are cleared within 15 minutes by a process mediated in whole or in part by primate complement (Cometta, et al, Human Gene Therapy, 7:15, 1990; Cometta, et al, Human Gene Therapy, 2:5, 1991). However, it has recently been discovered that retroviral resistance to complement inactivation by human serum is mediated, at least in some instances, by the packaging cell line from which the retroviral particles were produced. Retroviruses produced from various human packaging cell lines were resistant to inactivation by a component of human serum, presumably complement, but were sensitive to serum from baboons and macaques. See commonly owned U.S.S.N. 08/367,071, filed on December 30, 1994. Thus, in a preferred embodiment ofthe invention, recombinant retroviral particles are produced in human packaging cell lines, with packaging cell lines derived from HT1080 or 293 cells being particularly preferred. In addition to generating infectious, replication defective recombinant retroviruses as described above, at least two other alternative systems can be used to produce recombinant retroviruses carrying the vector construct. One such system (Webb, et al, BBRC, 190:536, 1 93) employs the insect virus, baculovirus, while the other takes advantage ofthe mammalian viruses vaccinia and adenovirus (Pavirani, et al, BBRC, 145:234, 1987) Each of these systems can make large amounts of any given protein for which the gene has been cloned. For example, see Smith, et al. (Mol. Cell. Biol, 3:12, 1983); Piccini, et al. (Meth. En∑ymology, 153.545, 1987); and Mansour et al. (Proc. Natl. Acad. Sci. USA, 52.1359, 1985) These retroviral vectors can be used to produce proteins in tissue culture cells by insertion of appropriate genes and, hence, could be adapted to make retroviral vector particles from tissue culture In an adenovirus system, genes can be inserted into vectors and used to express proteins in mammalian cells either by in vitro construction (Ballay, et al, 4:3861, 1985) or by recombination in cells (Thummel, et al, J. Mol. Appl. Genetics, 7:435, 1982). An alternative approach involves cell-free packaging systems. For instance, retroviral structural proteins can be made in a baculovirus system (or other protein production systems, such as yeast or E. coli) in a similar manner as described in Smith et al. (supra). Recombinant retroviral genomes are made by in vitro RNA synthesis (see, for example, Flamant and Sorge, J. Virol, 62: 1827, 1988). The structural proteins and RNA genomes are then mixed with tRNA, followed by the addition of liposomes with embedded env protein and cell extracts (typically from mouse cells) or purified components (which provide env and other necessary processing, and any or other necessary cell-derived functions). The mixture is then treated (e.g., by sonication, temperature manipulation, or rotary dialysis) to allow encapsidation of nascent retroviral particles. This procedure allows production of high titer, replication incompetent recombinant retroviruses without contamination with pathogenic retroviruses or replication-competent retroviruses. Another important factor to consider in the selection ofa packaging cell line is the viral titer produced therefrom following introduction ofa nucleic acid molecule from which the retroviral vector is produced. Many factors can limit viral titer. One ofthe most significant limiting factors is the expression level ofthe packaging proteins gag, pol, and env In the case of retroviral particles, expression of retroviral vector RNA from the provirus can also significantly limit titer. In order to select packaging cells and the resultant producer cells expressing high levels ofthe required products, an appropriate titering assay is required As described in greater detail below, a suitable PCR-based titering assay can be utilized In addition to preparing packaging and producer cell lines which supply proteins for packaging that are homologous for the backbone ofthe viral vector, e.g., retroviral gag, pol, and env proteins for packaging ofa retroviral vector, packaging and producer systems which result in chimeric viral particles, for instance a MoMLV-based retroviral vector packaged in a DNA virus capsid, may also be employed. Many other packaging and producer systems based on viruses unrelated to that ofthe viral vector can also be utilized, as those in the art will appreciate. Altering the Host Range of Recombinant Retroviral Particles Another aspect ofthe invention concerns recombinant xenotropic retroviral particles which have an altered host range as compared to retroviral particles containing amphotropic envelope proteins. The host cell range specificity ofa retrovirus is determined in part by the ewv gene products present in the lipid envelope. Interestingly, envelope proteins from one retrovirus can often substitute, to varying degrees, for that of another retrovirus, thereby altering host range ofthe resultant vector. Thus, packaging cell lines (PCLs) have been generated to express either amphotropic, ecotropic, xenotropic, polytropic, or other errveiope tropisms. Additionally, retroviruses according to the invention which contain "hybrid" or "chimeric" xenotropic envelope proteins can be similarly generated Retroviral particles produced from any of these packaging cell lines can be used to infect any cell which contains the corresponding distinct receptor (Rein and Schultz, Virology, 736:144, 1984). The assembly of retroviruses is characterized by selective inclusion ofthe retroviral genome and accessory proteins into a budding retroviral particle. Interestingly, envelope proteins from non-murine retrovirus sources can be used for pseudotyping (i.e., the encapsidation of viral RNA from one species by viral proteins of another species) a vector to alter its host range. Because a piece of cell membrane buds off to form the retroviral envelope, molecules normally in the membrane may be carried along on the viral envelope. Thus, a number of different potential ligands can be put on the surface of retroviral particles by manipulating the packaging cell line in which the vectors are produced or by choosing various types of cell lines with particular surface markers. Briefly, in this aspect the present invention provides for enveloped retroviral particles comprising: a nucleocapsid including nucleocapsid protein having an origin from a first vims, which is a retrovirus; a packagable nucleic acid molecule encoding a gene of interest associated with the nucleocapsid; and a membrane-associated xenotropic protein which determines a host range. In another preferred form ofthe present invention, the membrane-associated xenotropic protein ofthe vector particles is a chimeric or hybrid protein including an exterior receptor binding domain and a membrane-associated domain from a xenotropic envelope protein, at least a portion ofthe exterior receptor binding domain being derived from a different origin than at least a portion ofthe membrane-associated domain. The chimeric protein is preferably derived from two origins, wherein no more than one ofthe two origins is retroviral. Another embodiment of this aspect ofthe present invention concerns cell lines that produce the foregoing vector particles. Preferably, such cell lines are stably transfected with a nucleic acid molecule encoding the membrane-associated protein, whose expression is driven by an inducible promoter. Retroviral particles according to the invention may be targeted to a specific cell type by including in the retroviral particles a component, most frequently a polypeptide or carbohydrate, which binds to a cell surface receptor specific for that cell type. Such targeting may be accomplished by preparing a packaging cell line which expresses a chimeric env protein comprising a portion ofthe env protein required for viral particle assembly in conjunction with a cell-specific binding domain. In another embodiment, env proteins from more than one viral type may be employed, such that resultant viral particles contain more than one species of env proteins. (See WO 91/02805 entitled "Recombinant Retroviruses Delivering Vector Constmcts to Target Cells" and WO 95/31566 entitled "Compositions and Methods for Targeting Gene Delivery Vehicles", both of which are hereby incorporated by reference.) Yet another embodiment involves inclusion ofa cell specific ligand in the retroviral capsid or xenotropic envelope to provide target specificity. In a preferred embodiment at this aspect ofthe invention, the xenotropic env gene employed encodes all or a portion ofthe xenotropic env protein required for retroviral assembly in conjunction with a receptor binding domain ofa polypeptide ligand known to interact with a cell surface receptor whose tissue distribution is limited to the cell type(s) to be targeted, e.g., a hematopoietic stem cell. In this regard, it may be preferable to utilize a receptor binding domain which binds receptors expressed at high levels on the target cell's surface. Non-viral membrane-associated proteins may also be used to enhance targeting of recombinant retroviral particles to hematopoietic stem cells. Representative examples include polypeptides which act as ligands for hematopoietic stem cell surface receptors. Depending on the tissue distribution ofthe receptor for the protein in question, the recombinant retroviral particle could be targeted to a different subset of hematopoietic stem cells. When a ligand to be included within the xenotropic envelope is not a naturally occurring membrane-associated protein, it is necessary to associate the ligand with the membrane, preferably by making a "hybrid" or "chimeric" envelope protein. It is important to understand that such hybrid envelope proteins can contain extracellular domains from proteins other than other viral or retroviral env proteins. To accomplish this, the gene coding for the ligand can be functionally combined with sequences coding for a membrane- associated domain ofthe env protein. By "naturally occurring membrane associated protein", it is meant those proteins that in their native state exist in vivo in association with lipid membrane such as that found associated with a cell membrane or on a viral envelope As such, hybrid envelopes can be used to tailor the tropism (and effectively increase titers) of a retroviral vector according to the invention, as the extracellular component of env proteins SUBSTTTUTE SHEET (RULE 26) is responsible for specific receptor binding. The cytoplasmic domain of these proteins, on the other hand, play a role in virion formation. The present invention recognizes that numerous hybrid env gene products (i.e., specifically, retroviral env proteins having cytoplasmic regions and extracellular binding regions which do not naturally occur together) can be generated and may alter host range specificity. In a preferred embodiment, this is accompϋshetr by reuombuung the gene coding for the ligand (or part thereof conferring receptor binding activity) proximate ofthe membrane- binding domain ofthe envelope proteins that stably assemble with a given capsid protein. The resulting construct will code for a bifunctional chimeric protein capable of enhanced cell targeting and inclusion in a retroviral lipid envelope. Vector particles having non-native membrane-associated ligands as described herein, will, advantageously, have a host range determined by the ligand-receptor interaction ofthe membrane-associated protein. Thus, for targeted delivery to hematopoietic stem cells, a vector particle having altered host range can be produced using the methods ofthe present invention. The ligand will be selected to provide a host range including hematopoietic stem cells. Many different targeting strategies can be employed in connection with this aspect of the invention. For example, there are a number of progenitor cell types found in bone marrow that differentiate into blood cells. Many blood cells have relatively short life spans and therefore progenitor cells must continually divide and differentiate to replace the lost cells. In a preferred embodiment, gene therapy targets hematopoietic progenitor cells, including pluripotent stem cells. These progenitor cells are known to have unique cellular determinants that permit histological identification, separation from other cell types by various techniques, including fluorescence activated cell sorting (FACS) and positive and negative selection [see U.S. Patent No. 5,061,620], and which can be used as cell receptors for the membrane-associated proteins ofthe vector particles ofthe present invention. As used herein, a hematopoietic stem cell is a primitive, or immature, cell capable of self-renewal and which is capable of differentiating into precursor cells of all hematopoietic lineages, i.e., they are said to be "pluripotent." Recombinant vectors according to the invention may be introduced into such cells or any their more differentiated progeny, such as the various primitive progenitors and the more lineage committed precursor cells that give rise to the various hematopoietic cell lineages. One marker for such early hematopoietic cells is CD34, which can be identified using monoclonal antibodies. See U S patent 4,714,680, WO 93/25216, published December 23, 1993 WO 93/25216 describes a class of hematopoietic stem cells as having the phenotype CD34 " 7CD387HLA-DR' and lacking the lineage committed antigens CD33, CD10, CD5, and CD71 Representative examples of anti- SUBSTTTUTE SHEET (RULE 26) CD34 antibodies include 12.8 (Andrews, et al, Blood, 67:842, 1986) and MylO (Civin, et al, J. Immunol, 733:157, 1984, commercially available from Becton Dickinson under the designation HPCA-2). Other antibodies may be also utilized to target a selected cell type, such as anti-CD4 antibodies to target CD4+ T-cells and anti-CD8 antibodies to target CD8 + cells (see generally, Wilchek, et al. , Anal. Biochem. , 171:1, 1988). The vectors may be constmcted to target these cell types for gene delivery by including an expressible gene which encodes a membrane-associated protein that binds to a unique cellular determinant of such hematopoietic progenitor cell types. Examples of such progenitor cell types which could be targeted using recombinant retroviral particles ofthe present invention include pluripotent stem cells, erythroblasts, lymphoblasts, myeloblasts and megakaryocytes. Those in the art will also recognize that it is also possible to add ligand molecules exogenously to the retroviral particles which are either incorporated into the lipid envelope or which can be linked chemically to the lipid or protein constituents thereof. Targeting a retroviral vector carrying a gene of interest to a predetermined locus on a chromosome may also be employed. Clear advantages of such targeting include avoidance of insertional mutagenesis and assuring integration at sites known to be transcriptionally active. Techniques for targeting proviral integration to specific sites include integrase modification. See U.S.S.N. 08/156,789, supra. It is further envisioned that the therapy ofthe present invention be performed in vitro. For in vitro therapy (also referred to as "ex vivo therapy"), cells are removed and transduced in vitro. For recombinant particles having membrane-associated proteins to enhance hematopoietic stem cell targeting, the need to purify the cells to be targeted in vitro is optional because the vector would specifically transduce only the targeted cells. Thus, bone marrow samples could be removed from a subject and the desired cell type transduced without the need to perform one or more cell sorting procedures. The transduced cells could then be returned to the same patient or one who is HLA matched. In addition a wide variety of high affinity binding pairs can be used as targeting elements. Representative examples of include biotin/avidin with an affinity (Kp) of 10" 15 M (Richards, Meth. Enz., 184.3, 1990; Green, Adv. in Protein Chem., 29:85, 1985) and cystatin/papain with an affinity of 10" 14 M (Bjork, et al, Biochemistry , 29.1770, 1990). A wide variety of other high affinity binding pairs may also be developed, for example, by preparing and selecting antibodies which recognize a selected hematopoietic stem cell antigen with high affinity (see generally, U.S. Patent Nos RE 32,01 1 , 4,902,614, 4.543.439, and 4,41 1,993; see also Monoclonal Antibodies, Hvbridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol, eds., 1980, and Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratory Press, 1988). The binding pair for such antibodies, typically other antibodies or antibody fragments, may be produced by recombinant techniques (see Huse, et al, Science, 246:1275, 1989; see also Sastry, et al, Proc. Natl. Acad Sci. USA , 56:5728, 1989; and Michelle Akmg-Mees, et al. , Strategies in Molecular Biology , 3:1, 1990). As will be evident to one of ordinary skill in the art given the disclosure provided herein, either member (or molecule) ofthe affinity binding pair may be coupled to the retroviral particle. Nevertheless, within preferred embodiments ofthe invention, the larger of the two affinity binding pairs (e.g., avidin ofthe avidin/biotin pair) is coupled to the retroviral particle. As utilized within the context of targeting, the term "coupled" may refer to either noncovalent or covalent interactions, although generally covalent bonds are preferred. Numerous coupling methods may be utilized, including, for example, use of crosslinking agents such as N-succinimidyl-3-(2-pyridyl dithio) propionate ("SPDP"; Carlson, et al, J. Biochem., 773:723, 1978) and other such compounds known in the art. In particularly preferred embodiments ofthe invention, a member ofthe high affinity binding pair is either expressed on, or included as an integral part of, a retroviral particle, e.g., in the retroviral lipid envelope. For example, a member ofthe high affinity binding pair may be co-expressed with the envelope protein as a hybrid protein or expressed from an appropriate vector which targets the member ofthe high affinity binding pair to the cell membrane in the proper orientation. Uses of Recombinant Retroviral Particles The efficient use of recombinant retroviral particles to infect cells is dependent on the tropism ofthe envelope protein expressed on the surface ofthe retroviral particle . The use of a envelope enables infection of cells from different species. In one aspect, the present invention provides methods for inhibiting the growth of a selected tumor ("cancer") in a human, comprising the step of transducing hematopoietic stem cells ex vivo with a vector constmct which directs the expression of at least one anti-tumor agent Within the context ofthe present invention, "inhibiting the growth ofa selected tumor" refers to either (1) the direct inhibition of tumor cell division or metatstsis, or (2) immune cell mediated tumor cell lysis, or both, which leads to a suppression in the net expansion of tumor cells Inhibition of tumor growth by either of these two mechanisms may be readily determined by one of ordinary skill in the art based upon a number of well known methods, for example, by measuring the tumor size over time, such as by radiologic imaging methods (e.g., single photon and positron emission computerized tomography; see generally, "Nuclear Medicine in Clinical Oncology," Winkler, C. (ed.) Springer- Verlag, New York, 1986) or by a variety of imaging agents, including, for example, conventional imaging agents (e.g., Gallium-67 citrate) or specialized reagents for metabolite imaging, receptor imaging, or immunologic imaging. In addition, non-radioactive methods such as ultrasound (see, "Ultrasonic Differential Diagnosis of Tumors", Kossoff and Fukuda, (eds ), Igaku-Shoin, New York, 1984), may also be utilized to estimate tumor size. Alternatively, for other forms of cancer, inhibition of tumor growth may be determined based upon a change in the presence of a tumor marker, e.g. , prostate specific antigen ("PSA") for the detection of prostate cancer (see U.S. Patent No. Re. 33,405), and carcino-embryonic antigen ("CEA") for the detection of colorectal and certain breast cancers. For yet other types of cancers such as leukemia, inhibition of tumor growth may be determined based upon decreased numbers of leukemic cells in a representative blood cell count. Within the context ofthe present invention, "anti-tumor agent" refers to a compound or molecule which inhibits tumor growth. Representative examples of anti-tumor agents include immune activators and tumor proliferation inhibitors. Briefly, immune activators function by improving immune recognition of tumor-specific antigens such that the immune system becomes "primed." Priming may consist of lymphocyte proliferation, differentiation, or evolution to higher affinity interactions. The immune system thus primed will more effectively inhibit or kill tumor cells. Immune activation may be subcategorized into immune modulators (molecules which affect the interaction between lymphocyte and tumor cell) and lymphokines, that act to proliferate, activate, or differentiate immune effector cells. Representative examples of immune modulators include CD3, ICAM-1, ICAM-2, LFA-1, LFA-3, β-2-microglobulin, chaperones, alpha interferon and gamma interferon, B7/BB1 and major histocompatibility complex (MHC). Representative examples of lymphokines include gamma interferon, tumor necrosis factor, IL-1, IL-2, IL-3, IL-4, DL-5, IL-6, IL-7, TL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1, and G-CSF. In addition, RNA molecules having intrinsic biological activity may be utilized as anti-tumor agents. Sequences which encode anti-tumor agents may be obtained from a variety of sources. For example, plasmids that contain sequences which encode anti-tumor agents may be obtained from a depository such as the American Type Culture Collection (ATCC, Rockville, MD), or from commercial sources such as British Bio-Technology Limited (Cowley, Oxford, England) Alternatively, known cDNA sequences which encode anti- tumor agents may be obtained from cells which express or contain the sequences SUBSTTTUTE SHEET (RULE 26) Additionally, cDNA or mRNA libraries from specific cell sources can be purchased from commercial sources from which the desired sequences can be readily cloned by conventional techniques, e.g., PCR amplification. Sequences which encode anti-tumor agents may also be synthesized, for example, on an Applied Biosystems Inc. DNA synthesizer (e.g., ABI DNA synthesizer model 392, Foster City, CA). In addition to the anti-tumor agents described above, the present invention also provides anti-tumor agents which comprise a fusion protein of, for example, two or more cytokines, immune modulators, toxins or differentiation factors. Preferred anti-tumor agents in this regard include alpha interferon - Interleukin-2, GM-CSF - IL-4, GM-CSF - IL-2, GM- CSF - IL-3 (see U.S. Patent Nos. 5,082,927 and 5,108,910), GM-CSF - gamma interferon, and gamma interferon - IL-4, with gamma interferon - interleukin-2 being particularly preferred. Within another embodiments, the anti-tumor agent may further comprise a membrane anchor. The membrane anchor may be selected from a variety of sequences, including, for example, the transmembrane domain of well known proteins. Generally, membrane anchor sequences are regions ofa protein that anchor the protein to a membrane. Customarily, there are two types of anchor sequences that attach a protein to the outer surface ofa cell membrane: (1) transmembrane regions that span the lipid bilayer ofthe cell membrane (proteins containing such regions are referred to integral membrane proteins); and (2) domains which interact with an integral membrane protein or with the polar surface ofthe membrane (such proteins are referred to as peripheral, or extrinsic, proteins). Membrane anchors derived from integral membrane proteins are preferred. Membrane spanning regions typically have a similar structure, with a 20 to 25 amino-acid residue portion consisting almost entirely of hydrophobic residues located inside the membrane (see Eisenberg et al, Ann. Rev. Biochem. 53:595-623, 1984) Membrane spanning regions typically have an alpha helical structure (see Eisenberg et al. at 20; Heijne and Manoil at 109). Within a preferred embodiment, a membrane anchor is fused to the C- terminus of gamma interferon fusion protein, wherein the membrane anchor comprises the gamma-chain ofthe Fc receptor. Tumorigenicity of an anti-tumor agent can be assessed by various assays. Representative assays include tumor formation in nude mice or rats, colony formation in soft agar, and preparation of transgenic animals, such as transgenic mice. In addition to tumorigenicity studies, it is generally preferable to determine the toxicity of an anti-tumor agent. A variety of methods well known to those of skill in the art may be utilized to measure such toxicity, including for example, clinical chemistry assays which measure the systemic levels of various proteins and enzymes, as well as blood cell volume and number Once an anti-tumor agent has been selected, it is placed into a vector constmct according to the invention. Such a vector constmct can then be packaged into a recombinant retroviral vector and be used to transduce ex vivo hematopoietic stem ceils which are then re-mtrσduced into the patient. In the context ofthe present invention, it should be understood that the removed cells may not only be returned to the same patient, but may also be utilized to inhibit the growth of selected tumor cells in another allogeneic human. Preparation and Purification of Recombinant Retroviral Particles Another aspect ofthe invention concerns the preparation of recombinant retroviral particles. Recombinant retroviral particles according to the invention can be produced in a variety of ways, as those in the art will appreciate. For example, producer cells, i.e., cells containing all necessary components for retroviral vector packaging (including a nucleic acid molecule encoding the retroviral vector), can be grown in roller bottles, in bioreactors, in hollow fiber apparatus, and in cell hotels. Cells can be maintained either on a solid support in liquid medium, or grown as suspensions. A wide variety of bioreactor configurations and sizes can be used in the practice ofthe present invention. Cell factories (also termed "cell hotels") typically contain 2, 10, or 40 trays, are molded from virgin polystyrene, treated to provide a Nuclon D™ surface, and assembled by sonic welding one to another Generally, these factories have two port tubes which allow access to the chambers for adding reagents or removing culture fluid. A 10-layer factory provides 6000 cm 2 of surface area for growing cells, roughly the equivalent of 27 T-225 flasks Cell factories are available from a variety of manufacturers, including for example Nunc (Baxter, Sanform, ME). Most cell types are capable of producing high titer vector for 3 to 6 days, allowing for multiple harvests Each cell type is tested to determine the optimal harvest time after seeding and the optimal number of harvest days Cells are typically initially grown in DMEM supplemented with 2 to 20% FBS in roller bottles until the required number of cells for seeding a cell factory is obtained Cells are then seeded into the factories and 2 liters (L) of culture supernatant containing vector is harvested later at an appropriate time Fresh media is used to replenish the cultures Hollow fiber culture methods may also be used Briefly, high titer retroviral production using hollow fiber cultures is based on increasing viral concentration as the cells are being cultured to a high density in a reduced volume of media. Cells are fed nutrients and waste products are diluted using a larger volume of fresh media which circulates through the lumen of numerous capillary fibers. The cells are cultured on the exterior spaces ofthe capillary fibers in a bioreactor chamber where cell waste products are exchanged for nutrients by diffusion through 30 kD pores in the capillary fibers. Retroviruses which are produced from the cell lines are too large to pass through the pores, and thus concentrate in the hollow fiber bioreactor along side ofthe cells. The volume of media being cultured on the cell side is approximately 10 to 100 fold lower then volumes required for equivalent cell densities cultured in tissue culture dishes or flasks. This decrease in volume inversely correlates with the fold induction of titer when hollow fiber retroviral titers are compared to tissue culture dishes or flasks. This 10 to 100 fold induction in titer is seen when an individual retroviral producer cell line is amiable to hollow fiber growth conditions. To achieve maximum cell density, the individual cells must be able to grow in very close proximity and on top of each other. Many cell lines will not grow in this fashion and retroviral packaging cell lines based on these types of cell lines may not achieve 10 fold increases in titer. Cell lines which would grow very well would be non-adherent cell line and it is believed that a retroviral producer line based on a non-adherent cell line may reach 100 fold increases in titer compared to tissue culture dishes and flasks. Regardless ofthe retroviral particle and production method, high titer (from about lO O 1 ! cfu/mL) stocks can be prepared that will cause high level expression ofthe desired products upon introduction into appropriate cells. When all components required for retroviral particle assembly are present, high-level expression will occur, thereby producing high titer stocks. And while high titer stocks are preferred, retroviral preparations having titers ranging from about 10 3 to 10 6 cfu/mL may also be employed, although retroviral titers can be increased by various purification methods, as described below After production by an appropriate means, the infectious recombinant retroviral particles may be preserved in a cmde or purified form. Cmde retroviral particles are produced by cultivated infected cells, wherein retroviral particles are released from the cells into the culture media. The vims may be preserved in cmde form by first adding a sufficient amount ofa formulation buffer to the culture media containing the recombinant vims to form an aqueous suspension. Recombinant retroviral particles can also be preserved in a purified form. More specifically, prior to the addition of formulation buffer, the cmde retroviral preparation described above is clarified by passing it through a filter, and then concentrated, such as by a cross flow concentrating system (Filtron Technology Corp.. Nortborough. MA) Within one embodiment, DNase is added to the concentrate to digest exogenous DNA. The digest is then diafiltrated to remove excess media components and establish the recombinant vims in a more desirable buffered solution. The diafiltrate is then passed over a gel filtration column, such as a Sephadex S-500 gel column, and the purified recombinant vims is eluted. Cmde recombinant retroviral preparations can also be purified by ion exchange column chromatography, such as is described in more detail in U.S.S.N. Serial No. 08/093,436. In general, the cmde preparation is clarified by passing it through a filter, and the filtrate loaded onto a column containing a highly sulfonated cellulose matrix, wherein the amount of sulfate per gram of cellulose ranges from about 6 - 15 μg. The recombinant retrovims is eluted from the column in purified form by using a high salt buffer. The high salt buffer is then exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column. The purified preparation may then be formulated or stored, preferably at -70°C. Additionally, the preparations containing recombinant retroviruses according to the invention can be concentrated during purification in order to increase the titer of recombinant retrovims. A wide variety of methods may be utilized for increasing retroviral concentration, including for example, precipitation of recombinant retroviruses with ammonium sulfate, polyethylene glycol ("PEG") concentration, concentration by centrifiigation (either with or without gradients such as PERCOLL™, or "cushions" such as sucrose, use of concentration filters (e.g., Amicon filtration, Chicago, IL), and 2-phase separations. Briefly, to accomplish concentration by precipitation of recombinant retroviruses with ammonium sulfate, ammonium sulfate is added slowly to an appropriate concentration, followed by centrifiigation and removal ofthe ammonium sulfate either by dialysis or by separation on a hydrophobic column. Alternatively, recombinant retroviruses may be concentrated from culture medium with PEG (Green, et al, PNAS 67:385, 1970; Syrewicz, et al, Appl. Micro. 24:488, 1972) Such methods are rapid, simple, and inexpensive. However, like ammonium sulfate precipitation, use of PEG also concentrates other proteins from solution. Within other embodiments, recombinant retroviruses may be concentrated by centrifugation, and more particularly, low speed centrifiigation, which avoids difficulties associated with pelleting that accompanies high speed centrifugation (e.g., vims destmction or inactivation). Recombinant retrovimses according to the invention may also be concentrated by an aqueous two-phase separation method. Briefly, polymeric aqueous two-phase systems may be prepared by dissolving two different non-compatible polymers in water Many pairs of SUBSTTTUTE SHEET (RULE 26) water-soluble polymers may be utilized in the construction of such two-phase systems, including for example PEG or methylcellulose, and dextran or dextran sulfate (see Walter and Johansson, Anal. Biochem. 755:215, 1986; Albertsson, "Partition of Cell Particles and Macromolecules" Wiley, New York, 1960). As described in more detail below in Example 7, utilizing PEG at concentrations ranging from 5% to 8% (preferably 6.5%), and dextran sulfate at concemrations ranging from 0.4% to 1% (preferably 0.4%), an aqueous two-phase system may be established suitable for purifying recombinant retroviruses. Utilizing such procedures, approximate 100-fold concentration can be achieved with yields of approximately 50% or more ofthe total starting retrovims. For purposes of illustration, a representative concentration process which combines several concentration steps is set forth below. Briefly, recombinant retroviruses may be prepared either from roller bottles, cell factories, or bioreactors prior to concentration. Removed media containing the recombinant retrovims may be frozen at -70°C, or more preferably, stored at 2°C to 8°C in large pooled batches prior to processing. For material obtained from a bioreactor, the recombinant retrovims pool is first clarified through a 0.8 μm filter (1.2 μm glass fiber pre-filter, 0.8 μm cellulose acetate) connected in series with a 0.65 μm filter. This filter arrangement provides approximately 2 square feet (sq. ft.)of filter, and allows processing of about 15-20 L of pooled material before clogging. For material obtained from roller bottles or cell factories, a single 0.65 μm cartridge (2 sq. ft.) normally suffices for volumes up to 40 L. For 80 L cell factory processes, a 5 sq. ft. filter may be required. Preferably, after clarification the filter is rinsed with buffer (e.g., 150 mM NaCl, 25 mM Tris, pH 7.2-7.5). Following clarification, recombinant retroviruses are concentrated by tangential flow ultrafiltration utilizing cassettes with a 300,000 mw cut off. For bioreactor material (containing 12% to 16% FBS), 4 to 5 L of material may be concentrated per cassette. For roller bottles or cell factories at 12 to 16% FBS, 5 to 6 L of material may be concentrated per cassette. Finally, for cell factories containing 10% FBS, 8 to 9 L of material may be concentrated per cassette. Utilizing such procedures at an appropriate pressure differential between filtrate and retentate, up to 80 L of material may be concentrated to a volume of less than 500 mL in under two hours. This process also provides a yield of about 80%. Following the ultrafiltration step, DNAse may be added to a concentration of 50 U/mL, and recirculated at a lower pump speed with the filtrate line closed for 30 minutes Discontinuous diafiltration is then accomplished by adding additional buffer and utilizing the SUBSTTTUTE SHEET (RULE 26) same cross differential pressure as before. Generally, recovery after this step is approximately 70%. Concentrated material is then subjected to column chromatography on a Pharmacia S-500 HG size exclusion gel, utilizing 50 mM NaCl and 25 mM Tris pH 7.2-7.5 as minimum salt and ionic strength concentrations. Generally, recombinant retrovimses elute off in the first peak. Tangential flow filtration may once again be utilized to further reduce the volume of the preparation, after which the concentrated material is sterilized by filtration through a 0.2 μm Millipore filter (Philadelphia, PA). As an alternative to in vivo production, the retroviral packaging proteins may be produced, together or separately, from appropriate cells. However, instead of introducing a nucleic acid molecule enabling production ofthe viral vector, an in vitro packaging reaction is conducted comprising the gag, pol, and xeno env proteins, the retroviral vector, tRNA, and other necessary components. The resulting retroviral particles can then purified and, if desired, concentrated. Formulation Of Pharmaceutical Compositions Another aspect ofthe invention relates to pharmaceutical compositions comprising recombinant retroviral vectors as described above, in combination with a pharmaceutically acceptable carrier or diluent, while another aspect is directed toward a method for preserving an infectious recombinant retrovimses for subsequent reconstitution such that the recombinant retrovims is capable of infecting mammalian cells upon reconstitution. The methods described can be used to preserve a variety of different viruses, including recombinant type C retrovimses such as gibbon ape leukemia vims, feline leukemia vims and xeno-, poly- and amphotropic murine leukemia vims (Weiss, etal, RNA Tumor Viruses, 2d ed. 1985). See U.S.S.N. 08/153,342. Pharmaceutically acceptable carriers or diluents are nontoxic to recipients at the dosages and concentrations employed. Representative examples of carriers or diluents for injectable solutions include water, isotonic saline solutions, preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline), mannitol, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as human sem albumin (HSA) A particularly preferred composition comprises a recombinant retrovims in 10 mg/mL mannitol, 1 mg/mL HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl In this case, since the recombinant retroviral particle represents approximately 1 μg of material, it may be less than 1% of high molecular weight material, and less than 1/100,000 ofthe total material (including water). This composition is stable at -70°C for at least six months. Pharmaceutical compositions ofthe present invention may also additionally include factors which stimulate hematopoietic stem cell division, and hence, uptake and incorporation of vector constructs according to the invention. Particularly preferred methods and compositions for preserving recombinant retrovimses are described in U.S.S.N. 08/135,938, filed October 12, 1993, and U.S. Serial No. 8/153,342, filed November 15, 1993. The use of recombinant retrovimses to transduce hematopoietic stem cells useful in treating patients requires that the product be able to be transported and stored for long periods at a desired temperature such that infectivity and viability ofthe recombinant retrovims is retained. The difficulty of preserving recombinant retrovimses absent low temperature storage and transport presents problems in Third World countries, where adequate refrigeration capabilities are often lacking. The initial stabilization of materials in dry form to the preservation of antitoxins, antigens and bacteria has been described (Flosodort, et al, J. Immunol, 29:389, 1935). However, a limitation in this process included partial denaturation of proteins when dried from an aqueous state at ambient temperatures. Drying from the frozen state helped reduce this denaturation and led to efficient preservation of other biological materials, including bacteria and viruses (Stamp, et al, J. Gen. Microbiol, 7:251, 1947; Rowe, et al, Virology, 42:136, 1970; and Rowe, et al, Cryobiology, 5:153, 1971). More recently, sugars such as sucrose, raffinose, glucose and trehalose were added in various combinations as stabilizing agents prior to lyophilization of viruses. The use of sugars enhanced recovery of viable viruses, for research purposes which require that only some vims survive for later propagation. Recombinant retrovimses according to the invention can be stored in liquid, or preferably, lyophilized form. Factors influencing stability include the formulation (liquid, freeze dried, constituents thereof, etc.) and storage conditions, including temperature, storage container, exposure to light, etc. Alternatively, retroviral particles according to the invention can be stored as liquids at low temperatures. In a preferred embodiment, the recombinant retrovimses ofthe invention are formulated to preserve infectivity in a lyophilized form at elevated temperatures, and for this form to be suitable for injection into patients following reconstitution. Recombinant retroviral particles comprising retroviral vector constmcts according to the invention can be formulated in cmde or, preferably, purified form Cmde retroviral preparations may be produced by various cell culture methods, where retroviral particles are released from the cells into the culture media. Recombinant retroviral particles may be preserved in cmde form by adding a sufficient amount of formulation buffer. Typically, the formulation buffer is an aqueous solution containing various components, such as one or more saccharides, high molecular weight structural additives, buffering components, and/or amino acids. The recombinant retrovimses described herein can also be preserved in a purified form. For instance, prior to the addition of formulation buffer, cmde preparations as described above may be clarified by filtration, and then concentrated, such as by a cross flow concentrating system (Filtron Technology Corp., Nortborough, MA). DNase may be added to the concentrate to digest exogenous DNA, followed by diafiltration to remove excess media components and substitute in a more desirable buffered solution. The diafiltrate may then passed over a gel filtration column, such as a Sephadex™ S-500 gel column, and the eluted retroviral particles retained. A sufficient amount of formulation buffer may then be added to the eluate to reach a desired final concentration ofthe constituents and to minimally dilute the retroviral preparation. The aqueous suspension can then be stored, preferably at - 70°C, or immediately formulated. In an alternative procedure, the cmde preparation can be purified by ion exchange column chromatography, as described in co-owned U.S.S.N. 08/093,436, filed July 16, 1993 Briefly, the cmde recombinant retrovims is clarified by filtration and then loaded onto a column comprising a highly sulfonated cellulose matrix. Highly purified recombinant retrovims is eluted from the column using a high salt buffer, which is then exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column. After recovery, formulation buffer may then added to adjust the final concentration, as discussed above, followed by low temperature storage, preferably at -70°C, or immediate formulation When a dried formulation is desired, an aqueous preparation containing a cmde or purified retroviral preparation can be prepared by lyophilization or evaporation Lyophilization involves cooling the aqueous preparation below the glass transition temperature or below the eutectic point temperature ofthe solution, and removing water by sublimation. For example, a multistep freeze drying procedure as described by Phillips et al. (Cryobiology, 75:414, 1981) can be used to lyophilize the formulated recombinant vims, preferably from a temperature of -40°C to -45 °C The resulting composition should contain less than 10% water by weight Once lyophilized, such a preparation is stable and may be stored at -20°C to 25°C SUBSTITUTE 5HEET (RULE 26) In an evaporative method, water is removed by evaporation from the retroviral preparation aqueous suspension at ambient temperature. Evaporation can be accomplished by various techniques, including spray drying (see EP 520,748), where the preparation is delivered into a flow of preheated gas, usually air, whereupon water rapidly evaporates from droplets ofthe suspension. Spray drying apparatus are available from a number of manufacturers (e.g., Drytec, Ltd., Tonbridge, England; Lab-Piaπt, Ltd., Huddersfidd, England). Once dehydrated, the recombinant retroviral preparation is stable and may be stored at -20°C to 25°C. The resulting moisture content ofthe dried or lyophilized preparation may be determined through use of a Karl-Fischer apparatus (EM Science Aquastar' VIB volumetric titrator, Cherry Hill, NJ), or through a gravimetric method. Once dehydrated, the recombinant retrovims is stable and may be stored at -20°C to 25°C. As mentioned previously, aqueous preparations comprising retrovimses according to the invention used for formulation are typically composed of one or more saccharides, high molecular weight structural additives, buffering components, and water, and may also include one or more amino acids. It has been found that the combination of these components acts to preserve the activity ofthe recombinant retrovims upon freezing and lyophilization, or drying through evaporation. See co-owned U.S.S.N. 08/153,342, filed November 15, 1993. Various saccharides may be used alone or in combination, including sucrose, mannitol, glucose, trehalose, inositol, fructose, maltose, and galactose, with lactose being particularly preferred. The concentration ofthe saccharide can range from 0.1% to 30% by weight, preferably from about 1% to 12% by weight. A particularly preferred concentration of lactose is 3% to 4% by weight. Additionally, saccharide combinations can also be employed, including lactose and mannitol or sucrose and mannitol. It will also be evident to those skilled in the art that it may be preferable to use certain saccharides in the aqueous solution when the lyophilized formulation is intended for room temperature storage Specifically, disaccharides, such as lactose or trehalose, are preferred for such formulations One or more high molecular weight structural additives may be used to aid in preventing retroviral aggregation during freezing and provides stmctural support in the lyophilized or dried state. In the context ofthe present invention, stmctural additives are considered to be of "high molecular weight" if they are greater than 5000 daltons. A preferred high molecular weight stmctural additive is human semm albumin (HSA), although other substances may also be used, such as hydroxyethyl-cellulose, hydroxymethyl-cellulose. dextran, cellulose, gelatin, povidone, etc. Preferably, the concentration ofthe high molecular weight stmctural additive can range from 0.05% to 20%, with 0.1% to 10% by weight being preferred, and a concentration of 0.1% by weight HSA being particularly preferred SUBSTTTUTE SHEET (RULE 26) Amino acids, if present, tend to further preserve retroviral infectivity. In addition, amino acids function to further preserve retroviral infectivity during sublimation ofthe cooled aqueous suspension and while in the lyophilized state. A preferred amino acid is arginine, but other amino acids such as lysine, ornithine, serine, glycine, glutamine, asparagine, glutamic acid or aspartic acid can also be used. Preferably, the amino acid concentration ranges from 0.1% to 10% by weight. A particularly preferred arginine concentration is 0.1% by weight. A variety of buffering components may be used to maintain a relatively constant pH, depending on the pH range desired, preferably between 7.0 and 7.8. Suitable buffers include phosphate buffer and citrate buffer. A particularly preferred formulation pH is 7.4, and a preferred buffer is tromethamine. It may also be preferable to include in the formulation a neutral salt to adjust the final iso-osmotic salt concentration. Suitable neutral salts include sodium chloride, potassium chloride, and magnesium chloride, with sodium chloride being preferred. A particularly preferred method of preserving recombinant retrovimses in a lyophilized state for subsequent reconstitution comprises: (a) preparing an aqueous recombinant retroviral preparation comprising, in addition to the recombinant retrovims, about (i) 4% by weight of lactose, (ii) 0.1% by weight of HSA, (iii) 0.03% or less by weight of NaCl, (iv) 0.1% by weight of arginine, and a sufficient amount of tromethamine to provide a pH of approximately 7.4; (b) cooling the preparation to a temperature of about -40°C to -45°C to form a frozen preparation; and (c) removing water from the frozen preparation by sublimation to form a lyophilized composition having less than 2% water by weight. It is preferred that the recombinant retrovims be replication defective and suitable for administration into humans cells upon reconstitution. The lyophilized or dehydrated retrovimses ofthe subject invention may be reconstituted using a variety of substances, but are preferably reconstituted using water. In certain instances, dilute salt solutions which bring the final formulation to isotonicity may also be used. In addition, it may be advantageous to use aqueous solutions containing components known to enhance the activity ofthe reconstituted vims. Such components include cytokines, such as IL-2, polycations, such as protamine sulfate, or other components which enhance the transduction efficiency ofthe reconstituted vims. Lyophilized or dehydrated recombinant vims may be reconstituted with any convenient volume of water or the reconstituting agents noted above that allow substantial, and preferably total solubilization ofthe lyophilized or dehydrated sample. Administration of Recombinant Retroviral Particles In another aspect ofthe present invention, methods are provided for treating human patients afflicted with a variety of diseases, including a genetic disease, cancer, an infectious disease, an autoimmune disease, and inflammatory disease, a cardiovascular disease, and a degenerative disease. Examples of genetic diseases include but are not limited to; thalassemia, phenylketonuria, Lesch-Nyan syndrome, SCJJD, hemophilia A and B, cystic fibrosis, Duchenne's muscular dystrophy, inherited emphysema, familial hypercholesterolemia, and Gaucher's disease. Examples of cancers include but are not limited to; solid tumors, leukemias and lymphomas. Representative examples include melanomas, colorectal carcinomas, lung carcinomas (including large cell, small cell, squamous and adeno-carcinomas), renal cell carcinomas, cervical cancer, adult T-cell lymphoma leukemia, and breast adeno-carcinomas. Infectious diseases include but not limited to; hepatitis, tuberculosis, malaria, human immunodeficiency vims, herpes vims, tetanus, dysentery, shigella, FeLV, and FIV. Degenerative diseases include but are not limited to: Alzheimer's disease, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Inflammatory diseases include rheumatoid arthritis, spinal meningitis, and pancreatitis. Autoimmune diseases include diabetes, uveitis, HIV, and SCID. Cardiovascular diseases include, chronic rheumatic heart disease, arteriosclerosis, mitral valve and aortic stenosis, myocarditis, pericarditis, Marfan's syndrome, Ehlers-Danlos syndrome, Churg- Strauss syndrome, and scleroderma. Each of these methods comprise administering to a human a recombinant retroviral particle preparation as described above, such that a therapeutically efficacious amount of gene product encoded by the gene of interest carried on the vector constmct is produced. As used herein, a "therapeutically effective amount" of a gene product expressed from a vector constmct according to the invention is an amount that achieves a desired therapeutic benefit in a patient to an extent greater than that observed when the patient was not treated with the gene product. For instance, when the gene product is factor VM, a "therapeutically effective amount" refers to the amount of factor VIII needed to produce therapeutically beneficial clotting and will thus generally be determined by each patient's attending physician, although semm levels of about 0.2 ng/mL (about 0.1% of "normal" levels) or more will be therapeutically beneficial. When the gene product is an RNA molecule with intrinsic biological activity, such an antisense RNA or ribozyme, a "therapeutically effective amount" is an amount sufficient to achieve a clinically relevant change in the patient's condition through reduced expression ofthe harmful gene product, most often a protein In a preferred embodiment, the RNA molecule with intrinsic biological activity will be expressed in transduced hematopoietic stem cells in molar excess to the targeted RNA molecule. Expression levels ofthe heterologous and targeted RNAs can be determined by various assays, e.g., by PCR analysis. Typically, the dosage for ex vivo gene modified hematopoietic stem cells will be in the range of 10 5 to 10 8 cells per kilogram pateint body weight depending xφoπ the purity of stem cells in the starting population. Thus, for example, for CD34 + selected cells, usually 10 to 10 8 cells will be transduced ex vivo for re-infusion into a patient; for more highly enriched stem cell populations, such as CD34 + Thy- 1 Lin " selected cells, usually from 10 5 to 10 7 cells will be transduced and re-infused. The stem cell population will usually be transduced at mutiplicity of infection (MOI) of 10 to 1000, typically around 100 infectious recombinant retroviral particles per cell. Preferably, high titer preparations of retroviral vectors are used for ex vivo transduction. The volume that the high titer preparation is delivered in ex vivo is preferably not greater than 10% ofthe culture medium volume ofthe cell culture. More preferably, the volume ofthe high titer preparation is less than 1%, still more preferably less than 0 1%, and still more preferably less than 0.001% ofthe total cell culture volume. Additionally the retrovims is delivered in a medium that is free of agents that disturb or are toxic to the transduced cells in culture (eg. in an aqueous liquid with a composition similar to that of cell culture medium). Hematopoietic Stem Cells A pluripotent hematopoietic stem cell may be defined as follows: (1) a cell which gives rise to progeny in all defined hematolymphoid lineages; and (2) a cell which is capable of fully reconstituting a seriously immunocompromised host of all blood cell types and their progenitors, including the pluripotent hematopoietic stem cell by self renewal. "Hematopoietic stem cells" refers to a population of hematopoietic cells having all of the long-term engrafting potential in vivo. Animal models for long-term engrafting potential of candidate human hematopoietic stem cell populations include the SCJD-hu bone model (Kyoizumi et al. (1992) Blood 79: 1704; Murray et ai. (1995) Blood 85(2) 368-378) and the in utero sheep model (Zanjani et al. (1992) J. Clin. Invest. 89: 1179. For a review of animal models of human hematopoiesis, see Srour et al. (1992) J. Hematother. 1 : 143-1 3 and the references cited therein. At present, the best in vitro assay for stem cells is the long-term culture-initiating cell (LTCIC) assay, based on a limiting dilution analysis ofthe number of clonogenic cells produced in a stromal co-culture after 5-8 weeks. Sutherland et al. (1990) Proc. Natil Acad. Sci. 87:3584-3588. The LTCIC assay has been shown to correlate with another commonly used stem cell assay, the cobblestone area forming cell (CAFC) assay, and with long-term engrafting potential in vivo. Breems et al. (1994) Leukemia 8: 1095. For use in the present invention, a highly enriched stem cells population is preferred vπ order to maximize efficiency of gene transfer into the desired target cells. As described more fully below, an enriched stem cell population is exemplified by a population of cells selected by expression ofthe CD34 marker. In LTCIC assays, a population enriched in CD34+ cells will have an LTCIC frequency in the range of 1/50 to 1/500, more usually in the range of 1/50 to 1/200. Preferably, the stem cell population will be more highly enriched for stem cells than that provided by a population selected on the basis of CD34 + expression alone. By use of various techniques described more fully below, a highly enriched stem cell population may be obtained. A highly enriched stem cell population will typically have an LTCIC frequency in the range of 1/5 to 1/100, more usually in the range of 1/10 to 1/50. Preferably it will have an LTCIC frequency of at least 1/50. Exemplary ofa highly enriched stem cell population is a population having the CD34 + Thyl + Lin- phenotype as described in U.S. Patent No. 5,061,620. A population of this phenotype will typically have an average LTCIC frequency of approximately 1/20. Murray et al. (1995); Lansdorp et al. (1993) J. Exp. Med. 177: 1331. Hematopoietic stem cells may be isolated from any known source of stem cells, including bone marrow, mobilized peripheral blood (MPB), and umbilical cord blood. Initially, bone marrow cells may be obtained from a source of bone marrow, including ileum (e.g., from the hip bone via the iliac crest), tibia, femur, spine, or other bone cavities. Other sources of hematopoietic stem cells include embryonic yolk sac, fetal liver, and fetal spleen For isolation of bone marrow, an appropriate solution may be used to flush the bone, including saline solution, conveniently supplemented with fetal calf semm (FCS) or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from about 5 to 25 mM. Convenient buffers include HEPES, phosphate buffers and lactate buffers. Otherwise bone marrow may be aspirated from the bone in accordance with conventional techniques. Methods for mobilizing hematopoietic stem cells into the peripheral blood are known in the art and generally involve treatment with chemotherapeutic dmgs (e.g. , cyclophosphamide or etoposide), cytokines (e.g., GM-CSF, G-CSF or IL-3), or combinations thereof. Typically, apheresis for total white cells begins when the total white cell count reaches 500-2000 cells/ml and the platelet count reaches 50,000/ml. In order to SUBSTTTUTE SHEET (RULE 26) maximize hematopoietic stem cell recovery from MPB, daily aphoresis samples may be monitored for cells expressing CD34 and/or Thy-1. Desirably, the collection of peripheral blood leukocytes begins when an increase in CD34 + and or Thy-1 + cells is detected in order to obtain a sample during the peak of stem cell mobilization. While CD34 expression normally correlates with stem cell mobilization, in some cases it does not. Therefore, it is preferable to use the Thy-1 marker alone or in combination with CD34 to moπftσr stem cell mobilization (Murray, et al. 1995). Various techniques may be employed to separate the cells by initially removing cells of dedicated lineage ("lineage-committed" cells). Monoclonal antibodies and monoclonal antibody fragments are particularly useful for identifying markers associated with particular cell lineages and or stages of differentiation. The antibodies (or antibody fragments) may be attached to a solid support to allow for cmde separation. The separation techniques employed should maximize the viability ofthe fraction to be collected. The use of separation techniques include those based on differences in physical (density gradient centrifugation and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rhodamine 123 and DNA-binding dye Hoechst 33342). Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including complement and cytotoxins, and "panning" with antibody attached to a solid matrix or any other convenient technique. Techniques providing accurate separation include flow cytometry which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. A large proportion ofthe differentiated cells may be removed by initially using a relatively cmde separation, where major cell population lineages ofthe hematopoietic system, such as lymphocytic and myelomonocytic, are removed, as well as minor populations, such as megakaryocytic, mast cells, eosinophils and basophils Usually, at least about 70 to 90 percent ofthe hematopoietic cells will be removed. "Positive selection" refers to a selection procedure whereby the cell population of interest expresses the marker used as the basis for selection and, therefore, cells expressing the marker are retained and those not expressing the marker are discarded "Negative selection" refers to a selection procedure whereby the cell population of interest does not express the marker or have the characteristic that is used as the basis for selection and, therefore, cells having the specified marker or characteristic are discarded and those not having the specified marker or characteristic are retained Thus, positive selection SUBSTTTUTE SHEET (RULE 26) techniques may be employed to select stem cells based on, e.g., CD34 expression. One technique providing positive selection for CD34+ cells with high purity is described in PCT patent application No. WO94/02016, in which CD34+ cells are selected using a hapten conjugated anti-CD34 antibody and a hapten competition release system. Concomitantly or subsequent to a gross separation providing for positive selection, e.g., using the CD34 marker, a negative selection maybe carried out, where antibodies to lineage-specific markers present on dedicated cells are employed and the lineage-committed removed, e.g., by magnetic bead depletion or flow cytometer. For the most part, these markers include CD2 " , CD3\ CD7, CD8 ' , CD10 " , CD14 ' , CD15 ' , CD16 ' , CD19\ CD20 " , CD33 ' and glycophorin A-. Normally, negative selection will yield a cell population which is at least CD 14 ' and CDI 5 " , and preferably which is at least CD2 " , CD 14 ' , CD 15 ' , CD 16 ' , CD 19 ' and glycophorin A-. As used herein, Lin ' refers to a cell population lacking at least one lineage-specific marker. The hematopoietic cell composition may then be further separated using positive selection for the Thy-1 marker and or selection for rhodamine 10 , whereby a highly enriched hematopoietic stem cell population is achieved. See Table 1. The purified hematopoietic stem cells have low side scatter and low to medium forward scatter profiles by FACS analysis. Cytospin preparations show the enriched stem cells to have a size between mature lymphoid cells and mature granulocytes. Cells may be selected based on light-scatter properties as well as their expression of various cell surface antigens. While it is believed that the particular order of separation is not critical to this invention, the order indicated is preferred. Preferably, cells are initially separated by a cmde separation, followed by a fine separation, with positive selection of one or more markers associated with stem cells and negative selection for one or more markers associated with lineage committed cells. Compositions highly enriched in hematopoietic stem cells may be achieved in this manner. The desired hematopoietic stem cells are exemplified by a population with the CD34 + Thy-1 + Lin " phenotype. A hematopoietic stem cell composition is characterized by being able to be maintained in culture for extended periods of time, being capable of selection and transfer to secondary and higher order cultures, and being capable of differentiating into the various lymphocytic and myelomonocytic lineages, particularly B and T lymphocytes, monocytes, macrophages, neutrophils, erythrocytes and the like The stem cells may be grown in culture in an appropriate nutrient medium, including conditioned medium, a co-culture with an appropriate stromal cell line or a medium comprising a combination of growth factors sufficient to maintain the growth of SUBSTTTUTΕ SHEET (RULE 26) hematopoietic cells. For conditioned media or co-cultures, various stromal cell lines may be used. Since human stromal cell lines are not required, other stromal cell lines may be employed, including rodentiae, particularly murine stromal cell lines. Suitable murine stromal cell lines include AC3 and AC6, which are described in Whitlock, et al. Cell 48: 1009, 1987. Preferably, the stromal cell line used is a passage of AC6, AC6.21 (otherwise referred to as SySl). Cytokines may also be added, including, e.g., leukemia inhibitory factor (LIT), interleukins, colony stimulating factors, and stem cell factor (SCF, also known as steel factor, c-kit ligand, MGF). Of particular interest are LJT, stem cell factor, JL-3, JL-6, GM- CSF, G-CSF, MJP- lot, the flk2/flt3 ligand, and TPO/npl ligand. The factors which are employed may be naturally occurring or synthetic, e.g., prepared recombinantly, and preferably are human. The amount ofthe factors used will generally be in the range of about 1 ng/ml to 100 ng/ml. Generally, for LU, the concentration will be in the range of about 1 ng/ml to 100 ng/mg, more usually 5 ng/ml to 30 ng/ml; for JL-3, the concentration will be in the range of about 5 ng/ml to 100 ng/ml, more usually 5 ng/ml to 50 ng/ml; for JL-6, the concentration will be in the range of about 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 20 ng/ml, and for GM-CSF, the concentration will generally be 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 20 ng/ml, and for SCF the concentration will generally be 10 ng/ml to 150 ng/ml, and usually 50 ng/ml to 100 ng/ml. In one embodiment, the hematopoietic stem cells are optionally expanded prior to or after retroviral transduction. During expansion, the growth factors may be present only during the initial course ofthe stem cell growth and expansion, usually at least 24 hours, more usually at least about 48 hours to 4 days, or may be maintained during the course ofthe expansion. For use in clinical settings, it is preferable to transduce the hematopoietic stem cells without prior or subsequent expansion. In one embodiment therefore, the stem cells are cultured with or without cytokines in an appropriate medium, transduced with the appropriate vector, cultured for approximately 72 hours and reintroduced into the host. In one embodiment ofthe invention, at least about 0.1% ofthe hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle according to the invention. In another embodiment, at least about 1% ofthe hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle according to the invention, while in yet another embodiment ofthe invention, at least about 5% ofthe hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle according to the invention In a preferred embodiment ofthe invention, at least about 10% ofthe hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle as described herein. In another preferred embodiment, at least about 25% ofthe hematopoietic stem cells in a given hematopoietic cell population are transduced. In particularly preferred embodiments, at least about 50% ofthe hematopoietic stem cells in a given hematopoietic cell population are transduced. Even more preferred are embodiments where at least about 60%, 70%, 75%, 80%, 90%, or 95% ofthe hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle as described herein. Transduction may be accomplished by the direct co-culture of stem cells with recombinant retroviral particle producer cells, e.g., by the method of Bregni, et al, Blood 50:1418, 1992. For clinical applications, however, transduction by culturing the hematopoietic stem cells with recombinant retroviral supernatant alone or with purified retroviral preparations as described herein, in the absence of stromal cells, is preferred. Transductions may be performed by culturing the hematopoietic stem cells with the vims for from about four hours to six days. Preferably, transduction is carried out for three days, with the media replaced daily with media containing fresh recombinant retrovims particles. Alternatively, the stem cells may be cultured in the presence ofthe retrovims for several hours, e.g., four hours, daily for three to four days, with fresh media replacing the virus- containing media each day. In addition, the cell/vims preparations may be centrifuged. Typically, growth factors will be included in such amounts and concentration to maintain cell viability and induce cell cycling. Normally the cultures will include at least stem cell factor (SCF, also known as steel factor, MGF, and c-kit ligand), JL-3, and JL-6. Other cytokines of interest include leukemia inhibitory factor (LJF), G-CSF, GM-CSF, MUM, flk2/flt3 ligand, and TPO Polycations, such as protamine sulfate, polybrene and the like, will generally be included to promote binding. Protamine sulfate and polybrene are typically used preferably at a concentration of 4 ng/ml). Gene transfer into hematopoietic stem cells may be used to treat a variety of neoplastic, infectious or genetic diseases. For example, one may introduce genes that confer resistance to chemotherapeutic agents, thereby protecting the progeny hematopoietic cells, allowing higher doses of chemotherapy and thereby improving the therapeutic benefit. For instance, the mdrl gene (see U.S Patent No. 5,206,352) may be introduced into hematopoietic stem cells to provide increased resistance to a wide variety of chemotherapeutic dmgs which are exported by the mdrl gene product, in combination with the administration of chemotherapeutics such as taxol, e.g., for breast cancer treatment SUBSTTTUTE SHEET (RULE 26) Similarly, genes that provide increased resistance to alkylating agents, such as melphalan, may be introduced into hematopoietic stem cells in conjunction with high dose chemotherapy. For viral infections that primarily affect hematolymphoid cells, stem cells may be modified to endow the progeny with resistance to the infectious agent. In the case of human immunodeficiency vims (HIV), for example, specific, sense, aπtiseπse σrribxjzyme sequences may be introduced that interfere with viral infection or replication in the target cells. Alternatively, the introduced gene products may serve as "decoys" by binding essential viral proteins, thereby interfering with the normal viral life cycle and inhibiting replication. Alternatively, hematopoietic stem cells may be modified to produce a product to correct a genetic deficiency, or where the host has acquired a genetic deficiency through a subsequent disease. Genes that may correct a genetic deficiency include adenosine deaminase for the treatment of ADA " severe combined immunodeficiency; glucocerebrosidase for the treatment of Gaucher's disease; beta-globin for the treatment of sickle cell anemia; and factor VM or factor IX for the treatment of hemophilia. In many situations, cell immunotherapy involves removal of bone marrow or other source of hematopoietic stem cells from a human host, isolating the stem cells from the source and optionally expanding the stem cells. Meanwhile, the host may be treated to partially, substantially or completely ablate native hematopoietic capability. The isolated hematopoietic stem cells may be modified prior to or during this period of time, so as to provide hematopoietic stem cells having the desired genetic modification. After completion ofthe treatment ofthe host, the modified hematopoietic stem cells are reintroduced to the host to provide for expression ofthe foreign gene(s), and to reconstitute a functional hematopoietic system, if necessary. The methods of stem cell removal, host ablation and hematopoietic stem cell repopulation are known in the art. If necessary, the process may be repeated to ensure substantial repopulation ofthe modified stem cells. To ensure that the hematopoietic stem cells have been successfully modified, a vector-specific probe, or PCR using vector-specific primers, may be used to verify the presence ofthe vector constmct in the transduced stem cells or their progeny. In addition, the cells may be grown under various conditions to ensure that they are capable of maturation to all ofthe hematopoietic lineages while maintaining the capability, as appropriate, ofthe introduced DNA Various tests in vitro and in vivo may be employed to ensure that the pluripotent capability ofthe stem cells has been maintained The compositions comprising hematopoietic stem cells provide for production of myeloid cells and lymphoid cells in appropriate cultures In each ofthe cultures, mouse or SUBSTTTUTE SHEET (RULE 26) human stromal cells are provided, which may come from various sources, including, but not limited to, AC3, AC6 or stromal cells derived from mouse or human bone marrow by selection for the ability to maintain hematopoietic stem cells, and the like. Preferably, the stromal cells are AC6.21 and the ability to produce B lymphocytes and myeloid cells is determined in cultures supplied with LIF and JL-6. Generally, after 3 to 6 weeks of culture σιrAC6.21 stromal cells, the cells are analyzed by FACS for expression of CDI 9 (a B cell marker) andCD33 (a myeloid cell marker). Additionally, the hematopoietic stem cells can be analyzed for the ability to give rise to B cells, T cells and myelomonocytic cells in in vivo assays, as described below. To demonstrate differentiation to T cells, fetal thymus is isolated and cultured from 4 to 7 days at 25°C, so as to deplete substantially the lymphoid population. The cells to be tested for T-cell activity are then microinjected into the thymus tissue, where the HLA ofthe population which is injected is mismatched with the HLA ofthe thymus cells. The thymus tissue may then be transplanted into a scid/scid mouse as described in US Patent No. 5,147,784, particularly transplanting under the kidney capsule. Mice are sacrificed 6 to 7 weeks after transplantation and the thymus graft recovered and reduced to a single cell suspension. Donor-derived cells are detected by HLA staining and thymus differentiation analyzed by CD4 and CD8 staining and FACS analysis. Further demonstration ofthe sustained regenerative ability of hematopoietic stem cell populations may be accomphshed by the detection of continued myeloid and B-lymphoid cell production in the SCID-hu bone model. See Kyoizumi, et al, Blood 79:1704, 1992. Briefly, human fetal bone is isolated and a longitudinally sliced portion of this bone is transferred into the mammary fat pad ofa SCJD/SCJD animal. The bone cavity is diminished in endogenous cells by whole body irradiation ofthe mouse host prior to injection ofthe test donor hematopoietic cell population. The HLA ofthe population which is injected is mismatched with the HLA ofthe recipient bone cells. Stem cells from human hematopoietic sources will sustain B lymphopoiesis and myelopoiesis in such a SCID-hu bone model. To demonstrate the ability ofthe hematopoietic stem cell population to give rise to red blood cells, one may use conventional techniques to identify BFU-E units, for example, methylcellulose culture to show that the cells are capable of developing the erythroid lineage See Metcalf ( 1 77) In Recent Results in Cancer Research 61. Springer- Verlag, Berlin, pp. 1-227 Examples The following examples are included to more fully illustrate the present invention. Additionally, these examples provide preferred embodiments ofthe invention and are not meant to limit the scope thereof Standard methods for many ofthe procedures described in the following examples, or suitable alternative procedures, are provided' rrr wrdciy reorganized manuals of molecular biology, such as, for example "Molecular Cloning," Second Edition (Sambrook, et al, Cold Spring Harbor Laboratory Press, 1987) and "Current Protocols in Molecular Biology" (Ausubel, et al, eds. Greene Associates/Wiley Interscience, NY, 1990). EXAMPLE 1 PREPARATION OF RETROVIRAL VECTOR BACKBONES The following example describes the production of three retroviral vector backbones, designated KT-1, KT-3B, KT-3C. Vector KT-1 differs from KT-3B and KT-3C in that the former lacks a selectable marker which in KT-3B is neomycin resistance, whereas KT-3C confers phleomycin resistance. The Moloney murine leukemia vims (MoMLV) 5' long terminal repeat (LTR) EcoR I-EcoR I fragment, including gag sequences, from the N2 vector (Armentano et al, J. Vir. 61: 1647, 1987; Eglitas et al , Science 230: 1395, 1985) is ligated into the plasmid SK + (Stratagene, La Jolla, CA). The resulting constmct is designated N2R5. The N2R5 constmct is mutated by site-directed in vitro mutagenesis to change the ATG start codon to ATT preventing gag expression. This mutagenized fragment is 200 base pairs (bp) in length and flanked by Pst I restriction sites. The Pst I-Pst I mutated fragment is purified from the SK + plasmid and inserted into the Pst I site of N2 MoMLV 5' LTR in plasmid pUC31 to replace the non-mutated 200 bp fragment. The plasmid pUC31 is derived from pUCl9 (Stratagene, La Jolla, CA) in which additional restriction sites Xho I, Bgl II, BssH II and Nco I are inserted between the EcoR I and Sac I sites ofthe polylinker. This constmct is designated pUC31/N2R5gM. A 1.0 kilobase (Kb) MoMLV 3' LTR EcoR I-EcoR I fragment from N2 is cloned into plasmid SK + resulting in a constmct designated N2R3". A 1.0 Kb Cla I-Hind III fragment is purified from this constmct. The Cla I-Cla I dominant selectable marker gene fragment from pAFVXM retroviral vector (Kriegler et al, Cell 35:483, 1984; St. Louis et al, PNAS 85:3150, 1988), comprising a SV40 early promoter driving expression ofthe neomycin (neo) phosphotransferase gene, is cloned into the SK + plasmid. This constmct is designated SK + SV2-«eo A 1.3 Kb Cla I- BstB I gene fragment is purified from the SK + SV2-neo plasmid. KT-3B orKT-1 vectors are constructed by a three part ligatioπ in which the Xho I- Cla I fragment containing the gene of interest and the 1.0 Kb MoMLV 3' LTR Cla I-Hind III fragment are inserted into the Xho I-Hind M site of pUC3 l/N2R5gM plasmid. This gives a vector designated as having the KT-1 backbone. The 1.3 Kb Cla I-BstB I neo gene fragment from the pAFVXM retroviral vector is then inserted into the Cla I site of this plasmid in the sense orientation to yield a vector designated as having the KT-3B backbone. An alternative selectable marker, phleomycin resistance (Mulsant, et al. , Som. Cell andMol Gen., 14:243, 1988, available from Cayla, Cedex, FR) is used to make the retroviral backbone KT-3C as follows. The plasmid pUT507 (Mulsant, et al, supra) is digested with Nde I and the ends blunted with Klenow polymerase I. The sample is then further digested with Hpa I, Cla I linkers ligated to the mix of fragments, followed by digestion with Cla I to remove excess Cla I linkers. The 1.2 Kb Cla I fragment carrying the RSV LTR and the phleomycin resistance gene is isolated by agarose gel electrophoresis followed by purification using Gene Clean (BiolOl, San Diego, CA). This fragment is used in place ofthe 1.3 Kb Cla I-BstB I neomycin resistance fragment to give the backbone KT- 3C EXAMPLE 2 PREPARATION OF RETROVIRAL VECTOR CONSTRUCTS ENCODING PROTEINS The following example describes the preparation of various retroviral vector constmcts encoding different human genes of interest. More specifically, part (A) describes the production ofa vector constmct encoding the marker gene β galactosidase from E. coli, part (B) human interferon (hJF ), part (C) a retroviral vector constmct encoding human interleukin-2 (hJL-2), and part (D) the production of two retroviral vector constmcts coding for human factor VIII The first factor VIII constmct, codes for the B domain deleted form ofthe protein, while the second constmct codes for full length factor VIII A. Preparation of Cβ-gal. β-gal is obtained from the plasmid pSP65 as a HindM-Smal fragment. B. Preparation of KT-rhv-IFN. To obtain the human γ-IFN gene, the murine homologue is first cloned as follows: A mγ-IFN cDNA is cloned into the EcoR I site of pUC1813 essentially as set forth below. Briefly, pUC1813 (containing a sequence encoding γ-IFN) is prepared as essentially described by Kay et al. , Nucleic Acids Research 15:211%, 1987; and Gray et al, PNAS 50.5842, 1983) (Figure 1A). The mγ-IFN cDNA is retrieved by EcoR I digestion of pUCl813, and the isolated fragment is cloned into the EcoR I site of phosphatase-treated pSP73 (Promega; Madison, WI). This constmct is designated SP mγ-IFN. The orientation ofthe cDNA is verified by appropriate restriction enzyme digestion and DNA sequencing. In the sense orientation, the 5' end ofthe cDNA is adjacent to the Xho I site ofthe pSP73 polylinker and the 3' end adjacent to the Cla I site. The Xho I-Cla I fragment containing the mγ-IFN cDNA in either sense or antisense orientation is retrieved from SP mγ-IFN constmct and cloned into the Xho I-Cla I site ofthe KT-3 retroviral backbone. This constmct is designated KT mγ-IFN. 1. Preparation Of Sequences Encoding hγ-IFN Utilizing PCR (a) PHA Stimulation OfJurkat Cells Jurkat cells (ATCC No. CRL 8163) are resuspended at a concentration of 1 x 10 6 cells/ml in RPMI growth media (Irvine Scientific; Santa Ana, CA) with 5% fetal bovine semm (FBS) to a final volume of 158.0 ml. Phytohemoagglutinin ("PHA") (Curtis Mathes Scientific, Houston, TX) is added to the suspension to a final concentration of 1%. The suspension is incubated at 37°C in 5% CO2 overnight. The cells are harvested on the following day and aliquoted into three 50.0 ml centrifuge tubes. The three pellets are combined in 50 ml lx phosphate buffered saline (PBS, 145 mM, pH 7.0) and centrifuged at 1000 rpm for 5 minutes. The supernatant is decanted and the cells are washed with 50.0 ml PBS. The cells are collected for RNA isolation (b) RNA Isolation The PHA stimulated Jurkat cells are resuspended in 22.0 ml guanidinium solution (4 M guanidinium thiocyanate; 20 mM sodium acetate, pH 5.2; 0.1 M dithiothreitol, 0.5% sarcosyl). This cell-guanidinium suspension is then passed through a 20 gauge needle six times in order to disrupt cell membranes. A CsCl solution (5.7 M CsCl, 0.1 M EDTA) is then overlaid with 11.0 mL of the dismpted cell-guanidinium solution. The solution is centrifuged for 24 hours at 28,000 rpm in a SW28.1 rotor (Beckman, Fullerton, CA) at 20°C. After centrifugation the supernatant is carefully aspirated and the tubes blotted dry. The pellet is resuspended in a guanidinium-HCl solution (7.4 M guanidinium-HCl; 25 mM Tris-HCl, pH 7.5; 5 mM dithiothreitol) to a final volume of 500.0 μl. This solution is transferred to a microcentrifuge tube. Twelve and one-half microliters of concentrated Glacial acetic acid (HAc) and 250 μl of 100% EtOH are added to the microfuge tube. The solution is mixed and stored for several days at -20°C to precipitate RNA. After storage, the solution is centrifuged for 20 minutes at 14,000 rpm, 4°C. The pellet is then resuspended in 75% EtOH and centrifuged for 10 minutes in a microfuge at 14,000 rpm, 4°C. The pellet is dried by centrifugation under vacuum, and resuspended in 300 L deionized (DI) H2O. The concentration and purity ofthe RNA is determined by measuring optical densities at 260 and 280 nm. (c) Reverse Transcription Reaction Immediately before use, 5.0 1 (3.4 mg/mL) of purified Jurkat RNA is heat treated for 5 minutes at 90°C, and then placed on ice. A solution of 10.0 μl of lOx PCR buffer (500 mM KCl; 200 mM Tris-HCl, pH 8.4; 25 mM MgCl 2 ; 1 mg/ml bovine semm albumin (BSA)), lO.O μl of lO mM dATP, 10.0 μl of 10 mM dGTP, 10.0 μl of 10 mM dCTP, 10.0 μl of 10 mM dTTP, 2.5 μl RNasin (40,000 U/ml, Promega; Madison, WI) and 33.0 μl DI H2O, is added to the heat treated Jurkat cell RNA. To this solution 5.0 μl (10 8 nmol/mL) (Sequence ID No. 1), and 5.0 μl (200,000 U/ml) MoMLV reverse transcriptase (Bethesda Research Laboratories, EC 3.1.27.5, MD) is mixed in a microfuge tube and incubated at room temperature for 10 minutes. Following the room temperature incubation, the reaction mixture is incubated for 1 hour at 37°C, and then incubated for 5 minutes at 95°C The reverse transcription reaction mixture is then placed on ice in preparation for PCR SUBSTTTUTE SHEET (RULE 26) (d) PCR Amplification The PCR reaction mixture contains 100.0 μl reverse transcription reaction; 356.0 1 DI H 2 0; 40.0 μl lOx PCR buffer; 1.0 μl (137 nmol/mL) V-OLI #5 (Sequence ID No. 2); 0.5 μl (320 nmol/mL) V-OLI #6 (Sequence ID No. 3), and 2.5 μl, 5,000 U/ml, Taq polymerase (EC 2.7.7.7, Perkin-Elmer Cetus, CA). One hundred microliters of this mixture is arrqπσted into each of 5 tubes. (Sequence JD No. 1) 5' - 3': TAA TAA ATA GAT TTA GAT TTA This primer is complementary to a sequence ofthe mγ-IFN cDNA 30 base pairs downstream ofthe stop codon. V (Sequence ID No. 2) 5' - 3': GC CTC GAG ACG ATG AAA TAT ACA AGT TAT ATC TTG This primer is complementary to the 5' coding region ofthe mγ-EFN gene, beginning at the ATG start codon. The 5' end ofthe primer contains a Xho I restriction site. (Sequence ID No. 3) 5' - 3': GA ATC GAT CCA TTA CTG GGA TGC TCT TCG ACC TGG This primer is complementary to the 3' coding region ofthe mγ-JFN gene, ending at the TAA stop codon. The 5' end ofthe primer contains a Cla I restriction site. Each tube was overlaid with 100.0 μl mineral oil, and placed into a PCR machine (Ericomp Twin Block System, Ericomp, CA). The PCR program regulates the temperature ofthe reaction vessel first at 95°C for 1 minute, next at 67°C for 2 minutes and finally at 72°C for 2 minutes. This cycle is repeated 40 times. The last cycle regulates the temperature ofthe reaction vessel first at 95°C for 1 minute, next at 67°C for 2 minutes and finally at 72°C for 7 minutes. The completed PCR amplification reactions are stored at 4°C for 1 month in preparation for PCR DNA isolation. (e) Isolation Of PCR DNA The aqueous phase from the PCR amplification reactions are transferred into a single microfuge tube Fifty microliters of 3 M sodium acetate and 500 0 μl of chloroform isoamyl alcohol (24:1) is added to the solution. The solution is vortexed and then centrifuged at 14,000 rpm at room temperature for 5 minutes. The upper aqueous phase is transferred to a fresh microfuge tube and 1.0 mL of 100% EtOH is added. This solution is incubated for 4.5 hours at -20°C and then centrifuged at 14,000 rpm for 20 minutes. The supernatant is decanted, and the pellet is rinsed with 500.0 μl of 70% EtOH. The pellet is dried by centrifugation under a vacuum. The isolated hγ-IFN PCR DNA is resuspended in 10.0 μl DI H 2 O. 2. Constmction Of h-J N Retroviral Vectors (a) Creation A nd Isolation Of Blunt-Ended hg-lFN PCR DNA Fragments The hγ-INF PCR DNA is blunt ended using T4 DNA polymerase. Specifically, 10.0 μl of PCR amplified DNA; 2.0 μl, lOx, T4 DNA polymerase buffer (0.33 M Tris-acetate, pH 7.9, 0.66 M potassium acetate, 0.10 M magnesium acetate, 5 mM dithiothreitol, 1 mg/mL bovine semm albumin (BSA)); 1.0 μl, 2.5 mM dNTP (a mixture containing equal molar concentrations of dATP, dGTP, dTTP and dCTP); 7.0 μl DI H 2 O; 1.0 μl, 5000 U/mL, Klenow fragment (EC 2.7.7.7, New England Biolabs, MA); and 1.0 μl, 3000 U/ml, T4 DNA polymerase (EC 2.7.7.7, New England Biolabs, MA) are mixed together and incubated at 37°C for 15 minutes. The reaction mixture is then incubated at room temperature for 40 minutes and followed by an incubation at 68°C for 15 minutes. The blunt ended hγ-INF is isolated by agarose gel electrophoresis. Specifically, 2 0 μl of loading dye (0.25% bromophenol blue; 0.25% xylene cyanol; and 50% glycerol) is added to reaction mixture and 4.0 μl is loaded into each of 5 lanes ofa 1% agarose/Tris- borate-EDTA (TBE) gel containing ethidium bromide. Electrophoresis ofthe gel is performed for 1 hour at 100 volts The desired DNA band containing hγ-INF, approximately 500 base pairs in length, is visualized under ultraviolet light. This band is removed from the gel by electrophoretic transfer onto NA 45 paper (Schleicher and Schuell, Keene, NH. The paper is incubated at 68°C for 40 minutes in 400 0 μl of high salt NET buffer (1 M NaCl; 0.1 mM EDTA; and 20 mM Tris, pH 8 0) to elute the DNA The NA 45 paper is removed from solution and 400.0 μl of phenol chloroform. isoamyl alcohol (25 24.1) is added The solution is vortexed and centrifuged at 14,000 for 5 minutes The upper aqueous phase is transferred to a fresh tube and 400 0 μl of chloroform isoamyl alcohol (24 1 ) is added The mixture is vortexed and centrifuged for 5 minutes. The upper aqueous phase is transferred, a second time, to a fresh tube and 700.0 μl of 100% EtOH is added The tube is incubated at -20°C for 3 days Following incubation, the DNA is precipitated from the tube by centrifugation for 20 minutes at 14,000 rpm The supernatant is decanted and the pellet is rinsed with 500 0 μl of 70% EtOH The pellet, containing blunt ended hγ-JPN DNA, is dried by centrifugation under vaeuum and resuspended in 50.0 μl of DI H2O. The isolated blunt ended hγ-IFN DNA is phosphorylated using polynucleotide kinase Specifically, 25.0 μl of blunt-ended hγ-J N DNA, 3.0 μl of lOx kinase buffer (0 5 M Tris- HCl, pH 7.6, 0 1 M MgCl ; 50 mM dithiothreitol, 1 mM spermidine; 1 mM EDTA), 3 0 μl of 10 mM ATP, and 1.0 μl of T4 polynucleotide kinase (10,000 U/ml, EC 2.7 1.78, New England Biolabs, MD) is mixed and incubated at 37°C for 1 hour 45 minutes The enzyme is then heat inactivated by incubating at 68°C for 30 minutes (b) Ligation 0fhγ-4FNPCR DNA Into The SK + Vector An SK + plasmid is digested with Hinc II restriction endonuclease and purified by agarose gel electrophoresis as described below. Specifically, 5.9 μl (1 7 mg/mL) SK + plasmid DNA (Stratagene, San Diego, CA); 4.0 μl 1 Ox Universal buffer (Stratagene, San Diego, CA); 30.1 μl DI H2O, and 4.0 μl Hinc II, 10,000 U/mL, are mixed in a tube and incubated for 7 hours at 37°C. Following incubation, 4.0 μl of loading dye is added to the reaction mixture and 4.0 μl of this solution is added to each of 5 lanes ofa 1% agarose/TBE gel containing ethidium bromide. Electrophoresis ofthe gel is performed for 2 hours at 105 volts The Hinc II cut SK + plasmid, 2958 base pairs in length, is visualized with ultraviolet light The digested SK + plasmid is isolated by gel electrophoresis. Dephosphorylation ofthe Hinc JJ cleavage site ofthe plasmid is performed using calf intestine alkaline phosphatase Specifically, 50.0 μl digested SK + plasmid, 5 0 μl 1 M Tris, pH 8.0, 2 0 μl 5 mM EDTA, pH 8 0; 43.0 μl H 2 O and 2.0 μl, 1,000 U/mL, calf intestinal phosphatase ("CIP") (Boehringer Mannheim, Indianapolis, IN) are mixed in a tube and incubated at 37°C for 15 minutes Following incubation, 2 0 μl CIP is added and the solution is incubated at 55°C for 90 minutes Following this incubation, 2 5 μl 20% sodium dodecyl sulfate ("SDS"), 1 0 μl 0 5 M EDTA, pH 8 0, and 0 5 μl, 20 mg mL, prote ase K (EC 3 4 21 14, Boehringer Mannheim, Indianapolis, IN) are added, and the solution is incubated at 55°C for 2 hours This solution is cooled to room temperature, and 1 10 0 μl phenol chloroform isoamyl alcohol (25 24 1 ) is added The mixture is vortexed and centrifuged at 14,000 rpm for 5 minutes The upper aqueous phase is transferred to a fresh tube and 200.0 μl of 100% EtOH is added. This mixture is incubated at 70°C for 15 minutes. The tube is centrifuged and the pellet is rinsed with 500.0 μl of 70% EtOH. The pellet was then dried by centrifugation under a vacuum. The dephosphorylated SK + plasmid is resuspended in 40 μl DI H2O. The hγ-INF PCR DNA is ligated into the SK + plasmid using T4 DNA ligase. Specifically, 30.0 μl blunt ended, phosphorylated, hγ-IFN PCR DNA reaction mixture, 2.0 μl dephosphorylated SK + plasmid and 1.0 μl T4 DNA ligase are combined in a tube and incubated overnight at 16°C. DNA was isolated using a minprep procedure. More specifically, the bacterial strain DH5a (Gibco BRL, Gaithersburg, MD) is transformed with 15.0 μl of ligation reaction mixture, plated on Luria-Bertani agar plates (LB plates) containing ampicillin and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal, Gold Biotechnology; St. Louis, MO), and incubated overnight at 37°C. DNA is isolated from white bacterial colonies using the procedure described by Sambrook et al (Molecular Cloning, Cold Springs Harbor Press, 1989). The presence ofthe hγ-JFN gene is determined by restriction endonuclease cleavage with Xho I, Cla I, Ava JJ, Dra I, and Ssp I. The expected endonuclease restriction cleavage fragment sizes for plasmids containing the hγ- IFN gene are presented in Table 2. The isolated DNA plasmid is designated SK hγ-JFN and used in constmcting the retroviral vectors. Table 2 (c) Ligation Of hγ-IFN Gene Into Retroviral Vector The interferon gene is removed from SK hγ-IFN vector by digestion with Xho I and Cla I restriction endonucleases. The resulting fragment containing the hγ-IFN gene is approximately 500 bp in length, and is isolated in a 1% agarose/TBE gel electrophoresis The Xho I-Cla I hγ-IFN fragment is then ligated into the KT-3 retroviral backbone This constmct is designated KT hγ-IFN. The stmcture and presence expression of hγ-IFN is determined by transforming DH5a bacterial strain with the KT hγ-JFN constmct. Specifically, the bacteria is transformed with 15.0 μl of ligation reaction mixture The transformed bacterial cells are plated on LB plates containing ampicillin The plates are incubated overnight at 37°C and bacterial colonies are selected The DNA is isolated as described in (b) above, and digested with Xho I, Cla I, Dra I, Nde I, and Ssp I The expected endonuclease restriction cleavage fragment sizes for plasmids containing the hγ- JTN gene are presented in Table 3. Table 3 Subsequent sequencing of KT hγ-IFN, the retroviral vector, revealed the presence of a one base pair deletion within the hγ-IFN gene. This deletion is reversed using multi-step PCR procedure i. Sequence Selection Sequences are obtained from JJ3I Pustell sequence analysis program (Int Biotech, Inc , New Haven, CT) The following hγ-IFN primer sequences are used (Sequence ID No. 4) 5'-3': G CCT CGA GCT CGA GCG ATG AAA TAT ACA AGT TAT ATC TTG This primer is the sense sequence complimentary to the start codon ATG region extending seven codons upstream of hγ-IFN gene, and is designated hγ-IFN lb. (Sequence ID No. 5) 5'-3': GTC ATC TCG TTT CTT TTT GTT GCT ATT This primer is the anti-sense sequence complimentary to codons 106 to 120 ofthe hγ-IFN gene, and is designated hγ-IFN Rep B. (Sequence ID No. 6) 5'-3': AAT AGC AAC AAA AAG AAA CGA GAT GAC This primer is the sense sequence compUmentary to codons 106 to 120 ofthe hγ-IFN gene, and is designated hγ-IFN Rep A. (Sequence JJD No. 7) 5'-3': G CAT CGA TAT CGA TCA TTA CTG GGA TGC TCT TCG ACC TCG This primer is the anti-sense sequence complimentary to the stop codon ATT region and extending seven codons upstream ofthe hγ-IFN gene, and is designated hγ-EFN 3 b ii Initial PCR A solution of 1 x 10 6 KT hγ-IFN plasmid molecules in 398.0 μl, DI H2O; 50 μl, lOx PCR buffer (500 mM KCl and 200 mM Tris-HCl, pH 8.4, 25 mM MgCl 2; 1 0 mg/ml BSA), 5.0 μl, 2.5 mM dATP; 5.0 μl, 2.5 mM dGTP, 5.0 μl, 2.5 mM dCTP; 5.0 μl, 2.5 mM dTTP, 12.0 μl, 18 6 nmol/ml, oligonucleotide hγ-JJN lb; 15 0 μl, 24.6 nmol/ml, oligonucleotide hγ- IFN RepB; and 2.5 μl, Taq polymerase is mixed in a microfuge tube and 50 μl is aliquoted into 10 tubes Similarly, a solution of 1 x 10 6 KT hγ-IFN plasmid molecules in 395 0 μl, DI H 2 O; 50.0 μl, lOx PCR buffer (500 mM KCl, 200 mM Tris-HCl, pH 8 4, 25 mM MgCl , 1 mg/ml BSA), 5 0 μl, 2.5 mM dATP, 5.0 μl, 2 5 mM dGTP, 5 0 μl, 2 5 mM dCTP, 5 0 μl, 2 5 mM dTTP, 13 μl, 23 4 nmol ml, oligonucleotide hγ-IFN RepA, 17 Oμ 1, 18 0 nmol ml, oligonucleotide hγ-IFN 3b, and 2 5 μl Taq polymerase is mixed in a microfuge tube and 50 0 SUBSTTTUTE SHEET (RULE 26) μl is aliquoted into 10 tubes. The 20 tubes are placed in a PCR machine (Model 9600, Perkin Elmer Cetus; Los Angeles, CA). The PCR program regulates the temperature ofthe reaction vessel in the first cycle at 94°C for 2 minutes. The next 35 cycles are regulated at 94°C for 0.5 minutes, then at 55°C for 0.5 minutes and finally at 72°C for 1 minute. The final cycle is regulated at 72°C for 10 minutes. This cycling program is designated Program 10. Following PCR amplification, 225.0 μl of each reaction tube is mixed with 25.0 μl loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 50% glycerol, agarose gel loading dye) and loaded into the wells ofa 2% agarose gel containing ethidium bromide. The gel is electrophoresed at approximately 90 volts for 1 hour. Ultraviolet light is used to visualize the DNA band separation. Two bands are isolated, one fragment of 250 bp in size and the other of 150 bp in size by electrophoretic transfer onto NA 45 paper. Following precipitation, each ofthe two DNA pellets is resuspended in 20.0 1 DI H 2 O and prepared for further PCR amplification. ϋi. Annealing and Second Round PCR A solution of 20.0 μl ofthe 150 bp PCR DNA; 20.0 μl ofthe 350 bp PCR DNA: 161.5 μl, DI H 2 O; 25.0 μl, lOx PCR buffer (500 mM KCl; 200 mM Tris-HCl, pH 8.4, 25 mM MgCl 2; and 1 mg/ml BSA); 2.5 μl, 2.5 mM dATP; 2.5 μl, 2.5 mM dGTP; 2.5 μl, 2.5 mM dCTP; 2.5 μl, 2.5 mM dTTP; and 1.25 μl Taq polymerase is mixed in a microfuge tube and 47.3 μl aliquoted into each of 5 tubes. Each tube is placed in a PCR machine (Model 9600, Perkin-Elmer-Cetus, CA). The PCR program regulates the temperature ofthe reaction vessel for 5 cycles at 94°C for 0.5 minutes. The next cycle is regulated at 55 °C for 1 minute. Following this cycle, 1.2 μl hγ-IFN lb and 1.5 μl hγ-IFN 3b are added to the reaction mixture. The tubes are then PCR amplified using program 10. The product is designated rhγ-IFN. iv Creation and Isolation of Blunt-Ended rhg-IFN PCR DNA Fragment The PCR amplified hγ-JTN DNA is blunt ended using T4 polymerase. Specifically, 120.0 μl rhγ-IFN PCR solution is mixed with 1.25 μl, 2.5 mM dATP; 1.25 μl, 2 5 mM dGTP; 1.25 μl, 2.5 mM dCTP; 1.25 1, 2.5 mM dTTP; 1 1, T4 DNA polymerase; and 1.0 μl Klenow fragment. This mixture is incubated at room temperature for 10 minutes. Following incubation, 13.0 μl of agarose gel loading dye is added to the mixture and this solution is loaded into a 1% agarose gel. The gel is electrophoresed at approximately 90 volts for 1 hour. Ultraviolet light is used to visualize the DNA banding. A 500 bp band is isolated by electrophoretic transfer onto NA 45 paper as described above. Following precipitation, the DNA pellet is resuspended in 12.0 1 DI H2O. The isolated 500 bp fragment is blunt ended using T4 polynucleotide kinase. Specifically, 1.0 mg of this fragment is mixed with 1.5 μl lOx kinase buffer (0.5 mM Tris- HCl, pH 7.6; 0.1 mM MgCl 2 ; 50 mM dithiothreitrol; 1 mM spermidine; 1 mM EDTA); 1.5 μl, 10 mM ATP; and 1.0 μl, T4 polynucleotide kinase, and incubated at 37°C for 30 minutes v. Limtion ofrhv-IFNPCR DNA Into the SK + Vector The rhγ-JPN PCR DNA is ligated into the SK + vector. A solution of 2.0 μl hγ-JJN PCR DNA-kinase reaction mixture; 2.0 μl CJP treated SK + vector; and 1.0 μl, T4 DNA ligase is incubated at 16°C for 4 hours. DH5a bacteria is transformed as described above. vi. Ligation ofhv-IFN Gene Into Retroviral Vector Ligation of hγ-IFN gene into retroviral vector is performed as described above. The new vector is designated KT hγ-IFN. C. Preparation of KT-hJL-2. The method for cloning hJL-2 into KT-3 retroviral vector is essentially identical to the procedure for cloning hg-JTN into KT-3, with the exception that different primers are required for amplification ofthe hJL-2 DNA sequence. The following hTL-2 PCR primer sequences are used: V-OLI #55 (Sequence ID No. 8) 5'-3': ATAAATAGAAGGCCTGATATG This primer is complimentary to a sequence ofthe hJL-2 cDNA downstream ofthe stop codon V-OLI #1 (Sequence ID No. 9) 5'-3': GC CTC GAG ACA ATG TAC AGG ATG CAA CTC CTG TCT This primer is the sense sequence ofthe hJL-2 gene complimentary to the 5' coding region beginning at the ATG start codon. The 5' end ofthe primer contains a Xho I restriction site. V-OLI #2 (Sequence ID No. 10) 5'-3': GA ATC GAT TTA TCA AGT CAG TGT TGA GAT GAT GCT The primer is the anti-sense sequence ofthe hTL-2 gene complimentary to the 3' coding region ending at the TAA stop codon. The 5' end ofthe primer contains the Cla I restriction site. Preparation of Factor VM Vectors. The following is a description ofthe constmction of several retroviral vectors encoding factor VIII. Due to the size ofthe full length factor VM gene (7,056 bp), packaging constraints of retroviral vectors and because selection for transduced cells is not a requirement for ex vivo hematopoietic stem cell therapy, a retroviral backbone, e.g., KT-1, lacking a selectable marker gene is employed. A gene encoding full length factor VIII can be obtained from a variety of sources. One such source is the plasmid pCIS-F8 (EP 0 260 148 A2, published March 3, 1993), which contains a full length factor VM cDNA whose expression is under the control ofa CMV major immediate-early (CMV MJE) promoter and enhancer. The factor VIII cDNA contains approximately 80 bp of 5' untranslated sequence from the factor VIII gene and a 3' untranslated region of about 500 bp. In addition, between the CMV promoter and the factor VM sequence lies a CMV intron sequence, or "cis" element. The cis element, spanning about 280 bp, comprises a splice donor site from the CMV major immediate-early promoter about 140 bp upstream ofa splice acceptor from an immunoglobulin gene, with the intervening region being supplied by an Ig variable region intron. i. Constmction ofa Plasmid Encoding Retroviral Vector JW-2 A plasmid, pJW-2, encoding a retroviral vector for expressing full length factor VIII is constructed using the KT-1 backbone from pKT-1 To facilitate directional cloning ofthe factor VM cDNA insert into pKT-1, the unique Xho I site is converted to a Not I site by site directed mutagenesis. The resultant plasmid vector is then opened with Not I and Cla I. pCIS-F8 is digested to completion with Cla I and Eag I, for which there are two sites, to release the fragment encoding full length factor VM. This fragment is then ligated into the Not I/Cla I restricted vector to generate a plasmid designated pJW-2. ii. Construction ofa Plasmid Encoding Retroviral Vector ND-5. A plasmid vector encoding a tmncation of about 80% (approximately 370 bp) ofthe 3' untranslated region ofthe factor VM cDNA, designated pND-5, is constmcted in a pKT-1 vector as follows: As described for pJW-2, the pKT-1 vector employed has its Xho I restriction site replaced by that for Not I. The factor VM insert is generated by digesting pCIS-F8 with Cla I and Xba I, the latter enzyme cutting 5' ofthe factor VIII stop codon The approximately 7 kb fragment containing all but the 3' coding region ofthe factor VM gene is then purified. pCIS-F8 is also digested with Xba I and Pst I to release a 121 bp fragment containing the gene's termination codon. This fragment is also purified and then ligated in a three way ligation with the larger fragment encoding the rest ofthe factor VM gene and Cla I/Pst I restricted BLUESCP PT® KS + plasmid (Stratagene, San Diego, CA) to produce a plasmid designated pND-2. The unique Sma I site in pND-2 is then changed to a Cla I site by ligating Cla I linkers (New England Biolabs, Beverly, MA) under dilute conditions to the blunt ends created by a Sma I digest. After recircularization and ligation, plasmids containing two Cla 1 sites are identified and designated pND-3. The factor VM sequence in pND-3, bounded by Cla I sites and containing the full length gene with a tmncation of much ofthe 3' untranslated region, is cloned as follows into a plasmid backbone derived from a Not I/Cla I digest of pJW-1 [a pKT-1 derivative by cutting at the Xho I site, blunting with Klenow, and inserting a Not I linker (New England Biolabs)], which yields a 5.2 kb Not I/Cla I fragment. pCIS-F8 is cleaved with Eag I and Eco RV and the resulting fragment of about 4.2 kb, encoding the 5' portion ofthe full length factor VIII gene, is isolated. pND-3 is digested with Eco RV and Cla I and a 3.1 kb fragment is isolated. The two fragments containing portions ofthe factor VM gene are then ligated into the Not I Cla I digested vector backbone to produce a plasmid designated pND- 5. iii. Constmction of a B Domain-deleted Factor VM Vector The precursor DNA for the B-deleted FVIII is obtained from Miles Laboratory. This expression vector is designated p25D and has the exact backbone as pCISFδ above. The Hpa I site at the 3' ofthe FVJII8 cDNA in p25D is modified to Cla-I by oligolinkers. An Ace I to Cla I fragment is clipped out from the modified p25D plasmid. This fragment spans the B-domain deletion and includes the entire 3' two-thirds ofthe cDNA. An Ace I to Cla I fragment is removed from the pJW-2 above, and replaced with the modified B-domain deleted fragment just described. This constmct is designated B-del-1. As those in the art will appreciate, after constmction of plasmids encoding retroviral vectors such as those described above, such plasmids can then be used in the production of various cell lines from which infectious recombinant retrovimses can be produced. E. Preparation of MDR-1. Plasmid clones containing the the multi-dmg resistance- 1 (MDR-1) gene were obtained from ATCC (ATCC No. 61360 and 65704). The gene is isolated and purified using methods provided in Sambrook et al. (Molecular Cloning. A Laboratory Manual, 2d., 1989). Appropriate endonuclease restriction sites, Xho I and Cla I, (Molecular Cloning: A Laboratory Manual, 2d., 1989) are provided for insertion ofthe MDR-1 gene into KT-1 or KT-3B retroviral vector backbones. EXAMPLE 3 Isolation and Transduction of Bone Marrow Cells Pluripotent hematopoeitic stem cells, CD34 + are collected from the bone marrow of a patient by a syringe evacuation performed by known techniques. Alternatively, CD34 + cells may also be obtained from the cord blood of an infant if the patient is diagnosed before birth. Generally, 20 bone-marrow aspirations are obtained by puncturing femoral shafts or from the posterior iliac crest under local or general anesthesia. Bone marrow aspirations are then pooled and suspended in Hepes-buffered Hanks' balanced salt solution containing heparin sulfate at 100 Units/ml and deoxyribonuclease I at 100 μg/ml and then subjected to a Ficoll gradient separation. The buf y coated marrow cells are then collected and washed according to CEPRATE™ LC (CD34) Separation system (Cellpro, Bothell, WA). The washed buffy coated cells are then stained sequentially with anti-CD34 monoclonal antibody, washed, then stained with biotinylated secondary antibody supplied with the CEPRATE TM system. The cell mixture is then loaded onto the CEPRATE ,TM avidin column. The biotin- labeled cells are adsorbed onto the column while unlabeled cells pass through The column is then rinsed according to the CEPRATE system directions and CD34 + cells eluted by agitation ofthe column by manually squeezing the gel bed. Once the CD34 + cells are purified, the purified stem cells are counted and plated at a concentration of 1 x 10^ cells/ml in Iscove's modified Dulbecco's medium, IMDM (Irvine Scientific, Santa Ana, CA), containing 20% pooled non-heat inactivated human AB semm (hAB semm). After purification of CD34 + cells, several methods of transducing purified stem cells may be performed. One approach involves transduction ofthe purified stem cell population with vector containing supernatant cultures derived from vector producing cells. A second approach involves co-cultivation of an irradiated monolayer of vector producing cells with the purified population of non-adherent CD34 + cells. A third approach involves a similar co- cultivation approach, however the purified CD34 + cells are pre-stimulated with various cytokines and cultured 48 hours prior to the co-cultivation with the irradiated vector producing cells. Pre-stimulation prior to transduction increases effective gene transfer (Nolta et al, Exp. Hematol 20:1065; 1992). The increased level of transduction is attributed to increased proliferation ofthe stem cells necessary for efficient retroviral transduction. Stimulation of these cultures to proliferate also provides increased cell populations for re- infusion into the patient. EXAMPLE 4 Packaging Cell Production A MLV stmctural gene expression vectors To decrease the possibility of replication-competent vims being generated by genetic interactions between the MLV proviral vector DNA and the stmctural genes ofthe packaging cell line ("PCL"), separate expression vectors, each lacking the viral LTR, were generated to express the gag/pol and env genes independently. To further decrease the possibility of homologous recombination with MLV vectors and the resultant generation of replication- competent vims, minimal sequences other than the protein coding sequences were used. In SUBSTITUTE 5HEET (RULE 26) order to express high levels ofthe MLV stmctural proteins in the host cells, strong transcriptional promoters (CMV early and Ad5 major late promoters) were utilized. An example ofthe constmction ofa MoMLV gag/pol expression vector pSCVIO follows: 1. The 0.7 Kb HinCU/XmaM fragment encompassing the human cytomegalovirus (CMV) early transcriptional promoter (Boshart, et al, Cell 47:521, 1985) was isolated. 2. A 5.3 Kb Pstl(partial)/Scal fragment from the MoMLV proviral plasmid, ML V-K (Miller, et al., Mol CellBiol. 5:531, 1985) encompassing the entire gag/pol coding region was isolated. 3. A 0.35 Kb Dral fragment from S V40 DNA (residues 2717-2363) encompassing the SV40 late transcriptional termination signal was isolated. 4. Using linkers and other standard recombinant DNA techniques, the CMV promoter-MoMLV gag/pol-SV40 termination signal was ligated into the bluescript vector SK (Stratagene, San Diego, CA). An example ofthe constmction of an MLV xenotropic envelope expression vector follows. 1. A 2.2 Kb Nael/Nhel fragment containing the coding region ofthe xenotropic envelope obtained from clone NZB9-1 (O'Neill, et al, J. Virol. 55:100, 1985) was isolated. 2. Using linkers and other standard recombinant DNA techniques, the CMV early promoter and S V40 late termination signal described for the gag/pol expression above (pSCVIO) were ligated in the order CMV promoter-xeno env-termination signal (pCMV xeno . B Host Cell Selection Host cell lines were screened for their ability to efficiently (high titer) rescue a dmg resistance retroviral vector A alpha N2 (Armentano, et al, J. Vir. 61: 1641, 1987; and Eglitas, et al, Science 230: 1395, 1985) using replication competent retrovims to produce the gag/pol and env stmctural genes ("MA" vims). Titer was measured from confluent monolayers 16 h after a medium change by adding filtered supernatants (0 45 urn filters) to 5xl0 4 NTH 3T3 TK" cells on a 6 cm tissue culture plate in the presence of 4 ug ml polybrene followed by selection in G418 Among the non-murine cell lines which demonstrated the ability to package MoMLV-based vector with high titre were the cell lines CF2 (canine), D17 (canine), 293 (human), and HT1080 (human) These cell lines are preferred for production of packaging and producer cell lines, although many other cells may be tested and selected by such means C. Generation of Packaging Cell Lines (i) Preparation of gag/pol intermediates As examples ofthe generation of gag/pol intermediates for PCL production, D 17 (ATCC No. CCL-183), 293 (ATCC No. 1573), and HT1080 (ATCC No. CCL 121) cells were co-transfected with 1 ug ofthe methotrexate resistance vector, pFR400 (Graham and van der Eb, Virology 52:456, 1973), and 10 ug ofthe MoMLV gag/pol expression vector, pSCVIO (above) by calcium phosphate co-precipitation (D17 and HT1080, see Graham and van der Eb, supra), or lipofection (293, see Feigner, et al, Proc. Natl. Acad Sci., USA 54:7413, 1987). After selection for transfected cells in the presence ofthe drugs dipyrimidol and methotrexate, individual dmg resistant cell colonies were expanded and analyzed for MoMLV gag/pol expression by extracellular reverse transcriptase (RT) activity (modified from Goff, et al, J. Virol. 38:239, 1981) and intracellular p30 α, by Western blot using anti-p30 antibodies (goat antisemm #77S000087 from the National Cancer Institute). This method identified individual cell clones of each cell type which expressed 10-50x higher levels of both proteins compared with that ofthe packaging cell line PA317, as shown in Table 4. SUBSTTIUTE SHEET (RULE 26) TABLE 4 PROPERTIES OF MoMLV GAG/POL-EXPRESSING CELLS RT P30 8 "* LARNL CELL NAME ACTIVITY (CPM) EXPRESSION TΓΓRE (CFU ML) 3T3 800 N.D PA317 1350 +/- 1.2 x lO 3 D17 800 N.D. D17 4-15 5000 [ M M 1.2 X lO 4 D179020 2000 6.0 X 10 3 D17 9-9 2200 1.0 X lO 3 D17 9-16 6100 M M 1.5 X 10 4 D17 8-7 4000 N.D HT1080 900 N.D HTSCV21 16400 I I I I I 8.2 X 10 3 HTSCV25 7900 2.8 X 10 3 HTSCV42 11600 8.0 X lO 2 HTSCV26 4000 < 10 293 600 N.D 293 2-3 6500 I I I I I 7 x l0 4 293 5-2 7600 I I I I I N.D The biological activity of these proteins was tested by introducing a retroviral vector, LARNL which expresses both the amphotropic envelope and a Neo + marker which confers resistance to the drug G418. In every case, co-expression of gag/pol in the cell line and env from the vector allowed efficient packaging ofthe vector as determined by cell-free transfer of G418 resistance to 3T3 cells (titer). Titer was measured from confluent monolayers 16 h after a medium change by adding filtered supematants (0.45 μm filters) to 5x10 4 NIH353 TK + cells on a 6 cm tissue culture plate in the presence of 4 ug/ml polybrene followed by selection in G418 Significantly, the vector titers from the cell lines correlated with the levels of p3θfi ag (Table 4) Since the level of env should be the same in each clone and is comparable to the level found in PA317 (data not shown), this indicates that titre was limited by the lower levels oϊgaglpol in these cells (including PA317). The titre correlated more closely with the levels of p30^ than with the levels of RT. (ii) Conversion of gag/pol lines into xenotropic packaging cell lines. As examples ofthe generation of xenotropic PCLs, the gag/pol over-expressors for DI 7 (4-15) and HT1080 (SCV21) were co-transfected by the same techniques described above except that 1 μg of either the phleomycin resistance vector, pUT507 (for SCV21), or the hygromycin B resistance marker, pY3 (for 4-15, see Blochlinger and Diggelmann, Mol. Cell Biol. 4:2929, 1984), and 10 μg ofthe xenotropic envelope expression vector, pCMVxeno (above) was used. After selection for transfected cells in the presence of phleomycin or hygromycin, respectively, individual dmg resistant cell colonies were expanded and analyzed for intracellular expression of MLV 3 °% and gp75 β,7V proteins by Western blot using specific antisera. Clones were identified which expressed relatively high levels of both gag/pol and xeno env. A number of these xenotropic packaging cell lines were tested for their capacity to package retroviral vectors by measuring titre after the introduction of retroviral vectors. The results are presented in Table 5, below. TABLE 5 VECTOR TITRE ON XENOTROPIC PCLs KT-1 TITRE (CFU/ML) CELL CLONE ON FJ 1080 CELLS HT1080 SCV21 XF1 XF7 XF12 (HX) D17 4-15 X6 X10 (DX) X23 Highest titers are obtained when retroviral vectors are introduced into packaging cell lines by infection, as opposed to transfection (Miller, etal, Somat. Cell Mol. Genet., 12: 115, 1986). However, the xenotropic packaging cell lines described herein are blocked for infection by recombinant xenotropic retroviral particles since the cells express a xenotropic env protein (i.e., "viral interference"). To overcome the problem of "viral interference," whereby cell lines expressing a xenotropic envelope protein block later infection by xenotropic MLV vectors able to otherwise infect those cell types, vector particles containing other viral envelopes (such as VSV-g protein (Florikiewicz, et al, J. Cell Bio. 97. : 1381 , 1983; and Roman, et al, Exp. Cell Res. 175:316, 1988) which bind to cell receptors other than the xenotropic receptor) may be generated in the following manner 10 μg ofthe plasmid DNA encoding the retroviral vector constmct to be packaged is co-transfected into a cell line which expresses high levels of gag/pol with 10 μg of DNA from which a VSV-g protein is expressed. The resultant vector, containing VSV-g protein, is produced transiently in the co-transfected cells. Two days after transfection, cell free supematants are added to prospective xenotropic packaging cell lines (which express gag, pol, and env). Cell free supematants are then collected from the confluent monolayers and titered by PCR. Cell clones producing the highest titers are selected as packaging cell lines This procedure is sometimes referred to "G-hopping " VII Alternative Viral Vector Packaging Techniques Several additional alternative systems can be used to produce recombinant retrovims particles carrying a vector constmct according to the invention. Some of these systems take advantage ofthe fact that the insect vims, baculoviras, and the mammalian viruses, vaccinia and adenovirus, have been adapted to make large amounts of any given protein for which the corresponding gene has been cloned. For example, see Smith, et al. (Mol. Cell. Biol 3:12, 1983), Piccini, et al. (Meth. Enzymology, 153:545, 1987); and Mansour, et al. (Proc. Natl. Acad. Sci. USA 82.1359, 1985). These and similar viral vectors can be used to produce proteins in tissue culture cells by insertion of appropriate genes and, hence, could be adapted to make retroviral vector particles Adenovirus vectors are derived from nuclear replicating viruses and can be defective Genes can be inserted into vectors and used to express proteins in mammalian cells either by in vitro constmction (Ballay, et al, EMBO J. 4:3861, 1985) or by recombination in cells (Thummel, et al, J. Mol. Appl. Genetics 7 435, 1982) One preferred method is to constmct plasmids using the adenovirus Major Late Promoter (MLP) driving (1) gag/pol, (2) env, (3) a modified viral vector constmct. A modified viral vector constmct is possible because the U3 region ofthe 5' LTR, which contains the viral vector promoter, can be replaced by other promoter sequences (see, for example, Hartman, Nucl. Acids Res. 76:9345, 1988). This portion will be replaced after one round of reverse transcriptase by the U3 from the 3 * LTR. These plasmids can then be used to make adenovirus genomes n vitro (Ballay, et al. , supra), which are then transfected into 293 cells (a human cell line making adenovirus El A protein), for which the adenoviral vectors are defective, to yield pure stocks of gag/pol, env and retroviral vector carried separately in defective adenovirus vectors Since the titers of such vectors are typically Hfl-lO^lπά, these stocks can be used to infect tissue culture cells simultaneously at high multiplicity The cells will then be programmed to produce retroviral proteins and retroviral vector genomes at high levels Since the adenovirus vectors are defective, no large amounts of direct cell lysis will occur and retroviral vectors can be harvested from the cell supematants Other viral vectors such as those derived from unrelated retroviral vectors (e.g., RSV, MMTV or HJV) can be used in the same manner to generate vectors from primary cells In one embodiment, these adenoviral vectors are used in conjunction with primary cells, giving πse to retroviral vector preparations from primary cells Another alternative for making recombinant xenotropic retroviral particles is an in vitro packaging system. For example, such a system can be employ the following components: 1. gag/pol and env proteins made in the baculovirus system in a similar manner as described in Smith, et al, supra, or in other protein production systems, such as yeast or£. coli), 2. vector constmcts made using T7 or SP6 transcription systems or other suitable in vitro RNA-generating system (see, for example, Flamant and Sorge, J. Virol 62:1827, 1988); 3. tRNA made as in (2) or purified from yeast or mammalian cells; 4. liposomes (preferably with embedded env protein); and 5. cell extract or purified components (typically from mouse cells) to provide env processing, and any or other necessary cell-derived functions. Within this procedure, the components of (1), (2), and (3) are mixed. The env protein, cell extract and pre-liposome mix (in a suitable solvent) is then added. In a preferred embodiment, the env protein is embedded in the liposomes prior to adding the resulting liposome-embedded env to the mixture of (1), (2), and (3). The mix is treated (e.g., by sonication, temperature manipulation, or rotary dialysis) to allow encapsidation ofthe nascent viral particles with lipid plus embedded env protein in a manner similar to that for liposome encapsidation of pharmaceuticals, as described in Gould-Fogerite, et al, Anal. Biochem. 148:15, 1985). This procedure allows the production of high titers of replication incompetent recombinant retrovimses without contamination with pathogenic retrovimses or replication-competent retrovimses. D. Detection of Replication Competent Retrovimses (RCR) The propensity ofthe packaging cells described above to generate replication competent retrovims may be stringently tested by a variety of methods, two of which are described below. i. The Extended S + L' Assay The extended S + L * assay determines whether replication competent, infectious vims is present in the supernatant ofthe cell line of interest. The assay is based on the empirical observation that infectious retrovimses generate foci on the indicator cell line MiCl] (ATCC No. CCL 64 1) The MiCl] cell line is derived from the MvlLu mink cell line (ATCC No CCL 64) by transduction with Murine Sarcoma Vims (MSV). It is a non-producer, non- transformed, revertant clone containing a replication defective murine sarcoma provirus, S + , but not a replication competent murine leukemia provirus, L". Infection of MiCl i cells with replication competent retrovims "activates" the MSV genome to trigger "transformation" which results in foci formation. Supernatant is removed from the cell line to be tested for presence of replication competent retrovims and passed through a 0.45 μm filter to remove any cells. On day 1, MvlLu cells are seeded at 1.0 x 10 5 cells per well (one well per sample to be tested) on a 6 well plate in 2 mL Dulbecco's Modified Eagle Medium (DMEM), 10% FBS and 8 μg/mL polybrene. MvlLu cells are plated in the same manner for positive and negative controls on separate 6 well plates. The cells are incubated overnight at 37°C, 10% CO2. On day 2, 1.0 mL of test supernatant is added to the MvlLu cells. The negative control plates are incubated with 1.0 mL of media. The positive control consists of three dilutions (200 focus forming units (ffiα), 20 ffu and 2 ffu each in 1.0 mL media) of MA vims (Miller, et al. , Molec. and Cell Biol, 5:431, 1985) which is added to the cells in the positive control wells. The cells are incubated overnight. On day 3, the media is aspirated and 3.0 mL of fresh DMEM and 10% FBS is added to the cells. The cells are allowed to grow to confluency and are split 1 : 10 on day 6 and day 10, amplifying any replication competent retrovims. On day 13, the media on the MvlLu cells is aspirated and 2.0 mL DMEM and 10% FBS is added to the cells. In addition, the MiCli cells are seeded at 1.0 x 10 5 cells per well in 2.0 mL DMEM, 10% FBS and 8 μg/mL polybrene. On day 14, the supernatant from the MvlLu cells is transferred to the corresponding well ofthe MiCli cells and incubated overnight at 37°C, 10% CO2. On day 15, the media is aspirated and 3.0 mL of fresh DMEM and 10% FBS is added to the cells. On day 21, the cells are examined for focus formation (appearing as clustered, retractile cells that overgrow the monolayer and remain attached) on the monolayer of cells. The test article is determined to be contaminated with replication competent retrovims if foci appear on the MiCli cells. Cocultivation of Producer Lines and MdH Marker Rescue Assay As an alternate method to test for the presence of RCR in a retroviral particle producing cell line, producer cells are cocultivated with an equivalent number of Mus dunni cells (NTH NIAJJJ Bethesda, MD). Small scale co-cultivations are performed by mixing of 5 0 x 10 5 Mus dunni cells with 5.0 x 10 5 producer cells and seeding the mixture into 10 cm plates (10 mL standard culture media/plate, 4 μg/mL polybrene) at day 0 Every 3-4 days the cultures are split at a 1:10 ratio and 5.0 x 10 5 Mus dunni cells are added to each culture plate to effectively dilute out the producer cell line and provide maximum amplification of RCR. On day 14, culture supematants are harvested, passed through a 0.45 μm cellulose- acetate filter, and tested in the MdH marker rescue assay. Large scale co-cultivations are performed by seeding a mixture of 1.0 x 10 8 Mus dunni cells and 1.0 x 10 8 producer cells into a total of twenty T-150 flasks (30 mL standard culture media flask, 4 μg mL polybrene). Cultures are split at a ratio of 1 : 10 on days 3, 6, and 13 and at a ratio of 1 :20 on day 9. On day 15, the final supematants are harvested, filtered and a portion of each is tested in the MdH marker rescue assay. The MdH marker rescue cell line is cloned from a pool of Mus dunni cells transduced with LHL, a retroviral vector encoding the hygromycin B resistance gene (Palmer, et al, Proc. Nat'l. Acad. Sci. USA, 84:1055, 1987). The retroviral vector can be rescued from MdH cells upon infection ofthe cells with RCR. One mL of test sample is added to a well of a 6-well plate containing 1 x 10 5 MdH cells in 2 mL standard culture medium (DMEM with 10% FBS, 1% 200 mM L-glutamine, 1% non-essential amino acids) containing 4 μg/mL polybrene. Media is replaced after 24 hours with standard culture medium without polybrene. Two days later, the entire volume of MdH culture supernatant is passed through a 0.45 μm cellulose-acetate filter and transferred to a well ofa 6-well plate containing 5.0 x 10* Mus dunni target cells in 2 mL standard culture medium containing polybrene. After 24 hours, supematants are replaced with standard culture media containing 250 μg/mL of hygromycin B and subsequently replaced on days 2 and 5 with media containing 200 μg/mL of hygromycin B. Colonies resistant to hygromycin B appear and are visualized on day 9 post-selection, by staining with 0.2% Coomassie blue. EXAMPLE 5 Production of Recombinant Retroviral Particles The production of recombinant xenotropic retroviral particles carrying vector constmcts according to the invention, representative examples of which are described above, from the human xenotropic and canine xenotropic packaging cell lines HX and DX, respectively, is described below. A Transient Plasmid DNA Transfection of Packaging Cell Lines HX and DX The packaging cell line HX or DX is seeded at 5.0 x 10 5 cells on a 10 cm tissue culture dish on day 1 with DMEM and 10% fetal bovine semm (FBS). On day 2, the media is replaced with 5.0 mL fresh media 4 hours prior to transfection. Standard calcium pbewphate-DNA co-precipitations are performed by mixing 40.0 μl 2.5 M CaCtø, 10 μg of the plasmid encoding the vector to be packaged, and deionized H2O to a total volume of 400 μl. The DNA-CaC solutions are then added dropwise with constant agitation to 400 μl of precipitation buffer (50 mM HEPES-NaOH, pH 7.1; 0.25 M NaCl and 1.5 mM Na 2 HPO4- NaH2Pθ4). These mixtures are incubated at room temperature for 10 minutes. The resultant fine precipitates are added to different culture dishes of cells. The cells are incubated with the DNA precipitate overnight at 37°C. On day 3, the media is aspirated and fresh media is added. Supematants are removed on day 4, passed through 0.45 μm filters, and stored at -80°C. B. Packaging Cell Line Transduction DX or HX packaging cells are seeded at 1.0 x 10 5 cells/3 cm tissue culture dish in 2 mL DMEM and 10% FBS, 4 μg/mL polybrene (Sigma, St. Louis, MO) on day 1. On day 2, 3.0 mL, 1.0 mL and 0.2 mL of each ofa freshly collected supernatant containing VSV-g pseudotyped retroviral particles carrying the desired vector are added to the HX cells. The cells are incubated overnight at 37°C. On day 3, the pools of cells are cloned by limiting dilution by removing the cells from the plate and counting the cell suspension, diluting the cells suspension down to 10 cells/mL and adding 0.1 mL to each well (1 cell/well) of a 96 well plate (Corning, Corning, NY). Cells are incubated for 14 days at 37°C, 10% CO 2 . Several clones producing the desired recombinant xenotropic retrovims are selected and expanded up to 24 well plates, 6 well plates, and finally to 10 cm plates, at which time the clones are assayed for expression ofthe appropriate retroviral vector and the supematants are collected and assayed for retroviral titer. Using the procedures above, DX and HX cell lines may be derived that produce recombinant xenotropic retroviral vectors with titers greater than or equal to 1 x 10 6 cfu/ L in culture. SUBSTTTUTΈ SHEET (RULE 26) C. Titer Assays Normally vector titers are determined by transduction of target cells such as HT1080, with appropriate dilutions ofa vector preparation, followed by antibiotic selection and counting of surviving colonies (WO 91/02805). However, recombinant retroviral vectors carrying a desired vector constmct may not include a gene OTdrm ϋTa setectabie marker, as may be the case when the vector constmct encodes a large gene of interest, for instance, full length factor VIII, titering assays other than those based on selection of dmg resistant colonies are required. To this end, antibody and PCR assays, the latter of which is described below, may be employed to determine retroviral vector titer, i.e., the number of infectious particles comprising the retroviral vectors ofthe invention. While such a PCR assay may be required in the context ofa vector lacking a selectable marker, it is understood that such an assay can be employed for any given vector. To use PCR to amplify sequences unique to the retroviral vectors ofthe invention, various primers are required. Such primers can readily be designed by those skilled in the art and will depend on the retroviral vector backbone employed and the components thereof, the particular region(s) desired to be amplified, etc. Representative examples of particular primer pairs include those specific for LTR sequences, packaging signal sequences or other regions ofthe retroviral backbone, and also include primers specific for the gene of interest in the vector. Additional advantages in using such a PCR titering assay include the ability to assay for genome rearrangement, etc. In the practice ofthe present invention, the PCR titering assay is performed by growing a known number of HT 1080 cells, typically 1 x 10 5 cells, transduced with a retroviral vector capable of directing expression ofthe gene of interest on 6-well plates for at least 16 hr. before harvest. The retroviral vectors used for these transductions are preferably obtained from cell culture supematants. One well per plate is reserved for cell counting. Cells from the other wells are lysed and their contents isolated. DNA is prepared using a QIAmp Blood Kit for blood and cell culture PCR (QIAGEN, Inc., Chatsworth, CA). DNAs are resuspended at 5 x 10 6 cell equivalents/mL, where one cell equivalent is equal to the DNA content of one cell . To calculate titer, a standard curve is generated using DNA isolated from untransduced HT1080 cells (negative control) and HT1080 cells transduced with a known vector and having one copy of that vector per cell genome (positive control), such as may be prepared from packaging cell lines transduced with a retroviral vector encoding a selectable marker, e.g., neomycin resistance. For both the positive and negative controls, DNA is resuspended at 5 x 10 6 cell equivalents/mL. The standard curve is generated by combining different amounts ofthe positive and negative control DNA, while keeping the total amount of DNA constant, and ampUfying specific sequences therefrom by PCR using primers specific to a particular region ofthe retroviral vector. A representative group of mixtures for generating a standard curve is: Tube Positive Control (μL) Negative Control (μL) Distilled water (μL) 5.0 μL from each tube is placed into one of eight reaction tubes (duplicates are also prepared), with the remainder being stored at -20°C. 5.0 μL from each sample DNA preparation are placed into their own reaction tubes in duplicate. PCR reactions (50 μL total volume) are then initiated by adding 45.0 μL ofa reaction mix containing the following components per tube to be tested: 24.5 μL water, 5 μL 10X reaction PCR buffer, 4 μL of 25 mM MgCl 2 , 4 μL dNTPs (containing 2.5 mM of each of dATP, dGTP, dCTP, and dTTP), 5 μL of primer mix (100 ng or each primer), 0.25 μL Taq Start monoclonal antibody (Clontech Laboratories, Inc., Palo Alto, CA), 1.00 μL Taq Start buffer (Clontech Labs, Inc.), and 0.25 μL AmpliTaq DNA polymerase (Perkin-Elmer, Inc., Norwalk, CN). Just prior to aliquoting the reaction mix to the reaction tubes, 1 μL of a- 32 P dCTP (250 μCi; 3000 C/mmol, 10 mCi/mL, Amersham Corp., Arlington Heights, JL) is added into the reaction mix. After aliquoting 45.0 μL the reaction mix into each ofthe reaction tubes, the tubes are capped and placed into a thermocycler. The particular denaturation, annealing, elongation times and temperatures, and number of thermocycles will vary depending on size and nucleotide composition ofthe primer pair used. 20 to 25 amplification thermocycles are then performed. 5 μL of each reaction is then spotted on DE81 ion exchange chromatography paper (Whatman, Maidstone, England) and air dried for 10 min. The filter is then washed five times, 100 mL per wash, in 50 mM Na 2 PO 4 , pH 7, 200 mM NaCl, after which it is air dried and then sandwiched in Saran Wrap. Quantitation is performed on a Phospholmager SI (Molecular Dynamics, Sunnyvale, CA). Filters are typically exposed to a phosphor screen, which stores energy from ionizing radiation, for a suitable period, typically about 120 min. After exposure, the phosphor screen is scanned, whereby light is emitted in proportion to the radioactivity on the original filter The scanning results are then downloaded and plotted on a log scale as cpm (ordinate) versus percent positive control DNA (abscissa) Titers (infectious units/mL) for each sample are calculated by multiplying the number of cells from which DNA was isolated by the percentage (converted to decimal form) determined from the standard curve based on the detected radioactivity, divided by the volume of retroviral vector used to transduce the cells. As will be appreciated by those in the art, other methods of detection, such as colorimetric methods, may be employed to label the amplified products. EXAMPLE 6 Large Scale Production of Recombinant Xenotropic Retrovimses The recombinant retrovimses ofthe invention can be cultivated in a variety of modes, such as in a batch or continuous mode. In addition, various cell culture technologies can be employed to produce commercial scale quantities ofthe recombinant retrovimses according to the invention. Several such techniques are described below, although others known to those in the art may likewise be employed. A. Recombinant Retrovims Production From Hollow Fiber Cultures i. Culture Initiation To initiate a hollow fiber culture, the hollow fiber bioreactor (e.g., HFB; Cellco, Inc., Germantown, MD) is first conditioned for 48 hours prior to seeding by simulating a mn condition with 100-200 mL of complete growth media at 37°C. The growth media preferably is that to which the cell line has been adapted. All liquids in the HFB when originally shipped should be aspirated and replaced with the complete growth media. When seeding the bioreactor, the cells should not have been split more than 48 hours earlier and should be in log growth phase at the time of harvest for the seeding ofthe HFB. The cells typically are harvested by trypsinazation and pelleted by centrifugation. The cell pellet is then resuspended in 4 mL of 25% pre-conditioned media and delivered to the extra-capillary space by syringe using the side syringe ports found on the HFB. After seeding the HFB, the cells are allowed to adhere for 20 to 30 minutes before starting the circulation pump During this time, the media used to condition the HFB is replaced with 100-200 mL of 25% pre¬ conditioned media. The circulation feed pump is initiated with the starting flow rate set at 25 mL/min. (setting 5 with 2 long pump pins). After 1 hour from the time of switching the SUBSTHUTE SHEET RULE 26 pump on, a one mL sample of media is collected in order to record the initial levels of lactate and ammonia. On a daily schedule, 1 ml samples are collected every 24 hours to assay for the daily production of lactate and ammonia. The initial 100-200 mL of media is exchanged with fresh media when lactate levels begin to reach 2.0 g/L (or the equivalent to 22 mM/L). The same volume of media is replaced until the culture approaches daily levels of 20 mmol/L When daily levels of lactate reach 20 mmol L, the size of the reservoir bottle is increased to a 500 mL bottle containing 500 mL of fresh media. The flow feed rate is then increased to 50 mL/min. when the culture begins to produce 2.2 mmol/day of lactate. When daily 500 mL volumes reach 20 mmol/L of lactate, the original Cellco supplied reservoir feeding cap is exchanged for a larger reservoir cap (Unisyn-vender part #240820) adapted for the Cellco system with the addition of tubing and male luer lock fittings. This reservoir cap will accommodate 2 liter Corning bottles. (To avoid the exchange of reservoir caps during a culture mn, initiate the mn with a large reservoir cap which can also support smaller bottle sizes.) When daily lactate readings are assayed and recorded, the daily levels of lactate production ofthe culture can be used to determine when the culture reaches maximum cell density, .e., when the rate of lactate decreases and levels off. ii. Seeding Density for the 2X-β-gal To establish specific seeding requirements, two hollow fiber mns are performed, one mn seeded with a low number of cells, the other seeded with a high number of cells. Progress of each culture is tracked by analyzing the daily glucose consumption and lactate production levels. In this experiment, one HFB was seeded with 1.3 x 10 7 cells (representing the low seed culture), the other with 1.6 x 10 8 cells. Here, the cell line 2x-β-GALi 7 -i 4 was able to initiate a good hollow fiber mn under both seeding conditions. Initiating a mn with fewer cells is primarily convenient for reducing the effort required for generating the number of cells required to start a culture, although fewer cells initially extends the time it takes to reach optimal cell densities, which usually yield the highest titers. 2x-β-GAL 17.14 adapted well to hollow fiber culture, eventually requiring daily media changes of 500 mL in order to avoid accumulation of toxic levels of lactate. Plateauing of daily lactate production and drops in peak titer production correlated with maximum cell densities and the relative health ofthe culture iii Harvest Amounts β-gal titers for the above experiment were determined from frozen samples on 293 cells assayed 48 or 72 hours after transduction. The transduced cells were stained for β -gal activity and counted on a hemocytometer to yield a titer based on the number of blue cells /mL (BCT/mL) . Optimum titers were generally obtained on day 7 of a* high * seed" rfttm? at 1.8 xlO 8 BCT/mL from a 72 hour blue cell titer on 293 cells. A duplicate culture initially seeded at a 10 fold lower seeding density peaked at 5.2 xlO 7 BCT/mL from a 48 hour blue cell titer. Compared to flat stock cultures (from tissue culture dishes or flasks) titered using 48 hour blue cell titers on HT 1080 cells (calculated to be about 5 x 10 6 BCT/mL), the increase in titer by using hollow fiber systems is approximately ten fold higher. These maximum titers observed were reached prior to hitting 20 mmol L lactate levels, which appeared to reduce titers produced the following week. Cmde supematants can be harvested every 9 hours with out any loss of titer and three harvests per day should be possible with minimum titre loss. In addition, continuous hollow fiber cultures can be maintained for several weeks. When titers were compared between the low and the high seed culture, there was little differences by day 11 between the two seed cultures, both of which averaged 4 x 10 7 BCT/mL. EXAMPLE 7 Two-Phase Purification of Recombinant Retrovimses A. Concentration of DA/ND-7 recombinant particles 1400 ml of media (DMEM containing 5% Fetal Bovine Semm) containing DX/ND-7 vector at a titer of 1.25 x 10 6 cfu/ml is used as starting material. Three hundred milliliters of two-phase partitioning components (PEG-8000 (autoclaved), dextran-sulfate, and NaCl) are added to a final concentration of 6.5% PEG, 0.4% dextran-sulphate, and 0.3 M NaCl. The resultant solution is placed into a two-liter separatory funnel, and left in a cold room for 24 hours (including two mixing steps approximately 6 to 16 hours apart). Following the 24 hour period, the bottom layer (approximately 20 mL) is carefully eluted, and the interphase (approximately 1 mL) is collected in a 15 mL conical FALCON tube The interphase containing vector is diluted to 10 mL by addition of PBS, and incubated at 37°C in order to bring the solution to room temperature and destabilize the micelles. To one-half of the diluted interphase, KCl is added to a final concentration 0.4 M, and mixed well. The tube is then placed on ice for ten minutes, and spun for 2 minutes at 2,000 rpm in a bench-top centrifuge. The supernatant is removed and filtered through a 0.45 μm syringe filter. The other half of the interphase containing vector is separated by S-500 Sephadex chromatography in IX PBS. The results of these concentration processes, as determined in a BCFU assay, are shown below in Table 6: TABLE 6 PHASE QUANTITY OF VECTOR Cmde 1.75 x lO 9 bcfu Separation: Top phase 1.4 x lO 8 bcfu Separation: Interphase 7(+/-3) x 10** bcfii Separation: Bottom phase 2 x lθ6 bcfu Final step: KCl separation *6(+/-3) x 10 8 bcfu Final step: S-500 separation * 1.8(+/-0.3) x 10 8 bcfu * Note that since the sample was split into two halves, that these numbers were doubled in order to represent the level of purification that would be expected if the entire 1 mL interphase was separated as indicated. In summary, 1.4 liters of cmde research grade supernatant containing recombinant retroviral particles may be reduced to a 10 mL volume, with approximately 50% (+/-20%) being recovered when KCl separation is utilized as the final step. When S-500 chromatography is utilized as the final step, only about 10% ofthe initial recombinant retroviral particles are recovered in a 14 mL. In order to complete concentration ofthe retroviral vector particles, the vector- containing solution may be further subjected to concentration utilizing an MY-membrane Amicon filter, thereby reducing the volume from 10 to 14 mL, down to less than 1 mL EXAMPLE 8 Production of Vector from DX/ND7 β-gal Clone 87 Utilizing a Cell Factory DX/ND7 bgal clone 87, an expression vector, was grown in cell factories. Cells were grown in DMEM supplemented with Fetal Bovine Serum r roller bσtttes απtrl enough ceils to seed 20 10-layer cell factories (NUNC) at a 1:3 dilution were obtained. Each 10-layer cell factory is seeded with approximately 0.8 liters of cell medium. Cells were seeded into the cell factory by pouring media containing cells into the factory so that the suspensions evenly fill the 10 layers. The factory is then carefully tilted away from the port side to prevent the suspension from redistribution in the common tube. Finally, the cell factory is rotated into its final upright position. A hepavent filter is attached to each port. The factory was then placed in a CO 2 incubator. In three days, and for each ofthe next three days, supernatant containing vector was harvested. The cell factory is placed in a tissue culture hood. One filter is removed and sterile transfer tubing is connected to the open port. The factory is lifted so that supernatant drains into the tubing. Approximately 2 liters of supernatant is harvested from each factory. Fresh DMEM/FBS is used to replenish the lost medium. The transfer tubing is removed and the factory replaced in the incubator. From 20 cell factories, approximately 90 liters of cmde vector containing supernatant were obtained. Verification ofthe vector was performed by transduction of HT 1080 cells. These cells were harvested 2 days later and stained for b-gal protein. The titer ofthe supernatant was determined to be 2 x 10 7 /ml. EXAMPLE 9 Concentration of Recombinant Retrovims by Low-Speed Centrifugation A Retrovector Supernatant Preparation Producer cell lines DA/βgal and HX/DN-7 were cultured in a culture flask and a roller bottle, respectively, containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine semm plus lmM L-Glutamine, Sodium pymvate, non- essential amino acids and antibiotics Viral supernatant was harvested from the flask and roller bottle, and were filtered through a 0.45 urn syringe filter. The filtered supematants were stored either at 4°C (HX/ND7), or frozen at -70°C (DAβ-gal). B. Vims Concentration Viral supernatant was aliquoted into 50 ml sterile OAKRBDGE screw cap tubes, and placed into an SS34 rotor for use in a Sorvall centrifuge. The tubes were spun for 1 hour at 16,000 φm (25,000g-force) at 4°C. Upon completion ofthe spin, the tubes were removed, the supernatant decanted and a small opaque pellet resuspended in the DMEM media described above. C Vims Titration Concentrated vims was titered on HT1080 cells plated 24 hours earlier at a cell density of 2x10^ cells per well in a six well plate + 4 μg/ml polybrene. Briefly, vims preps were diluted from 1/10 to 1/10,000 and 50 μl of each dilution was used to infect one well from the six well plate. Plates were incubated overnight at 37°C. Forty-eight hours later, cells were fixed and stained with X-gal. The results are set forth below in Table 7. Table 7. Vims Concentration through Low Speed Centrifugation Experiment number Parameter description 1 Virus source DAβ-gal DAβ-gal HX/ND7 DAβ-gal HX/ND7 Titer of normal harvest 4.4 10 ' 2.1 x 10 ' 3.2 x 10 5x106 5x105 Titer of vims concentrate 6xl0 8 7.4xl0 7 3.2xl0 7 2.9x108 3.9x107 As is evident from Table 7, vims recovery ranged from 30% to 99%, with the best recovery being obtained from human producer cells (HX/ND7; recovery ranged from 91% to 99%). EXAMPLE 10 Concentration of Recombinant Retrovimses By Ultrafiltration S-500 purified supernatant containing the β-gal expressing recombinant retrovims DX/CB-bgal and partially concentrated supernatant containing the same vims were each filtered through a 0.45 urn filter, and loaded into a CENTRIPREP-100 filter (product #4308, Amicon, MA). The supematants were kept at a temperature of 4°C throughout this procedure, including during centrifugation. The CENTRTPREP filters were spun three times each for 45 to 60 minutes at 500 x G. Between each spin the filtrate was decanted. The retentate was thus sequentially reduced, such that the initial 15 mL (or 10 mL) volume was reduced to approximately 0.6 mL per unit. The resultant titer was determined by assaying HT1080 target cells set up at a concentration of 1 x 10 cells per well 24 hours prior to transduction ofthe viral sample. Cells were transduced in the presence of 8 μg/ml polybrene and 2 mL growth media (DMEM plus 10% FBS) per well. As shown in Table 8 below, approximately one hundred percent of the vims was recovered utilizing this procedure (note that titers are in BCFU/ml). Table 8 EXAMPLE 11 Preparation of Recombinant Retrovims in a Bioreactor Freezing protocol Producer cells are frozen in DMEM media containing 10% to 20% FBS, and 5 to 15% DMSO, at a concentration of 1 x 10 7 cells/ml/vial. Cells are frozen in a controlled rate freezer (Series PC, Controlled Rate Freezing System, Custom Biogenic Systems, Warren MI) at a rate of from 1 to 10°C per minute. Frozen cells are stored in liquid nitrogen. B. Bioreactor protocol Cells are thawed from frozen vials at 37°C, washed once with media to remove DMSO, and expanded into 850 cm 2 "FALCON" roller bottles (Corning, Corning, NY) Expanded cell culture is used to inoculate a "CELLIGEN PLUS" bioreactor (5 liter working volume; New Bmnswick, Edison, NJ). The cells are grown on microcarriers (i.e., Cytodex 1 or Cytodex 2; Pharmacia, Piscataway, N.J.) at a concentration of 3 to 15 g/L microcarrier. Initial inoculation densities are from 4 to 9 cells/bead at half to full volume for 2 to 24 hours. The media constituents for vims production are DMEM-high glucose (Irvine Scientific, Santa Ana, CA.) basal media supplemented with FBS (10 to 20%), Glutamine (8 to 15mM), glucose (4.5 to 6.5 g/L), Nonessential amino acids (IX), RPMI 1640 amino acids ( 0.2 to 9.6X), 10 mM HEPES, RPMI 1640 Vitamins (0.2 to 5X). During culture, pH (6.9 to 7.6) and dissolved oxygen ("DO" 5 to 90%) are controlled by the use ofa four gas system which includes air, oxygen, nitrogen, and carbon dioxide. After several days of batch growth the culture is then continuously perfused with fresh media with concurrent continuous harvesting in an escalating perfusion rate of 0.5 to 2.5 volumes/day. Cell retention is the result of differential sedimentation of cell covered beads in a decanting column. During operation the bioreactor is monitored for viable cells, titer, glucose, lactate, ammonia levels, and lack of contamination. Viable cells and titer range from 1 x 10 cells/ml to 1 x 10 cells/ml. Glucose ranges from 6 to 0.25 g/L, Lactate from 1 to 25 mM, and Ammonia ranges from 0.5 to 30 mM. Cells are incubated in the bioreactor for 5 to 25 days EXAMPLE 12 Cell sorting and analysis Apheresed samples were obtained with informed consent from multiple myeloma patients treated at the University of Arkansas Medical Center. The patients were treated on day 1 with cyclophosphamide at 6 g/m 2 (1.5 g/m 2 every 3 hours x 4 doses) From day 1 until the start of leukopheresis (usually 10-28 days), granulocyte macrophage colony stimulating factor (GM-CSF) was given at 0.25 mg/m 2 /day. Apheresis for total white cells was started when the peripheral blood white cell count was greater than 500 cells/ml and the platelet count was greater than 50,000 cells/ml. Patients were apheresed daily until from 6 x 10 mononuclear cells (MNC) were collected. Antibodies to CD 14 and CD 15 were obtained as FITC conjugates from Becton- Diekinson. Antibody to Thy-1 (GM201) was obtained from Dr. Wolfgang Rettrg (Lσdwig Institute, New York), and was detected with anti-γl-PE conjugate from Caltag. Antibody to CD34 (Tϋk 3) was obtained from Dr. Andreas Ziegler (University of Berlin), and detected with an anti-γ3 -Texas Red conjugate (Southern Biotechnologies). For cell sorting, fresh MPB samples were elutriated with a JE5.0 Beckman counterflow elutriator equipped with a Sanderson chamber (Beckman, Palo Alto, CA). Cells were resuspended in elutriation medium (Biowhittaker, Walkersviile, MD) at pH 7.2, supplemented with 0.5% human semm albumin (HSA). The rotor speed was set at 2000 RPM the cells were introduced, and the first fraction collected at a flow rate of 9.6 ml/min. Fractions 2 and 3 were collected at the respective flow rates of 14 and 16 ml/min. The larger cells remaining in the chamber were collected after stopping the rotor. Cells were resuspended in RPMI supplemented with 5% HSA, 10 μg/ml DNAse I and penicillin/streptomycin at 50 U/ml and 50 μg/ml, respectively. Fractions 2 and 3 were pooled and incubated with 1 mg/ml heat-inactivated human gamma-globulin to block non- specific Fc binding. Granulocytes were further depleted by incubation with CDI 5 conjugated to magnetic beads (Dynal M450, Oslo, Norway) followed by magnetic selection Anti-CD34 antibody or an IgG3 isotype matched control were added to cells in staining buffer (HBSS, 2% FCS, 10 mM HEPES) for 20 minutes on ice, together with anti- Thy-1 antibody at 5 mg/ml. Cells were washed with a FCS underlay, and then incubated with Texas Red conjugated goat anti-mouse IgG3 antibody and phycoerythrin-conjugated goat anti-mouse IgGl antibody for 20 minutes on ice. Blocking IgGl was then added for 10 minutes. After blocking, the FITC-conjugated lineage antibody panel (CD 14 and CDI 5) was added, and incubated for another 20 minutes on ice. After a final washing, cells were resuspended in staining buffer containing propidium iodide (PI). Cells were sorted either on the FACSTAR Plus cell sorter equipped with dual argon ion lasers, the primary laser emitting at 488 nm and a dye laser (Rhodamine 6G) emitting at 600 nm (Coherent Innova 90, Santa Cmz, CA) or on a high speed cell sorter as described in PCT patent application number PCT/US93/08205.. Residual erythrocytes, debris and dead cells were excluded by light scatter gating plus an FL3 (PI) low gate. Following isolation of a cell population by flow cytometry, the sample was diluted 1:1 in HBSS, pelleted, and resuspended in HBSS for hemocytometer counting. EXAMPLE 13 Transduction 1 x 10 CD34 + Thy + Lin ' (MPB) viable cells obtained as described above, were suspended in 1 ml of freshly thawed retroviral supernatant with cytokines at the following concentrations: c-kit ligand (Amgen) 100 ng/ml; JL-3 (Sandoz) 25 ng/ml; JL-6 (Sandoz) 50 ng/ml. Protamine sulfate was added at a final concentration of 4 ug/ml. At 24 and 48 hours, supernatant was replaced with freshly thawed retroviral supernatant. Cytokines and protamine sulfate were added at the concentrations listed above. After 72 hours, cells were harvested and placed in assays to determine transduction frequency. As a control, cells were cultured DMEM with cytokines and protamine sulfate as described above, but without retroviral supernatant. EXAMPLE 14 Methylcellulose assay In order to determine transduction frequency ofthe stem cells, the following experiment was performed. 5 x 10 3 or 2.5 x 10 3 cells from each transduction were added to 5 ml of methylcellulose (Stem Cell Technologies) containing the following cytokines: c-kit ligand 10 ng/ml; GM-CSF 25 ng/ml; G-CSF 25 ng/ml; JL-3 10 ng/ml; rhEPO 2 units/ml. 1.1 ml ofthe cell/cytokine methylcellulose mixture was plated onto four 3 cm gridded plates using a 5 ml syringe and 16.5 gauge needle, and the plates were placed in a 37°C incubator for 2 weeks. After 14 days, single methylcellulose colonies were picked and suspended in 50 μl Lysing Buffer (75 mM KCl, 10 mM Tris-HCl pH 9.25, 1.5 mM MgCl 2 , 0.5% Tween 20, 0.5% NP40, 1 mg/ml proteinase K) for PCR analysis. The lysates were amplified by PCR to determine the presence ofthe vector in the transduced cells. The PCR assay amplified a 134 bp fragment ofthe vector psi packaging sequence. A 40 cycle amplification (25 ul total volume) in Perkin-Elmer 9600 Cycler using 10 ul lysate and 0.20 uM each FI and B5 primers (Genset) was performed as follows: one cycle 95°C 30 sec; 40 cycles 95°C 10 sec, 64°C 15 sec, 72°C 15 sec; final extension 72°C 5 min. PCR products were visualized on ethidium bromide agarose gels. The results appear in Table 9. TABLE 9 Example 15 LTCIC Transduction Assay CD34+Thy+Lin- (MPB) viable cells were counted and transduced for 72 hours as described in Example 13 herein Viable cells seeded in 2-fold serial dilutions in a 96- well plate on pre-formed AC6.21 monolayers in Whitlock-Witte media (50/50 RPMI/IMDM, 10% Fetal Calf Semm, Pen-Strep. L-Glutamine, Sodium Pymvate, and 2-ME) supplemented with LIF at 50 ng/ml and IL-6 at 10 ng/ml. Final cell concentrations, set up in duplicate, were 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 cells/well respectively. The plates were cultured at 37°C, 5% CO 2 for 5 weeks, with the cells fed weekly by replacement of one-half the spent media with fresh media and cytokines. Bulk LTCIC Assay: After 5 weeks, plates were scored for cobblestone area forming cells (CAFC). LTCIC plates were prepared by harvesting wells containing multiple cobblestones and filtering the cultures through Nitex filters into 24-well plates. Cutlures were overlaid with approximately 0.6 mis methylcellulose containing 10 ng/ml c-kit ligand, 25 ng/ml GM-CSF, 25 ng/ml G-CSF, 10 ng/ml H-3, and 1.2 U/ml rhEPO, and cultured for 10-14 days at 37°C, 5% CO 2 (i.e., approximately 7 weeks culture from the time of transduction). Individual methylcellulose colonies were picked and analyzed by PCR for the presence ofthe vector psi packaging sequence as described in Example 14 herein. The results are shown in Table 10 below. The numbers represent the number of bulk LTCIC wells with at least one PCR positive colony/total number of bulk wells analyzed by PCR. The results show that the xenotropic vector did transduce stem cells as measured by LTCIC. The numbers do not represent a transduction frequency of LTCIC since the wells that were harvested contained multiple cobblestone areas, and the resulting methylcellulose colonies could have been derived from different cobblestone area forming cells and hence LTCIC. Clonal LTCIC Assay: The clonal LTCIC assay was performed as described above for the bulk LTCIC assa\ with the exception that individual wells were harvested where, based on limiting dilution, the wells harvested were most likely colonal (>95% probability based on Poisson distribution, i e , <37% ofthe wells at a given dilution contained a CAFC) The results are shown in Table 10 below. The numbers represent the number of clonogenic LTCIC wells with at least one positive colony by PCR/total number of clonal wells analyzed by PCR. The results from one experiment show that the xenotropic vector transduced 2 of 9 LTCIC. Since the cells from each well harvested were derived from a single cell, the results ofthe clonal LTCIC assay represents a tme LTCIC transduction frequency. TABLE 10 * Numbers indicate no. bulk LTCIC wells with at least one psi(+) positive colony/total number of bulk wells anaylzed by PCR. ** Numbers indicate no. clonogenic LTCIC wells with at least one psi(+) positive colony/total number clonal wells analyzed by PCR. While the present invention has been described above both generally and in terms of preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art in light ofthe description, supra. Therefore, it is intended that the appended claims cover all such variations coming within the scope ofthe invention as claimed. Additionally, the publications and other materials cited to illuminate the background of the invention, and in particular, to provide additional details concerning its practice as described in the detailed description and examples, are hereby incorporated by reference in their entirety. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANTS: CHIRON VIAGENE, INC. SYSTEMIX (ii) TITLE OF INVENTION: High Efficiency Ex Vivo Transduction of Hematopoietic Stem Cells by Recombinant Xenotropic Retroviral Preparations (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Chiron Viagene, Inc. (B) STREET: 4560 Horton Street (C) CITY: Emeryville (D) STATE: California (E) COUNTRY: U.S.A. (F) ZIP: 94608 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: Unassigned (B) FILING DATE: 19 April 1996 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Kruse, Norman J. (B) REGISTRATION NUMBER: 35,235 (C) REFERENCE/DOCKET NUMBER: 1156.100 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (510) 601-3520 (B) TELEFAX: (510) 655-3542 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TAATAAATAG ATTTAGATTT A 1 (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GCCTCGAGAC GATGAAATAT ACAAGTTATA TCTTG 35 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAATCGATCC ATTACTGGGA TGCTCTTCGA CCTGG 35 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCCTCGAGCT CGAGCGATGA AATATACAAG TTATATCTTG 40 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GTCATCTCGT TTCTTTTTGT TGCTATT 27 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AATAGCAACA AAAAGAAACG AGATGAC 27 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GCATCGATAT CGATCATTAC TGGGATGCTC TTCGACCTCG 40 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATAAATAGAA GGCCTGATAT G 21 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 baβe pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCCTCGAGAC AATGTACAGG ATGCAACTCC TGTCT 35 (2) INFORMATION FOR SEQ ID NO:10: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GAATCGAXTT ATCAAGTCAG TGTTGAGATG ATGCT 35

Claims: CLAIMS WE CLAIM: 1. A method of producing transduced hematopoietic stem cells, the method comprising: (a) obtaining a population of hematopoietic stem cells from a patient; and (b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovims, wherein the recombinant retroviral particles carry a vector constmct encoding a gene of interest. 2. The method of Claim 1 wherein the gene of interest encodes a protein or an active portion of a protein selected from the group consisting ofa cytokine, a colony stimulating factor, a clotting factor, and a hormone. 3. The method of claim 1 wherein said recombinant retroviral particle is a xenotropic recombinant retroviral particle. 4. The method of claim 1 wherein said hematopoietic stem cell is a CD34+Thy-1+Lin- hematopoietic stem cell. 5. The method of claim 1 wherein said recombinant retroviral vector particles are a high titer preparation of recombinant retroviral particles. 6. A composition comprising a population of hematopoietic stem cells transduced with recombinant retroviral particles substantially free from contamination with replication competent retrovims, wherein the recombinant retroviral particles carry a vector constmct encoding a gene of interest. 7. The composition of Claim 6 wherein the gene of interest encodes a protein or active portion ofa protein selected from the group consisting ofa cytokine, a colony stimulating factor, a clotting factor, and a hormone. 8. The composition of Claim 7 wherein said cytokine is selected from the group consisting of JL-2 and gamma-interferon. 9. The composition of Claim 6 wherein said recombinant retroviral particle is a xenotropic recombinant retroviral vector particle. 10. The composition of Claim 6 wherein said hematopoietic stem cell is a CD34+Thy- 1+Lin- hematopoietic stem cell. 11. The composition of claim 6 wherein said hematopoietic stem cells are transduced with a high titer preparation of hematopoietic stem cells. 12. A hematopoietic stem cell transduced with a recombinant retroviral particle substantially free from contamination with replication competent retrovims, wherein the recombinant retroviral particle carries a vector constmct encoding a gene of interest. 13. The hematopoietic stem cell of Claim 12 wherein the gene of interest constmct encodes a protein or active portion ofa protein selected from the group consisting ofa cytokine, a colony stimulating factor, a clotting factor, and a hormone. 14. The hematopoietic stem cell of claim 12 wherein said hematopoietic stem cell is a CD34+Thy-1+Lin- hematopoietic stem cell. 15 The hematopoietic stem cell of claim 12 wherein said recombinant retroviral vector particle is a xenotropic hematopoietic stem cell. 16 The hematopoietic stem cell of claim 15 wherein said hematopoietic stem cell is transduced by a high titer preparation of recombinant retroviral particles. 17. A method of treating a patient having a genetic disease, the method comprising: (a) obtaining a population of lieinaluporetic stem cells ft urn the patient; (b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovims, wherein the recombinant retroviral particles carry a vector constmct encoding a gene of interest useful in treating the genetic disease; and (c) re-introducing into the patient a therapeutically effective amount ofthe population of transduced hematopoietic stem cells. 18 The method of claim 17 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles. 19. The method of claim 17 wherein said hematopoietic stem cells are CD34+Thy-1+Lin- hematopoietic stem cells. 20. The method of claim 17 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles. 21 A method of treating a patient having cancer, the method comprising: (a) obtaining a population of hematopoietic stem cells from the patient; (b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovims, wherein the recombinant retroviral particles carry a vector constmct encoding a gene of interest useful in treating cancer; and (c) re-introducing into the patient a therapeutically effective amount ofthe population of transduced hematopoietic stem cells 22 The method of claim 21 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles. 23. The method of claim 21 wherein said hematopoietic stem cells are CD34+Thy-1+Lin- hematopoietic stem cells. 24. The method of claim 21 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particiw. 25. A method of treating a patient having an infectious disease, the method comprising: (a) obtaining a population of hematopoietic stem cells from the patient; (b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovims, wherein the recombinant retroviral particles carry a vector constmct encoding a gene of interest useful in treating the infectious disease; and (c) re-introducing into the patient a therapeutically effective amount ofthe population of transduced hematopoietic stem cells. 26. The method of claim 25 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles. 27. The method of claim 25 wherein said hematopoietic stem cells are CD34+Thy-1+Lin- hematopoietic stem cells. 28. The method of claim 25 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles. 29. A method of treating a patient having a degenerative disease, the method comprising (a) obtaining a population of hematopoietic stem cells from the patient; (b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovims, wherein the recombinant retroviral particles carry a vector constmct encoding a gene of interest useful in treating the degenerative disease; and (c) re-introducing into the patient a therapeutically effective amount ofthe population of transduced hematopoietic stem cells. 30. The method of claim 29 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles. 31. The method of claim 29 wherein said hematopoietic stem cells are CD34+Thy- 1 +Lin- hewfatopσietic stem cells. 32. The method of claim 29 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles. 33. A method of treating a patient having an inflammatory disease, the method comprising: (a) obtaining a population of hematopoietic stem cells from the patient; (b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovims, wherein the recombinant retroviral particles carry a vector constmct encoding a gene of interest useful in treating the inflammatory disease; and (c) re-introducing into the patient a therapeutically effective amount ofthe population of transduced hematopoietic stem cells. 34. The method of claim 33 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles. 35. The method of claim 33 wherein said hematopoietic stem cells are CD34+Thy-1+Lin- hematopoietic stem cells. 36. The method of claim 33 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles. 37. A method of treating a patient having a cardiovascular disease, the method comprising: (a) obtaining a population of hematopoietic stem cells from the patient; (b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovims, wherein the recombinant retroviral particles carry a vector constmct encoding a gene of interest useful in treating the cardiovascular disease; and (c) re-introducing into the patient a therapeutically effective amount ofthe population of transduced hematopoietic stem cells. 38. The method of claim 37 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles. 39. The method of claim 37 wherein said hematopoietic stem cells are CD34+Thy-1+Lin- hematopoietic stem cells. 40. The method of claim 37 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles. 41. A method of treating a patient having an autoimmune disease, the method comprising: (a) obtaining a population of hematopoietic stem cells from the patient; (b) transducing the population of hematopoietic stem cells with recombinant xenotropic retroviral particles substantially free from contamination with replication competent retrovims, wherein the recombinant xenotropic retroviral particles carry a vector constmct encoding a gene of interest useful in treating the autoimmune disease; and (c) re-introducing into the patient a therapeutically effective amount ofthe population of transduced hematopoietic stem cells. 42. The method of claim 41 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles. 43 The method of claim 41 wherein said hematopoietic stem cells are CD34+Thy-1+Lin- hematopoietic stem cells. 44. The method of claim 41 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles. 45. The method according to any one of claims 17, 21, 25, 29, 33, 37 or 44 further comprising expanding the transduced population of hematopoietic stem cells in vitro prior to re-introdtrction ofthe cells into the patient.

Inventors: JOLLY DOUGLAS J, KERR WILLIAM G, ROBBINS JOAN M, JOLLY, DOUGLAS, J., KERR, WILLIAM, G., ROBBINS, JOAN, M.
Applicants: SYSTEMIX, VIAGENE INC, CHIRON VIAGENE, INC.
Application year: 1996
Main classification: C12N15
IPC codes: A61K31, A61K35, A61K38, A61K48, A61P9, A61P37, C07K14, C12N5, C12N15