AU686181B2

AU686181B2 – Generation, concentration and efficient transfer of VSV-G pseudotyped retroviral vectors
– Google Patents

AU686181B2 – Generation, concentration and efficient transfer of VSV-G pseudotyped retroviral vectors
– Google Patents
Generation, concentration and efficient transfer of VSV-G pseudotyped retroviral vectors

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Publication number
AU686181B2

AU686181B2
AU70523/94A
AU7052394A
AU686181B2
AU 686181 B2
AU686181 B2
AU 686181B2
AU 70523/94 A
AU70523/94 A
AU 70523/94A
AU 7052394 A
AU7052394 A
AU 7052394A
AU 686181 B2
AU686181 B2
AU 686181B2
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nucleic acid
cells
protein
cell
vsv
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1993-06-04
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AU7052394A
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Jane C Burns
Theodore Friedmann
Jiing-Kuan Yee
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University of California

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University of California
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1993-06-04
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1994-06-03
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1998-02-05

1994-06-03
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1995-01-03
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patent/AU7052394A/en

1998-02-05
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1998-02-05
Publication of AU686181B2
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patent/AU686181B2/en

2014-06-03
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C12N15/09—Recombinant DNA-technology

C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts

C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

C12N15/86—Viral vectors

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C12N2740/10011—Retroviridae

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C12N2740/13041—Use of virus, viral particle or viral elements as a vector

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C12N2840/00—Vectors comprising a special translation-regulating system

C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Abstract

The present application discloses retrovirus-derived vectors in which the retroviral envelope glycoprotein has been replaced by the G glycoprotein of vesicular stomatitis virus, and the use of these vectors in the transfer of exogenous genes into the cells of a wide variety of non-mammalian organisms. Also disclosed is a method for the generation of retroviral vectors in high titers, wherein a recombinant, stable host cell line is provided which harbors the retroviral vector of interest without envelope protein. High-titer retroviral vector production is initiated by introducing nucleic acid encoding a functional membrane-associated protein into the cell line. The vectors disclosed in the present application can be concentrated by ultracentrifugation to titers greater than 109 cfu/ml which are especially useful in human gene therapy trials, and can also infect cells, such as hamster and fish cells, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein.

Description

GENERATION, CONCENTRATION AND EFFICIENT TRANSFER OF VSV-G PSEUDOTYPED RETROVIRAL VECTORS
Field of the Invention
The present invention relates generally to the field of viral vectors having an altered host range. More specifically, the present invention relates to the generation of high-titer pseudotyped retroviral vectors.
Government Interest in Invention
Certain aspects of the invention disclosed herein were made with United States government support under U.S. Public Health Service grants HD20034 and AI28945 from the National Institutes of Health. The United States government has certain rights in these aspects of the invention in the United States.
Background of the Invention
The assembly of enveloped animal viruses is characterized by selective inclusion of the viral genome and accessory viral proteins into a budding viral particle. Although the mechanisms for selective encapsidation or packaging are not well characterized, it has been postulated that the recognition of viral envelope proteins within the plasma membrane by the viral nucleocapsids represents one probable control point for packaging specificity.
Using internal image anti-idiotype antibodies, Vaux et al. (Nature (London) 336:36- 42 (1988)) have shown that the nucleocapsid of Semliki Forest virus (SFV) contains a specific receptor for the cytoplasmic tail of the virion E2 spike glycoprotein. Vaux et al. suggested that a specific receptor-iigand-like interaction between the two is likely to be critical in the organization of the budding of SFV and related viruses from infected cells.
In apparent contrast to the high degree of specificity of SFV for the E2 protein, is the well-known phenomenon of “pseudotype” formation, in which mixed infection of a cell by one virus and retroviruses results in the production of progeny virions bearing the genome of one of the viruses encapsidated by the envelope proteins of the other.
These phenotypically mixed viruses form plaques on appropriate indicator cells and can be neutralized by sera raised against the specific envelope protein. One virus known to participate in pseudotype formation is vesicular stomatitis virus (VSV), a member of the rhabdovirus family.
The mechanism for the inclusion of the envelope protein of one virus into the virions of an unrelated virus is uncertain. Sequence comparison of VSV G protein and retrovirus envelope proteins reveals no significant sequence similarity among these proteins. Heretofore, it has also been difficult to determine whether G protein alone in the absence of other VSV-encoded proteins can participate in pseudotype formation.

Pseudotypes do not form between VSV and alphaviruses such as SFV even though pseudotypes can form between two alphaviruses or between alphaviruses and related flaviviruses such as Japanese encephalitis virus.
In some cases, phenotypic mixing is unilateral, as in the case of VSV with fowl plaque virus (FPV) or VSV with Sindbis virus. The pseudotype virus particle VSV(FPV) containing the VSV genome encapsidated by the envelope protein of FPV and the pseudotype virus particle VSV(Sindbis) have been demonstrated, but the reverse pseudotypes, FPV(VSV) and Sindbis(VSV), containing FPV or Sindbis virus genome with the VSV G protein, have not been detected. Mixed infection of cells with retroviruses and VSV usually results in the formation of pseudotypes with much lower titers than that of VSV generated from the same cells. It is not clear whether this is due to the specificity of the interaction between the retroviral nucleocapsid and the G protein or due to other factors.
Retroviral vectors have been used to transfer genes efficiently by exploiting the viral infectious process. Foreign genes cloned into the retroviral genome can be delivered efficiently to cells susceptible to infection by the retrovirus. Through other genetic manipulations, the replicative capacity of the retroviral genome can be destroyed. The vectors introduce new genetic material to a cell but are unable to replicate. A helper virus or a packaging system can be used to permit vector particle assembly and egress. As used herein, the term “vector particle” refers to viral-like particles that are capable of introducing nucleic acid into a cell through a viral-like entry mechanism. Such vector particles, can under certain circumstances, mediate the transfer of genes into the cells they infect.
It is possible to alter the range of cells that these vectors can infect by including an envelope gene from another closely related virus. Miller et al. (Mol. Cell. Biol. 5:431 – 437 (1985)) constructed a MoMLV-derived vector to introduce a selectable marker, dihydrofolate reductase, into susceptible cells, and included the envelope region from the related amphotropic retrovirus 4070A to broaden the host range of the vector. Other investigators have described pseudotypes of retroviral vectors whose host cell range has been altered by substitution of envelope proteins from different viruses. Substitution of the gibbon ape leukemia virus envelope protein for the amphotropic retroviral envelope has resulted in vectors capable of infecting bovine and hamster cells, species not susceptible to infection with retroviral vectors containing the MoMLV envelope protein (Miller et al, J. Virol., 65:2220-2224 (1991 )). Similarly, substitution of the HTLV I envelope protein has been shown to restrict the host cell range of a MoMLV-based vector to cells infectable by HTLV I (Wilson et al., J. Virol., 63:2374-2378, (1 989)).

Retroviral vectors derived from Moloney murine leukemia virus (MoMLV) are important tools for stable gene transfer into mammalian cells. They have been used to study gene regulation and expression and to facilitate gene transfer for studies of human gene therapy. Two significant limitations to the use of these retroviral vectors are the restricted host cell range and the inability to produce high-titer virus. Infection with retroviral vectors results from specific interaction of the viral envelope glycoprotein with cellular receptors, defining the host range and determining the efficiency of infection. Attempts to concentrate retroviral vectors by centrifugation or other physical means generally result in loss of infectious virus with only minimal increases in titer. The instability of retroviral particles may be related to structural characteristics of the envelope protein and modification of envelope components might, therefore, result in a more stable particle.
As stated above, it is not clear what signals are required to direct the functional assembly of the vector particle, nor is it known what factors permit the nucleocapsid and the membrane-associated protein to interact and complete packaging. Accordingly, heretofore, alterations in the host range have not been effected by including heterologous membrane-associated proteins within a vector particle. By “heterologous membrane- associated protein” it is meant a membrane-associated protein having at least one origin other than a virus of the same viral family as the origin of the nucleocapsid protein of the vector particle. As used herein, viral “family” shall be used to refer to the taxonomic rank of family, as assigned by the International Committee on Taxonomy of Viruses.
Summary of the Invention According to one aspect of the invention, there is provided a method of introducing foreign nucleic acid into a cell of a non-mammalian species. The method includes infecting the cell with a vector particle. The vector particle includes the following components: the foreign nucleic acid, a nucleocapsid encapsidating the nucleic acid; the nucleocapsid comprising nucleocapsid protein of a retrovirus, and a membrane surrounding the nucleocapsid. The membrane has vesicular stomatitis virus (VSV) G protein associated therewith. In a preferred embodiment, the retrovirus is MoMLV. In another preferred embodiment, the nucleic acid sequence is operably linked to a promoter and encodes a gene that is expressible into a polypeptide. In one embodiment, the promoter comprises a tissue-specific promoter. Another embodiment comprises transcribing the nucleic acid sequence into complementary RNA. A further embodiment comprises expressing the polypeptide through translation of the RNA. Preferably, the nucleic acid sequence becomes integrated into the genome of the cell. In another preferred embodiment, the nucleic acid comprises a selectable marker gene. The

selectable marker can be a neomycin resistance gene. In other embodiments, the non- mammalian species is a fish such as a zebra fish, an insect such as a mosquito, or an amphibian such as a frog.
According to another aspect of the invention there is provided a method for concentrating vector particles. This aspect of the invention involves growing enveloped vector particles. The particles comprise a nucleocapsid including nucleocapsid protein having an origin from a retrovirus, a nucleic acid sequence encapsidated by the nucleocapsid protein, and a membrane-associated protein which is vesicular stomatitis virus (VSV) G protein. The method also comprises harvesting the vector particles, and concentrating the vector particles to form a pellet containing the viral particles. In preferred embodiments, the concentrating step is ultracentrifugation, filtration or chromatography. A preferred embodiment further comprises resuspending the pellet in a liquid and subjecting it to a second cycle of ultracentrifugation. In a further preferred embodiment, the liquid is TNE or 0.1 % Hank’s balanced salt solution. In a preferred embodiment of the method, the vector particles are concentrated to a titer of at least 108 cfu/ml or, in a particularly preferred embodiment, to a titer of at least 109 cfu/ml. In another embodiment, the pellet retains over 50% of the colony forming units present prior to ultracentrifugation. In yet another embodiment, the pellet retains over 90% of the colony forming units present prior to ultracentrifugation. According to another aspect of the invention, there is provided a solution of retroviral particles comprising a titer of at least 10s cfu/ml. In a preferred embodiment, the solution comprises a titer of at least 109 cfu/ml. In one embodiment of the solution, the retroviral particles comprise a membrane-associated protein that is VSV-G protein. In another embodiment of the solution, the retroviral particles comprise MoMLV nucleocapsid protein.
According to yet another aspect of the invention, there is provided a method of introducing foreign nucleic acid into a germ cell of a non-mammalian species. In this aspect of the invention, the method includes exposing embryos of an individual member of the non-mammalian species to a vector particle. The vector particle comprises the foreign nucleic acid, a nucleocapsid encapsidating the nucleic acid, and a membrane surrounding the nucleocapsid. The nucleocapsid comprises nucleocapsid protein of a retrovirus, and the membrane has VSV G protein associated therewith. The method further comprises growing the embryos into adults, breeding the adults to produce an F2 generation, and identifying adults that produce an F2 generation carrying the foreign nucleic acid. The adults contain the foreign nucleic acid in their germ line. In one embodiment of the invention, the retrovirus is MoMLV. In another embodiment, the

foreign nucleic acid is operably linked to a promoter and encodes a gene expressible into a polypeptide. The promoter can be a tissue-specific promoter. In a preferred embodiment, the foreign nucleic acid sequence is operably linked to a promoter and encodes a gene that can be transcribed into complementary RNA, and optionally further translated into polypeptide. In one embodiment, the nucleic acid sequence is integrated into the genome of the germ cells.
According to another aspect of the invention, there is provided a method of introducing foreign nucleic acid into a germ cell of a non-mammalian species. This method comprises exposing germ cells of the non-mammalian species to a vector particle. The vector particle comprises the foreign nucleic acid, a nucleocapsid encapsidating the nucleic acid, and a membrane surrounding the nucleocapsid. The nucleocapsid comprises nucleocapsid protein of a retrovirus, and the membrane has VSV G protein associated therewith. The method further comprises implanting the. germ cells into embryos of the non-mammalian species, growing the embryos into adults, breeding the adults to produce an F2 generation, and identifying adults that produce an F2 generation carrying the foreign nucleic acid. The adults contain the foreign nucleic acid in their germ line. In a preferred embodiment, the germ cells are identified using an antibody for germ cells of the non- mammalian species.
According to still another aspect of the present invention, there is provided a method for the generation of high-titer retroviral vectors. This method comprises obtaining host cells comprising a first nucleic acid sequence encoding the production of retroviral nucleocapsid protein, and introducing into the host cells a second nucleic acid sequence comprising retroviral long terminal repeats (LTRs) and a desired exogenous gene, thereby creating a recombinant host cell. A third nucleic acid sequence operably linked to a promoter is then introduced into the recombinant host cell. The third nucleic acid sequence encodes a membrane-associated protein having cytoplasmic, transmembrane and extracellular domains. The resulting retroviral particles comprise an envelope with the membrane-associated protein therein and a genome comprising the exogenous gene. In a preferred embodiment, the first nucleic acid sequence includes the retroviral gag and pol genes. In another embodiment, the retrovirus is MoMLV. In yet another preferred embodiment, the second nucleic acid sequence encodes a selectable marker. The selectable marker can be a neomycin resistance gene. In still another embodiment, the desired exogenous gene is expressible into a polypeptide. According to this aspect of the invention, the second nucleic acid sequence can be introduced into the host cells by infecting the host cell with a virus having a genome comprising the second nucleic acid sequence or by transfecting the cell with a plasmid comprising the

second nucleic acid sequence. The third nucleic acid sequence can be introduced into the recombinant host cells by infecting the host cell with a virus having a genome comprising the third nucleic acid sequence or by transfecting the cell with a plasmid comprising the third nucleic acid sequence. In a preferred embodiment of the invention, the promoter is derived from the human cytomegalovirus. In another embodiment, the protein is the vesicular stomatitis virus (VSV) G protein. In yet another embodiment, the protein is a non-viral protein, such as CD4 protein. An especially preferred form of this aspect of the present invention further comprises concentrating the retroviral particles. In a preferred embodiment, the concentrating step comprises ultracentrifugation. Another preferred form of this aspect of the invention further comprises infecting cells with the retroviral particles. In one embodiment, the cells are located in a living organism in vivo. In another embodiment, the cells are growing in vitro.
According to still another aspect of the present invention, there is provided a recombinant host cell comprising a first nucleic acid sequence encoding the production of retroviral nucleocapsid protein and a second nucleic acid sequence comprising retroviral long terminal repeats and a desired exogenous gene. In one embodiment, the host cell is a 293GP cell. In another embodiment, the second nucleic acid sequence encodes a selectable marker. The selectable marker can be a neomycin resistance gene.
In yet another embodiment, the desired exogenous gene is expressible into a polypeptide. According to yet another aspect of the present invention, there is provided a method of introducing a desired gene into a cell. This method comprises obtaining a recombinant host cell comprising a first nucleic acid sequence encoding the production of a retroviral nucleocapsid protein, and a second nucleic acid sequence comprising retroviral long terminal repeats and a desired exogenous gene. A vector carrying a gene encoding a membrane-associated protein is introduced into the host cell, producing retroviral particles in the recombinant host cells. These retroviral particles comprise an envelope with the membrane-associated protein therein and a genome comprising the exogenous gene. These retroviral particles are used to infect the cell. The cell can be located in vivo, or alternatively, can be located in vitro. In one embodiment, the first nucleic acid sequence encodes retroviral gag and pol proteins. In another embodiment, the retrovirus is MoMLV. In yet another embodiment, the second nucleic acid sequence encodes a selectable marker. This selectable marker can be a neomycin resistance gene. In a preferred embodiment, the desired exogenous gene is expressible into a polypeptide. In a preferred form of this aspect of the invention, the introducing step comprises infecting the recombinant host cell with a virus or transfecting the recombinant host cell with a plasmid. In a preferred embodiment, the membrane-associated protein is the

vesicular stomatitis virus (VSV) G protein. In another embodiment, the membrane- associated protein is a non-viral protein, such as CD4 protein. An especially preferred form of this aspect of the present invention further comprises concentrating the retroviral particles prior to infecting the cell. In a preferred embodiment, the concentrating step comprises ultracentrifugation.
Brief Description of the Figures Figure 1 is a schematic representation, not drawn to scale, of retroviral vectors and packaging constructs pLRNL, pLARNL, pLGRNL, pSVGP, and pSAM for the creation of retroviral vector particles. Figure 2 shows a Southern blot analysis of LGRNL infected cells using neomycin resistance gene as probe.
Figure 3 shows immunoprecipitates separated by SDS-PAGE and visualized by fluorography. Lanes 1 and 2 show the polyclonal cell line immunoprecipitated with anti- VSV G and normal rabbit serum, respectively. Lane 3 is mock infected MDCK cells immunoprecipitated with anti-VSV G antibody.
Figure 4 shows immunoprecipitates of selected cells pooled, grown for 1 week, and then reisolated by FACS sorting. Lane 1 shows the extracts of the twice-sorted cells immunoprecipitated with the anti-VSV-G antibody and lanes 2, 3 and 4 correspond to lanes 3, 2 and 1 of Figure 3, respectively. Figure 5 depicts the flow cytometric analysis of VSV-G protein expression on the surface of 293 producer cells stably transfected with the MoMLV gag and pol genes. The results for both negative control cells stained with mouse immunoglobulin (A) and cells stained with anti-VSV-G monoclonal antibody (B) are shown.
Figure 6 shows the hybridization (using a 32P-labelled neo PCR product probe) of amplified DNA extracted from 40 wild type embryos (Lanes 1 -2), 40 LSRNL-exposed embryos (Lanes 3-6), 50 LGRNL(VSV-G)-exposed embryos (Lanes 7-1 1 ), the no DNA negative control (Lane 12), 100 pg pLGRNL (Lane 13) and 0.1 pg pLSRNL (Lane 14).
Figure 7 shows the results of PCR of genomic DNA extracted from LGRLN(VSV-G) infected and control (non-infected) Xenopus A6 cells. Lane 1 shows the fragment from Sac I digestion of uninfected A6 cells. Lanes 2 and 3 show the fragment from Sac I digestion of plasmid pLGRNL infected cells. Lanes 5 and 6 show Bam HI digest of A6 clones. Lane 7 shows the fragment from Bam HI digestion of uninfected A6 cells, and Lane 8 shows the fragment from Bam HI digestion of plasmid pLGRNL infected cells. Figure 8 shows the structure of the pLSPONL retroviral vector. Figure 9 is a restriction map of the pHCMV-G plasmid.

Figure 10 depicts the G418 resistance titer of the pseudotyped virus LSPONL(G) relative to that of its amphotropic counterpart LSPONL(A) in the rat 208F fibroblast line and various human cell lines.
Figure 1 1 depicts the level of HBsAg secreted by primary hepatocytes after exposure to different multiplicity of infection (MOD of either LSPONL(G) or LSPONL(A).
Detailed Description of the Invention We have discovered that specific membrane-associated protein from unrelated virus can be incorporated into retroviral vector particles to provide an altered host range throughout a broad spectrum of cells. As a model system, we incorporated the VSV G membrane-associated protein into
Moloney murine leukemia virus (MoMLV)-based vector particles to generate a retroviral vector particle having a nucleocapsid derived from MLV and the membrane associated protein VSV G within its envelope. Such vector particles shall be referred to herein by the designation MLVIVSV G]. This designation shall be used herein generally to refer to vector particles having a nucleocapsid from one viral origin and a membrane-associated protein from another origin, with the non-bracketed portion of the designation referring to the capsid origin and the bracketed portion referring to the membrane-associated protein within the envelope of the vector particle.
For production of vector particles, the nucleic acid of the vector particles of the present invention can be used to transfect a suitable cell line. The vector particles are released into the supernatant from the transfected cells and used to infect a desired cell type. The nucleic acid used for transfection can either be in isolated form or can be already packaged into vector particles. In some instances, a helper virus is required to facilitate virion assembly. Thus, for example, the genes encoding the gag and pol genes can be incorporated into the nucleic acid of a helper virus and functional sequences from another virus can be incorporated into the nucleic acid which is packaged into the vector particles.
The gene encoding a membrane-associated envelope protein can be incorporated within the nucleic acid of either the vector particle or of the helper virus. Alternatively, the gene for this envelope protein could be expressed from a third fragment of nucleic acid or from the genome of the producer cell. In a preferred form of the present invention, the nucleic acid within the vector particle is integrated into the cellular genome of the cell infected by the vector particle, the envelope gene is located on a different fragment of nucleic acid than the nucleic acid that is vector particle genome. Thus, in this preferred embodiment, the membrane-associated protein will not be produced by the

vector particle infected cells containing the integrated nucleic acid from the vector particle.
As will be described hereinbelow, we have discovered that the VSV G protein alone is sufficient to interact with the nucleocapsid of MoMLV in the formation of MLV[VSV G] vector particles. The process of incorporation of the VSV G protein into the vector particles is efficient and results in the production of infectious vector particles with titers comparable to that of whole retroviruses. Thus, we believe that other heterologous membrane-associated proteins can also be efficiently incorporated into the envelopes of enveloped viruses. MoMLV is a murine retrovirus which has poor infectivity outside of mouse cells.
When this ecotropic virus is adapted to produce retroviral vector particles carrying, for example, the N2 vector genome, this vector will infect only mouse cells at appreciable efficiencies. The related amphotropic N2 virus will infect cells from human, mouse and other organisms. This difference is attributable to the substitution of the amphotropic envelope protein for the ecotropic envelope protein. Both types of viral vector particles have essentially identical nucleocapsids derived from MoMLV. However, neither ecotropic N2 nor amphotropic N2 virus will infect hamster cells. As shown in the Examples provided hereinbelow, we have discovered that hamster cells can be infected by MLVfVSV G] vector particles and that addition of anti-VSV serum to preparations of these viral particles completely abolished their infectivity. Thus, we have shown that the presence of VSV G protein in the vector particles results in vectors having a host range derived from VSV, the origin of the membrane-associated protein incorporated within their envelope.
In order to determine if vector particles containing heterologous membrane- associated protein could be efficiently assembled, we tested whether the VSV G protein could be assembled with the nucleocapsid of MoMLV. We first introduced the neomycin phosphotranferase gene (Neo) which provides neomycin resistance, as a selectable marker for the vector particles into the amphotropic packaging cell line PA317. The resultant vector particles were added to cells incapable of supporting amphotropic MLV infection. Baby Hamster Kidney (BHK) cells lack amphotropic cell surface receptors and are, therefore, not susceptible to infection with amphotropic MLV-based retroviruses, as shown below in the experiments of Example 1 .
In initial experiments, we confirmed that this cell line is indeed refractory to infection by amphotropic MLV retrovirus. The next set of initial experiments is shown in Example 1 . In these experiments, we confirmed that amphotropic N2 virus containing the Neo gene will grow in rat 208F fibroblasts, but not in BHK cells.

EXAMPLE 1
Growth of N2 Virus in Rat 208F Fibroblasts Amphotropic N2 virus containing the Neo gene inserted between the long terminal repeats (LTRs) of MoMLV was prepared from the producer cell line PA317/N2 and titered both on BHK cells and rat 208F fibroblasts, a cell line that is susceptible to MoMLV retrovirus infection. We observed a 105-fold decrease in Neo-resistant (Neor) colony forming units (CFU) in BHK cells compared with that in rat 208F cells; thus, the BHK cell we used failed to support infection by N2 virus containing the amphotropic retroviral envelope protein. With the initial studies completed, we produced enveloped vector particles containing heterologous membrane-associated proteins. As an example of such production, Example 2 is provided to show the production of amphotropic N2 MLV[VSV G] vector particles using an MLV-based retroviral vector encoding the VSV G protein. We assayed the MLVfVSV G] vector particles on BHK cells to determine if the host range of these vector particles had been altered relative to amphotropic MLV.
EXAMPLE 2 Generation of MLVfVSV G1 Vector particles To produce MLVfVSV G] vector particles, we used MoMLV-based retroviral vectors as illustrated in Figure 1 , which is not drawn to scale. In pLRNL, the Neor gene expressed from the Rous Sarcoma Virus (RSV) promoter was inserted between the LTRs of MoMLV. The MoMLV-based retroviral vector pLRNL contains the Neo-resistance (Neo) gene under control of the promoter of RSV. This vector is described in Li et al., Virology 171 :331 -341 (1989). A single BamH1 site is immediately upstream of the RSV promoter. Into this BamH1 site, a 1 .7-kilobase pair (kb) BamH1 fragment containing the entire coding region of VSV G gene was inserted, giving rise to the construct pLGRNL (ATCC No. 75473). This VSV G gene is described in Rose et al., Cell 30:753-762 (1982) and Rose et al., J. Virol., 39:519-528 (1981 ). The plasmid pSAM, also shown in Figure 1 , containing the gag region of MoMLV, a hybrid pol region between MoMLV and amphotropic virus 4070A and the env region of 4070A has been described in Miller et al., Science, 225:630-632.
HPRT-deficient 208F cells were derived from Fischer rat cells by selection in 6- thioguanine, as described in Quade, K. Virology, 98:461 -465 (1979). Thymidine kinase (Tk)-deficient BHK cells were derived from BHK21 cells by selection with 5- bromodeoxyuridine, as described in Littlefield et al., Nature 21 1 :250-252 (1 966). These BHK cells and 208F fibroblasts were grown in Dulbecco-modified Eagle medium (DME) with high glucose supplemented with 10% fetal calf serum (FCS).

To generate infectious vector particles, either pLGRNL or pLRNL was co- transfected into BHK cells with the helper vector pSAM expressing MoMLV gag protein, a polymerase gene and amphotropic envelope protein from the SV40 early promoter.
Supematants from transfected BHK cells were collected at 48 hours post-transfection and used to infect susceptible 208F cells and resistant BHK cells. To titer virus, cells were infected overnight with filtered supematants in the presence of 4 mg/ml polybrene.
Infected cells were selected in medium containing 400 mg/ml G418 and colonies were scored about 14 days after infection. The results of the experiment of this example are shown in Table 1. TABLE 1 :
Transient vector particle production from BHK cells following cotransfection by pSAM with either pLRNL or pLG RL
Vector Particle Cell infected Titer (CFU/ml)
LRNL 208F 480
BHK <10 LGRNL 208F 380 BHK 260 It can be seen from Table 1 that vector particles derived pLRNL lacking VSV G protein were able to infect cells efficiently but failed BHK cells. In contrast, pLGRNL, containing the VSV protein, could not only cells, also as indicated by appearance of Neor colonies. Thus, host range MLV[VSV G] had been altered relative amphotropic MLV. Accordingly, we believe produced pLGRNL- transfected Example 2 was incorporated into at least some retroviral particles, producing MLVfVSV capable infecting hamster cells. Since encoded pLGRNL is results of Example shown in indicate alone, without participation other VSV-encoded proteins, sufficient for formation particles. Accordingly, shows infectious assembled having nucleocapsid one virus and a heterologous membrane-associated protein. As stated above background invention, mixed infection retroviruses resulted lower titer than what obtained alone. Rather being result inefficient incorporation virions caused poor specificity between nucleocapsid, this due progressive inhibition cellular synthesis, including those proteins retroviruses, toxicity. Thus, order determine whether presence same family viruses origin necessary conducted experiments form any envelope an MLV. An example such experiment 3. EXAMPLE 3 Formation G1 Vector Env Gene Product For experiment, two additional vectors Figure , pLARNL pSVGP. manner similar production however, 2.7-kb Xbal fragment, (env) gene retrovirus 4070A inserted BamH1 site pLRNL. pSVGP constructed inserting 5.8 kb Hindlll-Scal fragment SV40 early promoter gag pol regions pSAM mammalian expression pcD, described Okayama et al., Mol. Cell. Biol., 3:280-289 (1983). "Poly A" indicates polyadenylation signal SV40. In example, co-transfected pLRNL, or with To generate 10 μg DNA together y_>Download PDF in English