June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Antisense oligonucleotide-based correction of the splicing defect caused by the c.769-784C>T variant in ABCA4.
Author Affiliations & Notes
  • Tomasz Zbigniew Tomkiewicz
    Department of Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Radboud Universiteit Donders Institute for Brain Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Sara Nieuwenhuis
    Department of Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Frans P Cremers
    Department of Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Radboud Universiteit Donders Institute for Brain Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Alejandro Garanto
    Department of Pediatrics and Human Genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Radboud Universiteit Radboud Institute for Molecular Life Sciences, 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   Tomasz Tomkiewicz None; Sara Nieuwenhuis None; Frans Cremers P6063546, Code P (Patent); Alejandro Garanto P6063546, Code P (Patent); Rob Collin ASTHERNA, Code O (Owner), P6063546, Code P (Patent)
  • Footnotes
    Support  Retina UK Foundation grant no. GR596 and UitZicht
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 71 – A0044. doi:
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    • Get Citation

      Tomasz Zbigniew Tomkiewicz, Sara Nieuwenhuis, Frans P Cremers, Alejandro Garanto, Rob WJ Collin; Antisense oligonucleotide-based correction of the splicing defect caused by the c.769-784C>T variant in ABCA4.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):71 – A0044.

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

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Abstract

Purpose : Stargardt disease is an inherited retinal disease caused by mutations in the photoreceptor-specific gene ATP-binding cassette transporter type A (ABCA4). Interestingly ABCA4 has a high occurrence of splicing-affecting mutations. Here, we screened 9 antisense oligonucleotides (AONs) designed to correct pseudoexon (PE) inclusion due to a recurrent deep-intronic variant (c.769-784C>T) in ABCA4.

Methods : AON efficacy to induce PE exclusion in the ABCA4 transcript was assessed in three cell models carrying the c.769-784C>T variant. First, AONs were tested at a final concentration of 0.5 µM in HEK293T cells transfected with an ABCA4 midigene (splice reporter vector) carrying the deep-intronic change. Next, AONs were tested in patient-derived fibroblasts at the same final concentration. Based on the splicing-correction efficacy of each individual AON, the three most efficacious AONs were selected for a final assessment in photoreceptor-progenitor cells (PPC). Selected AONs were delivered at a final concentration of 0.5 µM and 1 µM. One-way ANOVA test with subsequent Bonferroni correction was used for statistical analysis.

Results : The AON screening using the HEK293T-midigene model indicated high efficacy of all AONs. Given these results, the same pool of AONs was evaluated in the patient-derived fibroblasts. Based on PE correction AON2, AON7 and AON5 were selected for further assessment in the patient-derived PPC model. AON2-mediated splicing correction was the most efficacious at the lower concentration (0.5 µM, p<0.001), followed by AON7 (p<0.001). AON5 was least efficacious reaching statistical significance at final concentration 1 µM only (p=0.03).

Conclusions : PE exclusion is a thoroughly researched therapeutic approach that has been shown to rescue protein function. It can be achieved using AONs, however it is crucial to select the most efficacious therapeutic molecule for further testing by employing different models. Taking this into account, AON2 and AON7 showed a high degree of PE skipping in PPCs, which provide a retina-like cellular context. Further research of AON2 and AON7 includes protein rescue studies and safety profile assessment in advanced retina models to mimic the cellular environment in patients.

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

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