Using gene therapy to protect hematopoietic stem cells (HSC) from alkylating agents used in the treatment of malignant disease is an attractive strategy to alleviate prolonged neutropenia and thrombocytopenia that is dose-limiting. Both adult and pediatric patients with glioblastomas urgently need improved therapeutic strategies since even with aggressive treatment the median survival after diagnosis is approximately 12 months. Chemotherapy with the nitrosourea BCNU, the methylating agents procarbazine or temozolomide, and other alkylating agents is effective and can prolong survival but the therapeutic benefit is attenuated due to hematopoietic toxicity which limits dose-escalation of these drugs. We have previously demonstrated in the dog and non-human primate model efficient gene marking, in vivo selection and chemoprotection from temozolomide and BCNU following transplantation with MGMTP140K gene-modified cells. Thus, we wanted to explore the efficacy of autologous transplantation in macaques and baboons using cells gene-modified with an MLV-based gammaretrovirus vector developed for a pending clinical trial. This retroviral vector contains a myeloproliferative sarcoma virus LTR, negative control region deleted, dl587rev primer binding site (MND) vector backbone, which expresses MGMTP140K from the 5′ LTR promoter, and is pseudotyped with the gibbon ape leukemia virus (GALV) envelope produced from Phoenix-GALV packaging cells (PhGALV-MND.GRS.P140K c38). Following transduction the gene marking in pre-infusion colony forming units (CFUs) was 74.3% and 69.8% in the macaque and baboon respectively as determined by CFU-PCR. Intracellular MGMT-staining of cultured baboon cells 4 (76.4%) and 11 (89.9%) days after transduction confirmed high gene transfer levels. Both animals recovered neutrophil and platelet levels within expected time frames relative to historical controls and the average provirus copy number determined by real-time PCR approximately one month after transplantation was 0.14 and 0.72 in the macaque and baboon respectively. Retrovirus integration site analysis in the macaque 90 days after transplantation, and before chemotherapy, confirmed polyclonal hematopoietic reconstitution. There was no indication of progression to a pre-leukemic state. The macaque has been treated twice with O6-benzylguanine (O6BG) and BCNU and the gene marking has stably increased approximately 2.5-fold. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. In summary, we have been able to achieve efficient polyclonal gene marking with MGMTP140K gene-modified cells using a vector designed for clinical application in both macaques and baboons and have preliminary evidence of in vivo selection in the macaque. We believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy.

Disclosures: No relevant conflicts of interest to declare.

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