469. Development and Characterization of GPRG-Based Producer Cell Lines for the Bioproduction of Lentiviral Vectors for HIV Gene Therapy

Abstract

clinical production of self-inactivating lentiviral vectors (SIN-LVs). Here we sought to establish producer cell lines based on GPRG for the production of LVsh5/C46, a SIN-LV currently being assessed in the clinic for treatment of HIV-infected individuals. This vector encodes two viral entry inhibitors; sh5, a short hairpin RNA to the HIV coreceptor CCR5, and C46, a viral fusion inhibitor. We also sought to define the stability of GPRG packaging cell line, the GRPG-based LVsh5/C46 producer cell line, and LVsh5/C46 production following tetracycline induction as required for regulatory filling and clinical application of the GPRG system for bioproduction of LVsh5/C46. GPRG cells were cultured in D10 media with doxycycline (Dox) and puromycin (Puro). To generate LVsh5/C46 producer cells, GPRG cells were transfected with the transfer plasmid TL20-LVsh5/C46 and a Zeocin-resistance plasmid as a concatemeric array. Individual clones were evaluated for their ability to produce LVsh5/C46 vector and maintained in D10 media with Dox, Puro, and Zeocin. To assess the stability of the parental GPRG cell line for LV production, GPRG cells were transfected with transfer vector every 10 passages over a 3-month period (50+ total passages). Virus-containing media (VCM) was harvested 48h post-transfection and vector titer was assessed by complementary gene transduction assays. To assess the stability of LV production from the stable producer cell clones, cells were induced in D10 media without Dox. VCM was harvested 72h after induction and titer was similarly assessed over a range of vector dilutions. To analyze the stability of VSV-G expression following induction after long-term passage, GPRG cells were induced by Dox withdraw and then stained using a biotin-conjugated anti-VSV-G antibody, followed by a secondary staining with Streptavidin-Phycoerythrin. GPRG cells demonstrate stringent tetracycline-regulated expression of VSV-G. This packaging cell line was able to produce up to 107 LV transduction units (TU)/mL after transfection with the LV transfer vector and maintained high-level LV production for more than 50 passages in continuous culture. By utilizing concatemeric array transfection, we demonstrate efficient construction of a producer cell line based on GPRG for the production of LVsh5C46. This cell line consistently generated titers above 106 TU/mL. Further increases in titer could be achieved by recloning and selection of secondary producer cell lines. Titers peaked 2 to 5 days post-induction. We also show that the established stable producer cell lines could routinely maintain LVsh5/C46 production with titers exceeding 106 TU/mL during continuous culture exceeding 25 passages. The GPRG cell line efficiently expresses VSV-G on cell surfaces upon the removal of Dox. It can also generate high titers LVs after transfection of transfer vector plasmids. Moreover, this cell line allowed the derivation of high-titer producer cell lines for SIN-LVs. Producer cell lines demonstrated stable vector production during prolonged culture, and evaluation of the adaptability to adapt