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Single AAV-mediated CRISPR-Nme2Cas9 efficiently reduces mutant hTTR expression in a transgenic mouse model of transthyretin amyloidosis

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  • Jinkun Wen, Sun Yat-Sen University
  • ,
  • Tianqi Cao, Sun Yat-Sen University
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  • Jinni Wu, Sun Yat-Sen University
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  • Yuxi Chen, Sun Yat-Sen University
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  • Shengyao Zhi, Sun Yat-Sen University
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  • Yanming Huang, Sun Yat-Sen University
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  • Peilin Zhen, Sun Yat-Sen University
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  • Guanglan Wu, Sun Yat-Sen University
  • ,
  • Lars Aagaard
  • Jianxin Zhong, Sun Yat-Sen University
  • ,
  • Puping Liang, Sun Yat-Sen University
  • ,
  • Junjiu Huang, Sun Yat-Sen University

Transthyretin (TTR) amyloidosis is a hereditary life-threatening disease characterized by deposition of amyloid fibrils. The main causes of TTR amyloidosis are mutations in the TTR gene that lead to the production of misfolded TTR protein. Reducing the production of toxic protein in the liver is a validated strategy to treat TTR amyloidosis. In this study, we established a humanized mouse model that expresses mutant human TTR (hTTR; V30M) protein in the liver to model TTR amyloidosis. Then, we compared the efficiency of reducing the expression of mutant hTTR by dual adeno-associated virus 8 (AAV8)-mediated split SpCas9 with that by single AAV8-mediated Nme2Cas9 in this model. With two gRNAs targeting different exons, dual AAV-mediated split SpCas9 system achieved efficiencies of 37% and 34% reduction of hTTR mRNA and reporter GFP expression, respectively, in the liver. Surprisingly, single AAV-mediated Nme2Cas9 treatment resulted in 65% and 71% reduction of hTTR mRNA and reporter GFP, respectively. No significant editing was identified in predicted off-target sites in the mouse and human genomes after Nme2Cas9 targeting. Thus, we provide proof of principle for using single AAV-mediated CRISPR-Nme2Cas9 to effectively reduce mutant hTTR expression in vivo, which may translate into gene therapy for TTR amyloidosis.

Original languageEnglish
JournalMolecular Therapy
Pages (from-to)164-174
Number of pages11
Publication statusPublished - Jan 2022

    Research areas

  • AAV, gene therapy, mouse model, Nme2Cas9, TTR amyloidosis

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