Base editing restores the visual function of inherited retinal disease damage

retinal disease (IRD) is a group of blinding disorders caused by mutations in more than 250 different genes. Leber's congenital erythroidism (LCA) is one of the most common types of IRD, and most people with LCA have severe vision impairment throughout childhood, and become completely blind in middle age as the retina is sexually degenerative. There is currently no efficient treatment option.In 2017, the U.S. Food and Drug Administration (FDA) approved the first gene therapy for LCA patients with double allophic mutations in the RPE gene, which uses adeno-related viruses (AAVs) to transmit the correct RPE65 gene to the retina. Clinical trials have shown that visual function improves in the first year of treatment, but decreases in the long term. The researchers speculate that this may be due to a decrease in the level of GM expression delivered by AAV vectors over time, or the resistance of the REPE65 protein expressed by retinal degeneration to external sources caused by the lack of RPE65 proteins.On October 19, 2020, a team of researchers from the University of California, California, and case Western Reserve University published a study entitled "Restorationof visual function in adult mice with an inherited retinal disease via adenineb" in nature Biomedical Engineering. The results of the research, using adenine base editor (ABE), overcome the CRISPR-Cas9 system off-target mutations, low editing efficiency and other obstacles, successfully correct gene mutations, restore the visual function of adult mice with hereditary retinal disease.The researchers studied rd12 mice with a single base mutation on exon 3 of the RPE65 gene, causing the protein of the renewable 11-shun retinal and visual pigment to be unable to express as a clinically relevant model of LCA. First of all, in-body cells were evaluated to achieve the repair of mutation points by the same recombinant repair (HDR), and it was found that hdR was only 0.03% efficient, which could not restore the function of RPE65 and improve disease esophysiency.The researchers then tried to deliver ABE under the retina, correcting the pathological mutation with greater accuracy and minimal off-target rates by converting A from A on the complementary chain of the RPE65 gene to G. It designs ABE variants (ABEmax) with sgRNA-A5, sgRNA-A6 optimized with cocoons, and verifies that ABE's cryptoeloid optimization can produce more stable and higher protein expression through rd12 cell line, and ABEmax has a stronger base editing efficiency and can correct unrightetic mutations with minimal off-target rate.The researchers then produced three virulent vectors that expressed sgRNA and ABEmax by injection into pigment epithelioral (RPE) tissue in the retina of mice to assess the effectiveness of the base editing method. After five weeks of injection, the researchers tested the recovery of RPE65 protein in the eyes of treated and controlled mice using protein immunoprinting. It was found that the treatment of ABEmax with sgRNA-A5, sgRNA-A6 successfully expressed the correctly positioned RPE65 protein in mice. Analysis of the percentage of corrective cells per eye revealed that the A5 and A6 treatments corrected 32% and 17% of the cells, respectively.To further quantify its correction efficiency, the researchers sequenced DNA isolated in mice's RPE tissue in high-volume. The results show that the maximum correction rate of ABEmax treatment of sgRNA-A5 can be as high as 29%, and the maximum correction rate of ABEmax of sgRNA-A6 can be as high as 11%. The off-target activity of sgRNA-A5 and A6 was evaluated by chromosome outer ring DNA sequencing (CIRCLE-seq), and no off-target editing above the background level of the control RPE tissue was detected.The researchers then tested whether rd12 mice treated with the more efficient A5 could restore functional visual circulation to further evaluate the therapeutic effects of base editing. It was found that the eyes of rd12 mice treated by A5 showed a large amount of production of 11-shun resusceric yellow aldehyde after completely dark adaptation, the visual cycle was restored, and the newly generated 11-shun resusced yellow aldehyde can be light isomerized into full transverse resaldehyde immediately after flash stimulation, resulting in vision. In addition, mice treated with the gene editing have been tested to restore their retinal cells and visual functions, as well as the functional integrity of the visual path paths from the retina to the primary visual cortical cortical layer, and cortical responses.Krzysztof Palczewski, co-author of the study, said: 'In this proof-of-concept study, we provide evidence of the clinical potential of gene editing in correcting mutations that cause hereditary retinal disease and restoring visual function. After receiving gene editing therapy, the mice we studied were able to distinguish between visual changes based on direction, size, contrast, and frequency of time and space, and the results were very encouraging, representing a significant advance in the treatment of hereditary retinal diseases." (Biological Exploration):1.Restore of visual function in adult mice with an inherited retinal disease via adenine base