Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4.

Molecular therapy : the journal of the American Society of Gene Therapy 2024 Mar 06

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The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters the pre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1. Copyright © 2024. Published by Elsevier Inc.

Citation

Nuria Suárez-Herrera, Iris B Riswick, Irene Vázquez-Domínguez, Lonneke Duijkers, Dyah W Karjosukarso, Davide Piccolo, Miriam Bauwens, Elfride De Baere, Michael E Cheetham, Alejandro Garanto, Rob W J Collin. Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4. Molecular therapy : the journal of the American Society of Gene Therapy. 2024 Mar 06;32(3):837-851