Precision genome editing in the eye

Abstract

CRISPR-Cas-based genome editing technologies could, in principle, be used to treat a wide variety of inherited diseases, including genetic disorders of vision. Programmable CRISPR-Cas nucleases are effective tools for gene disruption, but they are poorly suited for precisely correcting pathogenic mutations in most therapeutic settings. Recently developed precision genome editing agents, including base editors and prime editors, have enabled precise gene correction and disease rescue in multiple preclinical models of genetic disorders. Additionally, new delivery technologies that transiently deliver precision genome editing agents in vivo offer minimized off-target editing and improved safety profiles. These improvements to precision genome editing and delivery technologies are expected to revolutionize the treatment of genetic disorders of vision and other diseases. In this Perspective, we describe current preclinical and clinical genome editing approaches for treating inherited retinal degenerative diseases, and we discuss important considerations that should be addressed as these approaches are translated into clinical practice.

Keywords: eye; genome editing; retina; retinal degeneration.

Fig. 1.

Conventional gene augmentation therapy and CRISPR-based genome editing approaches. (A) Gene augmentation therapy involves an AAV that carries and delivers a copy of the normal RPE65 cDNA to the RPE. (B) CRISPR-Cas-nuclease-mediated genome editing generates DSBs that are repaired by either NHEJ or HDR. NHEJ is the primary pathway for DSB repair throughout the cell cycle. Uncontrolled nucleotide insertions or deletions often occur as a result of NHEJ, whereas desired nucleotide changes specific to a donor DNA repair template are achieved via HDR. (C) BEs install targeted single-nucleotide conversions using either cytidine deaminase (CBE) or adenosine deaminase (ABE) domains tethered to a Cas9 nickase with an sgRNA. CBEs perform targeted C•G-to-T•A conversions, and ABEs perform targeted A•T-to-G•C conversions. (D) PEs utilize a reverse transcriptase tethered to a Cas9 nickase to write new DNA sequences into the target locus. Use of the Cas9 nickase avoids the formation of a DSB. A pegRNA, an extended sgRNA that contains the template sequence for reverse transcription, is utilized for nucleotide synthesis at the target locus. Red triangle indicates the site of DNA strand break.

Fig. 2.

Genes implicated in IRDs. Mutations in the genes predominantly expressed in the RPE, photoreceptors, bipolar cells, or retinal ganglion cells cause retinal degeneration. Depending on the type and site of mutation, selection of an appropriate genome editing agent and delivery vehicle can be optimized to target these genes efficiently.

Fig. 3.

Schematic representation of a genome editing approach for precision medicine. Personalized genome editing therapy would begin with the identification of a patient’s pathogenic mutation, followed by a computational prescreening process to generate a candidate library for experimental validation using a patient-derived cell line. The final custom-designed editing agent could be delivered to the patient as nucleic acid, mRNA, or RNP by choosing from various vehicles including AAV, lentivirus, engineered virus-like particles, lipid nanoparticles, gold nanoparticles, or others.