Science Translational Medicine. 2022 Jul 20
Qingquan Xiao, Zhijiao Xu, Yuanyuan Xue, Chunlong Xu, Lei Han, Yuanhua Liu, Fang Wang, Runze Zhang, Shuang Han, Xing Wang, Geng-Lin Li, Huawei Li, Hui Yang, Yilai Shu
Products used in the paper Details Operation
AAV vector packaging AAV-PHP.eB serotype was used in this study. The mxABE plasmid with target or nontarget gRNA was sequenced before packaging into AAV-PHP.eB vehicle, and the AAV vectors were packaged by PackGene Biotech. The vector titer was 2.86 × 10^13 and 3.99 × 10^13 genome copies/ml as determined by qPCR specific for the inverted terminal repeat of the AAV-PHP.eB–Myo6 and control virus. Request Quote

Research Field: hearing/ RNA editing

AAV Serotype: AAV-PHP.eB

Dose: With total injection volumes of 500 nl of mxABE-T or 358.4 nl of mxABE-NT virus per cochlea, about 1.43 × 10^10 genomes were delivered into each ear of Myo6C442Y/+ mouse pups at P0 to P2 with a rate of 3 nl/s.

Routes of Administration: inner ear injection

Targeted organ: inner ear

Animal or cell line strain: Myo6 gene-modified mice(Myo6-C442Y) produced in the C57BL/6J strain


Programmable RNA editing tools enable the reversible correction of mutant transcripts, reducing the potential risk associated with permanent genetic changes associated with the use of DNA editing tools. However, the potential of these RNA tools to treat disease remains unknown. Here, we evaluated RNA correction therapy with Cas13-based RNA base editors in the myosin VI p.C442Y heterozygous mutation (Myo6C442Y/+) mouse model that recapitulated the phenotypes of human dominant-inherited deafness. We first screened several variants of Cas13-based RNA base editors and guide RNAs (gRNAs) targeting Myo6C442Y in cultured cells and found that mini dCas13X.1-based adenosine base editor (mxABE), composed of truncated Cas13X.1 and the RNA editing enzyme adenosine deaminase acting on RNA 2 deaminase domain variant (ADAR2ddE488Q), exhibited both high efficiency of A > G conversion and low frequency of off-target edits. Single adeno-associated virus (AAV)-mediated delivery of mxABE in the cochlea corrected the mutated Myo6C442Y to Myo6WT allele in homozygous Myo6C442Y/C442Y mice and resulted in increased Myo6WT allele in the injected cochlea of Myo6C442Y/+ mice. The treatment rescued auditory function, including auditory brainstem response and distortion product otoacoustic emission up to 3 months after AAV-mxABE-Myo6 injection in Myo6C442Y/+ mice. We also observed increased survival rate of hair cells and decreased degeneration of hair bundle morphology in the treated compared to untreated control ears. These findings provide a proof-of-concept study for RNA editing tools as a therapeutic treatment for various semidominant forms of hearing loss and other diseases.

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