Liver-directed somatic gene correction rAAV system of regulatable Cas9/sgRNA

项目概要/摘要-项目3： 在体内纠正疾病基因突变的能力对两者都具有广泛的潜在效用。 治疗和基础研究。CRISPR/Cas9是一种强大的RNA指导的基因组编辑工具。 我们最近发现CRISPR/Cas9递送可以治愈成年小鼠肝脏中的遗传疾病 提供了基因矫正疗法的概念验证。这一分项目将相互配合， 与该tPPG的所有其他项目和核心合作，开发新的rAAV CRISPR工具来治疗α- 1例抗胰蛋白酶缺乏症（AATD）。该提案的主要目标是建立临床前rAAV CRISPR介导的AAT缺陷的校正的范例 突变的人AAT基因。该项目的影响是开发体细胞基因校正， rAAV系统（1）最大限度地提高CRISPR递送的效率和安全性，（2）最大限度地提高CRISPR递送的速率， 同源重组的基因校正，和（3）有效地纠正AAT突变的基因， 肝脏治疗小鼠肺部疾病。开发安全有效的运载工具， 纠正AAT缺陷的基因组编辑工具将指导未来的CRISPR临床试验- 介导的基因治疗AAT肺疾病。由于AAV血清型可以靶向广泛的 我们的方法具有广泛的基础研究和AATD以外的临床应用。 项目3有三个目标，侧重于肝脏定向体细胞AAT校正的不同方面： 目的1：开发肝导向rAAV载体，以最大限度地提高Z-AAT的效率和精度 基因组编辑在老鼠身上。目的2：研究rAAV HDR模板以校正Z-AAT突变。 小鼠肝脏。目的3：探讨Z-AAT在小鼠体内模型中的校正作用，以治疗肺部疾病。

Project Summary/Abstract – Project 3: The ability to correct disease gene mutations in vivo has broad potential utility for both therapy and basic research. CRISPR/Cas9 is a powerful RNA-guided tool for genome editing. Our recent discovery that CRISPR/Cas9 delivery can cure genetic disease in adult mouse liver provided proof-of-concept of gene correction therapy. This subproject will interact synergistically with all other Projects and Cores of this tPPG to develop new rAAV CRISPR tools to treat alpha- 1 antitrypsin deficiency (AATD). The main goal of this proposal is to establish a pre-clinical rAAV paradigm for CRISPR-mediated correction of AAT deficiency in mouse models carrying the mutant human AAT gene. The impact of this project is to develop somatic gene correction using rAAV systems to (1) maximize efficiency and safety of CRISPR delivery, (2) maximize the rate of homologous recombination for gene correction, and (3) efficiently correct AAT mutation in the liver to treat lung disease in mice. The development of safe and effective delivery vehicles and genome editing tools to correct AAT deficiency will guide future clinical trials for CRISPR- mediated gene therapy for AAT lung disease. Because AAV serotypes can target a wide range of tissues, our approach has broad basic research and clinical applications beyond AATD. Project 3 has three Aims that focus on different aspects of liver-directed somatic AAT correction: Aim 1: Develop liver-directed rAAV vehicles to maximize efficiency and precision of Z-AAT genome-editing in mice. Aim 2: Investigate rAAV HDR templates to correct Z-AAT mutation in mouse liver. Aim 3: Explore Z-AAT correction in mouse models in vivo to treat the lung disease.