In vivo protection of activated Tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate.

BACKGROUND: Nonmyeloablative allogeneic hematopoietic stem cell (HSC) transplantation can cure malignant and nonmalignant diseases affecting the hematopoietic system, such as severe combined immunodeficiencies, aplastic anemia and hemoglobinopathies. Although nonmyeloablative is favored over myeloablative transplantation for many patients, graft rejection remains problematic. One strategy for decreasing rejection is to protect donor activated T cells in the graft from methotrexate (MTX) by genetically modifying the cells to express MTX-resistant dihydrofolate reductase (Tyr22-DHFR), leaving the immunosuppressive effects of MTX to act solely on activated host T lymphocytes, shifting the balance to favor allogeneic engraftment. METHODS: To evaluate MTX resistance of Tyr22-DHFR(+) T lymphocytes in vivo, we transplanted dogs with autologous CD34(+) cells modified with yellow fluorescent protein (YFP) and DHFR-green fluorescent protein (GFP) lentivirus vectors. Dogs were then treated with a standard MTX regimen days 1, 3, 6 and 11) following immune activation with a foreign antigen as a surrogate assay to mimic early transplantation. RESULTS: DHFR-GFP(+) gene marking was maintained in CD3(+) CD25(+) and CD4(+) T lymphocytes after MTX treatment, whereas the level of T lymphocytes that expressed only a fluorescent reporter (YFP(+) ) decreased. These data show that Tyr22-DHFR expression protects T lymphocytes from MTX toxicity in dogs, highlighting a clinically relevant application for preserving donor T lymphocytes during post-transplantation immunosuppression. CONCLUSIONS: The findings of the present study have implications for the clinical translation of MTX-resistant T cells to facilitate engraftment of allogeneic cells following nonmyeloablative conditioning and to minimize the risk of rejection. In summary, Tyr22-DHFR expression in T lymphocytes provides chemoprotection from MTX-mediated elimination in the context of immune activation in vivo.

Characterization of the human artemis promoter by heterologous gene expression in vitro and in vivo.

Artemis is an endonucleolytic enzyme involved in nonhomologous double-strand break repair and V(D)J recombination. Deficiency of Artemis results in a B- T- radiosensitive severe combined immunodeficiency, which may potentially be treatable by Artemis gene transfer into hematopoietic stem cells. However, we recently found that overexpression of Artemis after lentiviral transduction resulted in global DNA damage and increased apoptosis. These results imply the necessity of effecting natural levels of Artemis expression, so we isolated a 1 kilobase DNA sequence upstream of the human Artemis gene to recover and characterize the Artemis promoter (APro). The sequence includes numerous potential transcription factor-binding sites, and several transcriptional start sites were mapped by 5' rapid amplification of cDNA ends. APro and deletion constructs conferred significant reporter gene expression in vitro that was markedly reduced in comparison to expression regulated by the human elongation factor 1-α promoter. Ex vivo lentiviral transduction of an APro-regulated green fluorescent protein (GFP) construct in mouse marrow supported GFP expression throughout hematopoeitic lineages in primary transplant recipients and was sustained in secondary recipients. The human Artemis promoter thus provides sustained and moderate levels of gene expression that will be of significant utility for therapeutic gene transfer into hematopoeitic stem cells.

Cytotoxicity associated with artemis overexpression after lentiviral vector-mediated gene transfer.

Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1alpha promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T(-)B(-) phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A.

Protection of mice from methotrexate toxicity by ex vivo transduction using lentivirus vectors expressing drug-resistant dihydrofolate reductase.

Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTX-treated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vector-modified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.

Methotrexate preconditioning allows sufficient engraftment to confer drug resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity.

Methotrexate (MTX) is an effective antitumor agent that has been demonstrated to be particularly useful in the treatment of hematopoietic neoplasms but causes substantial hematologic and gastrointestinal toxicity. We previously demonstrated that transplantation with transgenic marrow expressing drug-resistant dihydrofolate reductase (DHFR) into animals preconditioned by irradiation substantially protected recipient mice from the toxic side effects of methotrexate administration. Here we test the use of methotrexate itself as a preconditioning agent for engraftment of drug-resistant transgenic marrow, subsequently conferring drug resistance upon recipient animals. Administration of methotrexate beginning 1 or 2 weeks prior to or on the same day as transplantation with drug-resistant DHFR transgenic marrow did not allow sufficient engraftment to confer drug resistance to most unirradiated recipients. A small number of animals were curiously protected from lethal MTX toxicity but exhibited extremely low hematocrits and were not engrafted with stem cells, as indicated by low engraftment levels assessed in secondary transplant recipients. However, we subsequently found that MTX preconditioning allowed sufficient engraftment of DHFR transgenic marrow to confer drug resistance if MTX administration was withdrawn at the time of bone marrow transplantation (BMT) and withheld until 2 weeks post-transplant. Quantitative molecular analysis of primary and secondary recipients indicated a stem cell engraftment level of approximately 1%, consistent with previous studies demonstrating that a low level of DHFR transgenic cell engraftment was sufficient to confer drug resistance in recipient animals. We conclude that MTX can be used as a preconditioning agent for subsequent engraftment of hematopoietic stem cells, in this case conferring resistance to MTX.