Cas9 and AAV Vector-Based Strategies for X-linked Juvenile Retinoschisis (XLRS) Therapy

Abstract

Purpose : In order to model potential XLRS CRISPR therapeutics strategies in vivo,we explore an approach for the use of Cas9 in combination with adeno-associated virus (AAV) to rescue the non-secreting R141C variant of Rs1in mice.

Methods : A mouse line with constitutive expression of Cas9 protein (Rosa^Cas9/+) was crossed to mice containing a mutant copy of murine Retinoschisin (Rs1; Rs1^R141C/+)to generateRosa^Cas9/+, Rs1^R141C/Ystudy mice.Three AAV vectors were designed to test approaches to optimize gene insertion. All three utilized a guide targeting Rs1 intron 1, with different arrangements of a cDNA template to introduce human RS1 (exon 2-6), followed by a polyA to prevent transcription of mutant mouse Rs1. The viral vectors were delivered to Rosa^Cas9/+;Rs1^R141C/Ymouse eyesat postnatal day 21 bysub-retinal injection (right eye of each mouse, and the left eye was not injected as a control). At two months post injection, optical coherence tomography (OCT) and electroretinography (ERG) were conducted to evaluate the retinal structural and functional rescue, respectively, followed by retinal tissue harvest for detection of gene insertion by non-homologous end joining (NHEJ) characterization at the mouse Rs1 intron 1 locus and next-generation sequencing over the mutant region.

Results : Most of the injected eyes showed improved retinal organization to a varying extent in OCT imaging two months post sub-retinal delivery. All three viral vectors provided retinal structural protection by minimizing the inner retinal cavities significantly comparing to the control eyes, ERG data showed minimum but no significant gain of function though. Sequencing of Rs1 mRNA confirmed a mixed population of mutant and hybrid transcripts in study mice.
Basically, all mice showed varying levels of all modified transcripts for encoding hybrid mouse human proteinand varying but much lower levels of mutant mouse transcript.

Conclusions : All current three viral vectors are able to mediate gene insertion at the mouse Rs1 locus in the Cas9 mouse background and provide partial phenotypic protection.CRISPR Cas9 and AAV vector-based strategies show promise for XLRS gene therapy. Repeated studies with modified next generation vectors and optimized validation assays are underway.

This is a 2020 ARVO Annual Meeting abstract.