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

项目2 -项目概要/摘要 基因编辑有可能纠正突变，并为患者提供长期的治疗益处。 罕见的单基因疾病，如α-1抗胰蛋白酶缺乏症（AATD）。AATD是由AAT基因突变引起的。 (or PI）基因，其编码丝氨酸蛋白酶抑制剂，该丝氨酸蛋白酶抑制剂在肝细胞中产生并递送至肺， 中和中性粒细胞弹性蛋白酶。PI*Z突变编码突变体Z-AAT蛋白，其在细胞中聚集。 肝细胞，其可导致肝脏疾病并降低血清AAT。血清AAT降低导致进行性 呼吸道疾病和肺气肿。基因校正将解决AATD的两个方面。 CRISPR介导的同源定向修复（HDR）可用于部分纠正小鼠肝脏中的突变。 然而，HDR受限于递送长DNA修复模板的需要、其在非分裂或慢分裂中的低效率、以及其在非分裂或慢分裂中的低效率。 分裂细胞类型，以及其产生的遗传毒性双链断裂。为了解决这些局限性， 该提案将开发两种基于CRISPR的基因纠正策略，不需要双链断裂： 引物编辑和腺嘌呤碱基编辑。Prime editor（PE）由Cas9切口酶组成，Cas9切口酶融合到逆转录酶。 转录酶和一个扩展的指导RNA，该指导RNA兼作逆转录酶的模板以进行复制编辑 基因组靶点。腺嘌呤碱基编辑器（ABE），其由融合到多核苷酸的Cas9切口酶组成。 腺苷脱氨酶，可以纠正小鼠肝脏中的G到A点突变。PI*Z等位基因是由G到A 突变;因此，是通过ABE和PE进行基因校正的良好候选者。 本项目的目标是通过开发ABE优化PE和ABE工具用于体内AAT基因校正 和PE载体，其可被腺相关病毒（AAV）衣壳容纳;最大化靶向 编辑和最小化脱靶编辑;以及确定免疫应答如何影响编辑。目标1将 开发用于体内AAT基因校正的新型PE工具。将开发一个分离式AAV PE平台， 在体内启动编辑效率，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将加强 ABE对体内AAT基因校正的特异性。长期ABE表达可诱导脱靶编辑。 因此，新的ABE变体将被优化以增加活性并降低RNA编辑效应，并分裂 将在PI*Z模型中研究ABE的AAV递送。该目标还将开发自失活ABE， 减少脱靶效应。目的3将表征和减轻对PE和ABE的免疫应答， 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细胞应答，免疫应答如何调节编辑， 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。完成该项目将提高PE的效率和安全性， ABE在体内，为AATD和其他单基因疾病提供了HDR独立的基因编辑蓝图。

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.