Methylguanine Methyltransferase-Based In Vivo Selection Results in Only Transient Improvement in Long-Term Marking after Autologous Transplantation of Transduced Hematopoietic Stem Cells in Rhesus Macaques.
Authors: Larochelle Andre , Choi Uimook , Naumann Nora , Clevenger Josh R. , Malech Harry L. , Dunbar Cynthia E. 
Published: October 2019

Abstract
In vivo selective survival advantage of transduced cells contributed to clinically beneficial levels of genetic correction of lymphocytes following X-SCID gene therapy. For most blood disorders there will be no constitutive selective advantage of the gene-corrected cells. Alternatively, a selectable gene incorporated into the vector may provide selective survival advantage. The P140K mutant of human O6-methylguanine-DNA methyltransferase (MGMT*) is a candidate mammalian selectable gene for hematopoietic stem cell (HSC) gene therapy. AMD3100-mobilized CD34+ cells from 5 rhesus macaques were transduced daily from day 2 to 4 of culture using oncoretroviral (n=2 animals) or lentiviral (n=3 animals) vectors encoding the gp91phox-IRES-MGMT* cassette or the GFP-MGMT* fusion protein, respectively. Transduced CD34+ cells were selected after (in vivo, n=4) or before (ex vivo, n=1) autologous transplantation in rhesus macaques using the BG (120mg/m2)/TMZ 400 mg/m2 combination for in vivo selection and the BG (5uM)/BCNU (7.5uM) combination for ex vivo selection. Marking of peripheral blood (PB) cells was evaluated by FACS and/or real-time PCR. Bulk CD34+ cells were marked at 27–58% after transduction with oncoretroviral or lentiviral vectors. Four animals were transplanted with transduced non-selected CD34+ cells. Small fractions of cultured cells not transplanted were exposed to BG/BCNU resulting in an increase of marking to 88–97% in each case, confirming the in vitro survival advantage. Cells from animals #1 and #2 were transduced with oncoretroviral vectors and steady-state marking of 3.5% was obtained in PB. Animal #1 received BG/TMZ infusions at 3 and 6 months post-transplant. Marking declined to 3.3% and 1.1% after BG/TMZ treatment 1 and 2, respectively. Animal #2 received one cycle of BG/TMZ at 4 months post-transplant. Full hematopoietic recovery was not achieved and the animal died of infectious complications one month after treatment. Marking of 2% was detected in the PB at the time of death. Cells from animals #3 and #4 were transduced with lentiviral vectors. Animal #3 received 4 monthly infusions of BG/TMZ starting 5 months after transplantation. Marking increased from 0.1% at steady-state to 1.8% in PB after the first cycle but rapidly declined to 0.2%. Despite significant myelosuppression, additional cycles of BG/TMZ resulted in no significant improvement in marking. Animal #4 received 4 monthly infusions of BG/TMZ starting 3 months after transplantation. Marking increased from 3.3% at steady-state to 29.2% after the first cycle but rapidly declined to 6.2%. Each additional cycle of BG/TMZ resulted in a transient increase in marking with a peak increase gradually declining with each cycle. Animal #5 was transplanted with CD34+ cells transduced with lentiviral vector expressing GFP-MGMT* and exposed to BG/BCNU ex vivo before transplantation. At the time of reinfusion, 55% of the cells were vector positive. Stable hematopoietic recovery required one month, compared to an average recovery of 2 weeks in animals transplanted with transduced cells without ex vivo selection. Steady state marking in PB of only 0.7% was detected. These data combined with the theoretic concern that the use of cytotoxic drugs could increase the risk of leukemogenesis in the setting of drug-resistance gene therapy, raise concerns for the clinical applicability of this approach.