Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.
The Authors provide proof-of-concept of gene therapy for two inherited retinal degeneration conditions by using dual AAV vectors. This allows the expansion of AAV cargo capacity for gene therapy of syndromes that require the transfer of large genes.
- Dual AAV vectors significantly expand AAV cargo capacity in the retina.
- Unlike dual AAV overlapping vectors, trans-splicing and hybrid vectors efficiently transduce photoreceptors.
- Subretinal delivery of dual AAV vectors improves the retinal phenotype of murine models of inherited blinding conditions due to mutations in large genes.