CRISPR-mediated optogene expression from a cell-specific endogenous promoter in retinal ON-bipolar cells to restore vision

Abstract

Retinitis pigmentosa, an inherited form of retinal degeneration, is characterized by a progressive loss of rods and subsequent degeneration of cones, leading to blindness. However, the remaining neural portion of the retina (bipolar and ganglion cells) remains anatomically and functionally intact for an extended time. A possible treatment to restore the light sensitivity of the retina consists of rendering the remaining retinal cells photosensitive using optogenetic tools like, for example, Opto-mGluR6, a light-sensitive mGluR6 receptor. We have previously demonstrated that AAV vector-mediated expression of Opto-mGluR6 in ON-bipolar cells restores visual function in otherwise blind mice. However, classical gene supplementation therapy still suffers from high off-target expression rates and uncontrollable target gene expression levels that may lead to either cytotoxicity or lack of functional restoration. To address these issues and achieve cell-specific and endogenously controlled Opto-mGluR6 expression, we employed the CRISPR/Cas technology—in particular, homology-independent targeted integration (HITI) and microhomology-dependent targeted integration (MITI)—to knock-in the Opto-mGluR6 gene behind the ON-bipolar cell-specific GRM6 promoter. We compared four Cas systems in vitro and show that SpCas9 for HITI and LbCpf1 for MITI are well suited to promoting knock-in. As AAV2-mediated ON-bipolar cell transduction resulted in inefficiency, we evaluated Exo-AAVs as delivery vehicles and found Exo-AAV1 efficient for targeting ON-bipolar cells. We demonstrate that intravitreal injection of Exo-AAV1 carrying vectors that promote MITI significantly improved visual acuity in otherwise blind rd1 mice. We conclude by confirming and providing a qualitative evaluation of the MITI-mediated knock-in in the correct genomic locus.

Retinal Ciliopathies and Potential Gene Therapies: A Focus on Human iPSC-Derived Organoid Models

“…This strategy’s effectiveness was demonstrated in a retinitis pigmentosa rat model with a deletion in the mertk gene, and a significant phenotypic rescue was observed with MERTK protein expression in retinal pigment epithelium restored along with visual function. Moreover, the HITI technique was also recently used to knock-in the optogene Opto-mGluR6 into the ON-bipolar cells of rd1 mice, restoring light-sensitivity to the retina [ 162 ]. Another notable application of HITI was the treatment of an autosomal dominant RHO mutation.…”