Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors

Abstract

Purpose: Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to those inherited retinal diseases (IRDs), as Stargardt's disease (STGD) or Usher syndrome type IB (USH1B) that are due to mutations in genes larger than 5 Kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here we tested them in the large cone-enriched pig retina, which closely mimics human retina.

Methods: Three month-old Large White female pigs were injected subretinally with single or dual AAV vectors encoding either the reporter EGFP and RFP or the therapeutic ABCA4 or MYO7A transgenes, mutated in STGD and USH1B respectively. Transgene expression was evaluated one month later.

Results: We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that are about 2-3 folds lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than in mice and potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively.

Conclusions: Our data prove the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant pre-clinical model. The research leading to these results has received funding from the European Research Council funder the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. [282085] and from Italian Telethon Foundation Grant n. TGM11MT1.

Keywords: 538 gene transfer/gene therapy • 696 retinal degenerations: hereditary