Objective: The most common form of X-linked retinitis pigmentosais caused by mutations in the
RPGR gene, leading tophotoreceptor degeneration and loss of vision.
Gene therapy usingadeno-associated viral (AAV) vectors has proved its safetyin clinics. However, there is a potential to further improve theefficiency and the ease of AAV gene delivery by engineeringthe AAV capsid. The aim of this study is to evaluate the photoreceptortransduction efficiency of two AAV-RPGR vectorsharbouring engineered and wild type capsids following intravitrealand subretinal injection in mouse eye.Materials and Methods: We synthesized codon-optimizedhuman RPGRORF۱۵ gene cloned into an AAV vector with aCMV promoter. RPGRORF۱۵ transgene expression was analyzedby transfection into cells followed by western blottingusing an anti-RPGR antibody. The transgene was then clonedinto an AAV vector under control of the photoreceptor-specificGRK۱ promoter. AAV۲-(۷m۸) and AAV۸ capsid vectors wereused to introduce tyrosine to phenylalanine mutations by sitedirectedmutagenesis. To evaluate the function of mutant AAVs,we also produced an AAV shuttle plasmid encoding an EGFP reporter. AAVs were injected into the C۵۷BL/۶ mice vitreousand subretinal space to quantify whether efficient expression ofEGFP could be achieved.Results: The wild type and tyrosine mutant AAV۲-(۷m۸) andAAV۸ vectors were produced in HEK۲۹۳T cells, purified fromcell lysates by Heparin affinity chromatography as well as PEGprecipitation, concentrated and stored. Transduction efficienciesof capsid-mutant and wild type AAV vectors carrying theCMV promoter and the EGFP reporter were initially scored invitro. Capsid mutants exhibiting the highest transduction efficienciesrelative to the wild type vectors were then deliveredby Subretinal and intravitreal injection into mice eyes. Fundusphotographs from mouse eyes transduced with the mutant orwildtype AVV۲-(۷m۸) and AAV۸ vector expressing the GFPreporter and the fluorescence patterns demonstrated that thesecapsid modifications resulted in an increase in photoreceptortransduction compared to the unmodified AAV vectors wheninjected into the intravitreal or subretinal space, respectively.We will next test the safety and efficacy of AAV variants carryingGRK۱-RPGRORF۱۵ in C۵۷BL/۶ mice thorough bothintravitreal (AAV۲-YF) and subretinal (AAV۸-YF) injections.Conclusion: Developing stable AAVs capable of penetratinginto the target tissues will enhance their function and transductionefficiency not only for the gene therapy of retina but alsofor the treatment of solid tumors.