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

使用新颖的碱基编辑和 Prime 编辑技术编辑 AAT 基因座

基本信息

批准号：
10463808
负责人：
Wen Xue
金额：
$ 54.44万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-09 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10463808
关键词：
Address Adenine Adult Affect Airway Disease Alleles Animal Model Antibodies Antigens Blood Circulation Candidate Disease Gene Capsid Clustered Regularly Interspaced Short Palindromic Repeats DNA Repair Deaminase Dependovirus Disease Disease model Endonuclease I Engineering GTP-Binding Protein alpha Subunits, Gs Generations Genes Genomics Goals Guanosine Guide RNA Hepatocyte Human Genetics Immune Immune response Immunology Individual Inosine Leukocyte Elastase Liver Liver diseases Lung Measures Mediating Mendelian disorder Modeling Mus Mutation Organ Patients Phenotype Physiology Point Mutation Polymers Proteins Pulmonary Emphysema RNA Editing RNA-Directed DNA Polymerase Regulatory T-Lymphocyte Safety Serine Proteinase Inhibitors Serum Specificity T cell response Technology Therapeutic Transgenic Mice Variant Viral Work adenosine deaminase alpha 1-Antitrypsin Deficiency base base editing base editor cell type clinically relevant gene correction gene therapy genotoxicity improved in vivo mouse model mutant novel pre-clinical prime editing prime editor protein aggregation repaired response synergism tool vector

项目摘要

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.

项目 2 - 项目摘要/摘要基因编辑有可能纠正突变并为患有以下疾病的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 突变引起的（或 PI）基因，编码丝氨酸蛋白酶抑制剂，在肝细胞中产生并输送到肺中和中性粒细胞弹性蛋白酶。 PI*Z 突变编码突变 Z-AAT 蛋白，该蛋白聚集在肝细胞，可引起肝脏疾病并降低血清 AAT。血清 AAT 降低导致进行性气道疾病和肺气肿。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 受到需要提供长 DNA 修复模板的限制，其在非分裂或缓慢修复方面效率低下。分裂细胞类型及其基因毒性双链断裂的产生。为了解决这些限制，这该提案将开发两种基于 CRISPR 的基因校正策略，不需要双链断裂：底物编辑和腺嘌呤碱基编辑。 Prime editor (PE) 由融合反向的 Cas9 切口酶组成转录酶和扩展向导RNA，可兼作逆转录酶复制编辑的模板信息进入基因组目标。腺嘌呤碱基编辑器 (ABE)，由融合到腺苷脱氨酶，可以纠正小鼠肝脏中的 G 到 A 点突变。 PI*Z 等位基因是 G 到 A 的结果突变;因此，它是通过 ABE 和 PE 进行基因校正的良好候选者。该项目的目标是通过开发ABE来优化用于AAT基因体内校正的PE和ABE工具和可以被腺相关病毒（AAV）衣壳容纳的PE载体；最大化目标编辑并尽量减少脱靶编辑；并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。将开发一个分体式 AAV PE 平台，以最大化体内的主要编辑效率，然后将在 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 对体内 AAT 基因校正的特异性。长期 ABE 表达会导致脱靶编辑。因此，新的ABE变体将被优化以增加活性并减少RNA编辑效应，并分裂 AAV 的 ABE 传递将在 PI*Z 模型中进行研究。该目标还将开发自失活 ABE 减少脱靶效应。目标 3 将表征并减轻对 PE 和 ABE 的免疫反应，其中包含分别是病毒逆转录酶和细菌TadA蛋白以及已知抗原Cas9。这个目标将研究小鼠中抗体和 T 细胞对 PE 和 ABE 的反应，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。完成该项目将提高PE和PE的效率和安全性体内 ABE，为 AATD 和其他单基因疾病提供了独立于 HDR 的基因编辑蓝图。