Retrovirus Insertion Site Analysis Following In Vivo Drug Selection in a Phase I Clinical Trial Utilizing G156A MGMT for Enhanced Chemotherapy of Advanced Malignancies.

Abstract

The G156A mutant of the DNA repair gene O6-methylguanine DNA-methyltransferase (MGMT) confers hematopoietic resistance to O6-benzylguanine (BG) combined with DNA-alkylating agents BCNU or temozolomide, and allows for selective in vivo expansion with drug administration of murine hematopoietic progenitors transduced with G156A MGMT retrovirus. Here we report our latest findings on retroviral vector copy number and insertion site analysis following drug treatment from a Phase I clinical trial utilizing MGMT-mediated chemoprotection for enhanced treatment of advanced solid tumors. Seven patients have entered the trial and 6 have completed the cell infusion process. For all patients, autologous CD34+ cells were transduced ex vivo with an MFG retroviral vector containing the G156A MGMT gene (packaged with PG13 by the National Gene Vector Laboratory, Ken Cornetta, Director) in the presence of the fibronectin fragment CH-296 and the cytokines SCF, Tpo, and Flt-3 ligand for 72 hours with three additions of retroviral supernatant. At 72 hours following patient treatment with BG and BCNU, cells were re-infused. Prior to infusion, the average vector copy number by quantitative real-time PCR analysis for six patients was 0.34 copies per genome, with an average of 24% of CFUs transduced by standard PCR for G156A MGMT, and an average of 9% of CD34+ cells expressing the MGMT transgene by flow cytometry. In one patient with metastatic melanoma we have further analysis of insertions. For this patient, the pre-infusion vector copy number of the bulk CD34+ population was 0.54 copies per genome by real-time PCR, with 27% of CFUs transduced and 8% of CD34+ cells expressing the MGMT transgene prior to infusion. Linear amplification-mediated (LAM)-PCR analysis of retroviral insertion sites in pre-infusion CFUs from this patient confirmed a polyclonal population, with an average of 1.6 retroviral insertions per positive CFU. In this patient, BG (120 mg/m2) and BCNU (33 mg/m2) were administered at 6 weeks post-infusion, and temozolomide (300 mg/day for 5 days) was administered at 13 weeks. Peripheral blood (PB) and bone marrow (BM) granulocyte and mononuclear cells (MNCs) were collected at weeks 5, 11, 15, and 16 for DNA and CFU analysis. Vector copy number at all post-infusion time points was below the limit of detection of SYBR Green probe-based real-time PCR (<100 copies of G156A MGMT per 5000 genomes). LAM-PCR detected the vector in post-treatment samples based on an internal vector control band present in BM MNCs at week 11 and in BM granulocytes at week 16, although specific insertion sites were not detected. Standard PCR revealed 1 out of 100 CFUs from week 11 BM MNCs contained the vector, with 2 out of 30 CFUs from week 15 PB MNCs. LAM-PCR in a subset of week 11 CFUs confirmed a single insertion site present in the same PCR-positive CFU. Sequence analysis of clonal vector insertions pre- and post-infusion is ongoing, and thus far a number of sites have been characterized, adding to the emerging database of clinical retroviral insertions. These are the first data to show emergence of transduced mutant MGMT cells after nonmyeloablative conditioning in humans and suggest that despite a low frequency of vector-marked hematopoietic cells, clinical in vivo drug selection can be observed.