Vision rescue via unconstrained in vivo prime editing in degenerating neural retinas

Abstract

Retinitis pigmentosa (RP) is an inherited retinal dystrophy causing progressive and irreversible loss of retinal photoreceptors. Here, we developed a genome-editing tool characterized by the versatility of prime editors (PEs) and unconstrained PAM requirement of a SpCas9 variant (SpRY), referred to as PESpRY. The diseased retinas of Pde6b-associated RP mouse model were transduced via a dual AAV system packaging PESpRY for the in vivo genome editing through a non-NGG PAM (GTG). The progressing cell loss was reversed once the mutation was corrected, leading to substantial rescue of photoreceptors and production of functional PDE6β. The treated mice exhibited significant responses in electroretinogram and displayed good performance in both passive and active avoidance tests. Moreover, they presented an apparent improvement in visual stimuli-driven optomotor responses and efficiently completed visually guided water-maze tasks. Together, our study provides convincing evidence for the prevention of vision loss caused by RP-associated gene mutations via unconstrained in vivo prime editing in the degenerating retinas.

Eukaryotic-driven directed evolution of Cas9 nucleases

“…This variability in editing activity is not uncommon for enhanced Cas nucleases [ 15 , 47 , 48 ] and could be ascribed to modified preferences for target sequence composition, which is known to affect Cas activity [ 49 ]. Finally, the reduced molecular size of UltraCjCas9 is compatible with the packaging capacity of AAV vectors, the most commonly used delivery method for genome editing [ 50 , 51 ], consenting its delivery all-in-one with the sgRNA, as opposed to double AAVs for sgRNA and Cas9 for in vivo use [ 52 – 55 ].…”

Breaking genetic shackles: The advance of base editing in genetic disorder treatment

“…Initially manifesting as night blindness, RP gradually progresses to a more severe loss of vision, ultimately resulting in total blindness ( Hartong et al, 2006 ). To date, over 100 genes have been identified in various subtypes of RP with different genetic patterns, including the rhodopsin gene ( RHO ), the pre-mRNA processing factor 31 gene ( PRPF31 ), and the peripherin 2 gene ( PRPH2 ), among others ( Bhardwaj et al, 2022 ; Qin et al, 2023b ). Although Luxturna offers a treatment option for a minority of RP patients carrying RPE65 mutations, the genetic diversity of RP means that a universal cure for all patients is currently lacking ( Cross et al, 2022 ).…”

Eye on genome editing

“…Since its development as a platform for gene editing, a number of animal models of human diseases have demonstrated the utility of the CRISPR/Cas9 gene editing system. Using a genome editor (PE SpRY ), Qin et al (2023) corrected a pathogenic mutation in Pde6b , restoring retinal function and morphology. The authors report the first successful retinal photoreceptor prime editing of a clinically relevant gene ( Qin et al, 2023 ).…”

“…Using a genome editor (PE SpRY ), Qin et al (2023) corrected a pathogenic mutation in Pde6b , restoring retinal function and morphology. The authors report the first successful retinal photoreceptor prime editing of a clinically relevant gene ( Qin et al, 2023 ). The first human clinical trials have begun to report the results of both ex vivo and in vivo gene editing ( Gillmore et al, 2021 ).…”

“…To address these issues, Qin et al (2023) combined the prime editor effector domain (Moloney murine leukemia virus reverse transcriptase; Qin et al, 2023 ) with a recently described engineered Cas9 variant from Streptococcus pyogenes (SpRY; see Fig. 1; Walton et al, 2020 ).…”