July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Antisense oligonucleotide-based correction of deep-intronic ABCA4 splice mutations using patient-derived fibroblasts and photoreceptor precursor cells
Author Affiliations & Notes
  • Sarah Naessens
    Ghent University, Ghent, Belgium
  • Rob W J Collin
    Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands
  • Frauke Coppieters
    Ghent University, Ghent, Belgium
  • Miriam Bauwens
    Ghent University, Ghent, Belgium
  • Lonneke Duijkers
    Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands
  • Irina Balikova
    Ghent University, Ghent, Belgium
    Department of ophthalmology, Ghent University Hospital, Ghent, Belgium
  • Bart P Leroy
    Ghent University, Ghent, Belgium
    Department of ophthalmology, Ghent University Hospital, Ghent, Belgium
  • Elfride De Baere
    Ghent University, Ghent, Belgium
  • Alex Garanto
    Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands
  • Footnotes
    Commercial Relationships   Sarah Naessens, None; Rob Collin, None; Frauke Coppieters, None; Miriam Bauwens, None; Lonneke Duijkers, None; Irina Balikova, None; Bart Leroy, None; Elfride De Baere, None; Alex Garanto, None
  • Footnotes
    Support  FWO - Fund for Scientific Research Flanders
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4950. doi:https://doi.org/
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      Sarah Naessens, Rob W J Collin, Frauke Coppieters, Miriam Bauwens, Lonneke Duijkers, Irina Balikova, Bart P Leroy, Elfride De Baere, Alex Garanto; Antisense oligonucleotide-based correction of deep-intronic ABCA4 splice mutations using patient-derived fibroblasts and photoreceptor precursor cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4950. doi: https://doi.org/.

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

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Abstract

Purpose : Stargardt disease (STGD1) is one of the most common inherited retinal diseases. It can be caused by over 600 different coding mutations in the ABCA4 gene, explaining 70% of the cases. We and others reported non-coding, deep-intronic mutations in ABCA4 causing STGD1. Two of these (c.4539+1100A>G & c.4539+1106C>T) are neighboring mutations that create the same cryptic splice donor site, leading to inclusion of a 68- or 112-bp pseudo-exon and subsequent premature termination of ABCA4 protein synthesis. Here, we aimed to correct aberrant splicing caused by these mutations with antisense oligonucleotides (AONs), using patient-derived fibroblasts and photoreceptor precursor cells (PPCs).

Methods : We designed five AONs, targeting different regions of the cryptic donor site. Subsequently, these were transfected in control and patient-derived (c.4539+1106C>T) fibroblasts, and subjected to RNA isolation and RT-PCR analysis. Furthermore, we generated induced pluripotent stem cells (iPSCs), using the CytoTune iPS 2.0 Sendai Reprogramming Kit (Newcells Biotech), to obtain PPCs using a 2D-differentiation protocol. The two most potent AONs were selected for rescue experiments at the RNA and protein level in iPSC-derived PPCs.

Results : Two out of five AONs lead to a total or partial rescue of normal splicing in patient-derived fibroblasts (c.4539+1106C>T), in a dose-dependent manner. A sense oligonucleotide and a mutation-specific AON for c.4539+1100A>G were used as controls and did not show any restoration of normal splicing. This confirms the specific effect of the AONs and indicates that one mismatch is enough to abolish AON efficacy. Both AONs lead to a partial rescue of normal splicing in PPCs. Furthermore, mutation specific AONs for the c.4539+1100A>G mutation are currently being tested.

Conclusions : Overall, we describe the ability of AONs to correct aberrant splicing resulting from deep-intronic mutations in ABCA4. Furthermore, we show the importance of iPSC differentiation into PPCs, as this cell type is otherwise not accessible. This offers important therapeutic options for inherited retinal diseases leading to vision impairment and blindness.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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