Sustained Stable Engraftment of MGMT Gene-Modified Long-Term Repopulating Cells in Primary and Secondary Canine Recipients.
Authors: Beard Brian C. , Sud Reeteka , Gerull Sabine , Peterson Laura J. , Neff Tobias , Kiem Hans-Peter 
Published: October 2019

Abstract
Chemo-protection of gene-modified cells with mutant forms of methylguanine methyltransferase (MGMT) has the potential to increase the efficacy of clinical hematopoietic stem cell gene therapy. The therapeutic benefits of MGMT gene-modified cells are due to two factors: in vivo selection following transplantation to increase the level of cells that carry MGMT and a therapeutic transgene for genetic diseases or inhibitory transgenes for acquired diseases such as AIDS andhematopoietic protection from chemotherapy agents such as BCNU and temozolomide (TMZ) during treatment of malignant diseases.
In the allogeneic and autologous canine models we have demonstrated efficient engraftment, in vivo selection, and chemo-protection of MGMT gene-modified cells and increased gene-marking levels to >90%. Unequivocal proof that true ‘stem cells’ are the transduced population leading to long-term engraftment and gene-marking is elusive and the only reliable test is sustained engraftment in the large animal model (i.e. canine or non-human primates). To further test the durability and engraftment potential of MGMT gene-modified canine cells, we investigated three aspects:engraftment and repopulation in a primary recipient,durability during in vivo selection, andengraftment and repopulation in a secondary recipient.
Briefly, two DLA-identical allogeneic transplants were carried out with CD34-selected cells using an 18-hour transduction protocol. Cells were transduced with a VSVG-pseudotyped lentiviral vector expressing only the mutant version of MGMT(P140K). Cells were infused into primary recipients (G340 and G403) after pre-transplant conditioning with 200 cGy total body irradiation (TBI). G340 and G403 received multiple rounds of in vivo selection comprising 4 and 3 treatments with O6-benzylguanine (O6BG)/TMZ and 1 and 2 treatments with O6BG/BCNU respectively. Gene-marking stabilized in G340 and G403 after the final round of in vivo selection at about 60% and 4% respectively for at least 3 months before we proceeded to secondary transplantation. The original donors (G346 and G404) were conditioned with 920cGy TBI before receiving whole bone marrow cells from the primary recipients G340 and G403 respectively. Both G346 and G404 recovered neutrophil and platelet counts within expected time frames compared to historical controls. Shortly after transplantation the gene-marking in G346 stabilized around 80% and this level has been maintained for more than 1 year without in vivo selection. The gene-marking in G404 stabilized around 1% shortly after transplantation and this level was maintained until recently when in vivo selection was carried out in both G404 and G403 to increase gene-marking levels. Multiple clones contributed to hematopoietic repopulation in the primary and secondary recipients and we are in the process of tracking shared clones in the animals. These data establish the durability of MGMT(P140K) gene-modified cells and also present convincing evidence that true hematopoietic stem cells were transduced during the 18-hour transduction. This study demonstrates that MGMT(P140K) gene-modified cells repopulate a primary recipient and subsequently survive multiple rounds of in vivo selection while maintaining engraftment and repopulation potential in a secondary recipient.