Abstract
Purpose :
Retinitis pigmentosa (RP) is a group of inherited retinal diseases that present with progressive loss of photoreceptor cells, leading to blindness. Pde6a, which encodes a cGMP-specific phosphodiesterase, is a pathogenic gene for autosomal recessive retinitis pigmentosa (RP43), and currently, there are no effective therapy methods available. The compact CRISPR/SaCas9 system is widely recognized and preferred for gene therapy delivered through a single AAV. Here, we aim to investigate whether all-in-one AAV-SaCas9-mediated editing of the Nrl gene, a transcription factor controls the differentiation of rod photoreceptor cells, can rescue retinal degeneration in a RP mouse model carrying the Pde6anmf363/nmf363 mutation.
Methods :
The compact SaCas9 with optimal RNA (Nrl-sg2) was packaged into a single AAV using the AAV2.NN serotype. AAV-SaCas9-Nrl-sg2 was delivered to Pde6a mice by subretinal injection at postnatal day 7 (P7). The retinal DNA was extracted for deep sequencing at P60. Retinas from P60 mice were frozen-sectioned and immunostained using antibodies against HA tag, Rhodopsin, and Cone arrestin. The retinal functions were assessed by electroretinography (ERG) and optokinetic tracking response (OKR) at P60.
Results :
AAV-SaCas9-Nrl-sg2 treated mice exhibited effective Nrl editing in the retina, with efficiencies ranging from 15.3% to 57.2% (n=4). Immunofluorescence for HA tag confirmed the successful delivery and expression of SaCas9 to retinal photoreceptors. In comparison to the relatively weak signals of Rhodopsin and Cone arrestin in Pde6a untreated retinas, phototransduction cascade proteins are appropriately and effectively localized in the retinas treated with Nrl-sg2. The quantitative assay of retinal sections revealed that the ONL thickness in the Nrl-sg2 group measured 23.1 ± 2.6 μm, representing a 6.7-fold increase compared to the control group. The Nrl-sg2 group exhibited significantly improved photopic ERG b-wave (p < 0.05, Student t-test, n=3), supporting preserved cone function. Furthermore, the Nrl-sg2 group exhibited higher visual acuity compared to the control group by OKR test (p < 0.05, Student t-test, n=3).
Conclusions :
In summary, we utilized the state-of-the-art SaCas9 system that induced efficient Nrl editing in vivo by all-in-one AAV delivery. The Nrl-treated Pde6a RP mice exhibited efficient restoration of retinal morphology and visual function.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.