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

• 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
• 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; 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

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者提供长期治疗益处 罕见的单基因疾病，例如α-1抗胰蛋白酶缺乏症（AATD）。 AATD是由AAT突变引起的 （或PI）基因，该基因编码肝细胞中制成的丝氨酸蛋白酶抑制剂，并递送至肺部 中和中性粒细胞弹性酶。 PI*Z突变编码聚集在聚集的突变Z-AAT蛋白 肝细胞，可能导致肝病并降低血清AAT。减少血清AAT会导致渐进式 气道疾病和肺气肿。基因校正将解决AATD的两个方面。 CRISPR介导的同源性修复（HDR）可用于部分纠正小鼠肝脏中的突变。 然而，HDR受到提供长的DNA修复模板的需求，其在非分裂或慢速方面的效率低下 分裂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，这 建议将制定两个基于CRISPR的基因校正策略，这些策略不需要双链断裂： 主要编辑和腺嘌呤基础编辑。 Prime Editor（PE）由CAS9 Nickase组成，以逆转 转录酶和扩展指南RNA，它是逆转录酶复制编辑酶的模板的翻倍 信息进入基因组靶标。腺嘌呤基本编辑（ABE），由Cas9 nickase组成 腺苷脱氨酶可以纠正小鼠肝脏中的G-TO-A点突变。 pi*z等位基因由g-to-a产生 突变;因此，是通过ABE和PE进行基因校正的良好候选者。 该项目的目的是通过开发ABE优化体内AAT基因校正的PE和ABE工具 以及可以通过腺相关病毒（AAV）衣壳来容纳的PE载体；最大化目标 编辑和最小化脱靶编辑；并确定免疫反应如何影响编辑。目标1意志 开发用于体内AAT基因校正的新型PE工具。将开发一个拆分的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 转基因小鼠模型和与临床相关的AAT NULL/PI*Z小鼠模型。 AIM 2将增强 ABE在体内AAT基因校正方面的特异性。长期的安倍表达可以引起靶向编辑。 因此，将优化新的ABE变体以增加活动并降低RNA编辑效果，并分裂 AAV的ABE交付将在PI*Z模型中进行研究。这个目标还将发展为自动化的安倍 减少脱靶效应。 AIM 3将表征并减轻对PE和安倍的免疫反应 病毒逆转录酶和细菌TADA蛋白和已知抗原的Cas9。这个目标 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 该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 在体内安倍，为AATD和其他单基因疾病提供独立于HDR的基因编辑蓝图。