Viral Insertion Safety in Patients with Glioblastoma Who Received a Novel Lentiviral MGMT-P140K Gene Therapy to Protect Bone Marrow from Chemotherapy: No Dominant Clonal Evolution Observed with Chemo-Selection
Authors: Fung Hua , Sloan Andrew E , Reese Jane , William Basem M. , Lingas Karen , Rogers Lisa R , Embree Heather , Lazarus Hillard M. , Kane Donna , Dropulic Boro , Gerson Stanton L. 
Published: October 2019

INTRODUCTION: To protect normal bone marrow from chemotherapy in glioblastoma patients, we have developed a novel strategy by introducing a strong DNA repair protein, mutant (P140K) of human methylguanine methyltransferase (MGMT), into patients’ CD34+ hematopoietic progenitors (HPC) by lentiviral gene transfer leading to selective expansion of drug-resistant P140K-MGMT CD34+ cells and their myeloid and immune cell progeny.

METHODS: To achieve long-term stable expression of the P140K-MGMT gene, we used a lentiviral vector which integrates into the host genome. However, viral insertion mutagenesis has raised safety concerns; as the previous γ-retroviral vectors were associated with insertion mutations leading to development of acute lymphoblastic leukemia in 20% of treated patients. Nevertheless, new improved lentiviral vector with safe feature of insertion site far away from gene transcription start site has been developed. Here we evaluated the safety of a lentivirus vector under selection pressure of chemotherapy.

HYPOTHESIS: Our lentiviral vector is safer than traditional γ-retroviral vectors as evident by lack of early clonal dominance even with a chemo-selection.

RESULTS: Three glioblastoma patients were recruited and underwent resection, after which CD34+ HPC were mobilized with filgrastim, isolated by magnetic bead separation (Miltneyi CliniMACS), and transduced ex vivo with a GMP-grade lentiviral P140K-MGMT vector (Lentigen Corp). Subsequently, patients received radiation/temozolomide for 6 weeks and up to five cycles of monthly O6-benzylguanine/temozolomide (BG/TMZ) treatment. As a result, all three patients demonstrated a 5-15 fold selective expansion of P140K-MGMT positive HPC and their progeny granulocyte and mononuclear cells in peripheral blood and a small number of CFUs from bone marrow indicating a drug-selection mechanism. The viral insertion sites in the cells of these three patients were closely monitored in each chemotherapy cycle and the patients were followed for up to 1 year after the last therapy. We mapped a total of 17,308 viral insertion sites (VIS), for patient 1(6,146), patient 2(2,081) and patient 3(9,081) and the unique viral insertion sites (UVIS) was 382, i.e. 135, 76 and 171 for patient 1, patient 2 and patient 3 respectively. Overall, during the drug-treatment period, there were no persistent UVIS. Moreover, at the conclusion of BG/TMZ treatment, the VIS number sharply diminished.

CONCLUSION: Gene transfer of LV MGMTP140K vector into hematopoietic progenitor cells did not lead to clonal dominance during or after drug selection. Dose escalation of BG/TMZ will define hematopoietic tolerance and treatment response.

Disclosures

Embree: Lentigen Technology Inc: Employment. Dropulic:Lentigen Technology Inc: Employment, Patents & Royalties.