Advancements in pre-clinical development of gene editing-based therapies to treat inherited retinal diseases

Abstract

One of the major goals in the inherited retinal disease (IRD) field is to develop an effective therapy that can be applied to as many patients as possible. Significant progress has already been made toward this end, with gene editing at the forefront. The advancement of gene editing-based tools has been a recent focus of many research groups around the world. Here, we provide an update on the status of CRISPR/Cas-derived gene editors, promising options for delivery of these editing systems to the retina, and animal models that aid in pre-clinical testing of new IRD therapeutics.

Figure 1.

Benefits and limitations of major gene editor types and delivery methods.

Figure 2.

Schematic of lipid nanoparticle (LNP) delivery of EGFP mRNA and AAV delivery of EGFP cDNA to human retinal organoids, cone-rich species, and nonhuman primates. A schematic of EGFP expression is shown in photoreceptor cells and RPE both in vitro and in vivo.

Figure 3.

Allele-specific editing in hiPSCs harboring a dominant mutation in the CRX gene (from (Chirco et al., 2021)). The CRISPR/Cas9 dual-cutting target sites are mapped onto the mutant allele of the CRX gene (A). PCR was performed using primers shown in (A; purple triangles), revealing an additional 374-bp band representing the edited “knockout (KO) allele” after CRISPR/Cas9 editing (B). Sanger sequencing was also utilized to confirm loss of the K88Q mutation after CRISPR-mediated editing (C). Immunofluorescence staining using antibodies against SAG (green; D-I′), RCVRN (red; J-O′), and ARR3 (green; P-U′) are shown for control (CRX^WT; D-E′, J-K′, and P-Q′), CRX^K88Q/+ (F-G′, L-M′, and R-S′), and CRX^+/− (H-I′, N-O′, and T-U′) retinal organoids at D180 (n=3 organoids per line). Nuclei are counterstained with DAPI (blue). Scale bars (U and U′), 100μm. OPL, outer plexiform layer; INL/GCL, inner nuclear layer/ganglion cell layer.