Effective splicing restoration of a deep-intronic ABCA4 variant in cone photoreceptor precursor cells by CRISPR/SpCas9 approaches

• Published in: Molecular therapy. Nucleic acids Vol. 29; pp. 511 - 524
• Main Authors: De Angeli, Pietro, Reuter, Peggy, Hauser, Stefan, Schöls, Ludger, Stingl, Katarina, Wissinger, Bernd, Kohl, Susanne
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 13.09.2022; American Society of Gene & Cell Therapy; Elsevier
• Subjects: ABCA4; CRISPR-Cas9; deep-intronic variants; genome editing; inherited retinal dystrophy; MT: RNA/DNA editing; Original; Photoreceptor precursor cells; splicing; Stargardt disease; STGD1; genome editing; deep-intronic variants; inherited retinal dystrophy; CRISPR-Cas9; ABCA4; Photoreceptor precursor cells; MT: RNA/DNA editing; Stargardt disease; STGD1; splicing
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2022.07.023

Stargardt disease is an autosomal recessively inherited retinal disorder commonly caused by pathogenic variants in the ABCA4 gene encoding the ATP-binding cassette subfamily A member 4 (ABCA4) protein. Several deep-intronic variants in ABCA4 have been classified as disease causing. By strengthening a cryptic splice site, deep-intronic variant c.5197-557G>T induces the inclusion of a 188-bp intronic sequence in the mature mRNA, resulting in a premature termination codon. Here, we report the design and evaluation of three CRISPR-Cas9 approaches implementing Streptococcus pyogenes Cas9 (single and dual guide RNA) or Streptococcus pyogenes Cas9 nickase (dual guide RNA) for their potential to correct c.5197-557G>T-induced aberrant splicing in minigene splicing assays and patient-derived cone photoreceptor precursor cells. The different strategies were able to rescue correct splicing by up to 83% and increase the overall correctly spliced transcripts by 1.8-fold, demonstrating the successful CRISPR-Cas9-mediated rescue in patient-derived photoreceptor precursor cells of an ABCA4 splicing defect. The results provide initial evidence of possible permanent splicing correction for Stargardt disease, expanding the therapeutic toolbox to counteract deep-intronic pathogenic variants in ABCA4. [Display omitted] A mutation localized in the intronic part of ABCA4 able to affect the correct reading frame of the gene was targeted by three CRISPR-Cas9-based approaches. Rescue of the correct reading frame was achieved in mutant cone photoreceptor precursor cells, while no detrimental secondary effect was observed in control cells.