Enhanced genome editing to ameliorate a genetic metabolic liver disease through co-delivery of adeno-associated virus receptor  

作　　者：Shuming Yin Lie Ma Tingting Shao Mei Zhang Yuting Guan Liren Wang Yaqiang Hu Xi Chen Honghui Han Nan Shen Wenjuan Qiu Hongquan Geng Yongguo Yu Shichang Li Weishi Yu Mingyao Liu Dali Li 

机构地区：[1]Shanghai Key Laboratory of Regulatory Biology,Institute of Biomedical Sciences and School of Life Sciences,East China Normal University,Shanghai,200241,China [2]Bioray Laboratories Inc.,Shanghai,200241,China [3]Xinhua Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai,200092,China [4]College of Physical Education and Health,East China Normal University,Shanghai,200241,China [5]Key Laboratory of Adolescent Health Assessment and Exercise Intervention,Ministry of Education,College of Physical Education and Health,East China Normal University,Shanghai,200241,China [6]CIPHER GENE LLC,Beijing,100089,China

出　　处：《Science China(Life Sciences)》2022年第4期718-730,共13页中国科学（生命科学英文版）

基　　金：partially supported by grants from the National Key R&D Program of China (2019YFA0110802);the National Natural Science Foundation of China (81670470 and 81873685);grants from the Shanghai Municipal Commission for Science and Technology (18411953500 and 20140900201);a grant from the Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-05-E00054);the Fundamental Research Funds for the Central Universities

摘　　要：Genome editing through adeno-associated viral(AAV) vectors is a promising gene therapy strategy for various diseases,especially genetic disorders. However, homologous recombination(HR) efficiency is extremely low in adult animal models. We assumed that increasing AAV transduction efficiency could increase genome editing activity, especially HR efficiency, for in vivo gene therapy. Firstly, a mouse phenylketonuria(PKU) model carrying a pathogenic R408W mutation in phenylalanine hydroxylase(Pah) was generated. Through co-delivery of the general AAV receptor(AAVR), we found that AAVR could dramatically increase AAV transduction efficiency in vitro and in vivo. Furthermore, co-delivery of SaCas9/sgRNA/donor templates with AAVR via AAV8 vectors increased indel rate over 2-fold and HR rate over 15-fold for the correction of the single mutation in Pah;mice. Moreover, AAVR co-injection successfully increased the site-specific insertion rate of a 1.4 kb Pah cDNA by 11-fold, bringing the HR rate up to 7.3% without detectable global off-target effects. Insertion of Pah cDNA significantly decreased the Phe level and ameliorated PKU symptoms. This study demonstrates a novel strategy to dramatically increase AAV transduction which substantially enhanced in vivo genome editing efficiency in adult animal models, showing clinical potential for both conventional and genome editing-based gene therapy.

关 键 词：CRISPR/Cas9 gene therapy adeno-associated virus(AAV) AAVR PAH PKU 

分 类 号：Q78[生物学—分子生物学] R575[医药卫生—消化系统]