PDF(Pubmed)
Abstract：
Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3\' end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.
摘要：
正在研究通过CRISPR-Cas9在高度转录的白蛋白基因座处的肝定向腺相关病毒（AAV）载体介导的同源性非依赖性靶向整合（AAV-HITI），以在新生儿治疗后提供持续的转基因表达。我们表明，靶向白蛋白基因座的3'末端导致约15％的小鼠肝细胞的生产性整合，在两种遗传性疾病小鼠模型中达到系统蛋白的治疗水平。我们证明了全长HITI供体DNA在核酸酶切割后优先整合，尽管部分AAV基因组整合在靶基因座，在脱靶位点没有发现总的染色体重排或插入/缺失。与此相符,在1年的随访中没有发现肝细胞癌的证据.最后,AAV-HITI在被认为是安全的载体剂量下是有效的,如果直接翻译给人,除了新生儿之外,还在成人肝脏中提供治疗功效。总的来说,我们的数据支持这种基于肝脏定向AAV的敲入策略的发展.
