Regulatory-Approval Driven Safety and Efficacy of MGMTP140K Lentivirus Transduction of Human CD34+ Cells for a Phase I Clinical Trial for Upfront Treatment of Glioma,
Authors: Qing Yulan , Reese Jane , Liu Min , Desai Amar , Embree Heather , Dropulic Boro , Gerson Stanton L. 
Published: October 2019

Abstract
Abstract 4177
Temozolomide (TMZ) is a standard of care treatment for patients with glioma post surgical resection, but induces dose limiting myelosuppresion. Gene transfer of O6-benzylguanine (BG) resistant mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K) into hematopoietic stem cells (HSCs) protects hematopoiesis from alkylating agents and allows efficient in vivo selection of transduced HSCs in mice, dogs and primates. We designed a clinical trial to improve HSC tolerance of TMZ in patients with glioma [clinicaltrials.gov]. This trial uses a lentiviral backbone for gene delivery, and infuses cells during the active phase of treatment to maximize the potential for stem cell protection and selection.
The viral construct was synthesized by Lentigen, with an EF1-alpha promoter driving MGMTP140K (LV-P140K MGMT). Clinical grade vector was subject to safety and toxicity analysis at the request of both NCI and FDA. Transduction conditions were optimized towards a high rate of gene transfer and expression with ≤2 copies per cell to minimize adverse insertions. This study presents the validation of the vector by assessing the engraftment, drug resistance and copy number after gene transfer into human CD34+ cells followed by in vivo selection using a nonobese diabetic (NOD)/severe combined immunodeficient (SCID) gammaC (NSG) mice. We are currently analyzing the insertion sites of the clinical grade LV-P140K MGMT in human cord blood progenitors as a safety monitoring tool for LV-HSC gene therapy.
Engraftment of LV-P140K MGMT (LG631) transduced CD34+ cells in irradiated NSG mice, and the analysis of engrafted human CD45+ cells in peripheral blood, spleen and bone marrow showed consistent but slightly decreased (10%) engraftment of transduced cells compared to the freshly isolated non transduced cells and was multilineage. There was no toxicity to the mice or human HSC as a result of transduction with lentiviral vector. The NSG recipients of the LG631 transduced CD34+ cells received two rounds of 3-consecutive day treatments of 30 mg/kg of BG and 60 mg/kg of TMZ, and three weeks after the second round of treatment, expression of MGMT was examined by PCR in the CFUs from the BM of the recipient mice. Combined treatment with BG and TMZ showed efficient selection of human cells and human CFU expressing MGMT (100% after treatment vs 44+/−4.1% without treatment).
Copy number and characterization of insertion sites of the clinical grade LV-P140K MGMT in human CD34 cells serve as a critical safety monitoring tool for LV-HSC gene therapy. Insertion site analysis showed a polyclonal pattern and selection did not result in oligoclonality. The insertion database will continue to be made publically available as the data is generated prior to and during clinical trial application. We have fully characterized the preclinical safety and efficacy of a clinical grade lentiviral vector for clinical trial application in patients with glioma.

Disclosures:
No relevant conflicts of interest to declare.