MGMT (P140K) Allows for Efficient and Sustained In Vivo Selection in Non-Human Primates.
Authors: Beard Brian C. , Beebe Kate , Piasecki Julia , Gooch Christina , Dickerson David , Kiem Hans-Peter 
Published: October 2019

Abstract
In vivo selection strategies that convey a survival advantage to genetically modified cells carrying mutant forms of methylguanine methyltransferase (MGMT-P140K) have the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. For some applications such as genetic diseases or anti-HIV strategies, in vivo selection may be required to increase initially low levels of gene-modified cells while for malignant diseases hematopoietic stem cell (HSC) chemo-protection may be necessary during chemotherapy dose escalation. Thus we have explored the use of gammaretrovirally expressed MGMT(P140K) mutant in three baboons. Animals received CD34−enriched cells transduced with a GALV-pseudotyped retroviral vector expressing a bicistronic message containing P140K and GFP. Two of the animals were part of a competitive repopulation assay in which one half of the cells were gene-modified with a GALV-pseudotyped vector expressing only YFP. After stable engraftment all three baboons were treated with various regimens of O6-benzylguanine (O6BG) and temozolomide (TMZ) or BCNU. Following treatment with O6BG/TMZ the selection was transient for ‘protected’ cells gene-modified with MGMT(P140K)-GFP, and the expected negative selection of ‘unprotected’ gene-modified cells (YFP transgene alone) was subtle. Conversely, positive selection of MGMT(P140K)-GFP gene-modified cells and negative selection of YFP gene-modified cells was dramatic and sustained following treatment with only a single dose of O6BG/BCNU. The increase in gene-marking (up to ~85%) is stable following selection out to 22 months. Importantly, selection of hematopoietic cells was polyclonal and no evidence of insertional mutagenesis has been detected. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. We suspect that the delivery/absorption of TMZ in non-human primates is a contributing factor to transient selection because in animals with low levels (<1%) of MGMT(P140K) gene-modified cells no pronounced or sustained drop in white blood cell or platelet counts was observed following O6BG/TMZ. This is the case even up to TMZ dose levels of 700 mg/m2 that is above dose limiting toxicity in humans. In summary, MGMT selection is efficient and well tolerated in monkeys and we believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy.
Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow). Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow).