Production of optimized AAVs carrying the RPGR gene for X-linked retinitis pigmentosa type-۳ gene therapy

Backgrounds: The most common form of X-linked retinitis pigmentosa is caused by mutationsin the RPGR gene, leading to photoreceptor degeneration and loss of vision. Gene therapy usingadeno-associated viral (AAV) vectors has proved its safety in clinics and AAV serotype-۲(AAV۲) has been widely used for gene delivery into the retina. The aim of this study is toevaluate photoreceptor transduction efficiency of two rAAV۲-RPGR vectors with mutant capsidsfollowing intravitreal and sub-retinal delivery in mice.Materials and Methods: We synthesized codon-optimized human RPGRORF۱۵ gene cloned intoan AAV vector with CMV promoter. RPGRORF۱۵ transgene expression was analyzed bytransfection into cells followed by western blotting using an anti-RPGR antibody. The transgenewas cloned into an AAV vector under the control of photoreceptor-specific GRK۱ promoter.AAV۲-(۷m۸) and AAV۸ capsid vectors were used to introduce tyrosine to phenylalaninemutations by site-directed mutagenesis. To evaluate the function of mutant AAVs, we producedan AAV۲ shuttle plasmid encoding an EGFP reporter.Results: The recombinant AAV (۷m۸-YF)-EGFP particles were produced in HEK۲۹۳T cells,purified from cell lysates by Heparin affinity chromatography, concentrated and stored. Theactivity and titer of the AAV (۷m۸-YF)-EGFP variant has been assessed by transduction ofcultured cells showing a high transduction activity in vitro. We are now analyzing AAVRPGRORF۱۵mutant variants and will test the transduction efficiency of these mutant viruses inthe mouse retina.Conclusion: AAVs harboring capsid surface tyrosine mutations display increased stability andpenetration compared with the wild type counterparts.

کلیدواژه ها:

Retinitis Pigmentosa ، RPGR ، Gene therapy ، Adeno-associated virus