Fioretto, Federica (2026) MUTATION-INDEPENDENT GENOME EDITING APPROACHES FOR TREATMENT OF STARGARDT DISEASE. [Tesi di dottorato]

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Abstract

Stargardt disease (STGD1), the most common form of inherited macular degeneration, is caused by mutations in ABCA4, a large (coding sequence: 6822 bp), highly polymorphic gene, with more than 1200 different mutations described so far. No therapeutic option is currently available for STGD1 patients. Direct gene replacement represents an attractive therapeutic option for treatment of STGD1 since it is inherited as autosomal recessive, so adding a functional copy of the coding sequence has the potential to fully restore visual function, and degeneration of STGD1 retinal cells is relatively delayed, allowing a reasonable time window for therapeutic intervention. However, the identification of an efficient vector for the delivery of the ABCA4 gene to the retina has been hampered by the large size of the ABCA4 coding sequence, which prevents its delivery through the gold-standard vectors for gene therapy: AAVs. Targeted genome editing may represent an alternative to restore the wild-type ABCA4 sequence by correcting the disease-causing mutation within the endogenous locus. Genome editing via the CRISPR-Cas9 system is an exciting field of biomedical research which is gaining increasing interest for treatment of several diseases. However, classical genome editing approaches still have limitations, including sequence-specificity that limits applicability to diseases with high allelic heterogeneity. These limitations have thus far hindered development of effective gene editing approaches for several inherited retinal diseases, including STGD1. Therefore, in this project, we aimed to develop novel adeno-associated viral (AAV) vector-based genome editing strategies for treatment of STGD1 which rely on integration of large templates in ABCA4, to allow correction of multiple mutations with a single therapeutic DNA template. To achieve integration of the template we planned to 5 explore either the non-homologous end joining (NHEJ) or the microhomology�mediated end joining (MMEJ) repair pathways, which are active in post-mitotic cells like photoreceptors, the target cells for STGD1 treatment. Accordingly, we have developed multiple candidate sets of AAV-Abca4 vectors for targeting either the murine or the human ABCA4 gene. We side-by-side compared the different approaches in vitro, in murine and human cell lines, and found that they effectively mediate template integration at the endogenous locus, resulting in Abca4 expression. Subretinal delivery of the AAV-based approaches in the retina of Abca4-/- mice results in targeted DNA template integration, followed by productive reconstitution of Abca4 expression in transduced photoreceptors, demonstrating the therapeutic efficacy of these approaches in Abca4-/- mice. To assess translational potential, we further generated iPSC-derived retinal organoids from STGD1 patients’ cells and infected them with AAV2 vectors expressing Cas9 and donor templates. Genomic analysis demonstrated successful targeted integration within human photoreceptors, confirming the applicability of both NHEJ- and MMEJ-based editing strategies in disease-relevant models. Collectively, these findings establish a novel genome editing approach for correction of ABCA4-associated retinal degeneration, opening new therapeutic avenues for genome editing-based treatment of inherited retinal diseases due to mutations in large genes.

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