Production and purification of highly replication defective HSV-1 based gene therapy vectors.

摘要

Herpes Simplex Virus Type 1 (HSV-1) represents an attractive vehicle for a variety of gene therapy applications. To render this virus safe for clinical use, its cytotoxic genes must be removed without losing its ability to express transgenes efficiently. Therefore, complementing cell lines that can provide high titer virus are essential for clinical application. Manufacturing methods that can purify these vectors of host cell DNA and protein are also necessary for translation of this gene therapy strategy into clinic.;In this thesis, I have developed complementing cell lines that allow propagation to up to 1E6 PFU/ml routinely for triple as well as quadruple deleted vectors which are among the most difficult to culture to high titer. Replacement of the ICP4 promoter with the VP16 enhancer element enriched ICP0 IE gene promoter resulted in higher induction levels and faster kinetics of ICP4 expression and a 10 fold increase in vector yield.;Along these lines, I investigated the repressive nature of Vero cells to a quadruple IE mutant. A high throughput cell based chemical screen revealed a metal chelator, 1 10 phenanthroline, that was able to derepress EGFP expression not only from the quadruple backbone but of other ICP0 mutant viruses suggesting a unique cell state that was permissive for HSV-1 transgene expression. Interestingly, proteosome inhibition studies reveal that this pathway is essential for this effect not only in Vero cells but also normally permissive U2OS cells.;Finally, a systematic study into ion exchange chromatography for purification of these vectors reveals dramatic differences in infectious yield depending on the matrix chosen. Anion exchange resins bound the virus with high affinity and require high salt concentrations to recover adequate titers. Cation exchange chromatography was able to purify HSV-1 to moderate titers while removing a majority of the host contaminating DNA and protein in accordance with FDA standards for clinical grade viral based vaccines.