Dual adeno-associated virus vectors result in efficient in vitro and in vivo expression of an oversized gene, MYO7A.

Usher syndrome 1B (USH1B) is a severe, autosomal recessive, deaf-blind disorder caused by mutations in myosin 7A (MYO7A). Patients are born profoundly deaf and exhibit progressive loss of vision starting in their first decade. MYO7A is expressed in human photoreceptors and retinal pigment epithelium, but disease pathology begins in photoreceptors, highlighting the need to develop a gene replacement strategy that effectively targets this cell type. For its safety and efficacy in clinical trials and ability to transduce postmitotic photoreceptors, we have focused on developing a clinically applicable adeno-associated virus (AAV) platform for delivering full-length MYO7A cDNA (∼6.7 kb). Packaging of full-length MYO7A cDNA in AAV produces vectors with heterogeneous, fragmented genomes ("fAAV") capable of reconstituting full-length cDNA postinfection. We previously showed that fAAV vectors effectively delivered full-length MYO7A in vitro and in vivo. However, fAAV vectors are relatively inefficient and their heterogeneous genomes preclude definitive characterization, a drawback for clinical translatability. The aim of this study was to overcome these limitations by creating dual-AAV-vector platforms for USH1B with defined genomes. Human MYO7A was cloned in AAV vector pairs, each containing genomes <5 kb and intact inverted terminal repeats. One vector contained a promoter and 5' portion of the cDNA and the partner vector contained a 3' portion and polyadenylation signal. "Simple overlap" vectors share a central part of the MYO7A cDNA sequence. "Trans-splicing" and "hybrid" vectors utilize splice donor and acceptor sites with and without an additional central recombinogenic sequence, respectively. Vector pairs expressed full-length MYO7A in vitro and in vivo with equal or higher efficiency than fAAV, with a hybrid platform being most efficient. Importantly, analysis of MYO7A mRNA derived from each dual-vector platform revealed 100% fidelity to the predicted sequence. Our results suggest that dual AAV vectors with defined genetic payloads are a potential treatment option for USH1B.