An Optimized Adeno-Associated Virus (AAV) for Treatment of Leber Congenital Amaurosis (LCA-RPE65)

Abstract

Purpose:: We aimed to optimize RPE transduction through modifications of the transgene cassette and cis plasmid used to generate an adeno-associated virus (AAV) carrying wildtype human (h)RPE65 cDNA. Transduction characteristics of the optimized vector, AAV.CBA.hRPE65v2, were compared with those of early versions in vitro and in vivo.

Methods:: The Kozak sequence at the translational start site of hRPE65 was modified and we used a cis plasmid with kanamycin resistance and long (8.5 kb) stuffer sequence. The latter prevents reverse packaging from the AAV ITRs possible when the size of the vector plasmid backbone is less than the packaging limit of AAV. Transduction characteristics and biological effects of infection with AAV.CBA.hRPE65 (v1 and v2) were evaluated in primary RPE cultures from unaffected and affected dogs and in Rpe65-/- mice. Therapeutic efficacy and safety of AAV.CBA.hRPE65v2 was then evaluated in RPE65 mutant dogs.

Results:: Modifications of the transgene cassette increased transgene expression and preparations had optimal purity and transduction titer. Electroretinogram (ERG) testing showed therapeutic effect after treatment with AAV.CBA.hRPE65v2 (but not v1) in Rpe65-/- mice. Subretinal delivery of 1.5E11 GC of vector was well tolerated locally and systemically and treated animals showed improved visual behavior and reduced nystagmus within 2 weeks of injection. Nystagmus testing and ERGs performed 4-5 weeks and 3 months after injection showed reversal of the visual defects. There was minimal systemic distribution of vector sequences and immunohistochemically detectable RPE65 protein only in RPE cells exposed to virus. There was no evidence of retinal degeneration or inflammation. There were histopathological effects resulting from the subretinal injection, including dislodged RPE cells, localized persistent small detachments in the region of the bleb and outer segment changes.

Conclusions:: AAV.CBA.hRPE65v2 delivers the RPE65 transgene efficiently and quickly to RPE cells in vitro and in vivo and results in therapeutic effect in affected mice and dogs with minimal toxicity. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • nystagmus