Improved viral vector design for homology-independent targeted integration (HITI)

隐性疾病的基因治疗正在变得司空见惯，但对显性功能获得障碍的治疗研究不足，因为需要同时实现显性突变等位基因的击倒和正常功能的替换。该项目旨在设计高效的AAV载体，并增加解决显性疾病所需的复杂性。Cas9介导的同源独立靶向整合(HITI)有助于在任何选定的基因编辑核酸酶裂解的PAM位点插入任意序列(铃木2016年PMID：27851729)。HITI利用NHEJ-途径，这比传统的HDR途径更有效，因此提供了更现实的体内基因编辑策略。我们有一个‘Hiti Report’小鼠模型，可以在任何器官中对Hiti进行体内评估。我们对AAV Cas9 Hiti载体的初步数据显示，受控的、无错误的、PAM靶向的DNA插入体内的小鼠肝细胞中。作为疾病样本，我们计划使用HITI来治疗主要的肝脏疾病，即α-1抗胰蛋白酶(AAT)缺乏。具体地说，我们将针对常见的显性AAT基因变体(PIZ)，它导致肝细胞中AAT蛋白沉淀，导致肺中致病水平不足。根据项目进展，还有机会为其他器官开发Hiti载体平台。

Gene therapies for recessive disorders are becoming commonplace, but treatments for dominant gain-of-function disorders are under-researched due to the need to achieve both knock down of the dominant mutant allele and replacement of normal function. This project aims to design high-efficiency AAV vectors with the additional complexity required to tackle dominant disorders. Cas9-mediated Homology-Independent Targeted Integration (HITI) facilitates insertion of an arbitrary sequence at any selected gene editing nuclease cleaved PAM site (Suzuki 2016 PMID:27851729). HITI utilises the NHEJ-pathway, which is substantially more efficient than conventional HDR pathways, and therefore offers more realistic in vivo gene editing strategies. We have a 'HITI reporter' mouse model to allow in vivo HITI to be assessed in any organ. We have preliminary data with AAV Cas9 HITI vectors showing controlled, error-free, PAM-targeted, DNA insertion into mouse liver cells in vivo. As a disease exemplar, we plan to use HITI for the dominant liver disorder, alpha-1 antitrypsin (AAT) deficiency. Specifically, we will target the common, dominant AAT genetic variant (PiZ) that leads to AAT protein precipitation in hepatocytes causing deficient disease-causing levels in the lung. Depending on project progression there is also the opportunity to develop the HITI vector platform for other organs.