Padmanabhan, Arjun (2025) AAV-HITI for therapy of dominant Retinitis Pigmentosa. [Tesi di dottorato]

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Abstract

Retinitis Pigmentosa (RP) is a leading cause of inherited blindness that affects approximately 1 in 3,000 individuals worldwide, with 30-40% of cases resulting from autosomal dominant (AD) inheritance. The most frequent ADRP (RP4) is due to mutations in the gene encoding for rhodopsin (RHO), with several prevalent gain-of-function (GOF) mutations. Conventional gene therapy is ineffective for GOF mutations as removing the mutant RHO allele would be required. CRISPRCas9 based genome editing holds great promise to counteract limitations due to GOF mutations, where disruption of the mutated allele can be carried out leaving the healthy copy intact. However, the heterogenous nature of mutations in RHO restricts this approach as it requires the design of a guide RNA for every unique mutation and is also limited by the non-availability of a PAM site required for nuclease activity. To this end, my PhD project has been focused on the development of an adeno-associated viral vector-mediated homology-independent targeted integration (AAV-HITI) approach directed to RHO and optimized the HITI donor DNA design to achieve simultaneous knock-out of the endogenous RHO allele and knock-in of a correct RHO copy. Of note, the AAV-HITI system is designed to target the RHO locus regardless of the specific mutation. I have demonstrated the efficiency and efficacy of this system in a newly described humanized mouse model of ADRP. Following subretinal injection, AAV-HITI treated mice exhibited significant improvements in electrical function, visual acuity, and retinal morphology up to 1-year post-treatment. Moreover, I performed comprehensive molecular analysis, to further investigate all possible AAV-HITI outcomes within the on-target site, showing that HITI occurs predominantly in the desired direction of integration. Considering the human-centric nature of this system, I further investigated potential nuclease-driven off-target effects in human cells which revealed the overall safety of this platform. In conclusion, this AAV-HITI approach represents a significant step toward clinical translation, holding potential to transform treatments for ADRP patients regardless of their specific RHO mutations.

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