Abstract
Purpose :
Usher syndrome type 2A (USH2A) is a hereditary disorder characterized by a combination of sensorineural hearing loss and retinitis pigmentosa. Various types of mutations in the USH2A gene have been reported, contributing to the observed clinical heterogeneity among affected individuals. Effective therapeutic interventions directly targeting USH2A mutations remain elusive. The replacement of the mutation-containing 3'-prime part of the USH2A gene by a wild-type cDNA repair template via the Microhomology Mediated End Joining (MMEJ) DNA repair pathway following a Cas9-induced double-strand break represents a potential treatment option for various disease-causing mutations.
Methods :
Four different Cas9 target sites, located in USH2A exons 60-63, were characterized in an episomal BRET (Bioluminescence Resonance Energy Transfer)-based assay and verified using TIDE (Tracking of Indels by Decomposition) at the desired genomic locus. Wild-type cDNA repair templates containing varying microhomologous sequence (MHS) lengths (5, 10, 20 bp) and codon-optimized 5'-ends to prevent re-cleavage, were cloned and purified for transfection into human iPSCs (induced pluripotent stem cells) alongside corresponding Cas9/gRNA constructs. Subsequently, we analyzed MMEJ-based cDNA repair template integration using TIDER (TIDE template-directed editing) and next-generation amplicon sequencing.
Results :
Promising Cas9 cleavage sites in USH2A exon 60-63 were identified, inducing on-target frameshifting mutations of up to 63.0% in exon 61 and ranging between 15% to 35% in the remaining loci tested. TIDE, which assays frameshift mutations on a genomic level, validated the initial results, albeit at a lower rate due to higher background levels but with a similar ratio. Moreover, we achieved correct in-frame integration for the 20 bp MHS repair template based on MMEJ, albeit at very low frequencies.
Conclusions :
The results presented in this study lay the groundwork for an MMEJ-based strategy aimed at integrating a larger USH2A cDNA repair template to replace mutated regions. However, the integration efficiency remains low with the tested construct but holds potential for enhancement by utilizing a shorter MHS length alongside MMEJ repair pathway engineering targeting different repair pathway enzymes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.