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Yeh, Wei Hsi

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Yeh

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Wei Hsi

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Yeh, Wei Hsi
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    In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness
    (American Association for the Advancement of Science (AAAS), 2020-06-03) Yeh, Wei Hsi; Shubina-Oleinik, Olga; Levy, Jonathan; Pan, Bifeng; Newby, Gregory; Wornow, Michael; Burt, Rachel; Chen, Jonathan C.; Holt, Jeffrey R.; Liu, David
    Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide permanent therapeutic restoration of gene function. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G, resulting in the substitution p.Y182C) in transmembrane channel-like 1 (Tmc1) that causes deafness. Tmc1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction that causes profound deafness. To repair the Baringo mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual AAV vectors using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the Tmc1 c.A545G point mutation to wild type sequence (c.A545A) in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair-cell sensory transduction, hair-cell morphology, and partial low-frequency hearing four weeks post-injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.
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    Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses
    (Springer Science and Business Media LLC, 2020-01-14) Levy, Jonathan; Yeh, Wei Hsi; Pendse, Nachiket; Davis, Jessie; Hennessey, Erin; Butcher, Rossano; Luke, Koblan; Comander, Jason; Liu, Qin; Liu, David
    Base editors are widely used engineered proteins that introduce targeted point mutations into DNA without creating double-stranded DNA breaks. The application of base editors to study and treat genetic diseases is dependent on their in vivo delivery into relevant cell types. Delivery by adeno-associated virus (AAV), a clinically validated delivery method, poses special challenges because the size of base editors exceeds the AAV packaging limit. Here we describe the development and application of in vivo delivery methods for both cytosine base editors (CBEs) and adenine base editors (ABEs). Dual AAVs each provide one half of the editor and trans-splicing inteins reconstitute full base editor activity, circumventing the AAV packaging limit. We optimized each AAV component to greatly improve editing efficiency. The resulting AAVs enable in vivo base editing for the first time in mouse brain, retina, and heart, as well as the most efficient base editing to date in muscle and liver, with therapeutically relevant efficiencies at viral dosages known to be tolerated in humans. A single intravenous injection of split CBE in PHP.eB AAV resulted in editing of up to 59% of unsorted mouse cortical tissue. Intravenous injection of split CBE or ABE in AAV9 mediated up to 38%, 20%, and 9% base editing in unsorted mouse liver, heart, and skeletal muscle, respectively. Subretinal and intracerebroventricular injections of split CBE and ABE in Anc80, PHP.B/PHP.eB, and AAV9 viruses mediated mouse retina and brain editing in up to 38% and 50% of unsorted cells, respectively. We applied this system to directly correct in mouse brain tissue a mutation that causes the neurodegenerative ataxia Niemann-Pick disease type C (NPC), slowing neurodegeneration and increasing lifespan consistent with expectations based on mosaic NPC mice. These findings establish a broadly useful AAV platform for the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest for which in vivo base editing has not been previously reported.