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Adeno-associated virus vector enables safe and efficient Cas9 activation in neonatal and adult Cas9 knockin murine cochleae

Gene Therapy volume 27, pages 392–405 (2020)Cite this article

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Abstract

Adeno-associated virus (AAV)-mediated gene delivery systems have been shown to be effective tools for gene manipulation in the inner ear. For example, hair cells (HCs) and multiple other cell types can be transduced by the local injection of AAVs into the inner ear. However, application of the AAV-mediated CRISPR/Cas9 gene-editing approach to the inner ear in adult mice has not yet been studied. Based on our previous work, we investigated several AAV serotypes in neonatal and adult mice in parallel, and found that AAV8 had the top efficiency to transduce inner HCs. We then tested the ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knockin mice, and observed significant Cas9 activation in the inner ear of both neonatal and adult animals. Neither the AAV8 virus itself nor the surgical procedures used to deliver it—cochleostomy for neonatal mice and canalostomy for adult mice—caused any damage to HCs or impaired normal hearing. Our studies indicate that the local injection of AAV8-Cre can induce Cas9 activation to perform safe and efficient gene editing in the inner ear, expanding the repertoire of gene-editing tools for regulating gene expression in the inner ear as a part of efforts to rescue genetic hearing loss, initiate regeneration of HCs, or develop gene therapy techniques.

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Fig. 1: AAV vectors transduced diverse inner ear cell types in neonatal cochlea.
Fig. 2: AAV vectors transduced inner hair cells in adult cochleae.
Fig. 3: Inner ear cell subtypes infected by AAV8-Cre injected in neonatal Cas9-KI mice.
Fig. 4: AAV transduction in neonatal and adult Cas9-KI mice did not impair normal hearing and IHCs.
Fig. 5: Inner ear cell subtypes infected by AAV8-Cre injected into adult Cas9-KI mice.
Fig. 6: AAV8-Cre activated Cas9 protein expression in the inner ear specifically.

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Acknowledgements

HW was supported by the Key Project of National Natural Science Foundation of China (NSFC 81330023) and the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (SQ2017YFSF080012), Shanghai Key Laboratory of Translational Medicine on Ear and Nose diseases (14DZ2260300), and Innovative Research Team of High-level Local Universities in Shanghai. YT was supported by the National Natural Science Foundation of China (NSFC81800900), Shanghai Science and Technology Committee (18411953600, 18ZR1422100, and 18PJ1406900), Shanghai Municipal Health Commission (2018YQ59), and Shanghai Ninth People’s Hospital (QC201804). WZ was supported by National Natural Science Foundation of China (NSFC81700900).

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  1. These authors contributed equally: Wen Kang, Xingle Zhao

Authors and Affiliations

  1. Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, No. 639, Zhizaoju Road, Shanghai, 200011, PR China

    Wen Kang, Xingle Zhao, Zhuoer Sun, Tingting Dong, Chenxi Jin, Ling Tong, Weidong Zhu, Yong Tao & Hao Wu

  2. Ear Institute, Shanghai Jiaotong University School of Medicine, No. 115, Jinzun Road, Shanghai, 200011, PR China

    Wen Kang, Xingle Zhao, Zhuoer Sun, Tingting Dong, Chenxi Jin, Ling Tong, Weidong Zhu, Yong Tao & Hao Wu

  3. Shanghai Key Laboratory of Translation Medicine on Ear and Nose Disease, No. 115, Jinzun Road, Shanghai, 200011, PR China

    Wen Kang, Xingle Zhao, Zhuoer Sun, Tingting Dong, Chenxi Jin, Ling Tong, Weidong Zhu, Yong Tao & Hao Wu

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Contributions

WK and XZ conducted the experiments, analyzed data, and wrote the paper; ZS, WZ, TD, LT, and CJ performed western blot and IVIS, assisted with experimental design, performed experiments, and analyzed data; YT and HW designed and supervised the research, and wrote the paper. All the authors edited the paper.

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Correspondence to Yong Tao or Hao Wu.

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Kang, W., Zhao, X., Sun, Z. et al. Adeno-associated virus vector enables safe and efficient Cas9 activation in neonatal and adult Cas9 knockin murine cochleae. Gene Ther 27, 392–405 (2020). https://doi.org/10.1038/s41434-020-0124-1

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