79. Development and Validation of a Globally Portable Platform for Lentivirus Mediated Hematopoietic Stem Cell Gene Therapy
Authors: Jennifer E. Adair, Kevin G. Haworth, Guy Sauvageau, Shelly Heimfeld, Hans-Peter Kiem
Journal: Molecular Therapy (2015)

Lentivirus (LV) mediated gene therapy of CD34+ hematopoietic stem and progenitor cells (HSPCs) has demonstrated clinical success for a variety of diseases. However, current state-of-the-art requires ex vivo HSPC gene transfer in a dedicated Good Manufacturing Practices (GMP) facility, limiting treatment to highly developed countries capable of supporting GMP infrastructure. We developed a flexible, overnight platform for efficient isolation and LV gene modification of bone marrow and mobilized peripheral blood CD34+ HSPCs in a closed, table top system, the Prodigy CliniMACS™, with limited requirement for additional equipment. We performed all experiments with only a biosafety cabinet and personal protective equipment to simulate anticipated conditions in clinical facilities of underdeveloped countries. Given the economic burden of mobilization, we initially designed the process for bone marrow. A total of 7 custom programs were developed based on current device memory limitations: (1) hetastarch sedimentation to deplete red blood cells (RBCs), (2) labeling CD34+ cells in the RBC-depleted product, (3) immunomagnetic enrichment of CD34+ cells, (4) initial transduction (MOI = 20 IU/cell), (5) culture overnight, (6) second transduction (MOI = 20 IU/cell) and additional culture, and (7) harvest and formulation of the final product. Addition of a pyrimidoindole derivative, UM729, permitted efficient transduction of CD34+ HSPCs in £18 hours. The process took <36 hours from bone marrow collection to infusion of the gene modified cell product, with £3 hours hands-on time. Autologous, LV gene-modified CD34+ HSPCs from two nonhuman primates produced using this platform engrafted and supported multilineage hematopoietic repopulation after myeloablative total body irradiation. Total cell doses achieved were 27 × 107 and 5.4 × 107 CD34+ cells/kg body weight, respectively. Transduction efficiency in colony-forming units was 23% and 39%, respectively. We observed stable, persistent gene marking in peripheral blood cells up to 16% within one month after infusion. Neither animal displayed evidence for increased toxicity, including potential contamination from the cell product. We then validated processing of human mobilized apheresis products. We demonstrate efficient isolation of human CD34+ HSPCs and up to 60% transduction efficiency with a clinical-grade LV currently being tested in a phase I clinical trial for treatment of HIV-associated lymphoma. Immediate release criteria for product infusion were met (negative gram stain, endotoxin <5 EU/mL and cell viability >70%). Perform-and-report testing and xenotransplantation of these gene-modified cells into immunodeficient mice for further functional testing are in progress. These data demonstrate preclinical safety and feasibility of this portable strategy for ex vivo LV gene transfer into HSPCs, representing the first globally applicable advance in translation of HSPC gene therapy.