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rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination.
Ling, Chen; Yu, Chenghui; Wang, Cong; Yang, Ming; Yang, Hengbin; Yang, Keying; He, Yun; Shen, Yajie; Tang, Shiyi; Yu, Xiaomin; Zhou, Zhengjun; Zhou, Shaolai; Zhou, Jian; Zhu, Liqing; Li, Jixi.
Afiliação
  • Ling C; State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
  • Yu C; Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China.
  • Wang C; State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
  • Yang M; State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
  • Yang H; State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
  • Yang K; State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
  • He Y; State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
  • Shen Y; State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
  • Tang S; State Key Laboratory of Genetic Engineering, School of Life Sciences and Huashan Hospital, Shanghai Engineering Research Center of Industrial Microorganisms, MOE Engineering Research Center of Gene Technology, Fudan University, Shanghai 200438, China.
  • Yu X; Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China.
  • Zhou Z; Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China.
  • Zhou S; Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
  • Zhou J; Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
  • Zhu L; Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
  • Li J; Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China.
Nucleic Acids Res ; 52(11): 6518-6531, 2024 Jun 24.
Article em En | MEDLINE | ID: mdl-38783157
ABSTRACT
Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Regiões Promotoras Genéticas / Dependovirus / Recombinação Homóloga / Edição de Genes Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Regiões Promotoras Genéticas / Dependovirus / Recombinação Homóloga / Edição de Genes Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article