Gene editing ELANE to understand and treat severe congenital neutropenia

摘要 严重先天性中性粒细胞减少症（SCN）是一种危及生命的疾病，最常见于种系 ELANE基因突变，编码中性粒细胞弹性蛋白酶。显性作用的ELANE突变保留 表达，但改变中性粒细胞弹性蛋白酶蛋白结构，导致改变的蛋白质折叠和/或运输 骨髓细胞过度死亡基因编辑技术的最新进展使靶向遗传学成为可能。 造血干细胞我们假设提前终止密码子的引入 在ELANE的早期外显子内通过核酸酶介导的移码来检测PTCs，可以构成一个通用的、高度保守的基因组。 ELANE相关SCN的有效、简单的治疗方法。我们预测PTC会触发 无义介导的衰变（NMD）导致表达丧失以避免中性粒细胞前体细胞死亡。 我们最近开发了用于引入Cas9和向导RNA（sgRNA）的高效方法， 核糖核蛋白（RNP）复合物与CD 34+造血干细胞和祖细胞（HSPC）， 完整的靶向编辑，保留的造血干细胞（HSC）功能和不可检测的脱靶 编辑.我们建议开发来自SCN患者的HSC的高效和特异性编辑， 用于阐明ELANE突变SCN的分子病理学的工具，并作为潜在的治疗方法 模态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，但我们假设 功能性后果将是轻微的，如Papillon Lefevre综合征（PLS）-以丧失功能为特征， 不仅中性粒细胞弹性蛋白酶，而且组织蛋白酶C，蛋白酶3和丝氨酸蛋白酶4 -仅与 牙周炎无临床显著免疫缺陷。我们提出以下具体目标： 1.在CD 34 + HSPC中通过Cas9 RNP优化ELANE治疗性编辑。1a.定义最有利的站点， ELANE用于Cas9编辑以产生PTC。1b.使Cas9编辑特异性最大化，包括通过 全基因组脱靶评估。1c.研究ELANE编辑对HSC功能的影响，如通过 异种移植物测定。1d.比较健康供体和ELANE突变体CD 34 + HSPC上的编辑结果。 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 前兆应激反应2a.测量mRNA稳定性和翻译。2b.测量蛋白质功能， 贩卖人口2c.确定细胞对ELANE移码突变的反应。 3.确定ELANE缺陷中性粒细胞的功能能力。3a.评估潜在的蛋白酶- 依赖性功能包括ELANE定位、颗粒含量和脱粒、NETosis、趋化性， 和细胞因子的释放。3b.评估可能的蛋白酶非依赖性功能，包括氧化酶活性， 吞噬作用和微生物杀灭。3c.比较ELANE缺陷型与PLS中性粒细胞的功能。 这些研究的成功完成将阐明ELANE的生理和病理作用， 中性粒细胞的功能和发育，并使新的治疗性疗法的SCN的发展。

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.