CA2237000A1 - Stable packaging cell line producing pseudotyped retroviruses - Google Patents

Abstract

The present invention relates to a stable, pseudotyped retrovirus packaging cell line comprising packaging cells which generate helper-free recombinant pseudotyped retrovirus. The packaging cell line comprises one or more non-retroviral expression constructs, such as an expression construct with the human cytomegalovirus (CMV) immediate early promoter or derivatives of this promoter (e.g., pMD), which direct expression of: (a) retroviral gagpol genes and (b) a non-retroviral gene which is under the control of an inducible operator system and whose gene product pseudotypes retroviral core virions.
The present invention further relates to a method of making a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus. The present invention further relates to the particular packaging cell lines described herein (i.e., H29 gagpol, H29 new gagpol) and the particular cells and constructs (i.e., packaging elements) used to produce the stable, pseudotyped retrovirus packaging cell line described herein (e.g., H29 cells and pMD, pMDtet, pMDtet.G, PMD.gagpol, pMD.new gagpol constructs). The present invention relates to a retroviral vector for producing a cDNA library for expression in mammalian cells, comprising two retroviral long terminal repeats, a cloning site for insertion of cDNA and a cytomegalovirus promoter. The invention also relates to a cDNA
library for expression in mammalian cells, the library comprising retroviral vectors of the present invention. The present invention also relates to a method of expression cloning in mammalian cells. The present invention also relates to a method of cDNA expression cloning in mammalian cells. The present invention also relates to a method of identifying a gene defect responsible for a mutant phenotype using cDNA expression cloning by complementation in mammalian cells.

Description

CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 STABLE PACKAGING ~r~ T.TNF~ PRODUCING PSEUDC~
R~!; 1 KO~J 1KU ~

Rela~ Applications This application is a Continuation-in-Part of U.S.S.N.
08/555,15S, entitled ~Stable Packaging Cell Line Producing Pseudotyped Retroviruses For Gene Transfer", by Daniel S.
Ory, Michel Sadelain and Richard C. Mulligan, filed November 8, 1995 and of U.S.S.N. 08/651,050, entitled "A
Method For Generation Of Retroviral cDNA Expression Libraries With A Vesicular Stomatitis Virus-G (VSV-G) Host Range ~or Expression Cloning By Complementation", by Daniel S. Ory and Jean E. Schaffer, filed May 21, 1996. The teachings of each are incorporated herein by reference in their entirety.

Fundinq Statement Work described herein was supported by the National Institutes of Health grant, K11 HL02910. The U.S.
Government has certain rights in the invention.

Backqround Recombinant retroviruses are useful for in vivo and in vitro gene expression and for production of proteins of interest in eukaryotic host cells. Generally, recombinant retroviruses are produced by introducing a suitable proviral DNA vector into ~ ~lian cells that produce the necessary viral proteins for encapsidation of the desired recombinant RNA and generation of infectious recombinant virions. Since, for most gene transfer applications, the generation of pure stocks of recombinant virus free of replication-competent helper virus is desirable, there has CA 02237000 1998-0~-07 been considerable interest in developing cell lines that produce the necessary viral gene products for the generation of recombinant retrovirus, yet do not themselves either yield detectable helper virus or transfer viral genes (Coffin, J., In:RNA Tumor Viruses, Weiss, R. et al.
(ed.). Cold Spring Harbor Laboratory, Vol. 2, pp.36-73 (1985); Mann, R. et al ., Cell, 33:153-159 (1983); Watanabe, S., et al., Mol. Cell Biol., 3:2241-2249 (1983); Cone, R.D., et al., PNAS, USA, 81:6349-6353 (1984); Miller, A.D., et al., Mol. Cell Biol., 6:2895-2902 (1986); Bosselman, R.A., et al., Mol. Cell Biol., 7:1797-1806 (1986). One approach to doing so is to use mutated proviral genome to develop retroviral packaging cell lines. However, production of helper virus and/or transfer of packaging function (i.e., viral genes) may still occur.
Thus, an improved retroviral packaging cell line is needed which limits the potential for generation of helper virus.

Summary of the Invention The present invention relates to a stable packaging cell line which produces helper-free pseudotyped retroviruses and is of mammalian origin, preferably of non-murine origin, such as stable packaging human cell lines.
These are referred to herein respectively, as stable pseudotyped retrovirus packaging cell lines and stable pseudotyped retrovirus packaging human cell lines. The packaging cell line comprises one or more non-retroviral expression constructs, such as an expression construct with the human cytomegalovirus (CMV) immediate early promoter or derivatives of this promoter (e.g., pMD), which direct expression of: a) the retroviral gag gene and the retroviral pol gene, referred to as the retroviral gagpol genes, and b) a non-retroviral gene which is under the control of an inducible operator system and whose gene CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 product pseudotypes retroviral core virions. The gagpol gene products package the desired recombinant RNA into core virions, which are pseudotyped by the non-retroviral gene product, resulting in production of a stable, pseudotyped retrovirus packaging cell line capable of generating helper-free recombinant retrovirus.
In one ~-hoAi -nt, the present invention relates to a stable, pseudotyped retrovirus packaging cell line capable of generating helper-free recombinant pseudotyped retrovirus with a pantropic host range. These cell lines generate helper-free recombinant pseudotyped retrovirus.
The packaging cells comprise one or more non-retroviral expression constructs which direct expression of retroviral gagpol genes and a gene for the Vesicular Stomatitis Virus G (VSV-G) glycoprotein. The VSV-G glycoprotein, which is under the control of an inducible operator system (e.g., tet operator), provides an envelope protein that pseudotypes the retroviral core virion generated by the gagpol proteins. The result is a stable, pseudotyped retrovirus packaging cell line (e.g., H29 gagpol) which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range. In another embodiment, altered (e.g., mutated) retroviral gagpol genes are used to produce a stable, pseudotyped retrovirus packaging cell line (e.g., H29 new gagpol cell line).
The present invention further relates to a method of making a stable, pseudotyped retrovirus packaging cell line which generates helper-~ree recombinant pseudotyped retrovirus. In the method of the present invention, mammalian cells are co-transfected with one or more non-retroviral expression constructs which direct the expression of: a) retroviral gagpol genes and b) a non-retroviral gene which is under the control of an inducible operator system and provides a pseudotyped envelope for retroviral core virions. The gagpol proteins package the CA 02237000 1998-0~-07 desired recombinant RNA into core virions, which are pseudotyped by the non-retroviral gene product, resulting in production of a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus.
In one embodiment, the present invention relates to a method of making a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range. In this embodiment, mammalian cells are co-transfected with one or more non-retroviral expression constructs which direct the expression of retroviral gagpol genes (e.g., wild type or altered) and a VSV-G gene. The VSV-G gene, which is under the control of an inducible operator system, provides a pseudotyped envelope protein for the retroviral core virions, which are produced by the gagpol proteins.
This results in production of a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range.
In another embodiment, the present invention relates to a method of making a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range. In the method, mammalian host cells are co-transfected with a first non-retroviral construct which expresses the gene for tet transactivator fusion protein (tTA) (Gossen, M. and Bujard, M., Proc. Natl . Acad. Sci., 89:5547-5551 (1992)) and a second non-retroviral construct which expresses a gene for the VSV-G glycoprotein under the control of tet operator ( ;n;~l human CMV immediate early promoter incorporating tet binding sequences). The transfected cells are screened for tetracycline-inducible VSV-G
expression; VSV-G protein is not detected in the presence of tetracycline and is detected in the absence of CA 02237000 1998-0~-07 tetracycline. Such cells are transfected with a third non-retroviral construct which expresses the retroviral gagpol genes, and screened for production of retroviruses.
Transfected cells which produce retroviruses are stable, pseudotyped retrovirus packaging cells which generate helper-free recombinant pseudotyped retrovirus with a pantropic host range.
The present invention further relates to the particular packaging cell lines described herein (H29 gagpol or 293GPG cell line, H29 new gagpol cell line) and the particular cells and constructs (e.g., packaging elements) used to produce the stable, pseudotyped retrovirus packaging cell line described herein (H29 cells and pMD, pMDtet, pMDtet.G, PMD.gagpol, pMD.new gagpol constructs).
Another aspect of the invention is a retroviral vector for producing a cDNA library for expression in ~m~l ian cells. The retroviral vector comprises two retroviral long terminal repeats (LTRs) (e.g., a 5' retroviral LTR and a 3' retroviral LTR), a cloning site for insertion of cDNA and a cytomegalovirus (e.g., human) promoter. In one embodiment, the two LTRs are Moloney murine leukemia virus (MMLV). In a specific embodiment, the 3' MMLV LTR is unmodified and the 5' is a modified or chimeric MMLV LTR in which the U3 region of the 5' MMLV LTR is replaced with the cytomegalovirus (e.g., human) promoter or the cytomegalovirus enhancer-promoter.
The present invention also relates to a cDNA library for expression in mammalian cells. The library comprises retroviral vectors which comprise two retroviral LTRs, cDNA
and a cytomegalovirus promoter. The cDNA is positioned at a unique cloning site within the retroviral vector, preferably between the two LTRs, and is operably linked to the cytomegalovirus promoter.

CA 02237000 1998-0~-07 The present invention also relates to a method of expression cloning in - -lian cells. The method comprises the steps of introducing into mammalian cells a cDNA expression library comprising retroviral vectors of 5 the present invention and maintA; n ing the mammalian cells containing the expression library under conditions appropriate for expression of the cDNA expression library, whereby the cDNAs in the expression library are expressed in the ~ ~lian cells. In one embodiment, the cDNA
lO expression library is introduced into mammalian cells by infection with pseudotyped retroviruses produced in a stable ~mm~lian (e.g., human, murine) packaging cell line.
The stable mammalian packaging cell line can be selected to produce pseudotyped retroviruses with pantropic, ecotropic 15 or amphotropic host range, preferably pantropic host range.
In a particular embodiment, the present invention relates to a method of expression cloning in mammalian cells. The method comprises the steps of introducing a cDNA expression library comprising retroviral vectors which comprise two 20 retroviral LTRs, cDNA and a cytomegalovirus promoter into a packaging cell line which produces pseudotyped retroviruses; maintaining the packaging cell line containing the expression library under conditions appropriate for generation of pseudotyped retroviral 25 particles containing the cDNA expression library; infecting ~ lian cells with the pseudotyped retroviral particles, under conditions appropriate for infection of the m~mm~lian cells; and maintaining the resulting mammalian cells under conditions appropriate for expression of the cDNA in the 30 ~ ~lian cells. In a particular embodiment, the packaging cell line is a stable human embryonic kidney cell line and, specifically, a human 2s3-derived cell line.
The present invention also relates to a method of cDNA
expression cloning in m~Tnm~lian cells, wherein VSV-G
3S pseudotyped retrovirus particles which contain RNA produced CA 02237000 1998-0~-07 by transcription of cDNA in a cDNA library are produced in a packaging cell line. The cDNA library comprises vectors which, in turn, comprise two retroviral LTRs, cDNA and a cytomegalovirus promoter; the cDNA is positioned between the LTRs and operably linked to the promoter. Mammalian cells are then infected with the VSV-G pseudotyped retroviral particles produced under conditions appropriate for transcription of RNA contained in the VSV-G pseudotyped retrovirus particles and production of protein encoded by cDNA in the cDNA library (by translation of the RNA in the retrovirus particles) in the mammalian cells. Mammalian cells which contain the RNA contained in the VSV-G
pseudotyped retrovirus particles or protein encoded by the cDNA in the cDNA library are detected, using known methods.
For example, RNA can be detected using in situ hybridization. Alternatively, immunodetection can be used, where the cDNA encodes protein which is expressed at the cell surface and the expressed proteins can be detected using antibodies which bind the protein expressed by the cDNA of interest (see e.g., U.S. Patent No. 5,506,126). In addition, epitope tags can be used to detect the protein expressed. In addition, functional assays can be used to detect the function of a protein expressed by the cDNA of interest (e.g., a protein which confers an adhesive phenotype on a cell).
The present invention also relates to a method of identifying a gene defect responsible for a mutant phenotype using cDNA expression cloning by complementation in mammalian cells. In this method, VSV-G pseudotyped retrovirus particles which contain RNA produced by transcription of cDNA in a cDNA library are produced in a packaging cell line. The cDNA library comprises two retrovirus LTRs, cDNA and a cytomegalovirus promoter; the cDNA is positioned between the LTRs and operably linked to the promoter. Mammalian cells which display a mutant CA 02237000 1998-0~-07 phenotype are infected with the VSV-G pseudotyped retroviral particles under conditions appropriate for transcription of RNA contained in the VSV-G pseudotyped retrovirus particles and production of protein encoded by cDNA in the cDNA library (by translation of the RNA in the retrovirus particles). Mammalian cells with the mutant phenotype which display the wild type phenotype upon expression of the cDNA are identified. The cDNA which confers the wild type phenotype in the ~ lian cells is then identified, thereby determining the gene defect responsible for the mutant phenotype.
Development of the stable, pseudotyped retrovirus packaging cell lines described herein limits the formation of helper virus. As a result, stable pseudotyped retrovirus packaging cell lines are particularly valuable reagents for in vivo gene transfer studies aimed at cell lineage analysis and the development of human gene replacement therapies.
Use of the retroviral vectors of the present invention enables retroviral cDNA expression cloning in any mammalian cell type, obviating the need for specialized cells for efficient expression cloning. In any mutant mammalian cell type for which there is a phenotype distinct from the wild-type parental cell type (e.g., primary human cells derived from patients, primary or established cell lines derived from mutant animal strains, primary or established cell lines derived from knockout mice, mutant cell lines generated in cell culture) the genetic difference between the mutant and wild type cell (the genetic alteration(s) or defect(s)) can be rapidly identified by expression cloning by complementation using this invention.

CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 Brief Descxiption of the Fiqures Figure 1 is a schematic representation of the pMD
construct.
Figure 2 is a schematic representation of the pMDtet construct.
Figure 3 is a schematic representation of the pMD.G
construct.
Figure 4 is a schematic representation of the pMDtet.G
construct.
Figure 5 is a schematic representation of the pMD.gagpol construct.
Figure 6 is a schematic representation of the pMD.new gagpol construct.
Figure 7 is a schematic representation of the plasmid, pBC.tTA
Figure 8 is a schematic representation of the plasmid, MFG.SnlsLacZ.
Figure 9A is a schematic representation of the ~U3 retroviral construct, ~U3nlsLacZ
Figure sB is a schematic representation of the ~U3 retroviral construct, ~U3Bam.

Detailed Descri~tion of the Invention The present invention relates to a stable retrovirus packaging cell line of mammalian origin, preferably of non-murine origin, such as stable packaging human cell lines, which produce pseudotyped retroviruses. These are referred to herein, respectively, as stable pseudotyped retrovirus packaging cell lines and stable pseudotyped retrovirus packaging human cell lines for producing pseudotyped retroviruses for retroviral gene transfer. The packaging cell lines of the present invention comprise one or more non-retroviral constructs for expression of retroviral gagpol proteins, which produce a retroviral core virion, and a protein which provides a pseudotyped envelope for the CA 02237000 1998-0~-07 W O 97/174~7 PCTAJS96/17807 retroviral core virion. The protein which provides a pseudotyped envelope for retroviral core proteins is under control of an inducible operator system. That is, production of retrovirus by the packaging cell line described herein is controlled by the inducible expression of the protein which provides a pseudotyped envelope for a retroviral core virion. Packaging cell lines of the present invention l~- ~in viable when uninduced (e.g., in the presence of tetracycline when a tet operator is used) and express retroviral gagpol proteins which are non-enveloped; the uninduced packaging cell lines are capable of generating (producing) recombinant pseudotyped retroviral particles when induced (e.g., in the absence of tetracycline when a tet operator is used). Thus, the pseudotyped retrovirus packaging cell line of the present invention is stable. Once induced, the packaging cell lines generate (produce) recombinant pseudotyped retroviral particles.
In addition, the packaging cell line of the present invention limits the potential for generation of helper virus. Use of non-retroviral constructs and a non-retroviral protein which produces a pseudotyped envelope for the retroviral core virion contributes to the limited generation of helper virus. Potential for helper virus formation can be further limited by using non-murine cells (e.g., human cells). Murine cell lines (e.g., NIH 3T3 cells) are typically used to generate retrovirus packaging cell lines. However, the presence of endogenous murine retrovirus in the genome of murine cell lines, such as NIH 3T3 cells (Danos, et al., Proc. Natl. Acad. sci., 85:6460-6466 (1988), could facilitate recombination events between the host cell genome, the retroviral expression constructs and the retroviral vectors, thereby contributing to production of helper virus.

CA 02237000 1998-0~-07 In addition, the retroviral gagpol genes can be altered (e.g.,mutated), further limiting the potential for the production of helper virus. An example of mutated gagpol sequences (i.e., new 5' gagpol; new 3' gagpol) is described in Example 1.
As described in Example 2, packaging cell lines of the present invention can be derived from human 293 cells which incorporate a novel non-retroviral, human CMV immediate early promoter expression construct (pMD) to express the gagpol gene and pseudotyped envelope which limits the potential for generation of helper virus. In addition, silent mutagenesis of gagpol coding sequences minimizes homology with retroviral vector sequences, further limiting the potential for generation of helper virus. As further described in herein, the packaging cell lines of the present invention express the Vesicular Stomatitis Virus G
(VSV-G) glycoprotein which efficiently pseudotypes the retroviral core virions. The VSV-G glycoprotein has a broad host range. Therefore, VSV-G pseudotyped retroviruses demonstrate a broad host range (pantropic) and are able to efficiently infect cells that are resistant to infection by ecotropic and amphotropic retroviruses (Yee, J.-K., et al., Proc. Natl. Acad. sci . , 91:9564-9568 (1994)). High levels of expression of VSV-G are cytotoxic and therefore, VSV-G expression in the new packaging cell line is controlled by an inducible operator system, such as the inducible tet operator system, allowing for tight regulation of gene expression (i.e., generation of retroviral particles) by the concentration of tetracycline in the culture medium.
Finally, as demonstrated herein, VSV-G pseudotyped retroviral particles can be concentrated more than 100-fold by ultracentrifugation (Burns, J.C., et al., Proc. Natl.
Acad. Sci., 90:8033-8037 (1993)). The stable VSV-G
pseudotyped retrovirus packaging cell lines permit CA 02237000 1998-0~-07 WO 97/17457 PCT~US96/17807 generation of large scale viral preparations (e.g. from 10 to 50 liters supernatant) to yield retroviral stocks in the range of 107 to 10ll retroviral particles per ml.
The H29 cells that express the inducible VSV-G protein have been observed in cell culture for better than 20 passages. The H29 cells at passage 20 remain viable and continue to express in an inducible manner detectable VSV-G
protein (e.g., by cell fusion studies, Western blotting) at levels equivalent to cells at an early passage.
The expression construct for use in the present invention is a non-retroviral vector which directs expression of retroviral gagpol genes used to produce a retroviral core virion, and a protein which provides a pseudotyped envelope for the retroviral core virion. As described in Example 1, a suitable expression construct for use in the present invention is a human cytomegalovirus (CMV) ;~;ate early promoter construct. Other examples of constructs which can be used to practice the invention include constructs that use SV40, RSV and rat ~-actin promoters.
One or more of the non-retroviral expression constructs can be used to express the gagpol genes and the protein which provides a pseudotyped envelope, using skills known in the art. For example, the proteins can be expressed using one non-retroviral expression construct.
In addition, two non-retroviral expression constructs can be used wherein one construct expresses the gagpol genes and the other construct expresses the genes (VSV-G, tTA) which provide a pseudotyped envelope under control of an inducible operator. Alternatively, as described in Example 1, three non-retroviral constructs can be used: the first non-retroviral construct codes for the inducible tet transactivator protein (tTA) which controls expression of the gene that expresses a pseudotyped envelope, the second CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 non-retroviral construct expresses the genes which provides the pseudotyped envelope and the third non-retroviral construct expresses the gagpol genes. Further, the gag and pol sequences can be expressed separately, requiring a fourth non-retroviral construct (e.g., in which the third retroviral construct expresses the gag gene and the fourth retroviral expression construct expresses the pol gene).
As referred to herein, a "pseudotype envelope" is an envelope protein other than the one that naturally occurs with the retroviral core virion, which encapsidates the retroviral core virion (resulting in a phenotypically mixed virus). A suitable protein which provides a pseudotyped envelope is the Vesicular Stomatitis Virus G (VSV-G) glycoprotein, as described in Example 1. Any suitable serotype (e.g., Indiana, New Jersey, Chandipura, Piry) and strain (e.g., VSV Indiana, San Juan) of VSV-G can be used in the present invention. The protein chosen to pseudotype the core virion determines the host range of the packaging cell line. VSV-G interacts with a specific phospholipid on the surface of mammalian cells (Schlegel, R., et al., Cell, 32:639-646 (1983); Supertzi, F., et al., ~. Gen Virol., 68:387-399 (1987)). Thus, the packaging cell line which utilizes VSV-G to provide a pseudotyped envelope for the retroviral core virion has a broad host range (pantropic).
Other suitable proteins which can be used to provide a pseudotyped envelope for a retroviral core virion include type C murine retroviral envelope proteins; HTLV-1 envelope protein, Gibbon ape leukemia virus envelope protein, and derivatives of a suitable protein which provide a pseudotyped envelope (e.g., proteins which include insertions, deletions or mutations to prepare targeted envelope sequences such as ecotropic envelope with the EPO
- ligand, synthetic and/or other hybrid envelopes;
derivatives of the VSV-G glycoprotein). In addition, 3S derivatives of murine retroviral envelope proteins can be CA 02237000 1998-0~-07 used. For example, derivatives of the VSV-G protein can be obtained in which the portion of the VSV--G protein which is responsible for binding to the cell surface is replaced by a specific ligand and the portion of the VSV-G protein 5 responsible for membrane fusion is retained. The portion of the VSV-G protein responsible for binding to the cell surface is determined for example, by performing point mutation and deletion sequence analysis of the VSV-G
sequence. The ability of each mutated VSV--G protein to lO bind to the cell surface is determined using an appropriate binding assay. Retroviral particles incorporating such derivatives of VSV-G protein would now be able to be targeted to specific cell populations.
As discussed above, an inducible operator is used for 15 controlled expression of the gene which provides a pseudotyped envelope. For example, high levels of VSV--G
expression are cytotoxic (Yee, J.--K., et al., Proc. Natl.
Acad. Sci., 91:9564-9568 (1994) ) . Thus, an inducible tetracycline (i.e., tet) operator system is used to allow 20 for tight regulation of VSV-G expression by the concentration of tetracycline in the culture medium of the packaging cell line. That is, with the tet operator system, in the presence of tetracycline, the tetracycline is bound to the tet transactivator fusion protein (tTA), 25 preventing binding of tTA to the tet operator sequences and allowing expression of the gene under control of the tet operator sequences (Gossen, M. and Bujard, M., Proc. Natl.
Acad. Sci., 89:5547-5551 (1992) ) . In the absence of tetracycline, the tTA binds to the tet operator sequences 30 preventing expression of the gene under control of the tet operator. Examples of other inducible operator systems which can be used for controlled expression of the protein which provides a pseudotyped envelope are l) inducible eukaryotic promoters responsive to metal ions (e.g., the CA 02237000 1998-0~-07 metallothionein promoter), glucocorticoid hormones and 2) the lac repressor/operator/inducer system of E. coli.
The nucleotide sequences which are encoded by the non-retroviral constructs can be obtained from a variety of suitable sources for use in the present invention. For example, nucleotide sequences expressing the operator system, the pseudotyped envelope and the gagpol sequences can be purified from natural sources, produced by chemical synthesis or produced by recombinant DNA t~rhniques. For example, as described in Example 1, the gagpol sequence can be obtained using the pCripen~ construct.
The cells used to prepare the packaging cells are - ~lian cells, preferably non-murine cells. In a particular embodiment, the cells used to produce the packaging cell line are human cells (e.g., 293 cells, Graham, F., et al., J. Gen. Virol., 36:59-72 (1977); tsa 201 cells, Heinzel, S., et al ., J. Virol ., 62:3738 (1988)).
The packaging cell lines of the present invention can be used to produce recombinant pseudotyped retroviruses to enable gene transfer, in vitro and in vivo, for purposes of expressing all or a portion of a desired gene in eukaryotic cells. For example, using known techniques, the packaging cell lines described herein can be used to produce recombinant pseudotyped retroviruses which are used to introduce a gene which encodes a particular mRNA, protein or polypeptide (e.g., therapeutic proteins or polypeptides, such as insulin, human growth hormone, erythropoietin, gene replacement for cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor), ADA Deficiency (ADA), Gaucher's Disease (glucocerebrosidase), antisense therapy by expression of inhibitory mRNA sequences) into eukaryotic cells in order to produce the mRNA or protein in quantities which are useful in administration for therapeutic purposes or in a diagnostic context (Yee, J. -K., et al . , Proc. Natl .

CA 02237000 1998-0~-07 Acad. sci., 91:9564-9568 (1994); Dranoff, G., et al., Proc.
Natl. Acad. sci., 90:3539-3543 (1993); Miller, A.D., et al ., Meth. in Enz., 217:581-599 (1993)). That is, pseudotyped recombinant virus can be harvested from the packaging cell lines and used as viral stock to infect recipient cells in culture or in vivo using known methods.
In the case of secreted proteins or proteins expressed in hematopoietic cells, sensitive assays such as ELISA or Western blotting can be used to assess gene transfer efficiency. Alternatively, high titer viral stocks produced by packaging cell lines provide superior gene transfer efficiency in transducing cells (e.g., hematopoietic cells) and reduce contamination as compared with current co-cultivation techni~ues.
The packaging cell lines of the present invention can also be used to produce pseudotyped retroviruses containing DNA of interest for introducing DNA or genes of interest into mammalian cells, such as human cells, which will subsequently be at' i n; ~tered into localized areas of the body (e.g., ex vivo infection o~ autologous white blood cells for delivery of protein into localized ares the body, see e.g., U.S. Patent No. 5,399,346).
In addition, the packaging elements used to generate the stable, pseudotyped retrovirus packaging cell can be used in a variety of ways. For example, the H29 cell line, which demonstrates inducible VSV-G expression, can be used to generate retroviral libraries for expression cloning.
The potential for production of high titer viral stocks will improve the representation of rare cDNAs in a given library. The packaging cell lines of the present invention can also serve as the basis for further generation of pseudotyped packaging cell lines.
The packaging elements can be used as expression constructs for purposes of efficient constitutive (e.g., pMD) and inducible (pMDtet) gene expression. The pMDtet.G

CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 construct can be used for other applications for inducible expression of VSV-G. The pMD gagpol and pMD new gagpol can be used to develop new generations of retrovirus packaging cell lines. The pMD, pMDtet, pMDtet.G and pMD.gagpol constructs can be used for efficient expression of a heterologous gene.
Thus, as described herein, a stable cell line expressing VSV-G in an inducible fashion has been generated. In addition, a new CMV expression vector (pMD) and its derivatives (pMD.G, pMDtet.G, pMD.gagpol, pMD.new gagpol) which use genomic human ~-globin sequences for high levels of expression has been developed. As further described herein, a stable 293-based packaging cell line that uses CMV expression constructs, as compared with mutated proviral constructs, which limit the potential for helper virus has been developed and use of a mutated gagpol expression construct in a stable cell line to limit the potential for helper virus has been demonstrated.
The present invention also relates to a retroviral vector for producing a cDNA expression library, for expression in A ~lian cells, comprising two retroviral LTRs, a cloning site for insertion of cDNA, and a cytomegalovirus promoter. In one embodiment, the two LTRs are Moloney murine leukemia virus (MMLV). In a specific embodiment, the 3' MMLV LTR is unmodified and the 5' is a modified or ~-hi ~Aric MMLV LTR in which the U3 region of the 5' MMLV LTR is replaced with the cytomegalovirus promoter or the cytomegalovirus enhancer-promoter.
The retroviral LTRs can be derived from any suitable retroviral vector, preferably a retroviral vector which results in high titer or expression of retroviral proteins The two LTRs can be derived from the same retroviral vector or different retroviral vectors. For example, as described in Example 3, both retroviral LTRs can be derived from the Moloney murine leukemia virus (MMLV). Other suitable CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 retroviral LTRs include, for example, those derived from murine sarcoma virus (MSV), murine papillary sarcoma virus (MPSV), and Friend virus.
The cloning site of the retroviral vector can be a variety of cloning sites. For example, as described in Example 3, the cloning site can be a BamHI cloning site.
Other suitable cloning sites for use in the retroviral vectors of the present invention include, for example, any unique or infrequent restriction site within the gagpol or env genome, or within the U3 region.
The cytomegalovirus promoter can be obtained from any suitable source. For example, as described in Example 3, the complete cytomegalovirus enhancer-promoter is derived from the human cytomegalovirus (HCMV). Part or all of previously described CMV promoter could be used in the present invention. Other suitable sources for obtaining a cytomegalovirus promoter include commercial sources, such as Clontech, Invitrogen and Stratagene.
The retroviral vectors of the present invention can be used for expression cloning in mammalian cells, wherein a cDNA expression library comprising the retroviral vectors described herein are introduced into mammalian cells under conditions appropriate for expression of the cDNA
expression library. In one embodiment, the present invention relates to a cDNA expression library for expression in - ~ian cells, wherein the library comprises two retroviral LTRs, a cytomegalovirus promoter and cDNA, wherein the cDNA is positioned between the retroviral LTRs and is operably linked to the cytomegalovirus promoter.
The cDNA for use in the present invention is any cDNA
which is of interest for expression in mammalian cells.
The cDNA can be from any type of cells, such as blood cells, cells from tissue samples, or cultured cells.
Generally the cDNA will be from the same type of cell in CA 02237000 l998-0~-07 W O 97/17457 PCTrUS96/17807 which the cDNA is being expressed in those cases where expression cloning by complementation is being carried out.
The cDNA library for use in the present invention can be obtained using routine methods (e.g., Seed and Aruffo, Proc. Natl. Acad. Sci, USA, 84:3365-3369 (1987)). For example, mRNA can be prepared from any cell and the cDNA
synthesized using standard t~hniques (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) and commercially available cloning kits (e.g., Pharmacia, Invitrogen, Stratagene).
The retroviral vectors of the present invention can be introduced into mammalian cell using any technique which results in expression of the cDNA expression library in the cell (e.g., electroporation, calcium phosphate precipitation, cationic lipids, liposomes). In one embodiment, the cDNA expression library is introduced into a packaging cell line to produce retroviral particles, containing RNA transcribed from the cDNA expression library, which are used to infect mammalian cells resulting in expression of the cDNA expression library in the infected mammalian cell. In this embodiment, a packaging cell line (e.g., the 293GPG packaging cell line described herein) can be used to produce pseudotyped retroviral particles useful for expression cloning in mammalian cells, as described in Example 4. Other suitable packaging cell lines for use in the present invention include other human cell line derived (e.g., embryonic cell line derived) packaging cell lines and murine cell line derived packaging cell lines, such as Psi-2 cells (Mann, R., et al., Cell, 33:153-159 (1983); FLY (Cossett, F.L., et al., Virol., 193:385-395 (1993)), BOSC 23 cells (Pear, W.S., et al., Proc. Natl. Acad. sci, USA, 90:8392-8396 (1993), PA317 cells (Miller, A.D. and C. Buttimore, Molec. and Cell.
Biol., 6:2895-2902 (1986)), Kat cell line, (Finer, M.H., et al., Blood, 83 :43-50 (1994)) GP+E-86 cells and GP+EM12 CA 02237000 1998-0~-07 W O 97/174~7 PCTAUS96/17807 cells (Markowitz, D., et al ., J. Virol ., 62: 1120-1124 (1988), and Psi Crip and Psi Cre cells (U.S. Patent No. 5,449,614; Danos, 0. and Mulligan, R.C., PNAS, USA, 85:6460-6464 (1988)). Also see Yang, Y., et al ., Human Gene Ther., 6:1203-1213 (1995). The packaging cell lines for use in the present invention can produce retroviral particles having a pantropic amphotropic or ecotropic host range. Therefore, in this - h5~; ?nt, the cDNA expression library of the present invention can be expressed in any cell within the host range of the retroviral particle produced by the packaging cell line. Further, in this embodiment, the promoter of the retroviral vector of the present invention can be any promoter which produces sufficient levels of transcription of the retroviral vector in the particular packaging cell line (e.g., SV40 promoter, RSV promoter, ~-actin promoter).
The retroviral vectors of the present invention (e.g., the ~U3 retroviral vectors) can be used for transient transfection of packaging cell lines which produce pseudotyped retroviruses (e.g., the 293 GPG cells described herein, which are also referred to herein as H29 gagpol cells) for production of high titer pseudotyped (e.g., VSV-G) retrovirus. The retroviral vectors of the present invention permit construction of cDNA expression libraries (in the retroviral vectors) for transfection of retroviral packaging cell lines, in which pseudotyped retrovirus particles containing the cDNA expression libraries are produced. Each pseudotyped retroviral particle generally contains multiple mRNA molecules.
As described in Example 4, high titer VSV-G
pseudotyped virus using the ~U3 retroviral vectors have been produced using the 293GPG cells which can be used for expression cloning by complementation in any cell line having a VSV-G host range. The retroviral vector, ~U3nlsLZ, has been used to transfect 293GPG cells which CA 02237000 1998-0~-07 W O 97/174~7 PCTrUS96/17807 produce retroviruses at titers up to 3 x 106 infectious units (i.u.)/ml. In addition, as described in Example 5, the retroviral cDNA cloning vector, ~U3BAM, has been used to transfect 293GPG cells with levels of expression and viral titers ~_ -rable to the ~U3nlsLZ vector. Assessment of the effect of 5' untranslated sequences on cDNA
expression and viral titer showed that the viral ~U3Bam vector can acc~ odate up to 165 base pairs of 5' upstream non-coding DNA sequences with only a modest reduction in expression or viral titer (compared with a MFG-derived based retroviral vector which has been optimized for high cDNA expression and generation of high viral titers). The VSV-G retroviral pseudotypes produced by 293GPG cells described herein have broad host range and will permit infection of any mammalian type (Yee, J.-K, et al., PNAS, 91:9564-9568).
Thus, the present invention provides retroviral vectors which can be used with a variety of mammalian packaging cell lines to produce pseudotyped retroviral particles which can, in turn, be used to infect a variety of ~ -lian cells containing the expression product (protein, polypeptide) encoded by the cDNA. In a specific example, the retroviral vectors are used to transiently transfect 293GPG cells to produce high titer virus with a VSV-G host range. The ~U3BAM retroviral vector will allow cloning of any cDNA library into the vector. Transfection of the retroviral cDNA library into 293GPG cells produces retrovirus at titers >1o6 i.u./ml that are capable of infecting any mammalian cell type. Infection of host cells with this retroviral vector results in stable integration - of the proviral genome, facilitating long-term high level expression of the cDNA in the retroviral construct.
The novel methodology described herein makes use of retroviral vectors (e.g., ~U3Bam retroviral vectors) and a CA 02237000 1998-0~-07 W O 97tl7457 PCT~US96/17807 packaging cell line (e.g., the 293GPG cells), and provides for retroviral cDNA expression cloning in any mammalian cell type, obviating the need for specialized cells (e.g.
Cos7 cells, oocytes) for efficient expression cloning. In any mutant ~ ~lian cell type for which there is a phenotype distinct from the wild-type parental cell type (e.g., primary human cells derived from patients, primary or established cell lines derived from mutant animal strains, primary or established cell lines derived from knockout mice, mutant cell lines generated in cell culture) the gene defect(s) can be rapidly identified by expression cloning by complementation using this invention.
An extension of this methodology is the use of the retroviral vectors of the present invention to produce pseudotyped retrovirus in the packaging cells, for expression of candidate cDNA clones in cells that are derived from patients with genetic defects and established phenotypes. This will permit screening to determine the basis for genetic defects (e.g., altered expression of a gene involved in metabolism) in patients by complementation analysis. For example, numerous patients have been characterized biochemically and genetically to have single gene defects in fatty acid metabolism. However, the mutant genes have not been established by conventional methods. A
panel of retroviral constructs which encode candidate cDNAs for various enzymes in fatty acid metabolism can be tested, for example, for complementation in primary fibroblasts from these patients.
The retroviral vectors of the present invention are retroviral-derived vectors (e.g., Moloney murine leukemia virus-derived vectors) in which the retroviral enhancer-promoter (e.g., HCMV) has been precisely replaced with the cytomegalovirus enhancer-promoter in order to facilitate high levels of expression in the packaging cell lines (e.g., 293GPG).

CA 02237000 1998-0~-07 W O 97/17457 PCTrUS96/17807 Further, the retroviral vectors of the present invention can be used to generate retroviral cDNA
expression libraries to allow expression cloning in any - ~lian cell type. In addition, identification of mutant genes responsible for human genetic defects by expression cloning by complementation can be accomplished using retroviral vectors of the present invention. Expression cloning of mutant genes from cultured cell lines that have been mutagenized in culture and have a known phenotype or from primary or established cell lines derived from animals with mutant phenotypes can be performed. For example, the expression cDNA library of the present invention is introduced into a cell having a mutated phenotype and identification of the gene(s) which complements the defect is determined. Thus, an important advantage provided by the present invention is that expression cloning by complementation as described herein can be used to identify gene(s) responsible for a phenotype caused by a mutation and obtain proof of the function of the responsible gene(s). Further, the example of the present invention will allow identification in knockout mice of gene products which complement the introduced mutation, or of gene products which function within the pathway(s) affected by the mutation (i.e., identification of downstream effectors by suppressor analysis) can be determined with the retroviral vectors of the present invention. Retroviral cDNA libraries constructed in the retroviral vectors for commercial distribution are also provided.
The invention is further illustrated by the following examples, which are not intended to be limiting in any way.

CA 02237000 1998-0~-07 ExemPlification E~nle 1 ~xpression Vector Constructions pMD (see Figure 1) was constructed with the 3.1 kb EcoRI-BamHI fragment from pBC12.AB that includes the pXF3 backbone and human CMV ; -~iate early promoter regions and a 1.34 kb BamHI-Xbal fragment derived from pUCMd~s(R)S
(Sadelain, M., et al ., Proc. Natl . Acad . sci . USA, 92: 6728-6732 (1995)) that includes the genomic human ~-globin sequences from the BamHI site in exon 2 through 690 bp in the 3' untranslated region. The plasmid pUCMd~s(R)S, however, differs from the genomic sequence in that there is a 374 bp deletion in the second intron between the first and third RsaI sites. pBC12.AB is a derivative of pBC12/CMV/IL-2 lB. Cullen, Cell 46:973 (1986)) in which the IL-2 sequences (bp 756-1439) have been replaced with a polylinker. The 3.1 kb EcoRI-BamHI and 1.34 kb BamHI-Xbal fragments were ligated after the EcoRI and Xbal overhangs were blunt-ended by treatment with the Klenow fragments.
pMD.G (see Figure 3) was constructed with a 1.6 kb EcoRI fragment containing the VSV G gene that was derived from pSVGL (Rose and Bergman, Cell 34:513 (1983)) and was cloned into the EcoRI site in pMD which is within exon 3 of the genomic human ~-globin sequence.
pMDtet (see Figure 2) was generated with a 0.47 kb Xhol-BamHI fragment from pUHC 13-3 (Gossen and Bujard, Proc. Natl . Acad. sci. 89:5547-5551 (1992)), which contains the tet operator and ~in;~l CMV promoter sequences, the 1.34 BamHI-Xbal fragment from pUCMd~s(R)S and a 3.06 kb Xbal-Xhol fragment from pSL301 (Invitrogen).
To construct pMDtet.G (see Figure 4), the 1.6 kb EcoRI
fragment containing the VSV G gene (pSVGL) was cloned into the EcoRI site in pMD.tetG which is within exon 3 of the genomic human ~-globin sequence.

CA 02237000 1998-0~-07 To construct pMD.gagpol (see Figure 5), PCR was performed with pCRIPenv- (Danos and Mulligan, Proc. Natl.
Acad. Sci., 85:6460-6466 (1988)) and the following pairs of primers: 5'-CGGAATTCATGGGCCAGACTGTTACC-3' (SEQ ID No:1) and 5'-AGCAACTGGCGATAGTGG-3' (SEQ ID No:2), 5'-CGGAATTCTTAGGGGGCCTCGCGG-3~ (SEQ ID No:3) and 5'-ACTACATGCTGAACCGGG-3' (SEQ ID No:4). The PCR products were digested with EcoRI and Xhol and with EcoRI and HindIII, respectively, to generate 0.94 kb EcoRI-Xhol and 0.94 kb HindIII-EcoRI fragments. These fragments were ligated with the 3.3 kb Xhol-HindIII fragment from pCRIPenv- and pUC19 which had been linearized with EcoRI and phosphatase treated to produce pUCl9.gagpol. The 5.2 kb EcoRI fragment from pUCl9.gagpol was cloned into the EcoRI site in pMD, which is within exon 3 of the genomic human ~-globin sequence, to yield pMD.gagpol.
To construct pMD.new gagpol (see Figure 6), PCR was performed with pBCIL2.gagpol (Chung and Mulligan, unpublished results), which encodes a mutated gagpol sequence and the following pairs of primers: 5'-CGGAATTCATGGGTCAGACTGTTACTAC-3' (SEQ ID No: 5) and 5'-AGCAACTGGCGATAGTGG-3' (SEQ ID No: 2), 5'-CGGAATTCTTAGGGAGCTTCTCTTGTTAG-3' (SEQ ID No: 6) and 5'-ACTACATGCTGAACCGGG-3' (SEQ ID No: 4). The mutated gagpol sequences are as follows:

New 5'gagpol:
5'-ATGGGTCAGACTGTTACTACCCCTCTAAGTTTAACTTTGGGCCATTGGAAAGATGTAGAGAGGATCGCCCACAACCAGAGTGTAGACGTTAAGAAAAGACGTTGGGTCA~lllllGTT
CTGCAGAGTGGCCTACCTTCAACGTAGGCTGGCCAAGAGATGGTACTTTTAACAGAGAC
CTTATTACCCAGGTCAAGATCAAA~~ lAGTCCAGGCCCTCACGGACATCCAGATCA
GGTCCCTTACATTGTCACCTGGGAAGCTCTTGCCTTTGACCCTCCCCCTTGGGTGAAGC
CTTTTGTCCACCCTAAGCCCCCACCTCCCTTGCCTCCAAGTGCTCCTTCCCTCCCTCTT

CA 02237000 1998-0~-07 GAACCCCCTCGCAGTACTCCACCTCGATCCAGTCTCTATCCTGCCCTA-3' (SEQ ID
No: 7) New 3' gagpol:
5'-ATTCTATACGGAGCCCCTCCCCCTTTAGTTAACTTTCCAGACCCTGATATGACTAGAGTAACAAACTCTCCTAGTCTTCAGGCACACCTCCAAGCCCTGTACCTAGTCCAACATGAAG
TGTGGAGACCCTTAGCAGCTGCATACCAGGAACAGCTTGACAGGCCTGTAGTCCCCCAC
CCGTACAGAGTGGGAGACACTGTATGGGTCCGACGCCACCAAACAAAAAACTTAGAGCC
TCGATGGAAGGGCCCCTACACTGTACTACTCACAACCCCTACAGCCCTGAAGGTTGACG
GGATAGCTGCCTGGATTCACGCTGCACACGTGAAAGCAGCTGACCCTGGAGGGGGTCCC
TCTAGCAGATTAACCTGGCGCGTACAAAGATCCCAGAATCCTCTGAAAATCAGGCTAAC
AAGAGAAGCTCCCTAA-3' (SEQ ID No: 8) The PCR products were digested with EcoRI and XhoI and with EcoRI and HindIII, respectively, to generate 0.94 kb EcoRI-Xhol and 0.94 kb HindIII-EcoRI fragments. These fragments were ligated with the 3.3 kb Xhol-HindIII
fragment from pCRIPenv- and pUC19 which had been linearized with EcoRI and phosphatase treated to produce pUCl9.new gagpol. The 5. 2 kb EcoRI fragment from pUCl9.new gagpol was cloned into the EcoRI site in pMD, which is within exon 3 of the genomic human ~-globin sequence, to yield pMD.new gagpol.
A novel CMV expression vector (pMD) was constructed for expression of the wild-type gagpol (pMD.gagpol) and for the mutagenized gagpol (pMD.new gagpol). For pMD.gagpol, reverse transcriptase assays have been performed which demonstrate production of retroviral particles under conditions of both transient and stable expression. For pMD.new gagpol, reverse transcriptase assays have been performed which demonstrate production of retroviral particles under conditions of transient expression.

CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 ~rle 2 Construction of Packaqinq Cell Lines H29 cell line.
Human 293 cells (gift, B. Panning; Graham, F., et al . , J. Gen . Virol ., 36: 59-72 (1977)) were grown in DMEM with 5%
inactivated fetal bovine serum, supplemented with 2 mM L-glutamine, penicillin and streptomycin (293 growth media) and incubated at 37 C with 5% C02. The 293 cells were co-transfected by the calcium phosphate precipitation method (Pear, et al ., PNAS, 90: 8392-8396 (1994)) with 5 ~g pBC.tTA
(see Figure 7; T. Chung and R. Mulligan, unpublished results), 5 ~g pMDtet.G and 1 ~g pJ6~puro (gift, J.
Morgenstern). During transfection the 293 growth media was supplemented with 1.0 ~g/ml tetracycline (Sigma). The transfected cells were plated into selection 48 hours post-transfection in 293 growth media supplemented with l.0~g/ml tetracycline and 2 ~g/ml puromycin (Sigma). 72 independent clones were selected for clonal expansion and screened for tetracycline-inducible VSV-G expression. To screen the clones, each clone was plated in parallel into two 35 mm tissue culture dish (Corning) at 30% confluence.
The following day one plate was washed twice with 2 ml 293 growth media without tetracycline and the media changed to standard 293 media supplemented with 2 ~g/ml puromycin. At 48 hours the cells were harvested for total cellular protein and the paired samples run on a 7.5% SDS-polyacrylamide gel under reducing conditions. The gels were transferred onto nitrocellulose (Schleicher & Schuell, 0.45 mm) with a semi-dry electroblotter (Owl Scientific).
Western blotting was performed using standard procedures.
For the primary antibody a murine monoclonal anti-VSV-G IgG
(Sigma) was used at a dilution of 1:800. For the secondary antibody an HRP-coupled donkey anti-mouse IgG F(ab)2 fragment (Pharmingen) was used at a dilution of 1:10,000.
Chemiluminescent detection was performed with the Dupont CA 02237000 1998-0~-07 W O 97/17457 PCTnUS96/17807 NEN Renaissance kit. Positive cell lines (e.g. H29) were selected on the basis of no detectable VSV-G expression in the presence of tetracycline in the growth media and the detection of inducible VSV-G expression in the absence of tetracycline in the growth media.

H29 gaqPol cell line (293 GPG cell line).
H29 cells were grown in 293 growth media supplemented with 1.0 ~g/ml tetracycline and 2 ~g/ml puromycin (H29 media) and co-transfected by the calcium phosphate precipitation method with 10 ~g pMD.gagpol linearized with Scal and 2 ~g pSV2neo. During transfection the H29 media was supplemented with 1.0 ~g/ml tetracycline. The transfected H29 cells were plated into selection 48 hours post-transfection in H29 media supplemented with 1.0 ~g/ml tetracycline and 0.3 mg/ml G418 (Gibco). Sixty-nine independent clones were selected for clonal expansion and were screened for reverse transcriptase activity (Goff, et al ., ~. Virology, 38:239-248 (1981)).
Of the sixty-nine independent clones selected for clonal expansion, 10 clones had reverse transcriptase activity that exceeded the positive control (i.e., ~ Cre Cells). These 10 clones will be further characterized.

H29 new qaqpol cell line H29 cells are grown in 293 growth media supplemented with 1.0 ~g/ml tetracycline and 2 ~g/ml puromycin (H29 media) and co-transfected by the calcium phosphate precipitation method with 10 ~g pMD.new gagpol linearized with Scal and 2 ~g pSV2neo. During transfection the H29 media was supplemented with 1.0 ~g/ml tetracycline. The transfected H29 cells are plated into selection 48 hours post-transfection in H29 media supplemented with 1.0 ~g/ml tetracycline and 0.3 mg/ml G418 (Gibco). Independent clones are selected for clonal expansion and screened for CA 02237000 1998-0~-07 reverse transcriptase activity (Goff, et al., ~. Virology (1981)).

Discussion Thus, the derivative of pMD, pMDtet.G, provides for tetracycline inducible expression of VSV.G. Inducible expression of VSV-G has been demonstrated in a transient assay and in a stable cell line, H29. H29 is derived from 293 cells (Graham, F., et al., ~. Gen. Virol., 36:59-72 (1977)) and was selected after co-transfection with pBC.tTA
(the Tet transactivator), pMDtet.G and pJGnpuro. The H29 cells show inducible VSV-G expression by western blotting that is only 5-fold less than transient VSV-G expression in parental 293 cells. The H29 cells passaged in culture for 20 passages continue to demonstrate inducible VSV-G
expression.

Exam~le 3 Construction of the AU3 retroviral cloninq vectors The AU3nlsLacZ retroviral vector was constructed by precise replacement of the U3 region in the 5' LTR of MFG.SnlsLacZ (Berns, et al., Numan Gene Therapy, 6:347-368 (1995); see Figure 8) with the HCMV enhancer-promoter (nt -671 to -2) (Boshart, M., Cell, 41:521-530 (1985)). In ~U3nlsLacZ the entire 5' genomic flanking region and all but 65 bp from the 3' genomic flanking region from MFG.SnlsLacZ is eliminated.
The pMD plasmid was constructed as described in Example 1. For the construction of the ~U3nlsLacZ, a 701 bp fragment encoding the HCMV promoter was generated by PCR
with the pMD plasmid as the template with the pair of primers, 5'-GGGCCCAAGCTTCCCATTGCATACGTTGTATC-3' (SEQ ID
N0: 9) and 5'-GGACTGGCGCCGGTTCACTAAACGAGCTC-3' (SEQ ID
N0: 10), creating a 5' Hind III site and a 3' Ras I site.
The PCR product was digested with Hind III and Kas I to CA 02237000 1998-0~-07 yield a 677 bp fragment. The 91 bp ~as I-Sty I was isolated from the 3' LTR of MFG (Riviere, I., et al., PNAS, 92:6733-6737 (1995)). The 253 bp Sty I-Eag I and the 4994 bp Eag I-Sca I fragments were isolated from MFG.SNlsLacZ
(Berns, et al., Human Gene Therapy, 6:342-368 (1995)), and the backbone for ~U3nlsLacZ is a 2.65 kb ~ind III-Sma I
fragment from pUC18.
For the construction of ~U3Bam, a 561 bp fragment was generated by PCR with ~U3nlsLacZ as the template with the pair of primers, 5'-GTGACCTGGGAAGCCTTGGC-3' (SEQ ID N0: 11) and 5'CGGGATCCAGTCTAGAGGATGGTCCACC-3' SEQ ID N0: 12), creating a 5' Ras I site and a 3' Bam N I site. The PCR
product was digested with Ras I and Bam H I to yield a 389 bp fragment. The 389 bp Kas I-Bam ~ I fragment was ligated with 4466 bp Bam H I-Eag I and 695 bp Eag I-Kas I fragments that were derived from ~U3nlsLacZ.
Figure 9A displays the structure of the ~U3nlsLacZ
vector and Figure 9B displays the structure of the ~U3Bam vector.
~0 Exam~le 4 Production of VSV-G ~seudotyped retrovirus bY
transient transfection of 293GPG cells The plasmid pBC.tTA was constructed from pBC12/CMV/IL-2 (Cullen, B.R., Cell, 46:973-982 (1986)) by replacement of the IL-2 sequences (bp 756-1439) with the tet transactivator gene from pUHD10-1 (Gossen, M., et al., Proc. Natl. Acad. Sci., 89:5547-551 (1992)). Figure 7 displays the structure of the pBC.tTA plasmid.
The pMD, pMD.G, pMDtet, pMDtet.G, and pMD.gagpol constructs and the 293GPG cell line were constructed as described in Example 1.
Transient transfections with 293GPG cells were performed on 60 mm dishes where 4-5 x 106 cells were plated the night prior in 4 ml 293 GPG media. 4 ug of ~U3nlsLacZ

CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 was diluted into 300 ul opti-MEM (Gibco BRL) and incubated at room temperature for 30 minutes with 25 ul Lipofectamine ~Gibco BRL) diluted into 300 ul opti-MEM. 2.4 ml opti-MEM
was added to the DNA-Lipofectamine mixture and layered on top of the 293GPG cells, which had been rinsed 30 minutes prior to transfection and had media replaced with 2 ml opti-MEM. 2 ml 293 media was added at 7-8 hours post-transfection and the media was changed at 24 hours. The supernatant was harvested at 72 hours and viral titers determined as described below.

Assay for ~-Galactosidase ActivitY and Determination of Viral Titers To stain cells for ~-galactosidase activity, cells were washed with phosphate buffered saline supplemented with 1 mM magnesium (PBS+) and fixed with 1% glutaraldehyde in PBS+ for 10 minutes at 37~C (Lim, K., et al., Biotechniques, 7:576-579 (1989). The fixative was aspirated and the cells incubated with 3.3 mM potassium ferricyanide (Sigma), 3.3 mM potassium ferrocyanide (Sigma) and 0.2% X-gal (Molecular Probes) in PBS+ for 2 hours at 37~C. Quantitative ~-galatosdase activity was determined using a commercially available luminescent assay (Clontech). To determine viral titers, NIH 3T3 cells were plated at 1 x 105 cells per well in 6-well culture dishes 16 hours prior to infection and incubated for 24 hours with serial dilutions of viral supernatants containing 8 ug/ml polybrene (Sigma). Viral titer was determined as the average number of cells with blue nuclei (~-galactosidase-producing cells) per twenty 1 mm2 fields (2--3x 104 cells) multiplied by a factor to account for plate size, dilution of viral stock and division of target cells in tissue culture wells.

CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 Production of VSV-G pseudoty~ed retrovirus by transient transfections of 293GPG cells The 293GPG clone was used to produce VSV-G pseudotyped retrovirus by transient transfection, taking advantage of the high transfectability of 293 cells. For transient transfection of the 293GPG cells, the ~U3nlsLacZ retroviral construct was generated by substitution of the U3 region of the 5' LTR of MFG.nlsLacZ with the HCMV enhancer-promoter and deletion of the genomic 5' and 3' flanking regions of MFG.SnlsLacZ. When compared to MFG.SnlsLacZ, aU3nlsLacZ
enabled a 20-fold increase in expression in transient transfections. The 293GPG clone was transiently transfected by lipofectamine with an average efficiency of 40% with the ~U3nlsLacZ construct. A 48 hour virus supernatant was collected between 24 and 72 hours post-transfection and removal of tetracycline from the growth medium. Viral titers in the range of 1-3 x 106 i.u./ml were achieved transiently.

ExamPle 5 Effect of 5' Untranslated Sequences on cDNA
ex~ression and viral titer There are several differences between previously used vectors and the ~U3 retroviral cloning vectors. First, the AU3 vectors are specifically modified for high transient expression in 293-derived cell lines (e.g. 293GPG cells) by precise replacement of the U3 region in the 5' LTR by the complete human CMV enhancer-promoter (Boshart et al., Cell, 41:521-530 (1985)). Second, the ~U3 vectors are derived from MFG which is an established high titer and high expression vector. Finally, unlike the vectors used for previous retroviral expression cloning, the effect of 5' untranslated sequences on cDNA expression and cDNA viral titer was examined. This is important because in the course of construction of cDNA libraries (Seed, B. and CA 02237000 1998-0~-07 W O 97/17457 PCT~US96/17807 Aruffo, S., PNAS, 84: 3365-3369 (1987)) the cDNA inserts often include up to 200 base pairs of 5' untranslated sequences. Therefore, the retroviral cloning vector must be able to accommodate these additional sequences so that bias will not be introduced into the library.
The effect of 5' untranslated sequences on cDNA
expression and viral titer was ~ ;ned by insertion of the lacZ gene with 0-165 base pairs of 5' untranslated sequences into the ~U3Bam vector. The following is the data normalized to 0 base pairs of untranslated sequence (i.e., ~U3LacZ which does not contain the modified Bam H I
cloning site):
# of 5' expression (%) viral titer (%) untranslated bp These results demonstrate that the ~U3Bam vector can accommodate up to 165 bp of 5~ untranslated sequences within the cDNA insert with only a modest reduction in expression or viral titer as compared with a non-cloning MFG-based retroviral vector. This data supports the capability of the AU3Bam vector to promote efficient packaging of the cDNA inserts and efficient transfer of the cDNA to the target calls.

E~uivalents ~ 10 Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

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PSEUDOTYPED RETROVIRUSES
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(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: WUMS96-01 (B) FILING DATE: May 21, 1996 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: WHI95-07 (B) FILING DATE: N~v. ~er 8, 1995 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION
(A) NAME: Granahan, Patricia (B) REGISTRATION NUMBER: 32,227 (C) X~r~-~CE/DOCKET NUMBER: WHI95-07M PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 861-6240 (B) TELEFAX: (617) 861-9540 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 26 baQe pairs ~B TYPE: nucleic acid ,'CI STRANDEDNESS: single ~D, TOPOLOGY: linear CA 02237000 1998-0~-07 WO 97/17457 PCT~US96/17807 (ii) ~OLECULE TYPE: other nucleic acid (A) DESCRIPTION: /de~c ~ "oligonucleotide"

(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:1:

(2) INFORMATION FOR SEQ ID NO:2:
( i ) ~yU~N~: CHARACTERISTICS:
'A'I LENGTH: 18 ba~e pair~
BI TYPE: nucleic acid C, STRANDEDNESS: ~ingle ~D~ TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /de~c = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
~A'l LENGTH: 24 ba~e pair~
~BJ TYPE: nucleic acid ~CJ STRANDEDNESS: single ~D,, TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A'l LENGTH: 18 ba~e pairs IBI TYPE: nucleic acid ,C STRANDEDN~SS: ~ingle ~Dl TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID No:4:

CA 02237000 l998-0~-07 WO 97/17457 PCT~US96/17807 (2) INFORMATION FOR SEQ ID NO:5:
(i) s~Q~ CHARACTERISTICS:
~A' LENGTH: 28 ba~e pair~
~BJ TYPE: nucleic acid 'C, STRANDEDNESS: single ~D,l TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"

(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID No:6:
( i ) S~U~N~ CHARACTERISTICS:
'A'l LENGTH: 29 ba~e pairs BI TYPE: nucleic acid ~,C, STRANDEDNESS: ~ingle ~D, TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTIO~: /desc = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CGGAATTCTT AGGGAGCTTC ~l L~7 ~ lAG 29 (2) INFORMATION FOR SEQ ID NO:7:
(i) S~U~:N~ CHARACTERISTICS:
,'A'I LENGTH: 402 ba~e pair~
~Bl TYPE: nucleic acid ~C, STRANDEDNESS: single D; TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) S~U~N~ DESCRIPTION: SEQ ID NO:7:
ATGGGTCAGA CTGTTACTAC CC~l~lAAGT TTAACTTTGG GCCATTGGAA AGATGTAGAG 60 AGGATCGCCc A~Ac~r-A~7 TGTAGACGTT AA~.AAAA~AC GTTGGGTCAC 'Ll111'~71 l~'L 120 GCAGAGTGGC CTACCTTCAA CGTAGGCTGG CrAA~-~-ATG GTACTTTTAA cA~-A~-ACCTT 180 ATTACC~AGG TCAAGATCAA A~l~ AGT CCAGGCCCTC ACGGACATCC AGATCAGGTC 240 CCTTACATTG TCACCTGGGA AGCTCTTGCC TTTGACCCTC CCC~lLGGGT GAAGCCTTTT 300 GTCCACCCTA AGCCCC~ACC ~lCC~lLGCCT CCAAGTGCTC ~llCC~LCCC TCTTGAACCC 360 CA 02237000 1998-0~-07 W O 97/17457PCTtUS96tl7807 (2) lNr ~KMATION FOR SEQ ID NO:8:
U~N~ CHARACTERISTICS:
(A', LENGTH: 429 ba~e Pair~
,B TYPE: nUC1eiC aCid ~C~ STRANDEDNESS: ~ing1e ~DJ TOPOLOGY: 1inear (ii) MOLECULE TYPE: DNA (9~-- ;C) (Xi) ~U~N~: DESCRIPTION: SEQ ID NO:8:
ATTCTATACG GAGCCC~1CC CC~L.1AGTT AA~1~C~AG ACCCTGATAT GACTAGAGTA 60 AC~ArTCTC CTA~LC1--~A GGCACACCTC CAAGCCCTGT ACCTAGTCCA ACATGAAGTG 120 TGrArArCCT TAGCAGCTGC A~ACr~eGAA CAGCTTGACA GGCCTGTAGT CCCCrACCCG 180 TAe~rArTGG GAGACACTGT ATGGGTCCGA CGCr~Cr~A~ CAAAAAACTT AGAGCCTCGA 240 GCTGCCTGGA TTCACGCTGC ACACGTGAAA GCAGCTGACC CTGGAGGGGG 1CC~ ~AGC 360 AGATTAACCT GGCGCGTACA AAGATCCCAG AA~C~1~A A~ATCAGGCT ~CA~r~r~ 420 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
'A', LENGTH: 32 ba~e Pair~
,B, TYPE: nUC1eiC acid ~C~ STRANDEDNESS: ~ing1e ~,D~ TOPOLOGY: 1inear (ii) MOLECULE TYPE: DNA (genOmiC) (Xi) ~:QU~:N~ DESCRIPTION: SEQ ID NO:9:
GGGCCCAAGC TTCCCATTGC ATAC~ LA TC 32 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
~A~, LENGTH: 29 baSe Pair~
~B, TYPE: nUC1eiC ac id ~C STRANDEDNESS: qing1e ,D, TOPOLOGY: 1inear (ii) MOLECULE TYPE: DNA (genOmiC) W O 97/17457 PCTrUS96/17807 (xi) ~yu~:N~ DESCRIPTION: SEQ ID NO:lû:
GGACTGGCGC CGG~ACTA AACGAGCTC 29 t2) INFORMATION FOR SEQ ID NO:ll:
(i) s~Q~:N~ CHARACTERISTICS:
Aj LENGTH: 20 base p~irn BI TYPE: nucleic acid ~C, STRANDEDNESS: single ,DJ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (gen~ ic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

~2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
,A' LENGTH: 28 ba~e pairs BI TYPE: nucleic acid ~C, STRANDEDNESS: single fD TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Claims (35)

What is claimed is:

1. A stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus, wherein the packaging cell line comprises one or more non-retroviral expression constructs which direct expression of:
a) retroviral gagpol which produce a retroviral core virion; and b) a gene which encodes a pseudotyped envelope protein for the retroviral core virion and is under control of an inducible operator system, wherein the protein of b) provides a pseudotyped envelope protein for the retroviral core virion resulting in production of a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus.

2. A stable, pseudotyped retrovirus packaging cell line which is human derived and generates helper-free recombinant pseudotyped retrovirus with a pantropic host range, wherein the packaging cell line comprises one or more non-retroviral expression constructs which direct expression of:
a) retroviral gagpol genes which produce a retroviral core virion; and b) a gene which encodes a pseudotyped envelope protein for the retroviral core and is under control of an inducible operator system, wherein the Vesicular Stomatitis Virus G glycoprotein provides a pseudotyped envelope protein for the retroviral core virion resulting in production of a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range.

3. The stable, pseudotyped retrovirus packaging cell line of Claim 2 wherein the envelope protein of b) is Vesicular Stomatitis Virus G glycoprotein.

4. The packaging cell line of Claim 3 wherein the non-retroviral expression construct comprises a human cytomegalovirus immediate early promoter.

5. The packaging cell line of Claim 3 wherein the inducible operator system for expression of the Vesicular Stomatitis Virus G glycoprotein is a tet operator system.

6. The packaging cell line of Claim 3 wherein the retroviral gagpol genes are mutated.

7. The packaging cell line of Claim 3 wherein the cells are K29 gagpol cells comprising human cells which express VSV-G under the control of an inducible tet operator, tTA and gagpol.

8. The packaging cell line of Claim 6 wherein the cells are H29 new gagpol cells comprising human cells which express VSV-G under the control of an inducible tet operator, tTA and mutated gagpol genes.

9. A stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range, wherein the packaging cell line comprises one or more cytomegalovirus expression constructs which direct expression of:

a) retroviral gagpol genes which produce a retroviral core virion; and b) Vesicular Stomatitis Virus G glycoprotein under control of an inducible tet operator system, wherein the Vesicular Stomatitis Virus G glycoprotein provides a pseudotyped envelope protein for the retroviral core virion resulting in production of a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range.

10. The packaging cell line of Claim 9 wherein the cells are X29 gagpol cells comprising human cells which express VSV-G under the control of an inducible tet operator, tTA and gagpol.

11. A stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range, wherein the packaging cell line comprises one or more cytomegalovirus expression constructs which direct expression of:
a) mutated retroviral gagpol genes; and b) Vesicular Stomatitis Virus G glycoprotein under control of an inducible tet operator system, wherein the Vesicular Stomatitis Virus G glycoprotein provides a pseudotyped envelope protein which interacts with the retroviral gagpol proteins that are expressed from the mutated gagpol genes to generate helper-free recombinant pseudotyped retrovirus with a pantropic host range from a stable, pseudotyped retrovirus packaging cell line.

12. The packaging cell line of Claim 11 wherein the cells are H29 new gagpol cells comprising human cells which express VSV-G under the control of an inducible tet operator, tTA and mutated gagpol genes.

13. A method of making a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus, comprising the steps of transfecting mammalian cells with one or more non-retroviral expression constructs which direct the expression of a) retroviral gagpol genes which produce a retroviral core virion, and b) a protein which provides a pseudotyped envelope for the retroviral core virion and is under control of an inducible operator system, wherein the protein of b) provides a pseudotyped envelope protein for the retroviral core virion resulting in production of a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus.

14. A method of making a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range, comprising the steps of transfecting mammalian cells with one or more non-retroviral expression constructs which direct the expression of a) retroviral gagpol gones which produce a retroviral core virion, and b) Vesicular Stomatitis Virus G glycoprotein under control of an inducible operator system, wherein the Vesicular Stomatitis Virus G glycoprotein provides a pseudotyped envelope protein for the retroviral core virion resulting in production of a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range.

15. The method of Claim 14 wherein the non-retroviral expression construct is a cytomegalovirus construct.

16. The method of Claim 14 wherein the inducible operator system for expression of the Vesicular Stomatitis Virus G glycoprotein is a tet operator system.

17. The method of Claim 14 wherein the mammalian cells are H29 cells comprising human cells expressing VSV-G
under the control of an inducible tet operator and tTA.

18. The method of Claim 14 wherein the retroviral gagpol genes are mutated.

19. The method of Claim 18 wherein the mutated gagpol genes comprise the new 5' gagpol and the new 3' gagpol nucleotide sequences, SEQ. ID Nos: 7 and 8.

20. A method of making a stable, pseudotyped retrovirus packaging cell line which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range, comprising the steps of:
a) transfecting mammalian cells with a first non-retroviral construct which codes for the tet transactivator and a second non-retroviral construct which codes for the Vesicular Stomatitis Virus G glycoprotein under control of the tet transactivator;
b) screening the cells of a) for tetracycline-inducible VSV-G expression in which VSV-G is not detected in the presence of tetracycline and is detected in the absence of tetracycline;
c) transfecting the cells of b) with a third non-retroviral construct which codes for the retroviral gagpol proteins, and d) screening the cells of c) for production of retroviruses wherein the transfected cells of d) which produce retroviruses are stable, pseudotyped retrovirus packaging cells which generates helper-free recombinant pseudotyped retrovirus with a pantropic host range.

21. The method of Claim 20 wherein the second and third constructs are cytomegalovirus constructs.

22. The method of Claim 20 wherein the mammalian cells are H29 cells comprising human cells expressing VSV-G
under the control of an inducible tet operator and tTA.

23. The method of Claim 20 wherein the retroviral gagpol proteins are mutated.

24. The method of Claim 23 wherein the mutated gagpol proteins comprise the new 5' gag and the new 3' pol proteins, encoded by SEQ. ID No: 7 and 8.

25. H29 cell line comprising human cells expressing VSV-G
under the control of an inducible tet operator and tTA.

26. H29 gagpol cell line comprising human cells expressing VSV-G under the control of an inducible tet operator, tTA, and gagpol.

-46a-

27. H29 new gagpol cell line comprising human cells expressing VSV-G under the control of an inducible tet operator, tTA and mutated gagpol genes.

28. pMD comprising the vector of Figure 1.

29. pMDtet comprising the vector of Figure 2.

30. pMDtet.G comprising the vector of Figure 4.

31. pMD.gagpol comprising the vector of Figure 5.

32. pMD.new gagpol comprising the vector of Figure 6.

33. A retroviral vector for producing a cDNA library for expression in mammalian cells, comprising:
a) two retroviral LTR3;
b) a cloning site for insertion of cDNA; and c) a cytomegalovirus promoter.

34. The retroviral vector of Claim 33 wherein the retroviral LTRs are Moloney murine leukemia virus LTRs. ;

35. The retroviral vector of Claim 34 wherein one of the LTRs is a modified Moloney murine leukemia virus LTR
in which the U3 region of the Moloney murine leukemia virus LTR is replaced with the human cytomegalovirus promoter.