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)是一种威胁生命的疾病，最常见的原因是生殖系。 编码中性粒细胞弹性蛋白酶的Elane基因突变。主导作用的Elane突变保留了 表达但改变中性粒细胞弹性蛋白酶蛋白结构，导致蛋白折叠和/或运输改变 有过多的髓细胞死亡。基因编辑技术的最新进展使有针对性的基因 造血干细胞的修饰。我们假设提前终止密码子的引入 (PTCs)通过核酸酶介导的Elane早期外显子内的移码可以构成一种普遍的、高度的 Elane相关性SCN的有效、简单的治疗方法。我们预测PTCS将触发 无意义介导的衰退(NMD)导致表达缺失，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来引入Cas9和引导RNA(SgRNA)AS 核糖核蛋白(RNP)与CD34造血干细胞和祖细胞(HSPC)的复合物，与近 完整的靶向编辑，保存的造血干细胞(HSC)功能，以及无法检测到的脱靶 正在编辑。我们建议对SCN患者的HSCs进行高效和特异的编辑，作为一种 用于阐明Elane突变SCN的分子病理生物学和作为潜在治疗手段的工具 情态。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 功能后果将是轻微的，就像Papillon Lefevre综合征(PLS)-以损失为特征不是 只有中性粒细胞弹性蛋白酶，以及抗菌肽C、蛋白水解酶3和丝氨酸蛋白水解酶4-仅与 临床上无明显免疫缺陷的牙周炎。我们提出了以下具体目标： 1.优化了Cas9 RNP在CD34 HSPC中的Elane治疗性编辑。1A.确定最有利的地点 Elane for Cas9编辑以制作PTCS。1B.最大限度地提高Cas9编辑的特异性，包括通过以下方式进行评估 全基因组偏离目标的评估。1C。调查Elane编辑对HSC功能的影响 异种移植试验。1D。比较健康供者和Elane突变CD34 HSPC的编辑结果。 2.确定Elane移码突变对mRNA稳定性、蛋白功能和中性粒细胞的影响 前驱应激反应。2A。测量信使核糖核酸的稳定性和翻译。2B。测量蛋白质功能和 贩卖人口。2C。确定细胞对Elane移码突变的反应。 3.测定Elane缺乏性中性粒细胞的功能活性。3A.评估潜在的蛋白酶- 依从性功能包括Elane定位、颗粒含量和脱颗粒、网织、趋化、 和释放细胞激动素。3B.评估可能的不依赖于蛋白酶的功能，包括氧化酶活性， 吞噬和微生物杀灭。3C。比较Elane缺乏型和PLS型中性粒细胞的功能。 这些研究的成功完成将阐明Elane在心脏疾病中的生理和病理作用。 中性粒细胞的功能和发育，并使SCN的新治疗方法的开发成为可能。

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.