Genome-Editing Strategies for Treating Human Retinal Degenerations

Abstract

Inherited retinal degenerations (IRDs) are a leading cause of blindness. Although gene-supplementation therapies have been developed, they are only available for a small proportion of recessive IRD mutations. In contrast, genome editing using clustered-regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated (Cas) systems could provide alternative therapeutic avenues for treating a wide range of genetic retinal diseases through targeted knockdown or correction of mutant alleles. Progress in this rapidly evolving field has been highlighted by recent Food and Drug Administration clinical trial approval for EDIT-101 (Editas Medicine, Inc., Cambridge, MA), which has demonstrated efficacious genome editing in a mouse model of CEP290-associated Leber congenital amaurosis and safety in nonhuman primates. Nonetheless, there remains a significant number of challenges to developing clinically viable retinal genome-editing therapies. In particular, IRD-causing mutations occur in more than 200 known genes, with considerable heterogeneity in mutation type and position within each gene. Additionally, there are remaining safety concerns over long-term expression of Cas9 in vivo. This review highlights (i) the technological advances in gene-editing technology, (ii) major safety concerns associated with retinal genome editing, and (iii) potential strategies for overcoming these challenges to develop clinical therapies.

Keywords: CRISPR; gene editing; inherited retinal degenerations.

Figure 1.

Therapeutic CRISPR-based genome-editing strategies. (A) CRISPR-Cas9-induced double-strand breaks. In eukaryotic cells, breaks are repaired through either the error-prone non-homologous end-joining pathway, leading to indel mutations, or the high-fidelity homology-directed repair pathway, which may be leveraged to introduce precise edits in dividing cells. Black triangles indicate Cas9 cut sites. (B) DNA editing with Base Editors. Base editors consist of a Cas9 nickase fused to a DNA deaminase and use an sgRNA to locate a target sequence, where the deaminase either converts C to T, via U (cytosine base editors) or A to G, via I (adenine base editors). (C) DNA editing with Prime Editors. Prime Editors use a Cas9 nickase fused to a reverse transcriptase, with a pegRNA. The nicked DNA strand base pairs with the 3′ end of the pegRNA, which templates the desired DNA edit to be introduced by reverse transcription. (D) CRISPRi and CRISPRa. A catalytically dCas9 is guided by a sgRNA to locate a regulatory region of a gene of interest. CRISPRi involves either steric exclusion of RNAP by the dCas9 (left) or fusion of a transcriptional repressor (center) to reduce gene expression, whereas CRISPRa fuses a transcriptional activator to dCas9 to upregulate gene expression (right). CRISPR, clustered-regularly interspaced short palindromic repeats; Cas, CRISPR-associated; CRISPRi, CRISPR-interference; CRISPRa, CRISPR-activation pegRNA, prime editing guide RNA; RNAP, RNA polymerase; dCas9, dead Cas9; sgRNA, single guide RNA. Color images are available online.

Figure 2.

Strategies to mitigate the risks of off-target editing. (A) Delivery of Cas9-sgRNA RNPs. RNPs can be packaged into synthetic gold or lipid nanoparticles for delivery by injection to the subretinal space. Nanoparticles that are taken up by retinal cells release the RNPs to perform gene editing until they are naturally degraded, providing transient activity to minimise off-target editing. (B) Temporal control of gene editing with small-molecule drugs. Narrowing the time window for gene editor activity can significantly reduce off-target editing and may be achieved with small-molecule-inducible systems. Successfully implemented systems include: (I) inducible expression of editing components through drug-sensitive repressors; (II) inducible stabilization of Cas9 by insertion of a ligand-binding domain into Cas9; and (III) inducible complex assembly by insertion of a drug-responsive intein domain. (C) RNA editing with REPAIR/RESCUE. A catalytically dCas13b is fused to the RNA deaminase domain from ADAR (ADAR_DD). The fusion utilizes a gRNA to locate the target RNA sequence and allow the deaminase to convert a pathogenic point mutation (shown in red) from A to I or C to U in the case of REPAIR and RESCUE, respectively. Incorporating a mismatched C or U into the desired position of the guide sequence (shown in light blue) improves the specificity of editing. dCas13b, dead Cas13b; RNP, ribonucleoprotein. Color images are available online.

Figure 3.

Pathway for developing retinal gene-editing therapies.