Therapeutic adenine base editing corrects nonsense mutation and improves visual function in a mouse model of Leber congenital amaurosis

Abstract

Leber congenital amaurosis (LCA) is an inherited retinal degeneration that causes severe visual dysfunction in children and adolescents. In patients with LCA, pathogenic variants are evident in specific genes, such as RPE65, which are related to the functions of retinal pigment epithelium and photoreceptors. Base editing confers a way to correct pathogenic substitutions without double-stranded breaks in contrast to the original Cas9. In this study, we prepared dual adeno-associated virus vectors containing the split adenine base editors with trans-splicing intein (AAV-ABE) for in vivo adenine base editing in retinal degeneration 12 (rd12) mice, an animal model of LCA, which possess a nonsense mutation of C to T transition in the Rpe65 gene (p.R44X). AAV-ABE induced an A to G transition in retinal pigment epithelial cells of rd12 mice when injected into the subretinal space. The on-target editing was sufficient to recover wild-type mRNA, RPE65 protein, and light-induced electrical responses of retinal tissues. We suggest adenine base editing to correct pathogenic variants in the treatment of LCA.

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

“…A single subretinal injection of v4 BE-eVLPs in a mouse model of LCA efficiently corrected the disease-causing point mutation and restored visual function. In this model, once again, eVLPs achieved editing efficiencies and levels of rescue that are comparable with or higher than those previously achieved using viral delivery methods, including lentiviral BE delivery ( Suh et al., 2021 ) and AAV-mediated BE delivery ( Jo et al., 2021 ). The accessibility of the eyes and their immune-privileged status ( Taylor, 2009 ) may more readily enable the translation of new delivery modalities into preclinical and clinical studies.…”

“…BEs are known to exhibit low-level transcriptome-wide Cas-independent off-target RNA editing ( Anzalone et al., 2020 ). To investigate this possibility, we assessed off-target RNA editing by ABE-eVLPs and ABE-LVs by sequencing the Mcm3ap and Perp transcripts from treated eyes, two transcripts that were previously identified as potential candidates for off-target RNA editing based on their sequence similarity to the native TadA deaminase substrate ( Jo et al., 2021 ). We observed RNA off-target editing by ABE8e-NG-LV in both transcripts and low but detectable RNA off-target editing by ABE7.10-NG-LV at one adenine in Perp ( Figures S7 B and S7C).…”

Basic Principles and Clinical Applications of CRISPR-Based Genome Editing

“…The rd12 mouse model of LCA2 was subjected to subretinal injection of two AAVs encoding 1) SpCas9 and 2) sgRNA and donor DNA, resulting in the recovery of retinal function. 38 Recently, the same mouse model was successfully treated with subretinal injection of adenine base editors using RNPs 39 or intein-mediated split AAV vectors, 40 and prime editors using trans-splicing split AAV vectors, 41 showing promise in therapeutic genome editing with new genome editors.…”

Translational potential of base-editing tools for gene therapy of monogenic diseases

“…In two refs. ( Jo et al, 2021 ; Suh et al, 2021 ), in murine strain rd12 (carrying a nonsense mutation in exon 3: c.130C > T; p.R44X), which manifests the first signs of retinal degeneration at ∼3 weeks of age ( Pang et al, 2005 ), investigators were able to rescue retinal and visual function. Suh and others ( Suh et al, 2021 ) have tested the ABEmax system, which they have delivered into retinal cells by means of a lentivirus and achieved the following: RPE65 expression is restored in 32% of retinal cells, the total amount of 11- cis -retinal is 30% of the level in wild-type mice, and there is a 34% reduction in the concentration of all- trans- retinyl esters.…”

“…Suh and others ( Suh et al, 2021 ) have tested the ABEmax system, which they have delivered into retinal cells by means of a lentivirus and achieved the following: RPE65 expression is restored in 32% of retinal cells, the total amount of 11- cis -retinal is 30% of the level in wild-type mice, and there is a 34% reduction in the concentration of all- trans- retinyl esters. In another study ( Jo et al, 2021 ), investigators used the NG-ABEmax system as well as a dual AAV with trans-splicing intein as a vector for delivery to retinal cells; however, the efficiency of DNA editing was lower (13%) than that in ref. ( Suh et al, 2021 ).…”