668. Detailed Comparison of Self-Inactivating Lentiviral Vectors Produced By Transient Transfection and Vectors Produced By the Cytegrity™ Stable Cell Line System

Abstract

The Cytegrity™ system was used to generate a stable cell line for the production of LVsh5/C46, a self-inactivating lentiviral vector (SIN-LV) encoding a short hairpin RNA (shRNA) for down-regulation of the HIV-1 co-receptor CCR5, in combination with the HIV-1 fusion inhibitor, C46. This LV, produced by transient transfection, is currently being evaluated in clinical trials in HIV-infected individuals. Here we conducted a comparative analyses of LVsh5/C46 produced by transient transfection and LVsh5/C46 produced using the Cytegrity™ system to support the application of this system for clinical manufacturing SIN-LVs.LVs were produced by calcium phosphate transfection in 293T cells using the 4-plasmid system. Virus-containing media (VCM) was harvested 48h post-transfection and concentrated by ultracentrifugation through a 20% sucrose cushion. For cell line production, producer cells were induced in media without doxycycline (Dox), and the VCM was harvested at 72h and similarly concentrated by ultracentrifugation. LVs produced by each method were compared based on particle titer and using three independent assays for gene transduction potency on 293T and the TF-1a T cell line. These included FACS assays for cell surface C46 expression and shRNA-mediated knockdown of CCR5 expression, as well as a qPCR assay for vector copy number (VCN) per host cell genome. For all assays, titer was determined over a range of vector dilutions to define a linear relationship. The qPCR assay utilized genomic DNA extracted from transduced cells, and detect the C46 transgene and a sequence from the endogenous β-globin gene. As such, C46 VCN could be normalized to cellular genome.A higher concentration of p24 was observed in VCM produced by producer cell lines relative to transient transfection method. However, yield and potency of LVsh5/C46 produced using the two different systems was similar. Vectors were first evaluated for C46 titer by FACS using equal volumes of VCM. While vector produced by transient transfection had a modestly increased titer, when C46 titers were normalized and vector preparations were assed for gene transduction using the qPCR assay or via functional knock-down of CCR5, vector produced by the stable producer cell lines showed greater potency. Down-regulation of CCR5 expression and genomic C46 transgene (VCN) were each significantly higher in the target cells treated with LVsh5/C46 produced by the Cytegrity™ system than treated with vector produced by transient transfection.Based on three independent assays, we demonstrate that the Cytegrity™ stable LV production system is capable of generating similar quality and quantity of SIN-LVs compare to the transient transfection method. The higher CCR5 down-regulation efficacy and C46 VCN in transduced cells (normalized to C46 titer) indicate that LVsh5/C46 produced by producer cells has better potency than those vectors generated using the conventional 4-plasmid transient transfection. By removing the tedious transient transfection step, this production system can be easily adapted to cGMP conditions for the manufacture of clinical grade material.