(The) production of gutless adenovirus for gene therapy using site-specific recombinase system

Abstract

We wanted to optimize the production of gutless adenovirus (GAd) using site-specific recombinase system. GAds lack almost the entire viral genome, excluding the noncoding sequences responsible for packaging (yr) and inverted terminated repeats (ITRs) sequences, thereby resulting in improved safety and capacity (up to 36 kb). The GAds can be propagated in the presence of helper Ad (HAd) that retains the ability to replicate and provide the necessary proteins in trans for the replication and packaging of GAds. In order to construct a HAd for GAd production using site-specific recombinase system, two parallel excision elements flanking packaging signal (yr) should be placed onto helper genome. When HAds are coinfected with GAds into 293 complementing site-specific recombinase, the yr sequence in HAds is efficiently excised, thereby rendering the HAd DNA unpackable and still providing all the functions necessary in trans for the replication and packaging of GAds. There has been no report about comparison of GAd productions among different recombinase systems such as Cre/IoxP, FLP/FRT and FLPe/FRT. The comparison was done and best recombinase system (Cre) was adopted for optimization of GAd production finally. In this study, we constructed a HAd containing yi and enhanced GFP (EGFP) flanked by two parallel IoxP recognition sites (HAd/EGFP) as a model and investigated whether its use with flow cytometry enabled facilitating selection of 293Cre and titration of HAd during GAd amplification and production. Moreover, the effect of positioning a left excision element between LITR and packaging signal was investigated because it could have influence probably on excision efficiency of helper Ad. In final chapter, we optimized the procedures of transfection, amplification and production of GAd vector in order.