June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Combined AON-U7snRNA therapy decreases aberrant splicing caused by multiple deep-intronic ABCA4 variants in vitro
Author Affiliations & Notes
  • Nuria Suárez-Herrera
    Department of Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Radboud Universiteit Donders Institute for Brain Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Iris B Riswick
    Department of Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Radboud Universiteit Donders Institute for Brain Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Irene Vázquez-Domínguez
    Department of Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Radboud Universiteit Donders Institute for Brain Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Lonneke Duijkers
    Department of Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Davide Piccolo
    UCL Institute of Ophthalmology, London, London, United Kingdom
  • Michael E Cheetham
    UCL Institute of Ophthalmology, London, London, United Kingdom
  • Alejandro Garanto
    Department of Pediatrics, Radboudumc Radboud Institute for Molecular Life Sciences, Nijmegen, Gelderland, Netherlands
    Department of Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Rob WJ Collin
    Department of Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Radboud Universiteit Donders Institute for Brain Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Footnotes
    Commercial Relationships   Nuria Suárez-Herrera None; Iris Riswick None; Irene Vázquez-Domínguez None; Lonneke Duijkers None; Davide Piccolo None; Michael Cheetham ProQR Therapeutics, Alia Therapeutics, PYC, Code C (Consultant/Contractor), P6085273, Code P (Patent); Alejandro Garanto P6063546, P6085273, Code P (Patent); Rob Collin ASTHERNA, Code O (Owner), P6063546, P6085273, Code P (Patent)
  • Footnotes
    Support  Marie Sklodowska-Curie Innovative Training Networks (ITN) under grant No. 813490
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 64 – A0037. doi:
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      Nuria Suárez-Herrera, Iris B Riswick, Irene Vázquez-Domínguez, Lonneke Duijkers, Davide Piccolo, Michael E Cheetham, Alejandro Garanto, Rob WJ Collin; Combined AON-U7snRNA therapy decreases aberrant splicing caused by multiple deep-intronic ABCA4 variants in vitro. Invest. Ophthalmol. Vis. Sci. 2022;63(7):64 – A0037.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Stargardt disease is a progressive inherited retinal disease caused by ABCA4 mutations. Over the years, an increasing number of pathogenic intronic ABCA4 variants has been reported, usually causing splicing alteration at the pre-mRNA level. Antisense oligonucleotides (AONs) are an attractive therapeutic strategy to rescue these splicing defects, yet they are designed to target individual variants. In this study, we experimentally analyze the potential of combined AON-U7snRNA splicing modulation therapy to target multiple intronic ABCA4 variants using midigene-based splice assays in vitro.

Methods : HEK293T cells were transfected with wild-type or mutant ABCA4 midigenes harboring the genomic region where selected intronic variants are located. Twenty-four hours post-transfection, cells were left non-treated (NT) or transfected with single or multiple AON-U7snRNA cassette vectors, including the respective positive (naked AON, 0.5 µM) or negative (single/multiple empty U7snRNA cassette vectors) controls. RNA was isolated after 48 hours and transcripts were detected through RT-PCR (n=2). Levels of correct/aberrant splicing were measured by transcript band semi-quantification of each condition, using one-way ANOVA as statistical analysis.

Results : For an intron 30 variant, aberrant splicing was decreased from 97.95% (SD: 2.9) in NT to 26.17% (SD: 17.44) when transfected with the multiple-cassette vector (p<0.0001), whereas its respective single-cassette vector reduced aberrant levels to 17.17% (SD: 10.71 , p<0.0001) and naked AON achieved full rescue (0%, SD: 0, p<0.0001). In case of an intron 36 variant, aberrant splicing decrease was observed when delivering the naked AON (0%, SD: 0, p=0.0106), single (0.51%, SD: 0.72, p=0.0131) or multiple-cassette vector (0.87%, SD: 1.23, p=0.0152) compared to NT (16.61%, SD: 2.05). No relevant effect was observed after delivering empty controls or single-cassette vectors targeting a different region.

Conclusions : These splice assays showed significant reduction of aberrant ABCA4 transcripts through multiple AON-U7snRNA cassette delivery in vitro, illustrating the therapeutic potential of the system to correct different splicing defects caused by several intronic variants simultaneously. To corroborate the observed results, delivery of the U7snRNA system through AAV in patient-derived photoreceptor cells and retinal organoids is currently ongoing.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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