Production of optimized AAVs carrying the RPGR gene for X-linked retinitis pigmentosa type-۳ gene therapy
- سال انتشار: 1400
- محل انتشار: کنفرانس بین المللی ژنتیک و ژنومیکس انسانی
- کد COI اختصاصی: CHGGE01_365
- زبان مقاله: انگلیسی
- تعداد مشاهده: 181
نویسندگان
Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
چکیده
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 virusاطلاعات بیشتر در مورد COI
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