Abstract
Purpose :
Previous studies have shown that deletion or skipping of a mutant exon 13 to restore the reading frame of the USH2A gene could be an effective therapy for preventing the vision loss in animal models and clinical trial. Besides exon 13, there are 24 additional exons that are in-frame. Here we expanded our study in exon 13 to other in-frame exons to determine which exons are dispensable without losing their biological functions.
Methods :
We selected 6 in-frame human exons (exon 16, 18, 21, 38, 43, 45) as targets and 2 out-of-frame exons (exon 6 and 20) as controls. Dual sgRNAs targeting the flanking introns of the exon-of-interest were synthesized and co-injected with Cas9 protein into the mouse zygote to delete the targeted exons and generate the exon-deleted Ush2a mouse models. The genotype was validated by Sanger sequencing. The mRNA and protein products were assessed by qRT-PCR and immunohistochemistry. These mouse lines were crossed with Ush2a knockout mice to evaluate whether these exon-deleted usherin retain functions in retina and cochlea.
Results :
We successfully established all 8 exon-deleted Ush2a mouse models with up to 70% positive rate in F0 founders. Germline transmission was confirmed in F1 mice of all lines. Sequencing confirmed direct ligation of flanking introns in DNA and precise splicing of neighboring exons in mRNA. Usherin expression was detected in all 6 in-frame exon-deleted homozygous mice and localized correctly in the connecting cilia region of photoreceptors as that in wild type mice. No usherin was detected in out-of-frame control exon 5 and 19-deleted Ush2a mice, consistent with that of knockout mice.
Conclusions :
We confirmed that deletion of the 6 in-frame exons did not disrupt the correct splicing of the USH2A transcripts and lead to the expression of a shortened usherin protein in the mouse retina, indicating that these exons could be dispensable. Once the functional integrity of these exon-deleted usherin is validated in the retina and cochlea, CRISPR/Cas9-based or antisense oligonucleotides-mediated exon-deletion or exon-skipping therapies could be developed to treat USH2A patients with premature stop codon mutations in these 6 in-frame exons. Moreover, these approaches could be expanded to other exons of USH2A or other Usher syndrome genes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.