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.

NARRATIVE Severe congenital neutropenia is a life-threatening hereditary deficiency of neutrophils, white blood cells that are the first line of defense against bacterial and fungal infections. The disease is most often due to mutations in the ELANE gene. We propose to apply novel gene editing technologies to develop a highly efficient, simple therapeutic approach to better understand and eventually treat ELANE-associated severe congenital neutropenia.