CA2217297A1

CA2217297A1 - Transplantation of genetically modified cells having low levels of class i mhc proteins on the cell surface

Info

Publication number: CA2217297A1
Application number: CA002217297A
Authority: CA
Inventors: Gillis R. Otten
Original assignee: Individual
Current assignee: Cell Genesys Inc
Priority date: 1995-04-04
Filing date: 1996-04-03
Publication date: 1996-10-10
Also published as: NO974596D0; AU712415B2; KR19980703665A; NO974596L; WO1996031241A1; JPH11503024A; AU5533896A; EP0820311A4; EP0820311A1

Abstract

Mammalian cells are genetically modified by the introduction of genes encoding virus-derived MHC down-regulatory proteins. Suitable cells for transplantation are taken from a donor, and transfected with an expression vector encoding one or more virus-derived MHC down-regulatory proteins. The genetically modified donor cells are expanded ex vivo, if appropriate, and transplanted into a recipient mammalian host. The decreased levels of class I MHC protein allow the transplanted cells to survive under conditions where the genetically unmodified cells would be otherwise subject to attack by the recipient immune system. The donor cells may be additionally modified to target cells or to enhance effector function of the donor cells in the mammalian host.

Description

CA 022l7297 l997-l0-02 ~0 96/31241 PCT~U536/016~8 lRANSPLANTATION OF GENETlCALLY MODIFIED CELLS HAVING LOW

INTRODUCTION

15 Technical Field The field of the subject invention is the transplantation of cells and tissue that are genetically modified to express low levels of class I MHC
molecules for therapy.

Background To protect vertebrates from disease and infection, elaborate protective systems have evolved. In mammals, the immune system serves as the primary defense, with many different types of cells and mechanisms to protect the host, primarily Iymphoid and myeloid cells. The immune system results from cells of the Iymphoid lineages developing the ability to distinguish self from non-self antigens. Cells that are perceived to be "non-self" are destroyed by the immune system, whether that perception results from a viral infection, disease, aberrant expression of a tumor antigen,transplantation of foreign tissue, or other causes.
3~The major histocompatibility complex (MHC) proteins serve an important role in the system for self-versus-foreign recognition. Each individual has a set of several different Class I and ll MHC proteins. The MHC
proteins serve as an identifier of "self". Foreign antigens are generally recognized as "self + X", where the combination of self-MHC bound to a 3~i foreign peptide is recognized. When a transplantation is made from an allogeneic host, the transplant is recognized as foreign, and destroyed by the immune system, unless the transplant is MHC matched with the host, or the W O96/31241 PCTrUS~6/n~6~8 host is immunocompromised. When a transplant includes immunocompetent Iymphocytes or monocytes, a graft may attack the host as foreign, resulting in graft-versus-host disease.
There are many situations where one may wish to transplant cells into a recipient for therapy. When the host is immunocompromised, there may be an interest in transfusing specific white cells, particularly Iymphocytes such as T-cells, with or without genetic modifications for enhanced effector functionor for killing infected, diseased, or dysfunctional cells or cancer cells, to provide a protective immune response. In other cases, where certain cells 0 are lacking, such as islets of Langerhans in the case of diabetes; cells that secrete dopamine in the case of Parkinson's disease; bone marrow cells in various hematopoietic diseases; muscle cells in muscle wasting disease, or retinal epithelial cells in visual disorders, it would be desirable to be able to provide cells that can perform the desired function by expressing and secreting a gene product.
In order for transplanted cells to survive, they must be safe from attack by the host. It is therefore of interest to find effective ways to produce cellswhich will be functional after transplantation, while being safe from attack by the recipient's immune system.
Relevant Literature The down-regulation of MHC molecules on the cell surface by viral proteins is reviewed in Maudsley and Pound (1994) Immunology Today 12:429-431. The activity of adenovirus E3/19 kd protein is discussed in Routes et a/. (1993) J. Virol. 67:3176-3181 and Hermiston et a/. (1993) l Virol. 67:5289-5298. Expression of adenovirus E3/19 kd protein in a T-lymphoma cell lines is described in Korner and Burgert (1994) J. Virol.
68: 1442-1448.
The activity of the herpes simplex virus IC-447 protein in inhibiting antigen presentation to CD8+ T Iymphocytes is discussed in York et a/. (1994) Cell 77:525-535. The human cytomegalovirus proteins involved in MHC
protein down-regulation are described in Gilbert et a/. (1993) J. Virol. 67:3461 -3469 and Beersma et a/. (1993) J. Immunol. 151:4455-4464. Virus induced loss of class I MHC molecules from the surface of cells infected with myxoma virus is described in Boshnov et a/. (1992) J. Immunol.148:881-887.
Bare Iymphocyte syndrome is described in Sullivan et a/. (1985) J. Clin.
Invest. 76:75-79; Clement et al. (1988) J. Clin. Invest. 81:669-675; and Hume W O96/31241 PCTnUS96/n4648 et a/. (1989) Human Immunology 25:1-1 1.

SUMMARY OF THE INVENTION
Methods and compositions are provided for the transplantation of cells 5 having decreased levels of class I MHC molecules on their cell surface as a result of the expression of MHC down-regulatory proteins. Suitable cells for transplantation are taken from a donor, and transfected with an expression vector containing genes encoding one or more virus-derived MHC down-regulatory proteins. The cells are expanded ex vivo, if appropriate, and transplanted into a recipient host. The decreased levels of class I MHC
protein allow the transplanted cells to survive under conditions where the genetically unmodified cells would otherwise be subject to attack by the recipient host's immune system. The cells may be additionally genetically modified to comprise constructs for altered effector function or for targeting cs diseased or virally infected cells in the host.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a fluorescence activated cell sorter (FACS) profile of 143B
osteosarcoma cells transduced with retrovirus encoding US11, and stained 20 with FlTC-conjugated antibody specific for HLA Class I as described in Example 4, infra.
Figure 2 shows the Iysis of C1R-A2.1+ targets by HLA-A2-specific CTLs as described in Example 5, infra. The Y axis is percent specific Iysis and the X axis is the effector to target (E:T) ratio.

2~j Figure 3 is a FACS profile of C1R-A2.1 cells transduced with retrovirus encoding US11and CD4-zeta (top) or CD4-zeta (bottom) and labeled with PE-conjugated anti-CD4 antibody (Y axis) and FlTC-conjugated anti-HLA
antibody (X axis) as described in Example 5, infra.
Figure 4 is a FACS profile of C1 R-A2.1 clones transduced with retrovirus encoding US11 and stained with FlTC-conugated anti-HLA Class I
antibody as described in Example 5, infra.
Figure 5 (A) shows the resistance of C1R-A2.1 clones transduced with retrovirus encoding US11 to Iysis by HLA-A2-specific CTLs as described in Example 5, infra. Figure 5 (B) shows the susceptibility of C1R-A2.1 cells c 35 transduced with retrovirus encoding CD4-zeta to Iysis by HLA-A2-specific CTLs as described in Example 5, infra. The Y axis is percent specific Iysis and the X axis is the effector to target (E:T) ratio.

W O96/31241 PCT~US9~'0~6~8 DESCRIPTION OF SPECIFIC EMBODIMENTS
Cells are genetically modified to have decreased class I MHC proteins on their surface by the introduction of an expression construct encoding one 5 or more virus- derived MHC down-regulatory proteins. The modified cells are used in cell or tissue transplantation, because they are resistant to being killed by the transplant recipient's immune system.
The subject methods are particularly useful for modifying cells, such as hematopoietic stem cells and T Iymphocytes, that have been found to have 10 low levels of transduction or transformation. A significant down-regulation of Class I MHC molecules is achieved in such cells through the use of one or more techniques that enhance the effect of the subject methods. Techniques of interest include the use of combinations of down-regulatory genes, particularly in conjunction with vector elements that permit multiple ribosomal 5 entry in a single mRNA molecule (IRES elements); enhancement of transformation or transduction in vitro through the use of adhesion molecules or antibodies to adhesion molecules during in vitro culture; and selection for subpopulations of cells that express either low levels of MHC class I
molecules, or high levels of a marker indicative of l,ansrormed or transduced 20 cells. The utility of modified T cells may also be enhanced by in vitro culture techniques that reduce the susceptibility of the cells to Iysis by natural killer cells.
Rejection of transplanted "donor" cells is mediated by transplantation antigens expressed by the donor cells. The principal transplantation 25 antigens are the class I and class ll MHC proteins. Cells that are deficient in class I MHC molecules are generally not recognized by cytolytic T cells, and therefore can be transplanted and maintained in a variety of individuals.
Down regulation of class I MHC proteins in the subject invention is accomplished by introducing into host cells an expression vector comprising 30 virus-derived genes, whose products inhibit the cell surface expression of class I MHC proteins in the host cells.
The mammalian MHC class I molecule is a noncovalent, trimolecular complex that consists of an alpha chain encoded by genes within the MHC
locus, a 132-microglobulin light chain and a small peptide derived from the 35 intracellular proteolytic degradation of an endogenous cellular protein or foreign protein. The complete class I molecule is assembled from its components intracellularly, within the endoplasmic reticulum. Only upon W O96/31241 PCTrUS96/04648 assembly of the complete complex is the complete class I MHC molecule transported . to the plasma membrane. In the subject invention, post-translational inhibition of class I MHC expression is achieved by the introduction of virus-derived MHC down-regulatory proteins.
MHC down-regulatory proteins of interest include human adenovirus type 2 E3/19 kd gene (SEQ ID NO:1) and human adenovirus type 5 E3/19 kd gene (SEQ ID NO:2); herpes simplex virus ICP47 gene (SEQ ID NO:3);
human cytomegalovirus gene US11 (SEQ ID NO:4); human cytomegalovirus gene US5 (SEQ ID NO:5), and the like. Other viruses known to down-regulate 0 class I MHC expression include myxoma virus and rabbit fibroma virus, from which down-regulatory genes useful in the subject invention can be used.
The cells for modification may be any normal, i.e., non-transformed, mammalian cells of interest that find use in cell therapy, research, interactionwith other cells in vitro or the like. Suitable cells include epidermal cells such as keratinocytes; retinal epithelial cells; myoblasts; hematopoietic cells such as hematopoietic stem cells and T Iymphocytes; endothelial cells, including venule and arterial endothelial cells; myoblasts; 13-cells from the islets of Langerhans; and other cells that are readily manipulated in vitro. The cells can be maintained and expanded, if desired, in culture and may be introduced into a host where the cells will remain viable and functional for long periods of time.
Hematopoietic cells of interest include naive or mature Iymphocytes such as T-cells isolated from Iymph nodes, peripheral blood, etc. The T cells may be separated as to specific subsets or specificities after harvesting. Of particular interest are antigen specific T cells. Large quantities of T cells with a particular antigenic specificity may be produced by antigen stimulation and in vitro culture, as known in the art (see Lamers et a/. (1992) Int. J. Cancer 51:973-979). Of particular interest are allogeneic T cells genetically modified so as to have an altered effector function or to be reactive, for example cytotoxic, with a specific target. For example, T cells may be genetically modified to express a chimeric receptor as described in U.S. Patent number 5,359,046, issued October 25, 1994, to target diseased or infected cells, or cancer cells, in addition to expressing MHC down-regulatory genes.
Epidermal cells may be harvested from skin sections by disaggregation of the tissue sample, and separation of subsets of interest by conventional methods as known in the art.
The cells to be modified will be selected to achieve a particular function W O 96/31241 PCT~US9~'01648 when introduced into a mammalian host or used for research or other purpose. Also of interest will be the stem cells which act as the progenitors for any of the above cells, which may be the original progenitor or a progenitorcell that is already dedicated to a particular lineage.
The cells for modification are harvested from a suitable donor.
Generally the donor will be non-autologous, e.g. allogeneic, with respect to the recipient. However, in some cases, particularly those related to autoimmune disease, the donor will be autologous. The cells may be obtained from any mammalian host, including murine and other rodents, 0 lagomorphs, porcine, feline, bovine, canine, primate, efc., particularly human.
The method of harvesting will depend on the nature of the cells.
Hematopoietic cells may be fetal, neonate or adult, and are collected from fetal liver, bone marrow, Iymphoid tissue or blood, particularly peripheral blood that has been treated with progenitor cell mobilizing agents, e. g.
G-CSF, GM-CSF, etc. as known in the art. Separation of hematopoietic cells into subsets of interest may be performed by a number of techniques known in the art, including elutriation, density separation, leukophoresis, flow cytometry, high or low gradient magnetic separation, and the like.
Of particular interest are keratinocyte stem cells from the stratum 20 basalis. Normal myoblasts are obtained from tissue samples, which may include fetal, neonatal or adult tissue. Diverse muscles such as limb, trunk and extra-ocular may be used. The cells may be dissociated prior to genetic modification.
After harvesting, DNA or RNA constructs providing for expression of 25 virus-derived MHC down-regulatory genes are introduced into the cells using standard methods for introduction of nucleic acids. Such recombinant constructs will comprise at least one down-regulatory gene, as previously described. In many cases it will be desirable to introduce more than one down-regulatory protein, particularly to combine proteins that have different 30 modes of action, to provide an additive or synergistic effect. Combinations of interest include SEQ ID NO:1 or SEQ ID NO:2 combined with SEQ ID NO:3;
SEQ ID NO:1 or SEQ ID NO:2 combined with SEQ ID NO:4; SEQ ID NO:1 or SEQ ID NO:2 combined with SEQ ID NO:5; SEQ ID NO:3 combined with SEQ
ID NO:4; SEQ ID NO:3 combined with SEQ ID NO:5; and SEQ ID NO:4 35 combined with SEQ ID NO:5, and the like. The combined sequences may be present on a single vector, or may be introduced on separate vectors introduced sequentially or together into the cell.

W O 96/31241 PCT~US9~'0~6~8 The expression of the down-regulatory gene or combination of genes will preferably decrease the level of surface MHC class I molecules by at least about 70% as compared to the unmodified cell, more usually at least about 80%, and preferably by at least about 90%. The level of expression may be affected by the activation state of the cell, where quiescent cells may have reduced MHC molecules when compared to activated cells. Detection and quantitation of surface expression of MHC molecules may be performed by standard immunoassay, antibody staining and flow cytometry, etc., as known in the art.
0 In some cases it may be possible to achieve greater reduction in HLA
class I with a combination of two or more class I inhibiting genes than with a single gene. As a result, allogeneic cells may be even more resistant to class l-reactive CTL and may survive longer upon transplantation into an allogeneic recipient, being more resistant to immune rejection. The various class I inhibiting genes use distinct mechanisms to reduce HLA class expression. For example, US11 from cytomegalovirus (CMV) causes dislocation of HLA class I heavy chains from the endoplasmic reticulum to the cytosol where the heavy chains are rapidly degraded (Wiertz (1996) Cell 84:769; Jones (1995) J. Virol. 69:4830). ICP47 from Herpes Simplex Virus (HSV) binds to the TAP peptide transporter complex and blocks the cytosol to endoplasmic reticulum translocation of heavy chain binding peptides, thereby preventing the formation and transport of the heavy chain-peptide-,B2 microglobulin trimeric complex. As a consequence, class I heavy chains do not form stable complexes and are retained in the endoplasmic reticulum (Hill (1995) Nature 375:411; Fruh (1995) Nature 375:415; York et al. (1994) supra). The E3/19 proteins of Adenovirus type 2 and type 5 reside in the endoplasmic reticulum and bind to the lumenal domains of class I HLA heavy chains, thereby preventing further transport (Gooding (1990) Crit. Rev.
Immunol. 10:53; Paabo (1989) Adv. Cancer Res. 52:151; Wold (1989) Mol.
Biol. Med. 6:433; Wold (1991) Life Sci. Adv. 10:89; Wold (1991) Virol. 184:1).
As a consequence of the different mechanisms for the inhibition of HLA
Class 1, simultaneous expression of two genes may have multiplicative effect on class I reduction.
The constructs that will be employed will normally include a marker that allows for selection of cells into which the DNA carrying the C!ass I
inhbiting genes has been integrated, as against cells which have not integrated the recombinant construct. Various markers exist, particularly W O96/31241 PCTrUS~61n16q8 antibiotic resistance markers, such as resistance to G418, hygromycin, and the like. Alternatively, negative selection may be used, where the marker is theHSV-tk gene, which will make the cells sensitive to agents such as acyclovir and gancyclovir.
The constructs can be prepared in a variety of conventional ways.
Numerous vectors are now available which provide for the desired features, such as long terminal repeats, marker genes, and restriction sites. One may introduce the vector in an appropriate plasmid and manipulate the vector by restriction, insertion of the desired gene with appropriate transcriptional and translational initiation and termination regions, and then introduce the plasmid into an appropriate packaging host. At each of the manipulations, one may grow the plasmid in an appropriate prokaryotic host, analyze the construct to ensure that the desired construct has been obtained, and then subject the construct to further manipulation.. When completed, the plasmid 15 or excised virus may then be introduced into the packaging host for packagingand isolation of virus particles for use in the genetic modification.
The recombinant constructs may be further modified to include functional entities other than the down-regulatory sequences, which may find use in the preparation of the construct, amplification, transformation of the 20 host cell, etc.
Constructs useful for insertion of down-regulatory genes include retroviral vectors, adenoviral vectors and adeno-associated viral vectors. For retroviral vectors, combinations of retroviruses and an appropriate packaging line where the capsid proteins are functional for infecting human cells may be 25 suitable in the present invention. Various amphotropic virus-producing cell lines are known, such as PA12 (Miller et a/. (1985) Mol. Cell. Biol. 5:431-437);PA317 (Miller et a/. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos et a/. (1988) PNAS 85:6460-6464). Of particular interest is the kat retroviral system for transduction of primary human T Iymphocytes (Finer et a/. (1994) 30 Blood 83:43-50 and W094/29438).
Transcription of the down-regulatory genes may be controlled from the retrovirus LTR or from an internal promoter, for example, mammalian phosphoglycerate kinase, beta actin promoter or a hybrid SV40/HTLV-1 promoter, such as the SR-alpha promoter (Takebe et a/. (1988) Mol. and Cell.
35 Biol. 8:466). Retroviral vectors containing more than one down-regulatory gene or also containing another gene, such as a selectable marker, may be constructed by preparing retroviruses expressing a multicistronic messenger W O96/31241 PCT~US~'01G~18 RNA. These multicistronic mRNAs contain internal ribosome entry sites (IRES elements) that allow the efficient translation of more than one protein from a single RNA molecule. IRES elements may be derived from picornaviruses, such as poliovirus, encephalomyocarditis virus (ECMV) and related viruses (Pelletier and Sonenberg (1988) Nature 334: 320-325; Jang et al. (1989) J. Virol. 63: 1651-1660; Ghatta et a/. (1991) Mol. Cell. Biol.
11:5848-5859 or their functional equivalents in other viral or cellular genes.
Retroviruses containing two or more down-regulatory genes are of particular use to decrease the expression of Class I molecules in cells that are refractory to transduction, such as hematopoietic stem cells and other primary Iymphoid cells. IRES elements may also be used to construct retroviruses expressing additional proteins such as novel homing receptors, cytokines, cytokine receptors, cell survival genes, and suicide genes. For adenoviral vectors, methods well known in the art, such as those described in Wang et a/. (1995) Gene Therapy 2:775-783, may be used.
More than one down-regulating gene may also be introduced into target cells by sequential transduction of target cells with retroviruses encoding a single down-regulating gene by techniques well known in the art.
Alternatively, two or more down-regulating genes may be introduced into target cells by transducing the target cells simultaneously with recombinant retroviral vectors that are packaged into viruses comprising envelope proteins that interact with different receptors on the suRace of the target cells. For example, a construct encoding US11 may be packaged into a virus comprising amphotrophic envelope proteins and a construct encoding Ad2 E3/E19 may be packaged into a virus containing a Gibbon Ape Leukemia Virus envelope protein (U.S. Patent No. ~,470,726).
Transduction of the cells may be accomplished by the incubation of cells and virus for at least 24 hours in the culture medium. The cells are then allowed to grow in the culture medium for at least about two weeks, and may be allowed to grow for at least about five weeks or more, before transplantation.
After introduction of the subject DNA construct, those cells that show the desired phenotype may then be further analyzed by restriction analysis, electrophoresis, Southern blot analysis, polymerase chain reaction, staining 3s with antibodies specific for the donor MHC class I a chains or for 132-microglobulin, or the like. The resulting cells will then be screened to ensure that substantially no Class I MHC antigens are expressed on the W O96131241 PCT~US9''016q8 surface. If necessary, selection methods such as flow cytometry, high gradient magnetic separation, etc. may be used to enrich for cells that have low MHC class I expression.
As markers for separation, a wide variety of fluorescent or magnetically 5 labeled molecules can be employed, which may be conjugated as labels to antibodies specific for cellular markers that identify MHC antigens.
Fluorescent markers that are available include fluorescein, Texas Red, phycobiliproteins, allophycocyanin, cyanine derivatives, rhodamine, and tandem conjugates for surface markers.
The cells may then be grown in an appropriate nutrient medium for expansion, and used in a variety of ways. The cells may be used for transplantation, to become part of an existing tissue, or may be grown to form tissue for transplantation into a non-syngeneic host. For example, with keratinocytes, the cells may be used for replacement of skin in the case of burns, where keratinocytes may be grown to form a continuous layer prior to application. Similarly, the keratinocytes may be used in the case of plastic surgery to replace skin removed from the host for use at another site. Other uses for the keratinocytes include transplantation in decubitus ulcers. In the case of islets of Langerhans, they may be grown and introduced into 20 capsules or otherwise for insertion into a host for the production of insulin. In the case of retinal epithelial cells, they may be injected into the subretinal space of the eye to treat visual disorders, such as macular degeneration. In the case of immune cells, they may be injected into the bloodstream or elsewhere to treat immune deficiency. In the case of myoblasts, they may be 25 injected at various sites to treat muscle wasting diseases, such as Duchenne muscular dystrophy. For organ transplants, transplants of non-syngeneic tissue such as xenogeneic grafts of heart or liver may be performed between related species.
One of the obstacles to the use of genetically modified donor cells for 30 transplantation, specifically the transplantation of Iymphoid cells and hematopoietic stem cells, is the difficulty in obtaining efficient transduction of those cells in vitro. The use of adhesion molecules and antibodies to adhesion molecules as a coating for tissue culture plates during transduction serves to increase the efficiency of retroviral transduction (described in 35 co-pending U.S. patent application no. 08/517,488). For stem cells, the adhesion molecules may be fibronectin or the CS-1 domain of fibronectin.
Antibodies to VLA~, VLA-5, CD29, CD11a, CD11b and CD44 may also be W O96/31241 PCTrUS9~'0~61X
used. For T cells, ICAM-1 or LFA-3 adhesion molecules, or antibody to CD2 or LFA-1 may be used. For B cells, gp39 molecules or antibody to CD40 may be used.
The retroviral, adenoviral or AAV vector may also serve for introduction ci of therapeutic proteins in the transplanted cells, where the proteins may be retained intracellularly or be secreted. Production of proteins may include growth factors, e.g. G-, M-, and GM-CSF, epidermal growth factor, platelet derived growth factor, transforming growth factor, e~c.; Iymphokines, such as the interleukins; hormones, such as ACTH, somatomedin, insulin, angiotensin, etc., coagulation factors, such as Factor VIIIC; normal versions ofthe proteins associated with genetic diseases such as adenosine deaminase or the protein associated with cystic fibrosis; protective agents, such as a1-antitrypsin; regulatory proteins or enzymes associated with the production of amino acid free products, such as the expression of tyrosine ; hydroxylase for the production of L-dopamine, and the like. The genes may be under the transcriptional control of a constitutive promoter or inducible promoter (including enhancer sequence). In the latter situation, regulation may result by induction by a naturally occurring signal or as a result of introduction into the host of an exogenous signal.
In addition to therapeutic proteins, genes encoding proteins for redirecting the donor cells to target diseased or dysfunctional cells, or to enhance effector function may be included on the vector or on separate vectors for introduction into the donor cells.
Depending upon the nature of the cells, the therapy involved and the 2~ disorder, the cells may be employed as layers, introduced in containers formaintenance at a particular site, or as solid masses impregnated in inert matrices or independent of a matrix. The number of cells administered will vary widely, depending upon the particular application and the manner in which the cells are administered. Administration may be by injection, topical application, or incision and placement, in the appropriate location.
The level of immunosuppressive regimen which is employed with the modified cells will be substantially less rigorous than would normally be used in a comparable treatment with unmodified cells. The amount of immunosuppression required for maintenance of the modified cells will vary depending upon the nature of the match between the donor and recipient cells at minor histoco",patibility loci, the level of activity of the host's immune system, the manner in which the cells are introduced, the particular site at which the foreign cells are introduced, and the type and number of transplanted cells. The regimens which can be employed may be based on existing regimens associated with the transplantation of allogeneic tissue.
Therefore the dosage level, frequency of administration, manner of administration and formulations for different situations and patients will have been established.
The subject invention provides for a reduction in the adverse effects of immunosuppression, where the reduction may be as a result of lower dosages, reduced frequency of administration, delaying the initiation of administration of the drug, or combinations thereof. The immunosuppressive therapy can be maintained to prevent initiation of rejection by monitoring the transplant and/or immune system and regulating the administration of the immunosuppressive agent to maintain the graft at or preferably below initiation of rejection.
The immunosuppressive regimen may take many forms and may be combinations of forms. Immunosuppressive regimens include irradiation, chemotherapy, specific immunosuppressive agents, and the like. Of particular interest are immunosuppressive agents, such as cyclosporin A, cyclosporin G and FK-506; azathioprine, corticosteroids, e.g. prednisolone and methylprednisolone, monoclonal antibodies against various surface membrane proteins of the Iymphoid and/or myeloid lineage, etc.
One may initially use a level of immunosuppression comparable to, or somewhat less than what would normally be used as the initial level of administration and then rapidly reduce the level of administration to not more than 75% of the original level, preferably not more than about 60% of the original level. Frequently levels of 50% or less may be used.
The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL
Example 1 Construction of MHC Down-Regulatory Gene Expression Vector Isolation of US5 and US11 Human Cytomegalovirus (HCMV) Genes.
This example describes the construction of pTUS11.4N, which encodes the US11 gene.
107 human fibroblasts were infected with Towne strain of HCMV.
Three days later, at the time of 100% cytopathic effect, virus was collected ~WO96131241 PCTIU~''/0~6~8 from the culture supernatant by high speed centrifugation and then frozen at -70~C. Viral DNA was isolated from thawed virus by standard methods.
The isolated virus DNA was amplified by the polymerase chain reaction (PCR) with Taq polymerase, according to the manufacturers .~ directions. Oligonucleotide primers were designed and made to specifically amplify the US5 (SEQ ID NO:5) and US11 (SEQ ID NO:4) reading frames.
The primers for US5 were as follows: (SEQ ID NO:6) 5' GCC ACC ATG CAT
ACA CAA CGG GCC 3'; (SEQ ID NO:7) ~' TCC TAG CCA CCG GTT GTT A 3'.
The primers for US1 1were as follows: (SEQ ID NO:8) 5' GCC ACC ATG AAC
1~ crr GTA ATG C 3'; (SEQ ID NO:9) 5' TCA GTC TAT ATA TCA CCA CTG G 3'.
PCR amplified DNA products were electrophoresed on preparative agarose gels (0.8% agarose, 1 X TAE buffer). Bands at 648 and 381 bp were excised, extracted 2X with phenol chloroform, ethanol precipitated, and resuspended in 10 mM Tris, 1 mM EDTA. The isolated fragments were ligated to the PCR ll cloning vector (Invitrogen, according to the manufacturersinstructions) .
Competent E. coli One ShotTM (Invitrogen, San Diego, CA) were transformed with the ligated vector. Transformants were selected by growth in the presence of ampicillin. Transformants containing the appropriate 20 inserts were identified by PCR amplification with the appropriate primer set,followed by gel elec~rophoresis of the amplification product and detection of the appropriately sized band. Plasrnids containing the correct DNA sequence were then purified.

Isolation of the Herpes Simplex Virus (HSV-1) ICP47 gene. This example describes the construction of plCP47 which encodes the ICP47 gene.
107 Vero cells were infected with HSV-1 at an MOI of 0.01. Four days later virus was col!ected from the culture by high speed centrifugation and then frozen at -70 C. Viral DNA was isolated from thawed virus by standard methods. The ICP47 gene was amplified by PCR using primers corresponding to (SEQ ID NO:3, 1-20) and (SEQ ID NO:3, 247-267). The amplification product was ligated to the pCR ll vector as described above.

Isolation of Adenovirus Jype 2 (Ad2) E3/19 KD Gene. This example describes the construction of pCRll.Ad2.E3/19, which encodes the E3/19 gene from Adenovirus 2.

CA 022l7297 l997-l0-02 W O96/31241 PCTAUS96/01~18 The EcoRV C fragment comprising the E3 transcription unit of Ad2 was cloned as described in Kornereta/. (1992) P.N.A.S. 89:11857-11861. The E3 gene was amplified from the plasmid by PCR, using oligonucleotides that flank the complete E3/19 kd gene. The amplification product was ligated to 5the pCR ll vector as described above.

Isolation of Adenovirus Type 5 (Ad5) E3/19 KD gene. This example describes the construction of pCR3.Ad5 E3/19, which encodes the E3/19 gene from Adenovirus 5.
10The gene is isolated as described in Wold et a/. (1985) J. Biol. Chem.
260:2424-2431 and cloned into pCR3-Uni vector (Invitrogen).

Construction of Retrovirus Vectors and ProducUon of Virus. Retrovirus vectors and packaging cell lines similar to those described in Finer et al.
5(1994) Blood 83:43, are used. DNA fragments encoding SEQ ID NO:1; SEQ
ID NO:2; SEQ ID NO:3; SEQ ID NO:4; and SEQ ID NO:5 are excised from the plasmids by digestion with EcoRI restriction endonuclease, and purified by preparative gel electrophoresis. The purified DNA fragments are ligated to the retrovirus vector rkat2 (Finer et al., supra). Recombinant plasmids 20containing the down-regulatory gene inserted into the retroviral vector are identified by PCR amplification using the appropriate primer set, followed by gel electrophoresis of the amplification product, and identification of a band of the appropriate size. Recombinant plasmids containing the down-regulatory gene are prepared as described in Molecular Cloning: A Laboratory Manual, 252nd ed., J. Sambrook, E. F. Fritsch, T. Maniatis, CSHL, Cold Spring Harbor, NY,1989.

PurificaUon, Stimulation and Cu/ture of Human T Lymphocytes. T cells are isolated from a sample of peripheral blood from a suitable 30immunocompetent human donor. The mononuclear cells are isolated by Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient centrifugation (6009 for 20 min. at 20~ C). After centrifugation, interphase cells are collected, resuspended in buffer and sedimented at 300 x 9 and then once again resuspended in buffer and centrifuged at 200 x 9 to remove platelets.
35Polyclonal mouse anti-CD8 or anti-CD4 is added to the cell suspension and incubated for 15 minutes on ice. The cells are washed, and FITC conjugated anti-mouse antibodies added to the cells. The cells are incubated and CA 022l7297 l997-l0-02 W O96/31241 PCTrUS~6/0~618 washed, and sorted by flow cytometry for CD8 positive, and CD4 positive cells. Purified T cells are cultured in vitro with interleukin-2, and antibody reactive with the T cell receptorlCD3 complex, e.g. anti-CD3 as described in Lamers et a/. (1992) Int. J. Cancer 51:973-979, and Riddell and Greenberg (1990) J. Immunological Methods 128:189-201.

Refrovirus-mediated Gene Transfer. After about 5 days of culture, retrovirus is added to the cells together with 2-8 ,ug/ml polybrene at an MOI of1-10. One to three aliquots of virus are added at 1 day intervals during a period of 2-3 days. Cells are subsequently washed, and then recultured in the presence of IL-2. Within several days of transduction, T cells are analyzed for expression of the transduced gene, and expressing cells are separated from nonexpressing cells. Cells are stained for expression of cell surface class I MHC molecules using pan-specific, or donor specific anti-class I MHC
monoclonal antibodies. The gene-expressing, class I deficient transduced cells are separated from the non-expressing transduced cells by fluorescence activated cell sorting. Confirmation of the presence of the transduced gene is performed by PCR amplification using primers specific for the virus-derived gene.
Combination of Down-regulatory Genes. The T cells prepared above are then transduced with a second down-regulatory gene, using the same method of virus infection.

Example 2 Transplantation of Allogeneic Hematopoietic Stem Cells This example provides a protocol for the transplantation into a human recipient of allogeneic hematopoietic stem cells that have been genetically modified to down-regulate the class I MHC molecules on the cell surface.
The donor is given GM-CSF subcutaneously at a dose of 5 ~g/kg/day.
On days 6, 8 and 9 after mobilization, peripheral blood is collected.
Leukophoresis is performed using a 9 liter, 3 hour treatment set to collect peripheral blood mononuclear cells (see L. Campos, et a/. (1993) 1 eukemia 7:1409-15; A. Grigg, et a/. (1993) Bone Marrow Transplant 11, Suppl. 2:23-9).
On the day of transfection, CD34+ cells are isolated using a CellPro LC34 affinity column (CellPro, Bothell, WA). Recovered cells are plated out in Myelocult H5100 media (Stem Cell Technologies Inc., Vancouver, B.C.) W O96/31241 PCT~US96/04648 containing 100 nglml huSCF, 50 ng/ml hulL-3, 10 nglml hulL~, and 10-6M
hydrocortisone for a period of 48 hours for "pre-stimulation".
The transduction method for introducing the down-regulatory into stem cells is essentially the same as described in Example 1. After transduction the cells are expanded and differentiated in vitro in Myelocut medium with addition of 100 ng/ml hu SCF, 50 ng/ml hu IL-3, 10 ng/ml hu IL~, and 10 ~M
gancyclovir to inhibit 293 cell proliferation. These 293 cells will not survive under gancyclovir selection, due to their being transfected with the thymidine kinase gene.
At approximately 10 days after transfection, the cells are analyzed for MHC class I expression by staining with commercially available FITC
conjugated polyclonal anti-human ~2-microglobulin. If necessary, the cells are sorted to select for cells having low levels of surface class I molecules.
The recipient is administered with the genetically modified cells infused i.v. at a rate of 10 cc/min. of 106 cells/ml. The recipient is given a total of 108 cells in equal aliquots over a period of three days.
In accordance with the above results, donor cells can be provided that are resistant to immune destruction by host cytotoxic T Iymphocytes or HLA
Class I specific antibodies. The method of genetically modifying cells find use in transplantation for therapy, where transplanted allogeneic cells would otherwise be rejected by the recipient. The methods are also useful in modifying autologous cells from a patient suffering from an autoimmune disease, where the cells would otherwise be subject to attack by the host immune system. In this way, a wide range of diseases resulting from the loss of number and/or function of cells may be treated, where the introduced cells will survive, multiply and function.
Animal cells may be genetically modified by the subject methods and may then be used as a source of tissues and cells for transplantation, as a model for transplantation therapies, and experimentally to test for drug efficacy.

Example 3 Production of Retroviral Vectors This example describes the construction of retroviral vectors containing down-regulatory genes and the production of recombinant retroviruses to be used in the transduction of host cells. Table 1 lists the retroviral vectors produced from the plasmids described in Example 1. These retroviruses CA 022l7297 l997-l0-02 WO 96/31241 PCT/US9~'01618 produced encode either the down-regulatory gene alone (monocistronic retrovirus vector), or the down-regulatory gene in combination with another gene, the CD4/zeta gene, as a marker for transduction of the host cells (bicistronic retrovirus vector).
Table 1 Virus geneMonocistronic retrovirus Bicistronic retrovirus vector vector US11 pRT.US11 pCD4.US11 Ad2 E3/19 pCD4.Ad2 E3/19 Ad5 E3/19 pCD4.Ad5 E3/19 HSV-1 ICP47pRT.lCP47 pCD4.1CP47 A. Monocistronic retroviral vectors. Monocistronic vectors were constructed by insertion of cloned virus gene into the vector pRT43.267. This retroviral plasmid contains, from 5' to 3', a cytomegalovirus (CMV) immediate early enhancer/promoter region, a moloney murine sarcoma virus (MMSV) R/U5 10 region, a splice donor site, part of the murine moloney leukemia virus (MMLV)gag gene, an MMLV splice acceptor, the polylinker described below, and an MMLV 3' LTR. Sequences containing the US11 and ICP47 genes from the plasmids described in Example 1 were inserted into the polylinker as described in example C and G below.
The retrovirus vector pRT43.267 was derived from pRT43.2F3 (described in W094/29348) by the following steps: pRT43.2F3 was digested with EcoRI and Apal, and the vector fragment was isolated. An oligonucleotide containing a BamHI site, a Notl site and a Sall site was ligated to the vector fragment between the EcoRI and Apal sites on the vector 20 fragment.
B. Bicistronic retroviral vectors. Bicistronic vectors were constructed by insertion of cloned virus genes into pRT43.267TNFgsig.ic. This retroviral plasmid contains, from 5' to 3', a cytomegalovirus (CMV) immediate early enhancer/promoter region, an MMSV R/U5 region, a splice donor site, part of 25 the MMLV gag gene, an MMLV splice acceptor, the polylinker described below, an encephalomyocarditis virus (ECMV) IRES element, a CD4-zeta gene and an MMLV 3' LTR. Sequences containing the US11, ICP47, Ad2 E3/19, and ~ Ad5 E3/19 genes from the plasmids described in example 1 were inserted into the polylinker as described in examples D, E, F and H below to create 3~ bicistronic retrovirus vectors encoding both a down-regulatory gene and the W O96/31241 PCTrUS96104648 CD4-zeta gene.
The retroviral vector pRT43.267gsig.ic was derived from pRT43.2F3 with the following changes: the IRES element from EMCV (residues 286-871 from Genbank iocus EVCGAA, Accession no. X74312) was inserted immediately 5' to the ATG initiation codon of the CD4-zeta gene in pRT43.2F3.
102 bp of the 5' untranslated portion of the CD4-zeta gene upstream of the ATG initiation codon was deleted. A polylinker sequence containing the recognition sites for Bam Hl, Not 1, and Sall was inserted 5' of the IRES
element. These changes in pRT43.2F3 were accomplished using 0 techniques well known in the art.

C. Construction of pRT.US11. pUS11 was digested with Eco Rl and the 682 base pair US11 insert was isolated by gel electrophoresis. The pRT43.267 retroviral vector was digested with Eco Rl, treated with calf intestinal phosphatase and purified by gel electrophoresis. The US11 insert was joined to the vector using T4 DNA ligase. The ligation mixture was used to transform E. co/i, the cells were plated on LB plates with ampicillin and colonies were screened for inserts in the proper orientation.

D. ConstrucUon of pCD4.US11. The US11 insert was isolated as described above. The pRT43.267TNFgsig.ic retroviral vector was digested with Sal I and Not I to prepare a 9143 bp vector. The 9143 bp vector fragment was purified by gel electrophoresis and ligated to the US11 insert. Recombinant vector was isolated as described above.
F Construcfion of pCD4.Ad~ F3/19. pCRll.Ad. E31E19 was digested with Spe 1, treated with Klenow pol I and then digested with Not I to purify the 553 bp fragment that contains Ad2.E3/E18. The 9143 bp vector fragment was prepared from pRT43.267TNFgsig.ic as described above and then ligated to the Ad2.E3119 gene fragment. Recombinant vector was isolated as described above.

F. Construction of pCD4.Ad5 F3/19. pCR3.Ad5.E3119 with was digested with Hind lll and Xba I to prepare the 551 bp fragment containing the Ad5 E3/E19 gene. The 551 bp fragment was ligated to the 9143 bp vector fragment prepared from Not I and Sal I digestion of pRT43.267TNFgsig.ic and recombinant retroviral vector was isolated.

'WO 96/31241 PCTrUS9~/016~8 G. Construction of pRT.lCP47. plCP47 was digested with Eco Rl and the 293 bp fragment containing the ICP47 gene was purified. This fragment was then ligated to pRT43.267 that had been digested with Eco R1. Recombinant retroviral vector was prepared as described above.

H. Construction of pCD4.1CP47. plCP47 was digested with Hind lll and then treated with Klenow pol 1. After an additional digestion with Not 1, the 368 bp fragment containing the ICP47 gene was purified. This fragment was ligated 0 to the 9143 bp vector fragment prepared from pRT43.267TNFgsig.ic, and recombinant retroviral vector was isolated.

To produce recombinant retroviruses the above described retroviral vectors were co-transfected, along with the plK6.1MCVampac plasmid (which encodes gag-pol-amphotropic env as described in W0 94/29438) into TSA201 293 cells (Heinzel et al. (1988) J. Virol. 62(10):3738-3746).
Retrovirus containing cell culture supernatant was collected affer 48 hours, filtered, aliquoted and frozen. Titer was determined on NIH 3T3 cells and was approximately 1 x 106-1 x 107/ml.

Example 4 US11 inhibits expression of multiple class I alleles in several different human cell types This example describes the transduction of a variety of different human 2~ cell types with retroviral vectors containing the HCMV US11 down-regulatorygene and the reduction in HLA Class I expression in these cells upon expression of the US11 gene.

A. Bicisfronic refroviruses encoding US11 inhibit HLA expression 3û Celis were transduced with the bicistronic (IRES containing) retrovirus described in Example 3 encoding HCMV US11 and CD4/zeta. The following cells were transduced: 143B (human osteosarcoma cells (ATCC CRL-8303);
Raji human Burkitt's Iymphoma cells (ATCC CCL-86, J. Natl. Cancer Inst.
34:231, 1965); Jurkat human T cell leukemia cells (ATCC TIB-152, 3~i Immunol. 133: 123-128, 1984); and CD8+/CD4- IL-2-dependent normal human T cells (designated SJ27), which were derived as described in Finer et al., supra. and Morecki et a/. (1991) Cancer Immunol. Immother. 32:342.

W O96/31241 PCT~US96/04648 Retrovirus in the form of culture supe, llal~llt was added to cells in the presence of polybrene (2-2.5 llg/ml). Typically, 0.75 ml of virus (undiluted or diluted to about 1:4) was added to 1 x 106 cells. Infection was allowed to proceed overnight at 37~C. In some cases, to increase the number of 5 transduced cells, two additional fresh aliquots of virus were added at one day intervals. As a control, cells were transduced with retrovirus encoding CD4/zeta only. Four or more days after transduction, cells were stained by two color immunofluorescence for HLA class I and CD4/zeta and analyzed by flow cytometry. The antibody used to detect HLA class I reacts with 0 monomorphic determinants on the HLA-A, B and C class I molecules (1. Immunol. (1982) 128:129-135; Cell (1979) 14:9-20). The percentage of transduced cells was determined by the detection of CD4 expression of the surface of the cells and their HLA class I surface staining intensity was compared to that of the untransduced cells (CD4/zeta negative 15 subpopulation) to determine fold reduction of HLA Class I expression. The results are expressed in Table 2 below.
Table 2 Cell Linepercent HLA class I HLA class I fold reduction transduced transduced not transduced 143B 15% 1.5 60 40 osteosarcoma Raji B 4% 54 749 14 Jurkat T 7% 31 109 3.5 SJ27 normal T 1% 237 430 1.8 As shown in Table 2, the reduction in HLA class I expression was highest in the 143B osteoscarcoma cells and the Raji B cells and lowest in 20the Jurkat T cells and the SJ27 normal T cells (40 and 14 fold reduction, respectively versus 3.5 and 1.8 fold, respectively). In addition, CD4/zeta expression was also strongest in those cell lines (143B and Raji) with the greatest reduction in HLA class I expression, and weakest in the cells with the lowest reduction in HLA class I expression (Jurkat and SJ27 T cells).
25Since US11 and CD4/zeta are expressed from a bicistronic message, the level of CD4/zeta expression was used as an indirect measure of the level of US11 expression. Therefore, the reduction in HLA I expression directly VVO 96/31241 PCTrUS9G/0~648 correlates with the estimated level of expression of US11. The reason for weak expression in the T cell line is not known.
This example demonstrates that when US11 is efficently expressed, it is effective at inhibiting MHC class I expression in a variety of different celltypes.

B. Monocistronic retroviruses encoding US11 inhibit HLA expression This example demonstrates that retroviruses encoding US11 alone, in the absence of the IRES element and the expression of the CD4-zeta gene, are also able to inhibit the expression of HLA when transduced into host cells.
143B osteosarcoma cells were transduced with a retrovirus vector that encodes US11 only. After about 4 or more days of culture, cells were harvested and class I cell surface levels were determined by immunofluorescence and flow cytometry usin~ the W6/32 class l-specific mAb that reacts with a non-polymorphic HLA class I determinant described above. As demonstrated in Fig. 1, 17% of the cells expressed HLA class I at a level that was 30 fold less than that expressed by non-transduced 143B
cells, or by 143B cells that were transduced with a single gene retrovirus vector that encodes CD4/zeta only.

Example 5 US11 blockade of HLA-A2.1 expression and allorecoanition by allospecific CTLs This example demonstrates that the reduction of HLA expression of the surface of cell lines transduced with the down-regulatory US11 gene results in the resistance of these cells to the cytolytic effect of T cells specific for HLA.
C1R cells are an HLA-Anegative human B Iymphoblastoid cell line ((B-LCL), ATCC CRL-1993) which express no HLA-A and are HLA-B351~W and HLA-Cw4P~sitive (Proc. Natl. Acad Sc~. USA 86:2361-2364, 1989; J. Immunol.
148:1941, 1992). To provide cells that are susceptible to Iysis by HLA-A2-specific human cytolytic effector cells (CTLs), CIR cells were transfected with the HLA-A2.1 gene to yield an HLA-A2.1+ line, C1R-A2.1 (Hogan eta/. (1988) 1. Exp. Med. 168:72~). As shown in Fig. 2, C1 R cells were resistant to Iysis byHLA-A2-specific CTLs whereas the C1 R-A2.1 cell were susceptible to Iysis by the CTLs.
H LA-A2.1 positive C 1 R transfectants were transduced with the W O96131241 PCTrUS9'/0~618 bicistronic (IRES containing) retrovirus vector encoding both HCMV US11 and CD4/zeta. Cells were stained for MHC class I and CD4 molecules, and analyzed by two-color FACS. As shown in Fig. 3, the majority of transduced cells (CD4+) were found to have very low MHC class I compared to 5 untransduced (CD4-) cells. The CD4+ cells were cloned, and three clones were selected for further analysis. All of the clones had very low levels of HLAclass 1, as measured by flow cytometry (Fig. 4). In addition, clone 8 was found to have no detectable cell surface HLA-A2.1. The clones were labeled with 51Cr and used as targets in a four hour cytolytic assay using A2.1-specific 10 human T cell effectors. As shown in Fig. 5, Iysis of the US11-transduced C 1 R-A2 .1 + clones was equal to the background level of Iysis of the untransfected, parental (HLA-A2ne9ative) C1 R cells. Thus, it was demonstrated that US11 completely suppressed expression of HLA-A2.1 and blocked recognition by alloreactive CTL. Because the transducing retrovirus also encoded CD4/zeta it was important to show that CD4tzeta itself had no effect on HLA-A2.1. Indeed, C1R-A2.1 cells transduced with retrovirus vector for CD4/zeta showed no reduction in HLA-A2.1 and were as sensitive as untransduced C1R-A2.1 cells to CTL Iysis (Fig. 5).

Example 6 US11 blockade ~f HLA-A3 expression and recognition by allo-specific CTLs This example demonstrates the effect of the US11 gene on the expression of a different HLA allele and the effect on the resistance of the cells expressing the US11 gene to specific cytolysis by effector T cells specific for the second HLA allele.
An HLA-A3-specific human CTL line is derived by repeated stimulation of HLA-A3negative peripheral blood mononuclear celles (PBMC) with irradiated HLA-A3P~sitive allogeneic cells. The following cells are used as allogeneic stimulator cells: either PBMC (irradiated at 2000 R) (Jelachich, J. Immunol. 141: 1108, 1988), HLA-A3P~sitive C1R transfectants (10,000 R), Jurkat (10,000R) or Raji cells (10,000R). This CTL line is first tested for its ability to Iyse HLA-A3P~sitive C1R transfectants, but not parental (HLA-A3 ne9atiVe) C1R cells. HLA-A3P~sitiVe C1R cells are then transduced with the US11/CD4/zeta bicistronic retrovirus and CD4/zeta+, HLA-A31~W cells are purified and used as targets for the HLA-A3 reactive CTL. The US11-transduced HLA-A31~W C1R cells are Iysed minimally or not at all by the HLA-A3 specific CTLs.

~096/31241 PCTrUS96/04648 This example, in combination with Example 5, demonstrates that the US11 gene can down-regulate different HLA Class I alleles.

Example 7 US11 Expressing Cells Do Not Stimulate the Differentiation of Class l-specific CTL Precursors into CTL Effectors.
The previous examples demonstrated that differentiated CTL effector cells are unable to recognize US11-transduced, HLA class Idim or class Inegative target cells. In the transplant situation, nonlytic CTL precursors (CTLp) recognize donor allogeneic class I antigens and then dirrere"liate into Iytic CTL effectors. The differentiation of CTLp can be illustrated in vitro by the use of the "one-way" mixed Iymphocyte culture (MLC) or mixed Iymphocyte-tumor culture (MLTC). This example illustrates the use of the MLTC system to show that US11-transduced HLA class Idim or negative cells are unable to stimulate the differentiation of class l-specific CTLp into CTL effectors.
HLA-A2.1ne9ative or HLA-A3ne9ative human PBMC that contain CTLp are cultured either at a 1:1 ratio with irradiated (2000R) allogeneic HLA-A2+ orHLA-A3+ Iymphocytes that have been transduced with the US11 gene or at a 10:1 ratio with irradiated (10,000R) stimulator cell lines that have been transduced with the US11 gene. These stimulator cells may be derived from the HLA-A2.1+ C1R cell line, the HLA-A2+ JY cell line or from the HLA-A3+
C1R cell line (Takahashi et al, Proc. NaV. Acad. Sci 88: 10277, 1991). After 5-7 days of culture, cells are harvested and tested for cytolytic activity against HLA-A2.1 + targets (eg. HLA-A2.1 ' C1 R or JY) or HLA-A3+ targets (eg. HLA-A3+
C1R or Jurkat) in a 4 hr 51Cr release assay. Non-HLA specific Iytic CD56+
cells may be removed from the culture prior to the measurement of HLA-A2.1 or HLA-A3-specific CTL activity, as described in Kos ef al. J. Immunol.
155:578 (1995). CTL activity is measured to demo"sl,dte that little or no HLA
class l-specific CTL activity is seen in PBMC cultures that are stimulated with 3~ US11-transduced, HLAdim or negative cells. As a positive control to demonstrate the existence of class l-specific CTLp, PBMC are stimulated with MHC class I positive nontransduced cells.
In addition, the maturation of CTLp cells specific for different HLA
alleles is measured against suitable targets to demonstrate that the effect of US11 is not restricted to A2.1 or HLA-A3.

W O96/31241 PCTrUS~/01618 Example 8 Ad2 E3/19 and Ad5 E3/19 Block Expression of Multiple Class I Alleles Expressed by 143B Osteosarcoma Cells.
This example demonstrates that the adenovirus down-regulatory genes, Ad2 E3/19 and Ad5 E3/19, are also able to prevent the expression of HLA class I molecules on host cells when the host cells are transduced with retroviruses containing those genes.
143B is a human osteosarcoma cell line that expresses high levels of HLA class I on the cell surface. Cells were transduced with bicistronic 0 (Ad E3/19; CD4/zeta) retrovirus vectors, or with a control monocistronic vector encoding CD4/zeta molecules. After 4 or more days in culture, samples were taken and the level of cell surface HLA class I determined by immunofluorescence and flow cytometry. The percentage of cells that were transduced was measured by the expression of the CD4/zeta molecules on their cell surfaces. CD4Positive cells were then analyzed for HLA class I
expression. As shown in Table 3, the Ad5 E3/19 gene reduced HLA
expresssion by 6.6 fold and the Ad2 E3/19 gene reduced HLA expression 7.5 fold in the transduced (CD4+) population.

Table 3 Ad E3/19 % transduced HLA class 1,HLA class Ifold reduction gene not transduced transduced Ad2 E3/19 48% 38.5 5.1 7.5 Ad 5 E3/19 48% 39.2 5.9 6.6 Example 9 Blockade of HLA Expression by Combinations of Down-regulatory Genes This example illustrates the use of more than one down-regulatory 25 gene to inhibit the expression of HLA Class I in target cells. In particular, the following combinations are used: 1) HCMV US11 and Ad2 E3/19 or Ad5 E3/19; 2~ US11 and ICP47; and 3) ICP47 and Ad2 E3/19 or Ad5 E1/9.
Combinations of the recombinant retroviruses (either bicistronic or monocistronic) described in Example 1 are used to transduce mammalian 30 cells sequentially according to methods well known in the art. Alternatively,the cells are transduced simultaneously with the different retroviruses. For simultaneous infection, the retroviral vectors are packaged in cells expressing different viral envelope proteins that interact with different W O96/31241 PCTrUS~6/0~6~8 receptors. The plK6. 1 MCVampac packaging vector, which encodes amphotrophic env (as described in W094/29438), is used to package one of the recombinant viruses and pMOV-GaLV, which encodes the Gibbon Ape Leukemia SEATO virus envelope protein (as described in U.S. Patent no.
5,470,726) is used to package the other recombinant virus.
Further bicistronic vectors are prepared according to the protocols of Example 1 that encode the combinations of down-regulatory genes wherein the second down-regulatory gene is substituted for the CD4-zeta gene.
These bicistronic vectors are used to transduce the target cells as described in the preceding examples.

Example 10 Production of Class l-deficient Human Hematopoietic Stem Cells and T cells This example illustrates the production of human hematopoietic stem cells and T cells that have been transduced with vectors containing one or more down regulatory genes. To ensure efficient transduction of these cells and the loss of HLA Class I expression, the cells are plated on adhesion molecules or on antibodies to adhesion molecules during the transduction process.
A. High leve/ transduction of primary human hemafopoietic stem cells The ability to maintain both self-renewing and differentiating populations of cells derived from stem cells depends upon cell - cell contact of stem cells and stromal cells in the bone marrow (Gordon and Greaves, 2s Bone Marrow Transplantation._:335-338 (1989)). The contact of stromal cells and hematopoietic stem cells involves many molecules, including growth factors, exemplified by the kit ligand on stromal cells and c-kit receptor found on stem cells (Zsebo et al., Cell, 63:213-224 (1990)) and adhesion molecules such as fibronectin on stromal cells and VLA-4 on hematopoietic stem cells (Williams et al., Nature 352:438-441 (1991)). These contact molecules are either transmembrane or, if located extracellularly, they are proteins which contact transmembrane proteins and enable signals for either self-renewal or differentiation to be transmitted between the stromal cells and the stem cells.
3~ In order to improve retroviral gene transfer into hematopoietic stem cells by supernatant infection, recreation of the cell-cell cGnlacls as described in co-pending U.S. application no. 08/517,488, Finer et al., may be used.

CA 022l7297 l997-l0-02 W O 96/31241 PCTrUS9~'01618 Fibronectin, the CS-1 domain of fbronectin or antibody to VLA4 is added to the culture dishes as described below.

CD34+ cells are isolated from the peripheral blood of patients undergoing cyclophosphamide and G-CSF treatment. Mononuclear cells are isolated from leukophoresed blood by fractionation using a standard Ficoll gradient (Pharmacia, Piscataway, NJ). The CD34+ cells are isolated using positive selection on a CellPro CEPRATE LC affinity column (CellPro, Bothell, WA). This population of cells is then cultured for a period of 48-72 hours at a density of 0.5 - 1 X 106 cells/ml in ~prestimulation medium" which contains Myeloid Long Term Culture Medium supplied as a complete medium from Terry Fox Labs, (Vancouver, Canada) with the addition of 100 ng/ml human Stem Cell Factor (SCF), 50 ng/ml human IL-3, and 10 ng/ml human IL~
(Genzyme, Cambridge, MA).
Viral supernatant for infection of the CD34+ cells is produced as follows. 293 cells are transfected by first plating at a density of 1.4 X106 cells/10cm dish 24 hours prior to transfection, followed by co-transfection withthe retroviral vectors described in the previous examples and 7.5 ug of the packaging plasmid plK6.1 MCVampac. Eighteen hours later, transfection media is removed and replaced with 10 ml IMDM (JRH Biosciences, Woodland CA) + 10% FBS. Viral supernatant is then collected 24-36 hours later and 100ng/ml human SCF, 50 ng/ml human IL-3, 10 ng/ml human IL~, and 8 ug/ml polybrene are added.
To produce antibody-coated plates, 10 ~Lg of antibody or a combination of antibodies (Immunotech, Westbrook ME) is dissolved in 1 ml of PBS and incubated overnight in the tissue culture plates as discussed above.
Monoclonal antibodies against the adhesion molecules VLA-4, VLA-5, CD29, CD11a, CD11b, and/or CD44 are used for antibody-coating. After incubation the plates are washed gently with PBS, and cells and viral supernatant are added immediately. As a comparison, tissue culture plates are coated with fibronectin or a chymotryptic fragment of fibronectin, GS-1, as reported by Williams et al. (Nature 352: 438-441 (1991)) and Moritz et al. (J. Clin. Invest 93: 1451-1457 (1994)). Fibronectin and CS-1 coated plates are made by adding 30ug/ml PBS of fibronectin, derived from human plasma, or CS-1 (Sigma, St Louis, M0) to tissue culture plates. The plates are then incubated at 37- overnight and washed with PBS ("24 hour method"). Alternatively, the plate is placed under UV light for 1 hour with the lid off and then an additional W O96/31241 PCTrUS9'/01618 hour with the lid on, the PBS is removad, one ml of 2% BSA is added for 20 minutes, and the plates are washed with DPBS/0.2% HEPES ("2 hour method")(Williams et al. supra).

A. High level transduction of primary human T ce/ts Cell-cell contact plays an important role for the activation and growth of many cells of the hemapoietic lineage. For example, many cell-cell contacts have been identified that are essential for T cell activation (Bolhuis et al., Cancer Immunol. Immunother. 34:1-8 (1991)) including the interactions of 0 receptor/co-receptor pairs on T Iymphocytes and antigen presenting cells such as LFA-1 and ICAM-1, and CD-2 and LFA-3. In B Iymphocytes, the CD40 / gp39 interaction takes place between B Iymphocytes and T Iymphocytes and is necessary for B Iymphocyte activation (Armitage et a/., Sem. Immunol..
6:267-278 (1994)). Antibodies to CD2 (Springer et al., Nature 323:262 (1987)) or CD40 can substitute for the ligands and mediate cell-cell interaction and activation. The transduction of T and B Iymphocytes by supernatant infection has been reported to be of low efficiency (Hwu et a/., J. Immunol. 9:4104-4115 (1993); Baker et a/., Nucleic Acids Res. 20:5234 (1992)). Using an approach similar to that for stem cells, antibodies to the 2CI receptor present on the target cells (i.e., anti-CD2 or LFA1 antibody for T
Iymphocytes and anti-CD40 antibody for B Iymphocytes), which have been shown to activate their respective cell types, can also be used to enhance the supernatant transduction efficiency of these cells.
Primary human CD8+ T cells are purified from the peripheral blood of 2s healthy donors as follows: Peripheral blood mononucleocytes (PBMCs) are isolated from human blood by Ficoll-Hypaque density gradient centrifugation.
PBMCs are washed three times with D-PBSE/CMF (PBS containing 1mM
EDTA, Ca and Mg free), resuspended at 5x107 cells in 4 ml of D-PBSE/CMF
containing 0.5% of human gamma globulins, and incubated at room 30 temperature for at least 15 minutes. After incubation, CD8+ T cells are purified from the PBMC cell suspension by positive panning. Specifically, the PBMC suspension is loaded into a pre-washed T-25 tissue culture flask coated with an antibody specific for the human CD8 protein (AIS CD8 seiection flask (Applied Immune Sciences, Santa Clara, CA)) at a density of 3~j 5x107 cells per 4 ml per T-25 flask. Cells are incubated for one hour at room temperature, and the non-adherent cells removed by gentle pipetting and washing the flask three times with the D-PBSE/CMF. The CD8+ T cells are W O96/31241 PCTrUS~6/0~6~8 simultaneously released from the flask and activated by adding 10 ml of T cell medium containing 10 ng/ml OKT3 (Ortho Pharmaceuticals, Raritan, NJ) and 10% IL2 (Pharmacia). Alternatively, the physical separation is accomplished using complement mediated Iysis, fluorescence-activated cell sorting, magnetic beads, affinity chromatography, or the like. Cells are then incubated with T cell media for 48 hours, harvested from the flask, and washed once with T cell medium, and finally resuspended in fresh T cell medium (10%
FCS, Hyclone; RPMI1640, CellGro; 10mM Hepes buffer (Gibco); 1% Sodium pyruvate (Gibco); 1 % non-essential amino acids (Gibco); 2mM glutamine 0 (Gibco); 25 ~lM 2-mercaptoethanol (Sigma) and 1% streptomycin/penicillin) plus 10% IL2 at a density of 0.5-1.0x106/ml in 24 well plates.
To prepare recombinant retroviruses, 293 cells are plated at 1X106 cells/6 well plate, and then transfected with the appropriate construct after 48hours. 24 hours post transfection, the transfection media is removed and replaced with T cell growth media.
Antibody plates are prepared as described above. 0.5x106 purified and activated human CD8+ T cells prepared as described above (usually at day 4 or 5 post-purification/activation) are plated on the antibody coated plates and incubated with 1 ml of fresh T cell medium (plus 10% IL2 and 2 ~Lg/ml polybrene) together with 1 ml of viral supernatant obtained from the 293 transient transfection system described above. After an 8 hour incubation period, 1.5 ml of medium is removed from each well, and replaced with 0.5 ml of fresh T cell medium together with 1.0 ml of viral supernatant (polybrene at 2 ~Lg/ml and IL2 at 10%). After a 12 hour incubation period, the two step supernatant procedure is repeated.
The transduced CD8+ T cell population is subsequently maintained in T cell medium. T cells are periodically re-stimulated every 7 to 10 days by the addition of OKT3 at 10ng/ml or by exposing the cells to immobilized OKT3 in a T-25 tissue culture flask at a density of 1-2x107 CD8+ T cells/10 ml T cell medium plus 10% IL2. Cells are incubated for 48 hours, washed 1x with T
cell medium, and resuspended in fresh medium plus 10% IL2 at 0.5-1.0 x 106/ml.

The HLA class I deficient T cell and stem cell subpopulations prepared as described above are optionally further enriched by immunoaffinity techniques, e.g. HLA class I specific antibody plus complement mediated Iysis, fluorescence-activated cell sorting, adherence to a plastic substratum, CA 022l7297 l997-l0-02 ~NO 96/31241 PCT/US96/04648 magnetic beads, affinity chromatography, or the like. Pan-specific W6/32 anti-HLA Class I antibody and/or allele-specific BB7.2 anti-HLA-A2.1 class I
antibody are used (ATCC HB-82; Hum. Immunol. 3: 277-299, 1981).

Example 11 Protection of Class I Deficient Cells from Lysis by NK Cells This example describes a method to allow the survival of HLA Class I
deficient human T cells after transplantation. A potential obstacle to the use of HLA class I deficient human T cells is that Iymphoid cells that are deficientin cell surface class I HLA have been found to be susceptible to Iysis by natural killer (NK) cells . Moreover, it has been shown that human NK cells will recognize and Iyse class l-disparate allogeneic Iymphoblasts in vitro. To overcome this problem, HLA class I deficient human or murine T cells are incubated for prolonged periods in vitro (e.g. 5 days) prior to transplantation,to reduce their susceptibility to NK recognition and Iysis.
A. Resistance of normal human T cells to cytolysis by NK cells. Normal human T cells were isolated as described above and stimulated with the polyclonal activator PHA and were kept in culture for two or five days. The resultant T Iymphoblasts were then labeled with 51Cr and tested as targets for Iysis by freshly isolated allogeneic NK cells. It was observed that 5 day Iymphoblasts were resistant to NK Iysis, whereas 2 day Iymphoblasts were susceptible to Iysis.
B. Resistance of Class I deficient murine T cells to NK Iysis As a model for HLA class l-deficient human Iymphocytes, the 2~ susceptibility of class l-deficient murine T Iymphoyctes (derived from ~z-microglobulin knockout mice as described in WO93/16177) to NK Iysis was investigated. Surprisingly, it was found that class l-deficient mouse T
Iymphocytes were highly resistant to NK Iysis if the T Iymphocytes were maintained in culture for 5 days or more. In contrast, 2 day Iymphoblasts 3~ were susceptible, as expected from the prior art. On the other hand, HLA
class I deficient LPS-activated B Iymphoblasts remained completely susceptible to NK Iysis after 5 days of culture.

All publications and patent applications cited in this specification are 3'i herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

W O96/31241 PCTrUS~6/0~6~8 Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto 5 without departing from the spirit or scope of the appended claims.

CA 022l7297 l997-l0-02 W 0 96/31241 PCTrUS96/04648 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Cell Genesys, Inc.
(ii) TITLE OF INVENTION: Transplantation of Genetically Modified Cells Having Low Levels of Class I MHC Proteins on the Cell Surface (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: FLEHR, HOHBACH, TEST, ALBRITTON ~ HERBERT
(B) STREET: 4 Embarcadero Center, Suite 3400 (C) CITY: San Francisco (D) STATE: California (E~ COUNTRY: USA
(F) ZIP: 94111-4187 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy Disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(c) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sherwood, Pamela J
(B) REGISTRATION NUMBER: 36,677 (C) REFERENCE/DOCKET NUMBER: Cell 23-l (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-494-8700 (B) TELEFAX: 415-q9q-8771 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ==
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATGAGGTACA TGATTTTAGG ~l~l~C ~.l~GGCAG TCTGCAGCGC TGCCAAAAAG 60 ~l~llC~AAG GTGAAAATCG TAAAACTTTT ATGTATAAAT TTCCATTTTA TGAAATGTGC 300 GATATTACCA TGTACATGAG CAAACAGTAC AA~ll~lGGC CCCCACAAAA GTGTTTAGAG 360 AACACTGGCA C~llll~ll_ CA~Gel'~IG CTTATTACAG ~ll~lll GGTATGTACC 420 TTACTTTATC TCAAATACAA AAGCAGACGC AGTTTTATTG ATGAAAAGAA AATGCCTTGA q80 W O96131241 PCTADS96/Oi618 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 515 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA , (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 base pairs (B) TYPE: r.ucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
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AGAACCGCCG TGCACGACCC GGAGCGTCCC CTGCTGCGCT CTCCCGGGCT G~~ ,AA 180 AT~:GC~ ecA ACGCATCCTT GG~ ,IGt,cA CATCGAAGAA CCGGCGGGAC CGTGACCGAC 240 A~,~CCC~,lA ATCCGGTAAC CCGTTGA267 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 648 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

W O 96131241 PCTrUS9~'01~8 TTATCCTTGA ~r~IlllCGA TGAACCTCCG CCCTTGGTGG AGACGGAGCC GTTACCGCCT 120 ~ICC~ATG TTTCGGAGTA CCGAGTAGAG TATTCCGAGG C~lGC~I GCTCCGATCG 180 GGCGGTCGAC TGGAGGCTCT GTGGACCCTG CGCGGGAACC T~l~CGIGCC CACGCCGACA 240 c~CCGG~l~I ACTACCAGAC GCTGGAGGGC TACGCGGATC GAGTGCCGAC GCCGGTGGAG 300 GACGTCTCCG AAAGCCTCGT CGCAAAACGC TACTGGCTCC GGGACTATCG I~ll~CCCAA 360 CGCACAAAAC I~ ïlGTT CTACTTTTCC CCCTGCCACC AATGCCAAAC TTATTATGTA 420 GAGTGCGAAC CCCGGTGCCT CGTGCCTTGG ~ll_~cIGI GGAGCTCGTT AGAGGACATC 480 ~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 381 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:S:

ACCGGAGTTG TCTATCGCGA CATATCGTCG ACTATCGCGA CAGAAAAAAT A~C~ll~GIA 120 GAGAATGCCG TGTTGAAGGA A~G~llll ATTGAGACGA TAAAACAGCA TCAGGAGCCA 180 ACAACCAGCA GGGTAATCCC GCAACATAAA TACAAAGTCA CAGCGAAGAA T~l~lC~l 300 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sin~le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

~GCCACCAIGCATACACAACGGGCC 24 (2) INFORMATION FOR SEQ ID NO:7:

W O96/31241 PCTrUS96/04648 ~i) SEQUENCE CHARACTERISTICS:
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TCCTAGCCACCGGTTGTTA 19 , (2) INFORMATION FOR SEQ ID NO:8:
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(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Claims

WHAT IS CLAIMED IS:

1. A method of transplanting non-autologous cells into a mammalian host, the method comprising:
harvesting nontransformed cells from other than said recipient;
introducing a nucleic acid construct comprising one or more virus-derived MHC class I down-regulatory genes into said cells to form donor cells, wherein surface expression of MHC class I molecules is decreased on said cells by at least 70% as a result of introducing said nucleic acid construct; and transplanting said donor cells into said mammalian host.

2. A method according to Claim 1, wherein said virus-derived MHC
class I down-regulatory gene is selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

3. A method according to Claim 2, wherein said nucleic acid construct comprises a combination of virus-derived MHC class I down-regulatory genes selected from the group consisting of SEQ ID NO:1 or SEQ
ID NO:2 with SEQ ID NO:3; SEQ ID NO:1 or SEQ ID NO:2 with SEQ ID NO:4;
SEQ ID NO:1 or SEQ ID NO:2 with SEQ ID NO:5; SEQ ID NO:3 with SEQ ID
NO:4; SEQ ID NO:3 with SEQ ID NO:5; and SEQ ID NO:4 with SEQ ID NO:5.

4. A method according to Claim 3, wherein said nucleic acid construct comprises an IRES element positioned between said virus-derived MHC class I down-regulatory genes.

5. A method according to Claim 2, wherein said nucleic acid construct comprises a retrovirus vector.

6. A method according to Claim 2, wherein said mammalian host is a human.

7. A method according to Claim 6, wherein said cells are hematopoietic cells.

8. A method according to Claim 7, wherein said hematopoietic cells are transduced with said retrovirus vector on a culture surface comprising at least one of adhesion molecules and antibodies specific for adhesion molecules.

9. A method according to Claim 8, wherein said adhesion molecules and antibodies specific for adhesion molecules are selected from the group consisting of fibronectin, CS-1 domain of fibronectin, anti-VLA-4, anti-VLA-5, anti-CD29, anti-CD11 a, anti-CD11b, anti-CD44, anti-LFA-1, anti-ICAM-1, anti-LFA-3, anti-CD2 and anti-CD40.

10. A method according to Claim 7, wherein said hematopoietic cells are hematopoietic stem cells.

11. A method according to Claim 7, wherein said cells are T cells.

12. A method according to Claim 6, further comprising the step of:
culturing said cells in vitro prior to said transplanting.

13. A method according to Claim 1, wherein said donor cells further comprise a construct comprising a gene encoding at least one protein for directing said cells to target diseased or infected cells, or cancer cells in the mammalian host.

14. A method according to Claim 1, wherein said donor cells further comprise a construct comprising a gene encoding at least one protein for enhancing effector function of said donor cells.

15. A nontransformed mammalian donor cell comprising:
a nucleic acid construct comprising one or more virus-derived MHC
class I down-regulatory genes, wherein surface expression of MHC class I
molecules is decreased on said cell by at least 70% as a result of introducing said nucleic acid construct.

16. The donor cell of Claim 15, wherein said nucleic acid construct comprises a retrovirus vector.

17. A nontransformed mammalian donor cell according to Claim 15, wherein said cell is human.

18. A donor cell according to Claim 15, wherein said virus-derived MHC class I down-regulatory gene is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

19. A donor cell according to Claim 18, wherein said nucleic acid construct comprises a combination of virus-derived MHC class I down-regulatory genes selected from the group consisting of SEQ ID NO:1 or SEQ
ID NO:2 with SEQ ID NO:3; SEQ ID NO:1 or SEQ ID NO:2 with SEQ ID NO:4;
SEQ ID NO:1 or SEQ ID NO:2 with SEQ ID NO:5; SEQ ID NO:3 with SEQ ID
NO:4; SEQ ID NO:3 with SEQ ID NO:5; and SEQ ID NO:4 with SEQ ID NO:5.

20. A donor cell according to Claim 19, wherein said nucleic acid construct comprises an IRES element positioned between said virus-derived MHC class I down-regulatory genes.

21. A donor cell according to Claim 17, wherein said cell is a hematopoietic cell.

22. A donor cell according to Claim 21, wherein said hematopoietic cells have been transduced with said retrovirus vector on a culture surface comprising at least one of adhesion molecules and antibodies specific for adhesion molecules.

23. A donor cell according to Claim 22, wherein said adhesion molecules and antibodies specific for adhesion molecules are selected from the group consisting of fibronectin, CS-1 domain of fibronectin, anti-VLA-4, anti-VLA-5, anti-CD29, anti-CD11a, anti-CD11b, anti-CD44, anti-LFA-1, anti-ICAM-1, anti-LFA-3, anti-CD2 and anti-CD40.

24. A donor cell according to Claim 21, wherein said cell is a hematopoietic stem cell.

25. A donor cell according to Claim 21, wherein said cell is a T cell.

26. A donor cell according to Claim 15 further comprising a nucleic acid construct comprising a gene encoding a protein for directing said cells to target diseased or infected cells or cancer cells in a mammalian host receiving said donor cell.

27. A donor cell according to Claim 15 further comprising a nucleic acid construct comprising a gene encoding a protein for enhancing effector function of said donor cell.

28. A method for decreasing the susceptibility of a mammalian T
cell to lysis by natural killer (NK) cells, the method comprising:
maintaining said T cells in in vitro culture for at least about 5 days.