WO1997010006A1

WO1997010006A1 - Method for inhibiting cell-mediated killing of target cells

Info

Publication number: WO1997010006A1
Application number: PCT/US1996/014571
Authority: WO
Inventors: R. Chris Bleackley; Grant Mcfadden; Richard W. Moyer
Original assignee: The Governors Of The University Of Alberta; University Of Florida Research Foundation, Inc.
Priority date: 1995-09-11
Filing date: 1996-09-11
Publication date: 1997-03-20
Also published as: CA2231804A1; JP2001519644A; EP0859638A1; AU7108496A

Abstract

Disclosed is a method for inhibiting cell-mediated (e.g., CTL-mediated) killing of a mammalian target cell by expressing in the target cell a poxvirus serpin proteinase inhibitor (SPI)-1. If desired, SPI-2 can be expressed in the cell in addition to SPI-1. Cells expressing these proteinase inhibitors are resistant to cell-mediated killing and apoptosis.

Description

METHOD FOR INHIBITING CELL-MEDIATED

KILLING OF TARGET CELLS

1. Cross Reference To Related Applications

This application claims priority under 35 U.S.C. § 119 from U.S. Provisional Application Serial No.60/003,665, filed September 11 , 1995.

2. Statement as to Federally Sponsored Research

This invention was made at least in part with funds from the Federal Government under National Institutes of Health Grant No. 5 RO1 Al 15722. The Government therefore has certain rights in the invention. 3. Background of the Invention

Cell-mediated killing of target cells can be undesirable in vivo. For example, cell-mediated killing of grafts is thought to be responsible for rejection of a graft by a host Similarly, autoimmune diseases, such as diabetes, are thought to be arise from the undesirable induction of cell-mediated killing of an individual's cells (e.g., pancreatic islet cells) . Cell-mediated killing has also hampered progress in the field of gene therapy. Many gene therapy methods entail the use of a viral vector to express a therapeutic gene in a target cell. Generally, such viral vectors also encode viral antigens that induce the patient's immune system to effectuate cell-mediated killing of target cells that are transfected with the virus and therapeutic gene. Thus, these examples illustrate that the induction of an immune response can be undesirable.

Cell-mediated killing by cytotoxic T lymphocytes (CTL) is thought to occur by secretion of cytoplasmic granules containing perform and granzymes, or by signaling via the Fas pathway (Berke, 1994, Ann. Rev. Immun. 12:735-773 and Nagata et al., 1995, Science 267:1449-1456). Summary of the Invention

Applicants have discovered that cell-mediated killing of a target cell can be inhibited by expressing in the target cell a proteinase inhibitor of an orthopoxvirus (e.g., SPI-1).

Accordingly, the invention features a method for inhibiting cell-mediated cell death of a mammalian target cell by expressing in the target cell an orthopoxvirus proteinase inhibitor, thereby inhibiting cell-mediated cell death. In particular, the invention can be used to inhibit CTL-mediated cell death and apoptosis. The invention is thus useful for suppressing the immune system of a patient, e.g., for treating an autoimmune disease, inhibiting graft rejection, or inhibiting the death of cells that are targeted in gene therapy methods.

Brief Description of the Drawings Figs. 1A and 1B are histograms representing the inhibition of granule-meditated cytolysis of cells infected with CPV or RPV. L1210 (Fig. 1A) or EL4 (Fig. 1B) cells were either mock-infected or infected with CPV or RPV, and cytolysis was assayed by incubation with stimulated CTL21.9 effector cells in a standard 4 hour chromium release assay at an effector to target ratio of 5:1. To facilitate comparison of multiple experiments, the values for % specific lysis for mock-infected cells were set to 100%, and other data are shown relative to mock-infected cells. Each bar represents the mean and standard deviation calculated from 3-5 independent experiments, each performed in triplicate.

Effector cells (CTL21.9 - Havele, et al., 1986, Journal of Immunology 137: 1448-1454) were stimulated for 24 hours prior to assay at 37°C with a 1/250 dilution of

2C1 1 hybridoma supernatant (anti-CD3 monoclonal antibody - Leo, et al., 1987, Proceedings of the National Academy of Sciences USA 84:1374-4274). L1210 cells (a murine T lymphoma derived from DBA/2 mice, obtained from Dr. Pierre Goldstein, CNRS, Marseille, France or EL4 cells (a murine T lymphoma cell line) were infected at a multiplicity of 10 plaque forming units per cell with either CPV (Brighton Red strain) or RPV (Utrecht strain), or were mock-infected in RPMI (RPMI 1640 containing 5% fetal bovine serum (Gibco) and 10^-4 M β-mercaptoethanol) for 12 hours at 37°C.

Cells were labeled with ⁵¹Cr (100μCi per 2 × 10⁶ cells, Dupont NEN) at 37°C for 90 minutes, washed three times, and added to V-bottomed 96-well plates (1 × 10⁴ cells per well) with stimulated CTL21.9 effector cells (5 × 10⁴ cells per wall). Plates were centrifuged at 500 rpm for 5 minutes to promote cell contact, and then incubated for 4 hours at 37°C in RPMI . Supernatants were then removed and counted in a gamma counter. Each assay was performed in triplicate Specific lysis and relative % Specific Lysis were calculated as follows: % Specific Lysis = (sample -spontaneous release)/(total - spontaneous release)×100 Relative % Specific Lysis =

% Specific Lysis (virus infected cells)/% Specific Lysis (mock infected cells).

Figs.2A - 2D are a series of histograms representing inhibition of Fas-mediated cytolysis of virus-infected L1210-Fas (Fig. 2A), EL4 (Fig. 2B) and YAC-1 (Fig. 2C) cells. L1210-Fas, EL4 and YAC-1 cells were either mock-infected or infected with the indicated virus as described in the legend to figure 1. Cytolysis of target cells was assayed by incubation with stimulated PMM-1 effector cells in a standard 4 hour chromium release assay at an effector to target ratio of 5:1. To facilitate companson of multiple expenments, the values for % specific lysis for mock-infected cells were set to 100%, and other data are shown relative to mock infected cells. Each bar represents the mean and standard deviation calculated from 3-5 independent experiments, each performed in triplicate. Fig. 2D is the histogram obtained after L1210-FAS and L1210 cells were either mock-infected or infected with CPV or RPV, and incubated with stimulated PMM-1 effector cells at an effector to target ratio of 5:1 and the % specific chromium release was determined. Representative data from a single experiment performed in triplicate are shown. Virus infections, chromium release assays, and calculations were performed as described in the legend to Figs. 1A and 1 B PMM-1 effector cells (Kaufmann, et al., 1981 , Proceedings of the National Academy of Sciences USA 78:2502) (a BALB/c derived peritoneal exudate lymphocyte CTL hybridoma; obtained from Dr. G. Berke, Weizmann Institute of Science, Rehovot, Israel, were stimulated prior at assay by incubating with PMA (10 ng/ml; Sigma) and lonomyαn (3#μg/ml; Sigma) at 37°C for three hours L1210-FAS is an L1210 cell transfectant expressing the murine Fas antigen (Rouvier, et al., 1993, Journal of Experimental Medicine 177:195-200) (obtained from Dr. Pierre Goldstein,

CNRS, Marseille, France), and YAC-1 is murine Lymphoma cell line.

Figs. 3A-3F are a series of histograms representing Fas-mediated cytolysis of target cells infected with virus mutants in the SPI-1 or SPI-2 genes. L1210-FAS (Figs. 3A and 3D), EL4 (Figs. 3B and 3E), or YAC-1 (Figs. 3C and 3F) were either mock-infected or infected with either wild type CPV, a CPV mutant in the SPI-1 gene

(Thompson, et al., 1993, Virology 197:328-338) (CPVΔSPI-1), a CPV mutant in the SPI-2 gene (Ali, et al., 1994, Virology 202:305-314) (CPVΔSPI-2), wild type RPV, RPV mutants in the SPI-1, SPI-2 (All, et al., 1994, Virology 202:305-314), or both SPI-1 and SPI-2 genes (RPVΔSPI-1, RPVΔSPI-2, RPVΔSPI-1/2, respectively). Data are shown as the % specific lysis relative to mock infected cells and are the mean and standard deviations of three independent experiments. Virus infections, chromium release assays, and calculations were performed as described in the legends to Figs. 1A, 1 B, and 2A-2D. RPV mutant in both SPI-1 and SPI-2 was constructed from RPVΔSPI-1 by homologous recombination leading to replacement of a 447 bp region of the SPI-2 open reading frame with the E.coli lacZ gene driven by the vaccinia virus p1 1 promoter. Figs. 4A-4D are a series of histograms representing Granule-mediated cytolysis of target cells infected with virus mutants in the SPI-1 or SPI-2 genes. L1210 (Figs. 4A and 4C) or EL4 (Figs.4B and 4D) cells were infected with wild type viruses or virus mutants in either the SPI-1 or SPI-2 genes, or with RPV containing mutations in both the SPI-1 and the SPI-2 genes (RPVΔSPI-1/2, described above). Data are shown as the % specific lysis relative to mock infected cells and represent the mean and standard deviations of three independent experiments. Virus infections, chromium release assays, and calculations were performed as described in the legends to Figs. 1A, 1 B, and 2A-2D.

Fig. 5 is a listing of the DNA and amino acid sequences for SPI-1 (SEQ ID Nos:

1 and 2, respectively).

Fig 6 is a listing of the DNA and amino acid sequences for SPI-2 (SEQ ID NOs: 3 and 4, respectively).

Detailed Description of the Invention The invention provides a method for inhibiting cell-mediated killing of a target cell

"Cell-mediated" killing refers to ability of a cell (e.g., a cytotoxic T lymphocyte (CTL)) to effectuate the death of a target cell, e.g., by cytolysis or apoptosis. In practicing the invention, cell death is inhibited by expressing in the target cell a proteinase inhibitor of a pox virus, such as an orthopoxvirus, thereby inhibiting cell-mediated (e.g., CTL-mediated) killing of the target cell.

If desired, the method can include identifying the target cell as a target of cell-mediated killing. Numerous cells are known to be targets of cell-mediated cell death Preferably, the cell is a human cell. For example, cells of heterologous or autologous grafts are targets for cell-mediated killing, and such cells can be used in the invention. Bone marrow cells are particularly suitable for use in the invention, as such cells can readily be obtained, manipulated in vitro, and then introduced into a patient. Other suitable cells include cells that are associated as tissues (e.g., liver tissue) or organs (e.g., hearts) that can be grafted in methods of transplantation. Likewise, cells that are destroyed due to autoimmune diseases, e.g., pancreatic islet cells of diabetes patients, also are targets for cell-mediated killing. In addition, CD4⁺ lymphocytes of patients infected with Human Immunodeficiency Virus (HIV) are considered target cells in the invention. Such CD4⁺ lymphocytes are thought to be killed by apoptosis that is induced by stimulation of the CD4 molecule on the lymphocytes by a complex of gp120 and antibody. The Fas antigen/Fas ligand system is thought to mediate the death of CD4⁺ cells even though the cells are not thought to be infected with HIV. Thus, cell-mediated killing of these cells results in the massive depletion of CD4 cells that is observed in AIDS patients. When the invention is used in a method of treating a patient, the cell can be autologous or heterologous to the patient.

In a preferred embodiment, the invention can be used to inhibit cell-mediated killing of cells that are the targets of conventional gene therapy methods. Many conventional gene therapy methods employ viral vectors to express a therapeutic gene in a cell of a mammal. A "therapeutic" gene is any gene that, when expressed, confers a beneficial effect on a cell. In vivo, such a gene is one that ameliorates a sign or symptom of a disorder, or confers a desired phenotype on a cell or another cell of the patient. Such a therapeutic gene can be, for example, a gene that corrects a deficiency in gene expression (e.g., an insulin gene for correcting a deficiency in insulin expression). Traditional gene therapy methods are hindered because viral vectors carrying therapeutic genes also express viral antigens that induce cell-mediated killing of target cells that are transfected with the viral vectors. The invention thus provides a method for inhibiting cell-mediated death of these target cells, with inhibition being accomplished by expressing a poxvirus proteinase inhibitor in the target cell. If desired, the proteinase inhibitor can be expressed from the same vector, or a different vector, as the vector used to express the therapeutic gene. Additional cells that are targeted for cell-mediated cell killing can readily be identified in conventional in vitro CTL cytotoxicity assays, such as chromium release assays.

Conventional gene delivery and gene expression methods can be used to express a proteinase inhibitor in a cell. For example, vectors derived from mammalian viruses, such as retrovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors, are well known in the art and can be used in the invention. Other art-known gene delivery and expression techniques can also be used in the invention. For example, the proteinase inhibitor can be expressed from a genetic construct (i.e., any nucleic acid, such as a plasmid or cosmid, engineered to express a gene). If desired, the vector can be engineered to contain a "suicide" gene. Such genes are known in the art for their ability to encode a factor that renders the cell sensitive to a substance that can be administered to the cell in the event that subsequent killing of the target cell should be desired.

The various genetic constructs can be introduced into a cell by conventional methods, such as liposome-based methods, direct uptake, electroporation, CaCl-based methods, and the like. When liposome-based methods are used, the liposomes can be engineered to have on their surface desired markers, e.g., receptors, antibodies, lectins, or carbohydrates.

Typically, the nucleic acid encoding the proteinase inhibitor is operably linked to a promoter that is active in a mammalian cell (e.g ., a promoter that naturally drives expression of a gene in a mammalian cell or a promoter of a virus that infects a mammalian cell). Preferably, the promoter is a promoter of an poxvirus, such as a promoter that naturally drives expression of a proteinase inhibitor. If desired, the promoter can be a cell-specific promoter, a tissue-specific promoter, or a stage-specific promoter. Such promoters are known in the art and can be used to confer specificity in the practicing the invention. When tissues or organs are used as the target cells in the invention, conventional organ perfusion methods are well suited for delivering the nucleic acid encoding the proteinase inhibitor to the cells of the tissue or organ.

Preferably, the proteinase inhibitor is a serpin proteinase inhibitor, such as SPI-1 (SEQ ID NO: 2; GenBank Accession No. UO7766; Ali et al., supra), SPI-2 (SEQ ID

NO: 4 ; GenBank Accession No. UO7763; Ali et al., supra (SPI-2 is also known as crmA in CPV), or SPI-3 (Turner et al., 1995, Viroceptors, Virokines, and Related Immune Modulators Encoded by DNA Viruses, McFadden ed, R.G. Landes Company, Austin, TX). The proteinase inhibitor can be derived from any pox virus, preferably, the virus is an orthopox virus, such as a cowpox virus, rabbitpox virus, smallpox virus, or vaccinia virus. Such virus can be obtained from ATCC. Each of these viruses naturally encodes members of the serpin family of proteinase inhibitors, which can be used in the assay. Mutant strains of these viruses that have decreased pathogenicity while nonetheless expressing a proteinase inhibitor can used. In addition, variants (e.g., conservative variants) and mutants of these proteinase inhibitors can be used, provided that the resulting polypeptide retains a detectable ability to inhibit CTL-mediated cell death (e.g., as determined in a CTL cytotoxicity assay as described herein).

By "conservative variant" is meant a polypeptide having an amino acid substitution where the native amino acid and the substituted amino acid are of approximately the same charge and polarity. Such substitutions typically include, e.g., substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine, methionine, aspartic acid, glutamic acid; asparagine, glutamine, serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In general, such conservative amino acid substitutions do not significantly affect the function of the polypeptide. If desired, the ability of a variant or mutant to function as a proteinase inhibitor can be measured in a CTL cytotoxicity assay as described herein, using the wild-type proteinase inhibitor as a standard.

The invention also presumes the use of nucleic acids encoding a protease inhibitor where the nucleic acid is a degenerate variant of the nucleic acid sequences expressly described herein. By "degenerate variants" of a nucleotide sequence is meant nucleotide sequences that encode the same amino acid sequence as a given nucleotide sequence, but in which at least one codon in the nucleotide sequence is different. Degenerate variants occur due to the degeneracy of the genetic code, whereby two or more different codons can encode the same amino acid. Applicants have discovered that the presence of nucleotide encoding SP-1 polypeptide and nucleotide encoding SP-2 polypeptide provide enhanced inhibition of cell-mediated killing.

Included within the invention is an isolated target cell that is resistant to cell-mediated killing. Such cells contain a nucleic acid that consists essentially of a nucleic acid that expresses a poxvirus SPI-1 polypeptide, as described herein. Such cells can also include a nucleic acid that consists essentially of a nucleic acid that expresses an SPI-2 polypeptide. If desired, these cells can also express a therapeutic gene. The term "isolated" target cell means that a gene is delivered to a cell and expressed non-systemically in a population of cells (e.g., in liver cells or pancreatic islet cells). Such cells can be in the form of a cell suspension (e.g., bone marrow cells) or they can be in the form of a tissue or organ (e.g., a liver) for use in organ transplantation methods. Encompassed by this term are cell populations of a mammal that are targeted for SPI-1 expression, without systemic expression of SPI-1 in the mammal. For example, liver cells of a mammal that are targeted for SPI-1 expression are considered "isolated," even when the cells are contained within the mammal.

The invention also provides a method for determining whether a nucleic acid (i.e., a "test" nucleic acid) encodes a polypeptide that inhibits cell-mediated killing of a target cell. Thus, this aspect of the invention provides a method for identifying additional proteinase inhibitors that can be used in the methods described above.

One method entails introducing into a target cell a mutant poxvirus that does not express a functional SPI-2 polypeptide; expressing in the target cell the "test" nucleic acid; and detecting inhibition of cell-mediated killing of the target cell as an indication that the test nucleic acid encodes a polypeptide that inhibits cell-mediated killing. In this sense, the "test" nucleic acid is tested for its ability to complement the SPI-2 deletion. Mutant viruses that fail to encode functional SPI-2 are known in the art (Ali et al., infra), and additional viruses can be produced using conventional mutagenesis methods. It is expected that the majority of such polypeptides will be proteinase inhibitors that, like SPI-2, belong to the serpin family of proteinase inhibitors . Typically, the polypeptide encoded by the "test" nucleic acid is further characterized by testing its ability to confer resistance to cell-mediated killing in the presence of a poxvirus that does not encode a functional SPI-1 polypeptide. Such an SPI-1 mutant has been described (Thompson et al., infra), and additional mutant viruses can be produced using conventional gene manipulation techniques. A "test" nucleic acid encoding a polypeptide that confers resistance to cell-mediated killing in both of the aforementioned genetic settings is considered a new inhibitor of cell-mediated killing.

The nucleic acid encoding such a polypeptide can then be used in the invention to confer on a target cell resistance to cell-mediated killing.

Examples

The following examples and meant to illustrate, not limit, the invention, the metes and bounds of which are determined by the claims. While the methods described in these examples are typical of those that can be used, other procedures known to those skilled in the art may alternatively be used.

Example I: Cowpox Virus and Rabbitpox Virus Inhibit CTL-Mediated Cytolysis This example shows that cells infected with an orthopoxvirus are resistant to

CTL-mediated cytolysis. In this example, target cells were infected with wild-type cowpox virus (CPV (Brighton Red strain) or rabbbitpox virus (RPV (Utrecht strain)), (American Type Culture Collection, Rockville, MD.), at a multiplicity of infection (moi) of ten plaque forming units (pfu)/cell for twelve hours. Each of these viruses expresses a serpin proteinase inhibitor in the infected cells. The target cells were then assayed for cytolysis by CTL21.9 effector cells in a conventional chromium release assay. In this example, the target cells were L1210 (ATCC) and EL4 cells (thymoma cells). These target cells are known to be efficiently lysed by CTL21 9, and lysis occurs in a calcium-dependent reaction that involves the secretion of cytoplasmic granules (Garner et al., 1994, J Immun. 153:5413-5421 ).

Infection of L1210 cells with CPV resulted in a dramatic and reproducible inhibition of cytolysis by CTL21 .0 (Fig. 1A), as compared with mock-infected cells. Infection with RPV also resulted in significant inhibition of CTL-mediated cytolysis (Fig. 1A). Infection of EL4 cells with either CPV or RPV resulted in an approximately 50% reduction in the level of cytolysis by CTL21.9 (Fig. 1B). The calcium-dependent nature of cytolysis of these cells, was confirmed by carrying out the assays in the presence of EGTA (data not shown). In sum, this example illustrates that infection by CPV and RPV each can inhibit the ability of CTL to lyse target cells.

Example II: CPV and RPV Each Inhibit the Fas Pathway of Cytolysis

This example demonstrates that cytolysis that occurs via the Fas pathway can be inhibited by infecting a target cell with CPV or RPV. In this example, PMM-1 cells were used as the cytolytic effectors. PMM-1 is a cytolytic hybrdi oma cell line that does not express perform or granzymes (Kaufmann et al., 1981 , PNAS 78:2502 and

Kelgason et al., 1992, Eur. J. Immunol. 22:3187-3190). These CTL lyse target cells via the Fas pathway (Garner et al., 1994, 153:5413-5421 ). Each of the target cells in this experiment expressed Fas. These cells were L1210-Fas (an L1210 cell transfected with sequences expressing murine Fas (Rouvier et al., 1993, J. Exp. Med. 177: 195-200)), EL4 cells, and YAC-1 cells (ATCC).

Infection of the Fas-expressing target cells with CPV or RPV inhibited the ability of PMM-1 cells to lyse the target cells via the Fas pathway (Figs. 2A-2C). As a control, L1210 cells that do not express Fas were used in the experiment. L1210 cells, unlike L1210-Fas cells, are not lysed efficiently, indicating that cytolysis by PMM-1 cells is dependent upon expression of Fas (Fig. 2D). In addition, the mechanism of killing is independent of calcium concentration, as determined by the observation that killing is unaffected by the presence of EGTA. In sum, this example illustrates that expression of CPV of RPV in a target cell can inhibit CTL-mediated cell death the occurs by the Fas pathway of cytolysis. Example III: CPV SPI-2 and SPI-1 Inhibit Fas-mediated Cytolysis

This example demonstrates that expression of SPI-2 and SPI-1 are responsible for the ability of CPV and RPV to inhibit CTL-mediated cytolysis. Mutated versions of SPI-1 and SPI-2 were used in these experiments to demonstrate the role of SPI-1 and SPI-2. The SPI-1 mutant, CPVΔSPI-1 , has been described previously (Thompson et al., 1993, Virology 197:328-338). Likewise, the SPI-2 mutant,

CPVΔSPI-2, has been described (Ali et al., 1994, Virology 202:305-314). Cells infected with CPV containing a mutation in the SPI-2 gene were lysed by PMM-1 effectors at a level comparable to the level of lysis obtained with mock-infected cells (Figs. 3A-3C). Cells infected with a CPV mutant having a mutation in the SPI01 gene showed comparable inhibition to wild-type CPV-infected cells. Similarly, mutation of the RPV SPI-2 gene almost completely relieved virus-mediated inhibition of cytolysis of infected L1210-FAS cells. In addition, mutation of SPI-2 partially relieved virus-mediated inhibition of EL4 cells and YAC-1 cells (Figs.3D-3F). In these experiments, mutation of both the SPI-1 and SPI-2 genes completely abrogated RPV-mediated inhibition of cytolysis in EL4 and YAC-1 cells (Figs.3E and 3F). These data indicate that both SPI-1 and SPI-2 confer resistance to CTL-mediated cytolysis that occurs via the Fas pathway.

Example IV: Use of SPI-1 and SPI-2 in Combination to inhibit Granzyme-Mediated Cytolysis

In contrast to the results obtained with PMM-1 effector cells, mutations in either the SPI-1 or SPI-2 genes alone for both CPV and RPV did not completely relieve virus-mediated inhibition of granule-mediated cytolysis by CTL21 9 effector cells

(Figs.4A-4D). However, an RPV strain that contained a mutation in both the SPI-1 and SPI-2 genes completely lacked the ability to inhibit cytolysis by CTL21 9 cells (Figs.4C and 4D). These data indicate that, while SPI-1 and SPI-2 individually are able to inhibit cytolysis via the Fas pathway, complete inhibition of granule-mediated cytolysis is best achieved by expression of both SPI-1 and SPI-2.

The description provided herein is meant to illustrate, but not limit, the scope of the invention. Indeed, following the guidance provided herein, those of ordinary skill in the art can readily practice various additional embodiments of the invention.

Claims

What is claimed is:

1. A method for inhibiting cell-mediated killing of a mammalian target cell the method comprising introducing into the target cell a nucleic acid consisting essentially of a nucleic acid expressing a poxvirus serpin proteinase inhibitor (SPI)-1 polypeptide, thereby inhibiting cell-mediated death of the target cell.

2. The method of claim 1 , further comprising introducing into the target cell a nucleic acid consisting essentially of a nucleic acid expressing a poxvirus SPI-2 polypeptide.

3. The method of claim 1 , further comprising introducing into the target cell a nucleic acid consisting essentially of a nucleic acid expressing a therapeutic polypeptide.

4. The method of claim 1 , wherein the poxvirus is an orthopoxvirus.

5. The method of claim 4, wherein the orthopoxvirus is cowpox virus.

6. The method of claim 4, wherein the orthopoxvirus is rabbitpox virus.

7. The method of claim 4, wherein the orthopoxvirus is a smallpox virus.

8. The method of claim 4, wherein the orthopoxvirus is a vaccinia virus.

9. The method of claim 1 , wherein SPI-1 has the amino acid sequence of SEQ ID NO: 2 or conservative variants thereof.

10. The method of claim 2, wherein SPI-2 has the amino acid sequence of SEQ ID NO: 3 or conservative variants thereof.

11. The method of claim 1 , further comprising introducing into the cell a nucleic acid consisting essentially of a nucleic acid expressing SPI-3 or conservative variants thereof.

12. The method of claim 1 , wherein introducing occurs in vitro..

13. The method of claim 1 , wherein introducing occurs in vivo.

14. The method of claim 1 , further comprising administering the target cell to a patient.

15. The method of claim 14, wherein the target cell is heterologous to the patient.

16. The method of claim 14, wherein the target cell is autologous to the patient.

17. The method of claim 1 , wherein the introduced nucleic acid is contained within a liposome.

18. The method of claim 1 , wherein the introduced nucleic acid comprises a viral vector.

19. The method of claim 18, wherein the viral vector is derived from a mammalian virus.

20. The method of claim 19, wherein the mammalian virus is selected from the group consisting of retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses.

21. The method of claim 1 , wherein the target cell is a human cell.

22. The method of claim 1 , wherein the target cell is a pancreatic islet cell.

23. The method of claim 1 , wherein the target cell is a bone marrow cell.

24. The method of claim 1 , wherein the target cell is a CD4⁺ lymphocyte.

25. The method of claim 24, wherein the CD4⁺ lymphocyte is obtained from a patient infected with a human immunodeficiency virus.

26. The method of claim 24, wherein introducing comprises perfusing the a tissue with a liquid comprising a nucleic acid encoding the SPI-1.

27. An isolated target cell that is resistant to cell-mediated death the cell comprising a nucleic acid consisting essentially of a nucleic acid expressing a poxvirus serpin proteinase inhibitor (SPI)-1 polypeptide.

28. The cell of claim 27, further comprising a nucleic acid consisting essentially of a nucleic acid expressing an SPI-2 polypeptide.

29. A method for determining whether a test nucleic acid encodes a polypeptide that inhibits cell-mediated killing of a target cell, the method comprising

introducing into a target cell a poxvirus that is defective in the expression of at least one serpin proteinase inhibitor;

expressing in the target cell the test nucleic acid; and

detecting inhibition of killing of the target cell as an indication that the test nucleic acid encodes a polypeptide that inhibits cell-mediated killing.

30. The method of claim 29, wherein the poxvirus is an orthopox virus.

31. The method of claim 29, wherein the proteinase inhibitor is selected from the group consisting of SPI-1 and SPI-2.

32. The method of claim 29, wherein detecting is in vitro.

33. The method of claim 29, wherein detecting is in vivo.

34. The cell of claim 27 further comprising a therapeutic gene.

35. The cell of claim 28 further comprising a therapeutic gene.