The goal of this application is to develop a safe and effective dual Adeno associated virus (AAV)-based gene therapy for the treatment of Usher Syndrome 1B (USH1B). USH1B is a recessively inherited disease that presents with deafness and vestibular defects from birth, progressive retinal degeneration, and vision loss within the first decade. A major obstacle to developing treatments for vision loss in USH1B patients is the lack of animal models that faithfully recapitulate the retinal phenotype. Previously characterized, naturally-occurring shaker1 strains display only subtle changes in retinal function and no retinal degeneration. They are also difficult to work with due to the variable MYO7A expression levels observed among shaker1 strains that have different underlying mutations. To overcome these limitations, we engineered two new mouse models of USH1B with 1) a complete knockout of MYO7A (Myo7a-/-) or 2) conditional, retina-only knockout of MYO7A (CKO Myo7a-/-).
In Aim 1, I will characterize retinal structure and function in these novel models to identify potential outcome measures for MYO7A gene therapy. These mice provide the opportunity to evaluate gene supplementation in a retina with no endogenous MYO7A, to gain key insights into the mechanism of the disease, and observe any differences that may exist between the roles of MYO7A in mouse vs human retina. Retina only, CKO Myo7a-/- mice allow for separation of the vestibular defect and the ability, for the first time, to conduct visually-guided behavior tests in a model of USH1B. AAV has emerged as the gold standard in retinal gene therapy. However, the size of the MYO7A cDNA exceeds its packaging capacity (~5kb). To overcome this hurdle, dual AAV vector platforms have been developed wherein large genes are split into two AAV vectors, with the 5' and 3' halves of the gene packaged into separate capsids. Upon co-injection, the 5' and 3' `halves' recombine to form full-length gene and encode full length protein. We have already demonstrated the ability to deliver full length MYO7A in vivo using these dual AAV vector platforms. However, safety and efficacy concerns remain. Our previous results show that dual AAV-MYO7A promote toxicity in subretinally injected C57BL/6 mice that we believe is caused by formation of truncated protein from the 5' gene `half'.
In Aim 2, I will eliminate formation of truncated protein by changing the split point of MYO7A and investigating AAV capsid mutants to ?silence? the 5' vector when it is not recombined with the 3' vector. A concern with dual AAV vectors is the low rate of recombination between the 5' and 3' gene halves. I will increase recombination efficiency by directing concatemerization of gene halves with zinc fingers through the incorporation of unique zinc finger binding domains into our dual vector system. I hypothesize this will ?pull? our dual vectors together in their proper orientation, thus increasing recombination and transgene expression. The increased recombination rate/overall MYO7A expression will allow for a reduction in the total vector dose, an added safety feature. By improving the safety and efficiency of dual AAV-MYO7A vectors, we can gather the necessary IND-enabling data to support clinical application of a gene therapy for USH1B.
I propose to develop a safe and effective gene therapy for vision loss associated with Myosin VIIA Usher syndrome (USH1B). I will characterize novel mouse models to identify outcome measures, and develop dual AAV vectors that efficiently deliver only full-length therapeutic protein for use in IND-enabling studies to treat this disease.
