Analysis of retroviral production and transduction systems.

摘要

Gene therapy has been focused as one of the most feasible strategies in curing fatal diseases by correcting genetic disorders or introducing new genetic information. Retroviral gene delivery systems consist of two distinct steps: Vector preparation and gene transfer. As an emerging technology, gene therapy processes have not been optimized completely to promise clinical successes, which motivated this study.; A mathematical model was set up to characterize and the compare various retroviral production systems. Results showed higher and more stable production of ecotropic retrovirus from GP+E86/LNCX producer cells than vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped retrovirus from 293GPG/EGFP producer cells. It was also shown that 10% FBS and 37°C is the optimized conditions to get maximized number of infectious ecotropic retrovirus for long-term production. These findings were applied to design a production system of circulating culture medium through cold reservoir.; Retroviral transduction systems were analyzed and infectious retroviral concentration and transduction rate constant were determined by curve-fitting experimental data to the mathematical model. The result showed two-order of magnitude difference of infectious retroviral concentration from the titer. From the analysis developed in this study, an efficiency parameter representing tranduction rate constant was extracted and showed its usefulness in quantifying various retroviral transduction system under various conditions. VSV-G pseudotyped retrovirus has shown to be more efficient vector than ecotropic and amphotropic retrovirus by comparing transductin rate constants. By analyzing the effect of factors involved in retroviral transduction, it was shown that Polybrene increased the efficiency with higher concentration, while chondroitin sulfate C increases nonspecific binding efficiency at low concentration but decreases at high concentration. Furthermore, the correlation of amphotropic retroviral transduction efficiency with receptor expression was clearly verified under phosphate-depleted condition.; This study clearly showed the usefulness of engineering approach to retroviral production and transduction systems in maximizing the efficiency.