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

Yan, Shihai; Zheng, Xiao; Lin, Yingqi; Li, Caijuan; Liu, Zhaoming; Li, Jiawei; Tu, Zhuchi; Zhao, Yu; Huang, Cuiqing; Chen, Yizhi; Jun, Li; Song, Xichen; Han, Bofeng; Wang, Wei; Liang, Weien; Lai, Liangxue; Li, Shengbo Eben; Li, Shihua

doi:10.1038/s41551-023-01007-3

Cited by 37 publications

(15 citation statements)

References 78 publications

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“…Although work is needed to improve the infection efficiency in P2 injected mice, the efficiency of contractions is comparable to what has been seen by others using sgRNAs flanking the repeats. By contrast, here, we recovered repeat contractions rather than a collection of uncontrolled mutations 15,16,24,25 . Thus, our approach is as efficient as excising the repeat tract, while remaining more precise and avoiding both on-target and off-target mutations.…”

Section: Discussionmentioning

confidence: 69%

Cas9 nickase-mediated contraction of CAG/CTG repeats at multiple disease loci

Murillo,

Alpaugh,

Larin

et al. 2024

Preprint

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Expanded CAG/CTG repeats cause at least 15 different neurodegenerative and neuromuscular diseases that all remain without an effective disease modifying treatment. Because the size of the repeat tract accounts for the majority of the variation in disease severity, contracting them presents an attractive therapeutic avenue. Here, we show that the CRISPR-Cas9 nickase targeting the CAG/CTG repeat itself leads to efficient contractions in Huntington’s disease patient-derived neurons and astrocytes, as well as in myotonic dystrophy type 1 patient-derived neurons. Using single-cell DNA sequencing, PCR-free whole genome sequencing, and targeted long-read sequencing of theHTTlocus, we found no off-target mutations above background in neurons and astrocytes. Furthermore, we delivered the Cas9 nickase and sgRNA stereotactically to a mouse model of Huntington’s disease using adeno-associated viruses, and found contractions accumulating in over half of the infected cells over a period of 5 months. We also found that the Cas9 nickase was prone to silencing, further improving the safety of the approach. Our results provide the proof of concept for using the Cas9 nickase to contract the repeat tract safely in multiple cell types and diseases.

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Section: Discussionmentioning

confidence: 69%

Cas9 nickase-mediated contraction of CAG/CTG repeats at multiple disease loci

Murillo,

Alpaugh,

Larin

et al. 2024

Preprint

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“…Systemic delivery of genome editors with glucose-conjugated silica nanoparticles and AAV9 can lead to modest levels of editing in the brain, sufficient for therapeutic benefit 44,45 . In this study, we found that the Cas9-AAV distributed further through the brain than Cas9-RNPs, although the RNP delivery approach was more effective for high levels of localized neuronal editing.…”

Section: Discussionmentioning

confidence: 99%

Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs

Stahl

Sabo

Kang

et al. 2023

Preprint

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Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here we tested the ability of cell penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection enhanced delivery system. These transient Cas9 RNPs showed greater local editing of neurons and reduced adaptive immune responses relative to Cas9 delivered using AAV serotype 9. The production of ultra-low-endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

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“…Treatment can be achieved through a single brain stereotaxic injection or intravenous delivery of CRISPR/Cas9 with partial replacement of m HTT ( Yan et al, 2023 ). Intravenous delivery of CRISPR/Cas9 is typically performed in newborn animals before the blood-brain barrier is fully established.…”

Section: Animal Models Of Hd For Treatmentmentioning

confidence: 99%

“…While there are no existing treatments to modify the course of the disease, extensive research in the field of polyglutamine (polyQ) diseases has uncovered a broad range of deleterious effects of expanded polyQ on diverse cellular mechanisms and functions ( Bradford et al, 2010 ; Friedman et al, 2007 ). This understanding has led to the prevailing theory that inhibiting the expression of expanded polyQ proteins may be an effective approach to curing these diseases ( Yan et al, 2023 ).…”

Section: Introductionmentioning

confidence: 99%

Large animal models for Huntington’s disease research

Han,

Liang,

et al. 2024

Zoological Research

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Huntington’s disease (HD) is a hereditary neurodegenerative disorder for which there is currently no effective treatment available. Consequently, the development of appropriate disease models is critical to thoroughly investigate disease progression. The genetic basis of HD involves the abnormal expansion of CAG repeats in the huntingtin ( HTT ) gene, leading to the expansion of a polyglutamine repeat in the HTT protein. Mutant HTT carrying the expanded polyglutamine repeat undergoes misfolding and forms aggregates in the brain, which precipitate selective neuronal loss in specific brain regions. Animal models play an important role in elucidating the pathogenesis of neurodegenerative disorders such as HD and in identifying potential therapeutic targets. Due to the marked species differences between rodents and larger animals, substantial efforts have been directed toward establishing large animal models for HD research. These models are pivotal for advancing the discovery of novel therapeutic targets, enhancing effective drug delivery methods, and improving treatment outcomes. We have explored the advantages of utilizing large animal models, particularly pigs, in previous reviews. Since then, however, significant progress has been made in developing more sophisticated animal models that faithfully replicate the typical pathology of HD. In the current review, we provide a comprehensive overview of large animal models of HD, incorporating recent findings regarding the establishment of HD knock-in (KI) pigs and their genetic therapy. We also explore the utilization of large animal models in HD research, with a focus on sheep, non-human primates (NHPs), and pigs. Our objective is to provide valuable insights into the application of these large animal models for the investigation and treatment of neurodegenerative disorders.

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

Cited by 37 publications

References 78 publications

Cas9 nickase-mediated contraction of CAG/CTG repeats at multiple disease loci

Cas9 nickase-mediated contraction of CAG/CTG repeats at multiple disease loci

Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs

Large animal models for Huntington’s disease research

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