No abstract

The prime editor (PE) can edit genomes with almost any intended changes, including all 12 possible types of base substitutions, small insertions and deletions, and their combinations, without the requirement for double strand breaks or exogenous donor templates. PE demonstrates the possibility of correcting a variety of disease-causing mutations and might expand the therapeutic application of gene editing. In this study, PE was optimized based on a dual-adeno-associated virus (AAV) split-intein system in vitro by screening different split sites and split inteins. We found that splitting PE before amino acid 1105(Ser) of SpCas9 with Rma intein resulted in the highest on-target editing. The orientations of pegRNA and nicking sgRNA in the AAV vector were further optimized. To test the in vivo performance of the optimized dual-AAV split-PE3, it was delivered by subretinal injection in rd12 mice with inherited retinal disease Leber congenital amaurosis. The prime editors corrected the pathogenic mutation with up to 16% efficiency in a precise way, with no detectable off-target edits, restored RPE65 expression, rescued retinal and visual function, and preserved photoceptors. Our findings establish a framework for the preclinical development of PE and motivate further testing of PE for the treatment of inherited retinal diseases caused by various mutations.

No abstract

Inhibition of vascular endothelial growth factor (VEGF) is the standard therapy for neovascular age-related macular degeneration (nAMD). However, anti-VEGF agents used in the clinic require repeated injections, causing adverse effects. Gene therapy could provide sustained anti-VEGF levels after a single injection, thereby drastically decreasing the treatment burden and improving visual outcomes. In this study, we developed a novel VEGF Trap, nVEGFi, containing domains 1 and 2 of VEGFR1 and domain 3 of VEGFR2 fused to the Fc portion of human IgG. The nVEGFi had a higher expression level than aflibercept under the same expression cassettes of adeno-associated virus (AAV)8 in vitro and in vivo . nVEGFi was found to be noninferior to aflibercept in binding and blocking VEGF in vitro . AAV8-mediated expression of nVEGFi was maintained for at least 12 weeks by subretinal delivery in C57BL/6J mice. In a mouse laser-induced choroidal neovascularization (CNV) model, 4 × 10 8 genome copies of AAV8-nVEGFi exhibited a significantly increased reduction in the CNV area compared with AAV8-aflibercept (78.1% vs. 63.9%, p < 0.05), while causing no structural or functional changes to the retina. In conclusion, this preclinical study showed that subretinal injection of AAV8-nVEGFi was long lasting, well tolerated, and effective for nAMD treatment, supporting future translation to the clinic.

Mucopolysaccharidosis type I (MPS I) is a severe disease caused by loss-of-function mutations variants in the α-L-iduronidase (IDUA) gene. In vivo genome editing represents a promising strategy to correct IDUA mutations, and has the potential to permanently restore IDUA function over the lifespan of the patients. Here, we used adenine base editing to directly convert A>G (TAG>TGG) in newborn murine model harboring Idua-W392X mutation, which recapitulates the human condition and is analogous to the highly prevalent human W402X mutation. We engineered a split-intein dual-adeno-associated virus (AAV) 9 in vivo adenine base editor to circumvent the package size limit of AAV vectors. Intravenous injection of AAV9-base editor system into MPS I newborn mice led to sustained enzyme expression sufficient for correction of metabolic disease (GAGs substrate accumulation) and prevention of neurobehavioral deficits. We observed a reversion of the W392X mutation in 22.46 plus-or-minus sign 6.74% of hepatocytes, 11.18 plus-or-minus sign 5.25% of heart and 0.34 plus-or-minus sign 0.12% of brain, along with decreased GAGs storage in peripheral organs (liver, spleen, lung and kidney). Collectively, these data showed the promise of a base editing approach to precisely correct a common genetic cause of MPS I in vivo and could be broadly applicable to the treatment of a wide array of monogenic diseases.