Gene Editing vs Neutrophil Elastase Inhibitors for Treatment of ELANE Associated Neutropenia

项目摘要 中性粒细胞弹性蛋白酶（NE）基因ELANE的突变是两种周期性高血压的最常见原因。 中性粒细胞减少症（CyN）和严重先天性中性粒细胞减少症（SCN）。突变体ELANE的产物，突变体 嗜中性粒细胞弹性蛋白酶以显性的细胞内在方式破坏嗜中性粒细胞的形成。突变 酶通过未折叠蛋白反应（UPR）启动细胞凋亡。将突变体ELANE转染到a. 髓样细胞系HL 60细胞，导致髓样祖细胞成熟失败和细胞死亡， 凋亡来自SCN和CyN患者的骨髓原代髓系细胞或iPSC具有相同的缺陷， 中性粒细胞的形成、成熟和存活。 粒细胞集落刺激因子是一种非常有效的皮下治疗大多数患者，但有 对于它会增加患者发生MDS/AML的风险的担忧挥之不去。一种细胞可渗透的， 中性粒细胞弹性蛋白酶的口服活性抑制剂和敲低突变基因的表达可以纠正 ELANE相关的中性粒细胞减少症在这种疾病的实验室模型。然而，这些令人兴奋的发现是 基于在SCN和CyN患者中发现的130多种已知的ELANE突变中的几种。 这是非常重要的，以显示药理学或遗传沉默是有效的整个频谱， 突变，因为基因型-表型研究和临床观察表明，一些突变是远 比其他突变更严重。拟议的研究将利用两种细胞模型，iPSC和HL 60 细胞，以显示每种策略的潜在优点和缺点，重点是疾病的严重程度 与突变有关。 目的：加速SCN和CyN的新疗法的开发。 具体目标： 目标1.在表达更广泛的CRISPR/Cas9基因的HL 60细胞系中产生CRISPR/Cas9介导的突变ELANE敲入（KI）， 突变ELANE的多样性。在这些细胞系中进行CRISPR/Cas9敲除（KO）。比较有效性 MK-0339与KO的相互作用促进细胞增殖、存活和分化， 突变，例如，根据突变类型、群体频率和相关临床严重程度进行选择。 目标二。从具有不同ELANE突变的SCN/CyN患者中创建额外的iPSC系，并产生 在这些细胞系中，CRISPR/Cas9介导的ELANE KO。比较KO与MK-0339的有效性，以纠正 这些细胞系中细胞增殖、成熟和存活的缺陷。 目标3：比较ELANE KO与MK-0339产生的中性粒细胞的功能特性。

PROJECT SUMMARY Mutations in ELANE, the gene for neutrophil elastase (NE), are the most frequent cause of both cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). The product of mutant ELANE, mutant neutrophil elastase, acts in a dominant, cell-intrinsic fashion to disrupt neutrophil formation. The mutant enzyme initiates apoptosis via the unfolded protein response (UPR). Transfection of mutant ELANE into a myeloid cell line, HL60 cells, leads to a failure of maturation of myeloid progenitor cells and cell death by apoptosis. Bone marrow primary myeloid cells or iPSCs from patients with SCN and CyN have the same defect in neutrophil formation, maturation and survival. Granulocyte colony-stimulating factor is a very effective subcutaneous treatment for most patients, but there are lingering concerns about its contribution to the patients' risk of developing MDS/AML. A cell permeable, orally active inhibitor of neutrophil elastase and knocking down expression of the mutant gene can correct ELANE associated neutropenia in laboratory models of this disorder. However, these exciting findings are based on testing only a few of more than 130 known mutations in ELANE found in patients with SCN and CyN. It is very important to show that pharmacological or genetic silencing is effective across the spectrum of mutants, because genotype-phenotype studies and clinical observations indicate that some mutations are far more serious than other mutations. The proposed studies will utilize two cellular models, iPSCs and HL60 cells, to show potential advantages and disadvantage of each strategy focusing on the severity of disease associated with the mutations. Goal: To hasten development of novel therapies for SCN and CyN. Specific Aims: Aim 1. Generate CRISPR/Cas9 mediated mutant ELANE knock-ins (KI) in HL60 cell lines expressing a wider diversity of mutant ELANEs. Perform CRISPR/Cas9 knock-out (KO) in these lines. Compare the effectiveness of MK-0339 versus KO to promote cell proliferation, survival and differentiation across the spectrum of these mutations, e.g., selected by type of mutation, frequency in populations and associated clinical severity. Aim 2. Create additional iPSC lines from SCN/CyN patients with diverse ELANE mutations and generate CRISPR/Cas9 mediated ELANE KO in these lines. Compare effectiveness of KO versus MK-0339 to correct defects in cell proliferation, maturation and survival in these cell lines. Aim 3. Compare the functional properties of neutrophils produced by ELANE KO versus MK-0339.