AAV-CRISPR/Cas9-Mediated Gene Rescue in USH2A Knock-out hiPSC-Derived Retinal Organoids

Abstract

Purpose : Mutations in the human USH2A gene have been linked to the pathogenesis of Usher Syndrome type II, characterized by progressive visual, vestibular, and auditory impairment. Here, we generated USH2A knock-out (KO) human induced pluripotent stem cells (hiPSCs)-derived retinal organoids as an in vitro model to investigate adeno-associated viral (AAV) vector-mediated gene rescue. The DNA knock-in in the mutant organoids exploits the Homology-Independent Targeted Integration (HITI) gene editing strategy.

Methods : Homozygous nonsense mutations have been introduced in USH2A mutational hotspots, exons 13 and 61, by CRISPR/SpCas9. These USH2A KO hiPSC clones were validated by sequencing, checked for karyotypic stability and absence of copy number variations, and then differentiated into retinal organoids along with isogenic controls. After 225 days, they were collected for single-cell RNA sequencing (scRNAseq), transmission electron microscopy, and immunohistochemistry to examine any phenotypic variations at the photoreceptor and cilial level. We designed an AAV construct to rescue the expression of Usherin in the retinal organoids with USH2A KO at exon 13 via inserting an USH2A cDNA spanning exons 2 to 17 into the intron 17 of USH2A. Similarly, the retinal organoids with USH2A KO at exon 61 will be transduced with an AAV incorporating the USH2A cDNA exons 60 to 72 into the intron 59 of USH2A. The insertion efficiency is first being tested in HeLa cells via junction PCR and immunocytochemistry.

Results : The disruption of the USH2A reading frame is expected to result in a non-functional protein, whose expression would be restored by the AAV-mediated HITI strategy. Analysis is ongoing to assess if the KO of USH2A has a morphological impact on the photoreceptors, and specifically on the cilium, where Usherin localizes. Moreover, scRNAseq data will be applied to identify differentially expressed genes and affected pathways in the USH2A mutant organoids compared to the controls. Transfection of HeLa cells with the dual AAV system is being performed to get preliminary insight on the HITI strategy efficiency.

Conclusions : We successfully generated USH2A KO hiPSC-derived retinal organoids. The HITI-mediated AAV-based gene editing approach will rescue full length-Usherin expression in mutant organoids, offering a promising therapeutic strategy for 70% of Usher Syndrome type II patients carrying mutations in the USH2A gene.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.