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：
10338097
负责人：
Daniel Evan Bauer
金额：
$ 73.13万
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 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10338097
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 Genetic Translation 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 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 trafficking

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.

抽象的严重先天性中性粒细胞减少症 (SCN) 是一种危及生命的疾病，最常见的原因是种系编码中性粒细胞弹性蛋白酶的 ELANE 基因突变保留了显性作用的 ELANE 突变。表达但改变中性粒细胞弹性蛋白酶蛋白质结构，导致蛋白质折叠和/或运输基因编辑技术的最新进展使得靶向基因成为可能。我们着迷于造血干细胞的提前终止密码子的引入。 ELANE 早期外显子内核酸酶介导的移码（PTC）可能构成一种普遍的、高度我们预测 PTC 将触发 ELANE 相关 SCN 的有效、简单的治疗方法。无义介导的衰变（NMD）导致表达缺失，从而避免中性粒细胞前体细胞死亡。我们最近开发了高效的方法来引入 Cas9 和向导 RNA (sgRNA) 核糖核蛋白 (RNP) 与 CD34+ 造血干细胞和祖细胞 (HSPC) 形成复合物，几乎完整的靶向编辑，保留造血干细胞（HSC）功能，并且无法检测到脱靶我们建议对来自 SCN 患者的 HSC 进行高度、特异的高效编辑，作为一种方法。阐明 ELANE 突变 SCN 分子病理学的工具并作为潜在的治疗方法这种编辑策略会导致中性粒细胞缺乏中性粒细胞弹性蛋白酶，但我们感兴趣功能性后果将是轻微的，如 Papillon Lefevre 综合征 (PLS) – 其特点是损失不仅与中性粒细胞弹性蛋白酶有关，还与组织蛋白酶 C、蛋白酶 3 和丝氨酸蛋白酶 4 相关 - 仅与我们提出以下具体目标： 1. 在 CD34+ HSPC 中通过 Cas9 RNP 优化 ELANE 治疗编辑 1a。用于 Cas9 编辑以产生 PTC 的 ELANE 最大化 Cas9 编辑特异性，包括评估。 1c. 研究 ELANE 编辑对 HSC 功能的影响。 1d. 比较健康供体和 ELANE 突变体 CD34+ HSPC 的编辑结果。 2.确定ELANE移码突变对mRNA稳定性、蛋白质功能和中性粒细胞的影响 2a. 测量 mRNA 稳定性和翻译。 2c.确定细胞对ELANE移码突变的反应。 3. 确定 ELANE 缺陷的中性粒细胞的功能能力 3a。依赖功能，包括 ELANE 定位、颗粒含量和脱颗粒、NETosis、趋化性、 3b. 评估可能的蛋白酶独立功能，包括氧化酶活性， 3c. 比较 ELANE 缺陷型和 PLS 中性粒细胞的吞噬作用和杀灭作用。这些研究的成功完成将阐明 ELANE 在中性粒细胞功能和发育，促进 SCN 新型治疗方法的开发。