Developing chimeric AAV viruses for intravitreal delivery of RPGR for X-linked Retinitis Pigmentosa gene therapy
Maryam Haghshenas1 , Sina Mozaffari-Jovin1 *
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Abstract: The most common form of X-linked retinitis pigmentosa is caused by mutations in the RPGR gene, leading to photoreceptor degeneration and loss of vision. The aim of this study is to evaluate the photoreceptor transduction efficacy of a capsid-engineered AAV2-RPGR virus for intravitreal injection in mouse eyes and compare it with the subretinal injection of the AAV8-RPGR.
Methods: We synthesized the codon-optimized human RPGRORF15 gene cloned into an AAV vector with a CMV promoter. The RPGRORF15 expression was analysed by transfection into cells followed by Western blotting using an anti-RPGR antibody. The transgene was then cloned into an AAV vector under control of the photoreceptor-specific promoter GRK1. AAV2-(7m8) and AAV8 capsid vectors were used to introduce tyrosine to phenylalanine (Y-F) and threonine to valine (T-V) mutations. To evaluate the function of mutant AAVs, we also produced an AAV shuttle plasmid encoding an EGFP reporter. The wild-type and tyrosine mutant AAV2-(7m8) and AAV8 viruses were produced and purified from cell lysates by heparin affinity chromatography or PEG precipitation and stored for injection into mouse eyes.
Results: The transduction efficiency of mutant and wild-type AAV vectors carrying the CMV promoter and the EGFP reporter were initially analysed in cells. Purified AAV8 and AAV2 viruses were then injected into subretinal and intravitreal spaces of mouse eyes, respectively. Fundus photographs from mouse eyes transduced with the [Y-F]-[T-V] mutant or wild-type AAV2(7m8) and AAV8 expressing the GFP reporter and the fluorescent intensities of the prepared retina flat mounts demonstrated that capsid modifications resulted in an increase in retinal cells transduction compared to the control AAVs. We next tested the efficacy and specificity of AAV variants carrying GRK1-RPGRORF15 delivered to mouse eyes via intravitreal and subretinal injections. Both AAVs showed comparably strong and specific transduction efficiencies specifically in photoreceptors.
Conclusion: we developed AAVs with chimeric capsids capable of penetrating into the retina via delivery to the eye vitreous. The RPGR transgene packaged in this chimeric AAV could be specifically expressed in photoreceptor cells after intravitreal injections. This will allow us to design gene therapy clinical trials for the X-linked-RP using the less invasive intravitreal delivery method, in future.
