Gene editing ELANE to understand and treat severe congenital neutropenia

基因编辑 ELANE 了解和治疗严重先天性中性粒细胞减少症

基本信息

批准号：
10580862
负责人：
Daniel Evan Bauer
金额：
$ 76.61万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10580862
关键词：
Abbreviations Acute Myelocytic Leukemia Affect Alleles Bacterial Infections Biological Assay Bypass CD34 gene Cell Cycle Cell Death Chemotaxis Colony-Stimulating Factor Therapy Competence Complex Cytoplasmic Granules Dependence Development Disease Dysmyelopoietic Syndromes Engineering Etiology Evaluation Exons Frameshift Mutation Functional disorder Genes Genetic Germ-Line Mutation Granulocyte Colony-Stimulating Factor Guide RNA Hematological Disease Hematopoietic stem cells Immunologic Deficiency Syndromes Individual Inherited Injections Leukocyte Elastase Leukocytes Life Measures Mediating Messenger RNA Methods Minor Modality Modification Molecular Mutation Mycoses Myeloid Cells Neutropenia Nonsense-Mediated Decay Outcome Oxidases Papillon-Lefevre Disease Pathogenicity Pathologic Pathway interactions Patients Peptide Hydrolases Periodontitis Phagocytosis Physiological Progranulocytes Proteinase 3 Proteins Protocols documentation Regulation Ribonucleoproteins Role Serine Protease Site Specificity Structure Technology Terminator Codon Therapeutic Xenograft procedure biological adaptation to stress clinically significant curative treatments design genome-wide granulocyte high risk homologous recombination insertion/deletion mutation mRNA Stability mRNA Translation microbial mutant neutrophil novel novel therapeutics nuclease precursor cell premature preservation protein folding protein function protein structure protein transport repaired response stem cell function therapeutic genome editing tool

项目摘要

ABSTRACT Severe congenital neutropenia (SCN) is a life-threatening disorder due most commonly to germline mutation of the ELANE gene, encoding neutrophil elastase. Dominantly acting ELANE mutations preserve expression but alter neutrophil elastase protein structure resulting in altered protein folding and/or trafficking with excess myeloid cell death. Recent advances in gene editing technologies have enabled targeted genetic modification of hematopoietic stem cells. We hypothesize that introduction of premature termination codons (PTCs) by nuclease-mediated frameshifts within early exons of ELANE could constitute a universal, highly efficient, simple therapeutic approach for ELANE-associated SCN. We predict that the PTCs would trigger nonsense-mediated decay (NMD) resulting in loss of expression to circumvent neutrophil precursor cell death. We have recently developed highly efficient methods for the introduction of Cas9 and guide RNA (sgRNA) as ribonucleoprotein (RNP) complexes to CD34+ hematopoietic stem and progenitor cells (HSPCs), with nearly complete on-target editing, preserved hematopoietic stem cell (HSC) function, and undetectable off-target editing. We propose to develop highly efficient and specific editing of HSCs from patients with SCN, both as a tool for elucidation of the molecular pathobiology of ELANE-mutant SCN and as a potential therapeutic modality. This editing strategy would result in neutrophils deficient for neutrophil elastase, but we hypothesize that the functional consequences will be minor, as Papillon Lefevre syndrome (PLS) – featuring the loss not only of neutrophil elastase but also cathespin C, proteinase 3, and serine protease 4 – is associated only with periodontitis without clinically significant immunodeficiency. We propose the following specific aims: 1. Optimize ELANE therapeutic editing by Cas9 RNP in CD34+ HSPCs. 1a. Define the most favorable sites in ELANE for Cas9 editing to produce PTCs. 1b. Maximize Cas9 editing specificity, including evaluation by genome-wide off-target assessment. 1c. Investigate impact of ELANE editing on HSC function as measured by xenograft assay. 1d. Compare outcomes of editing on healthy donor and ELANE mutant CD34+ HSPCs. 2. Determine effects of ELANE frameshift mutation on mRNA stability, protein function, and neutrophil precursor stress response. 2a. Measure mRNA stability and translation. 2b. Measure protein function and trafficking. 2c. Determine cellular response to frameshift mutations of ELANE. 3. Determine the functional competence of ELANE-deficient neutrophils. 3a. Assess potential protease- dependent functions including ELANE localization, granule content and degranulation, NETosis, chemotaxis, and release of cyokines. 3b. Assess likely protease-independent functions including oxidase activity, phagocytosis and microbial killing. 3c. Compare the function of ELANE-deficient to PLS neutrophils. Successful completion of these studies would illuminate the physiologic and pathologic roles of ELANE in neutrophil function and development and enable the development of novel curative therapies for SCN.

抽象的严重的先天性中额（SCN）是一种危及生命的疾病 Elane基因的突变，编码中性粒细胞弹性酶。主动表演岩石突变保留表达但会改变中性粒细胞弹性酶蛋白质结构，从而改变蛋白质折叠和/或运输超过髓样细胞死亡。基因编辑技术的最新进展已实现目标通用修饰造血干细胞。我们假设引入过早终止密码子（PTC）通过核酸酶介导的埃莱恩早期外显子内的移架可能构成通用，高度高效的，简单的治疗方法，用于Elane相关的SCN。我们预测PTC会触发废话介导的衰减（NMD）导致表达丧失，以绕过中性粒细胞前体细胞死亡。我们最近开发了高效的方法，用于引入Cas9和引导RNA（SGRNA）作为核糖核蛋白（RNP）复合物至CD34+造血茎和祖细胞（HSPC），几乎具有完整的目标编辑，保留的造血干细胞（HSC）功能和不可检测的靶向编辑。我们建议从SCN患者中开发高效和特定的HSC的编辑，均为阐明Elane突变SCN分子病理学的工具，并作为潜在疗法方式。这种编辑策略将导致中性粒细胞缺乏中性粒细胞弹性酶，但我们假设作为Papillon Lefevre综合征（PLS），功能后果将很小仅中性粒细胞弹性蛋白酶，但也仅由cathespin c，蛋白酶3和丝氨酸蛋白酶4 - 仅与牙周炎没有临床明显的免疫缺陷。我们提出以下具体目标： 1。通过CAS9 RNP在CD34+ HSPC中优化Elane治疗编辑。 1a。定义最有利的网站 Cas9编辑的Elane生成PTC。 1B。最大化CAS9编辑特异性，包括通过全基因组脱离目标评估。 1C。研究Elane编辑对HSC功能的影响异种移植分析。 1d比较在健康供体和Elane突变体CD34+ HSPC上进行编辑的结果。 2。确定Elane Frameshift突变对mRNA稳定性，蛋白质功能和中性粒细胞的影响前体应力反应。 2a。测量mRNA稳定性和翻译。 2b。测量蛋白质功能和贩运。 2C。确定细胞对Elane的移码突变的反应。 3。确定贫血缺陷性嗜中性粒细胞的功能能力。 3a。评估潜在蛋白酶 - 依赖功能，包括浮力定位，颗粒含量和脱粒，Netosis，趋化性，趋化性，并释放氰基。 3b。像蛋白酶非依赖性功能一样评估，包括氧化酶活性，吞噬作用和微生物杀伤。 3C。比较Elane缺乏症的功能与PLS中性粒细胞。这些研究的成功完成将阐明Elane在中性粒细胞功能和发育，并使SCN的新型治疗疗法的发展。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Dissecting ELANE neutropenia pathogenicity by human HSC gene editing.

DOI：
10.1016/j.stem.2020.12.015
发表时间：
2021-05-06
期刊：
Cell stem cell
影响因子：
23.9
作者：
Rao S;Yao Y;Soares de Brito J;Yao Q;Shen AH;Watkinson RE;Kennedy AL;Coyne S;Ren C;Zeng J;Serbin AV;Studer S;Ballotti K;Harris CE;Luk K;Stevens CS;Armant M;Pinello L;Wolfe SA;Chiarle R;Shimamura A;Lee B;Newburger PE;Bauer DE
通讯作者：
Bauer DE