Prime Editing for Inherited Retinal Diseases

Abstract

Inherited retinal diseases (IRDs) are chronic, hereditary disorders that lead to progressive degeneration of the retina. Disease etiology originates from a genetic mutation-inherited or de novo-with a majority of IRDs resulting from point mutations. Given the plethora of IRDs, to date, mutations that cause these dystrophies have been found in approximately 280 genes. However, there is currently only one FDA-approved gene augmentation therapy, Luxturna (voretigene neparvovec-rzyl), available to patients with RPE65-mediated retinitis pigmentosa (RP). Although clinical trials for other genes are underway, these techniques typically involve gene augmentation rather than genome surgery. While gene augmentation therapy delivers a healthy copy of DNA to the cells of the retina, genome surgery uses clustered regularly interspaced short palindromic repeats (CRISPR)-based technology to correct a specific genetic mutation within the endogenous genome sequence. A new technique known as prime editing (PE) applies a CRISPR-based technology that possesses the potential to correct all twelve possible transition and transversion mutations as well as small insertions and deletions. EDIT-101, a CRISPR-based therapy that is currently in clinical trials, uses double-strand breaks and nonhomologous end joining to remove the IVS26 mutation in the CEP290 gene. Preferably, PE does not cause double-strand breaks nor does it require any donor DNA repair template, highlighting its unparalleled efficiency. Instead, PE uses reverse transcriptase and Cas9 nickase to repair mutations in the genome. While this technique is still developing, with several challenges yet to be addressed, it offers promising implications for the future of IRD treatment.

Keywords: CRISPR/Cas9 systems; Ophthalmology; adeno-associated viral (AAV) vectors; gene editing; inherited retinal diseases (IRD); prime editing; retinal degeneration.

FIGURE 1

Overview of prime editing (PE). (A) Illustrations of all 12 kinds of DNA substitutions. (B) The machinery of PE. From the 5′ to the 3′ end, the pegRNA contains the spacer, gRNA scaffold, reverse transcription template (RTT), and primer binding sequence (PBS). “Created with BioRender”.

FIGURE 2

Overview of prime editing mechanism. The spacer (red line) anneals with its complementary strand of the DNA (1) directing the H840A SpCas9 nickase to nick the PAM-containing strand (black arrow) of the target DNA (2). The primer binding sequence (PBS) then hybridizes with the nicked DNA (3) initiating the elongation of the free 3′ end according to the reverse transcription template (RTT) sequence that carries the intended edit (4). The newly synthesized strand leads to either 3′ or 5′ flap excision. The excision of the 5′ flap is favored, and it leads to the heteroduplex formation (5). The replacement of the original sequence via endogenous DNA mismatch repair mechanism incorporates the desired mutation at the target site (6). “Created with BioRender”.