36 Chemoprotection Of Long-Term Repopulating Hematopoietic Stem Cells From Alkylator Therapy: in vivo Comparison of Gene-Transfer Vectors that Express a DNA Repair Protein.

摘要

Dose-intensification of alkylator-based chemotherapy in cancer patients can result in life-threatening cytopenia. The DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), repairs DNA damage mediated by alkylating agents in hematopoietic stem cells (HSC). Two viral vector systems for the transfer and expression of the MGMT DNA repair protein in HSC were compared in vivo. A foamy virus vector that can transduce noncycling HSC was compared to an oncoretroviral vector that can transduce HSC induced into cell cycle by cytokine stimulation. The virus vectors express a mutant form of MGMT called MGMTP140K that protects HSC from high-dose alkylator therapy. Lineage-depleted bone marrow (BM) from C57BL/6 mice was transduced for 10-16 hours with the foamy virus vector or following a 2-day pre-stimulation with the oncoretrovirus vector. The bulk transduction efficiency using the foamy virus vector ranged from 12-25% and the progenitor transduction efficiency was 55-57%. Transductions with the oncoretrovirus vectors resulted in similar bulk and progenitor transduction efficiencies (55-60%). Data are from three primary and two secondary transplant experiments. Transplantation of oncoretroviral vector-transduced cells resulted in 90-95% of the cells expressing MGMTP140K in the PB and BM in primary and secondary recipient mice following 2-3 cycles of alkylator therapy. In mice transplanted with foamy virus-transduced cells, MGMTP140K was elevated at 6 months post-transplantation but protection was not as robust as in mice transplanted with the oncoretrovirus-transduced cells. Approximately 50% of the progenitor colonies contained the foamy provirus following in vivo selection. In secondary reconstitution experiments, MGMTP140K expression remained elevated in the BM of mice transplanted with oncoretrovirus- or foamy virus vector-transduced cells. These data demonstrate that although foamy virus transduction is not quite as efficient as the oncoretrovirus transduction strategy, a simple overnight protocol using a foamy virus vector can be used to transduce minimally stimulated HSC. Studies are in progress to further dissect the requirements for efficient transduction of HSC with foamy virus vectors.