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Cas9-mediated replacement of expanded CAG repeats in a pig model of Huntington’s disease

Nature Biomedical Engineering volume 7, pages 629–646 (2023)Cite this article

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Abstract

The monogenic nature of Huntington’s disease (HD) and other neurodegenerative diseases caused by the expansion of glutamine-encoding CAG repeats makes them particularly amenable to gene therapy. Here we show the feasibility of replacing expanded CAG repeats in the mutant HTT allele with a normal CAG repeat in genetically engineered pigs mimicking the selective neurodegeneration seen in patients with HD. A single intracranial or intravenous injection of adeno-associated virus encoding for Cas9, a single-guide RNA targeting the HTT gene, and donor DNA containing the normal CAG repeat led to the depletion of mutant HTT in the animals and to substantial reductions in the dysregulated expression and neurotoxicity of mutant HTT and in neurological symptoms. Our findings support the further translational development of virally delivered Cas9-based gene therapies for the treatment of genetic neurodegenerative diseases.

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Fig. 1: Analysis of the expression of Cas9 and GFP protein in pig brain.
Fig. 2: RNA-seq analysis of pig striatal tissues injected with AAV-GFP or AAV-Cas9.
Fig. 3: Analysis of HD KI pig brains injected with AAV9 HTT-gRNA-RFP-20Q/Cas9.
Fig. 4: Analysis of intravenous delivery of AAV HTT-gRNA-RFP-20Q/Cas9 in HD KI pigs.
Fig. 5: Intravenous injection of CRISPR/Cas9 in HD KI pigs alleviates motor function deficits.
Fig. 6: Analysis of the neuropathology in HD KI pigs after AAV-CRISPR/Cas9 treatment.
Fig. 7: RNA-seq analysis of CRISPR/Cas9 treatment in HD KI pigs.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw data from whole-genome sequencing are available from the NCBI Sequence Read Archive (SRA), with accession code PRJNA886395. The raw RNA-seq data are available from the NCBI Sequenced Read Archive (SRA), with accession code PRJNA886382. The other raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Z. Ouyang, C. Lai, N. Li, S. Gou, K. Wang, Q. Jin and H. Shi for technical assistance, and D. Wu and Y. Ai for animal care. This work was supported by The National Natural Science Foundation of China (81830032, 31872779, 81922026, 82071421, 82171244,32170981); the National Key Research and Development Program of China (2021YFA0805300,2022YFA1105403); the Guangzhou Key Research Program on Brain Science (202007030008, 202007030003), Key Field Research and Development Program of Guangdong province (2018B030337001); the National Key Research and Development Program of China Stem Cell and Translational Research (2017YFA0105102, 2017YFA0105101, 2017YFA0105103, 2017YFA0105104), Department of Science and Technology of Guangdong Province (2021ZT09Y007, 2020B121201006).

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  1. Guangdong Key Laboratory of Non-human Primate Research, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China

    Sen Yan, Xiao Zheng, Yingqi Lin, Caijuan Li, Jiawei Li, Zhuchi Tu, Chunhui Huang, Yizhi Chen, Jun Li, Xichen Song, Bofeng Han, Wei Wang, Weien Liang, Xiao-Jiang Li & Shihua Li

  2. China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

    Zhaoming Liu, Yu Zhao & Liangxue Lai

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Contributions

S.Y., X.-J.L., S.L. and L.L. designed the research; S.Y., X.Z., Y.L., C.L., Z.L., J.L., Z.T., Y.Z., C.H., Jun Li, X.S., B.H., Y.C., W.W. and W.L. performed the research; S.Y., X.-J.L., S.L., L.L. and X.Z. analysed the data; J.L. performed bioinformatics analysis; S.Y., X.-J.L., S.L. and L.L. wrote the paper with input from all authors.

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Correspondence to Sen Yan, Liangxue Lai, Xiao-Jiang Li or Shihua Li.

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Footprints in sand of HD KI pigs treated with a brain injection of AAV-Cas9 and control gRNA, and of a KI pig treated with a brain injection of AAV-Cas9 and HTT gRNA-20Q.

Footprints in sand of an HD KI pig treated with an intravenous injection of AAV-Cas9 and control gRNA, and of a KI pig treated with an intravenous injection of AAV-Cas9 and HTT gRNA-20Q.

Treadmill performance of an HD KI pig after an intravenous control and of a 5-month-old KI pig treated with an intravenous treatment injection.

Treadmill performance of an HD KI pig treated with a brain-injection control and of a 7-month-old KI pig with a treatment brain injection.

Treadmill performance of an HD KI pig before (at 3 months of age) and after (at 7 months of age) brain injection treatment.

Treadmill performance of WT pigs and brain-injection-treated 2-yr-old KI pigs.

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Yan, S., Zheng, X., Lin, Y. et al. Cas9-mediated replacement of expanded CAG repeats in a pig model of Huntington’s disease. Nat. Biomed. Eng 7, 629–646 (2023). https://doi.org/10.1038/s41551-023-01007-3

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