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Gene therapy of Usher syndrome type 1B (USH1B) due to mutations in the large Myosin VIIA ( MYO7A ) gene is limited by the packaging capacity of adeno-associated viral (AAV) vectors. To overcome this, we have previously developed dual AAV8 vectors which encode human MYO7A (dual AAV8. MYO7A ). Here we show that subretinal administration of 1.37E+9 to 1.37E+10 genome copies of a good-manufacturing-practice-like lot of dual AAV8. MYO7A improves the retinal defects of a mouse model of USH1B. The same lot was used in non-human primates at doses 1.6× and 4.3× the highest dose proposed for the clinical trial which was based on mouse efficacy data. Long-lasting alterations in retinal function and morphology were observed following subretinal administration of dual AAV8. MYO7A at the high dose. These findings were modest and improved over time in the low-dose group, as also observed in other studies involving the use of AAV8 in non-human primates and humans. Biodistribution and shedding studies confirmed the presence of vector DNA mainly in the visual pathway. Accordingly, we detected human MYO7A mRNA expression predominantly in the retina. Overall, these studies pave the way for the clinical translation of subretinal administration of dual AAV vectors in USH1B subjects., Competing Interests: A.A. is Scientific Founder and Chief Scientific Officer of AAVantgarde BIO Srl, a company that has licensed dual AAV8.MYO7A and is further developing this program into the clinic. R.F. is currently an employee of AAVantgarde BIO Srl. J.-B.M. is an employee of Genosafe, a contract research organization involved in the further development of this program into the clinic. M.F., A.N., F.G., and V.A. are employees of Reithera Srl, a contract development and manufacturing organization involved in the further development of this program into the clinic. S.C. is Scientific Founder and Chief of Technology of Reithera Srl., (© 2023 The Authors.)

Split intein-mediated protein trans- splicing expands AAV transfer capacity, thus overcoming the limited AAV cargo. However, non-mammalian inteins persist as trans- splicing by-products, and this could raise safety concerns for AAV intein clinical applications. In this study, we tested the ability of several degrons to selectively decrease levels of inteins after protein trans- splicing and found that a version of E. coli dihydrofolate reductase, which we have shortened to better fit into the AAV vector, is the most effective. We show that subretinal administration of AAV intein armed with this short degron is both safe and effective in a mouse model of Stargardt disease (STGD1), which is the most common form of inherited macular degeneration in humans. This supports the use of optimized AAV intein for gene therapy of both STGD1 and other conditions that require transfer of large genes., Competing Interests: A.A., P.T., and I.T. are co-inventors on the patent application number PCT/EP2019/0708020 entitled “Intein proteins and uses thereof.” A.A. is founder, shareholder, and consultant of InnovaVector srl and AAVantgarde Bio. The other authors declare that they have no competing interests., (© 2021 The Authors.)

Retinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)