Editing of the AAT locus using novel base editing and prime editing technologies

项目2--项目摘要/摘要 基因编辑有可能纠正突变，并为慢性阻塞性肺疾病患者提供长期治疗益处 罕见的单基因疾病，如α-1抗胰蛋白酶缺乏症(AATD)。AATD是由AAT的突变引起的 (或PI)基因，它编码一种丝氨酸蛋白酶抑制物，由肝细胞制造，并被输送到肺组织以 中和中性粒细胞弹性蛋白酶。Pi*Z突变编码突变的Z-AAT蛋白，该蛋白聚集在 肝细胞，可引起肝病，降低血清AAT。血清AAT降低导致进展性 呼吸道疾病和肺气肿。基因矫正将解决AATD的这两个方面。 CRISPR介导的同源定向修复(HDR)可用于部分纠正小鼠肝脏的突变。 然而，HDR受到以下因素的限制：需要提供长的DNA修复模板，其在非分割或慢速- 分裂的细胞类型，及其产生的遗传毒性双链断裂。为了解决这些限制，此 Proposal将开发两种基于CRISPR的不需要双链断裂的基因校正策略： 主要编辑和腺嘌呤碱基编辑。主编(PE)由融合了反转的Cas9昵称组成 转录酶和一个延伸的引导RNA，它兼有逆转录酶复制编辑的模板作用 将信息输入到基因组目标。腺嘌呤碱基编辑程序(ABE)，由Cas9尼克酶与 腺苷脱氨酶，可纠正小鼠肝脏G-to-A点突变。PI*Z等位基因是由G-to-A引起的 突变；因此，是通过ABE和PE进行基因校正的一个很好的候选者。 本项目的目标是通过开发ABE来优化用于体内AAT基因校正的PE和ABE工具 和可被腺相关病毒(AAV)衣壳容纳的PE载体；最大限度地提高目标 编辑和最小化非目标编辑；以及确定免疫反应如何影响编辑。目标1将 开发用于体内AAT基因校正的新型PE工具。将开发一个拆分式AAV PE平台，以最大限度地 体内最佳编辑效率，然后在PI*Z中测量PE基因校正和肺表型 转基因小鼠模型和临床相关的AAT Null/PI*Z小鼠模型。目标2将增强 ABE用于体内AAT基因纠正的特异性。长期的ABE表达可以诱导非靶点编辑。 因此，新的ABE变体将进行优化，以增加活性并减少RNA编辑效应，并将其拆分 AAV对安倍的投放将在PI*Z模型中进行调查。这一目标也将使自我失活的安倍发展到 减少偏离目标的影响。目标3将表征和缓解对PE和ABE的免疫反应，这两种病毒 病毒逆转录酶和细菌TADA蛋白，以及已知抗原Cas9。这一目标将 研究小鼠对PE和ABE的抗体和T细胞反应，免疫反应如何调节编辑，以及 CAR-Treg是否可以减轻免疫反应。项目2将受益于与 本P01中的其他项目和核心。该项目的完成将提高体育和体育的效率和安全性 ABE在体内，为AATD和其他单基因疾病提供了不依赖HDR的基因编辑蓝图。

PROJECT 2 - Project Summary/Abstract Gene editing has the potential to correct mutations and provide long-term therapeutic benefit for patients with rare monogenic diseases like alpha-1 antitrypsin deficiency (AATD). AATD is caused by mutations in the AAT (or PI) gene, which encodes a serine protease inhibitor that is made in hepatocytes and delivered to lung to neutralize neutrophil elastase. The PI*Z mutation encodes mutant Z-AAT protein that aggregates in hepatocytes, which can cause liver disease and reduces serum AAT. Reduced serum AAT causes progressive airway disease and emphysema. Gene correction would address both aspects of AATD. CRISPR-mediated homology directed repair (HDR) can be used to partially correct mutations in mouse liver. Yet, HDR is limited by the need to deliver a long DNA repair template, its inefficiency in non-dividing or slow- dividing cell types, and its generation of genotoxic double-strand breaks. To address these limitations, this proposal will develop two CRISPR-based gene correction strategies that do not require a double-strand break: prime editing and adenine base editing. Prime editor (PE) is comprised of Cas9 nickase fused to reverse transcriptase and an extended guide RNA that doubles as a template for reverse transcriptase to copy editing information into the genomic target. Adenine base editor (ABE), comprised of a Cas9 nickase fused to an adenosine deaminase, can correct G-to-A point mutations in mouse liver. The PI*Z allele results from a G-to-A mutation; and thus, is a good candidate for gene correction via ABE and PE. The goal of this project is to optimize PE and ABE tools for AAT gene correction in vivo by developing ABE and PE vectors that can be accommodated by adeno-associated virus (AAV) capsids; maximizing on-target editing and minimizing off-target editing; and determining how immune responses affect editing. Aim 1 will develop novel PE tools for in vivo AAT gene correction. A split AAV PE platform will be developed to maximize prime editing efficiency in vivo, then PE gene correction and lung phenotype will be measured in a PI*Z transgenic mouse model and a clinically-relevant AAT null/PI*Z mouse model. Aim 2 will enhance the specificity of ABE for in vivo AAT gene correction. Long-term ABE expression can induce off-target editing. Therefore, new ABE variants will be optimized to increase activity and reduce RNA editing effects, and split AAV delivery of ABE will be investigated in a PI*Z model. This Aim will also develop self-inactivating ABE to reduce off-target effects. Aim 3 will characterize and mitigate immune responses to PE and ABE, which harbor viral reverse transcriptase and bacterial TadA protein, respectively, and Cas9, a known antigen. This Aim will investigate antibody and T cell response to PE and ABE in mice, how immune response regulates editing, and whether CAR-Treg can mitigate immune response. Project 2 will benefit from extensive interactions with the other projects and cores in this P01. Completing this project will improve the efficiency and safety of PE and ABE in vivo, providing an HDR-independent gene editing blueprint for AATD, and other monogenic diseases.