Nme2 Cas9-mediated therapeutic editing in inhibiting angiogenesis after wet age-related macular degeneration onset.

BACKGROUND: Age-related macular degeneration (AMD), particularly wet AMD characterised by choroidal neovascularization (CNV), is a leading cause of vision loss in the elderly. The hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) pathway contributes to CNV pathogenesis. Previous gene editing research indicated that disrupting these genes in retinal pigment epithelial cells could have a preventive effect on CNV progression. However, no studies have yet been conducted using gene editing to disrupt VEGF signalling after CNV induction for therapeutic validation, which is critical to the clinical application of wet AMD gene editing therapies. METHOD: Here, we employed the single-adeno-associated virus-mediated Nme2 Cas9 to disrupt key molecules in VEGF signalling, Hif1α, Vegfa and Vegfr2 after inducing CNV and estimated their therapeutic effects. RESULTS: We found that Nme2 Cas9 made efficient editing in target genes up to 71.8% post 11 days in vivo. And only Nme2 Cas9-Vegfa treatment during the early stage of CNV development reduced the CNV lesion area by 49.5%, compared to the negative control, while Nme2 Cas9-Hif1α or Nme2 Cas9-Vegfr2 treatment did not show therapeutic effect. Besides, no off-target effects were observed in Nme2 Cas9-mediated gene editing in vivo. CONCLUSIONS: This study provides proof-of-concept possibility of employing Nme2 Cas9 for potential anti-angiogenesis therapy in wet AMD.

Screening an Effective Dual-Adeno-Associated Virus Split-Cytosine Base Editor System for C-to-T Conversion In Vivo.

The cytosine base editor (CBE) has shown promise as a gene editing tool for gene therapy, as it can convert cytidine to thymidine. Adeno-associated virus (AAV) has been widely used for in vivo gene therapy, but its limited 4.7 kb packing capacity presents challenges in delivering CBE by a single AAV. To address this, one feasible solution is to split CBE into two sections for dual-AAV delivery. In this study, we utilized BE3 as an example and constructed 22 potential split-BE3 pairs with the combination of 11 splitting sites and two split-inteins (Npu and Rma). These split-BE3 pairs were initially screened in the green fluorescent protein (GFP) reporter system, with six split-BE3 pairs selected for further evaluation. The subsequent screening of split-BE3 pairs was performed at two endogenous sites in 293T and HeLa cells, revealing that the split-BE3-Rma674, split-BE3-Rma713, and split-BE3-Rma1005 displayed effective C-to-T conversion after transfection. The effectiveness of dual-AAV split-BE3 was further validated in culture cells and adult mouse eyes. Of note, the split-BE3-Rma674 demonstrated the most efficient C-to-T editing after AAV infection, with a maximal editing efficiency of 23.29% ± 10.98% in the mouse retinal pigment epithelium cells in vivo. Overall, our study presents a novel split-BE3 system with effective C-to-T conversion, which could be applied to CBE-based in vivo gene therapy.