CRISPR/Cas9 Mediated ELANE Knock-out Restores Survival and Granulocytic Differentiation of HL60 Cells Expressing Mutant Neutrophil Elastase: Is Neutrophil Elastase a Dispensible Granulocyte Protease?
Authors: Makaryan Vahagn , Archibald Isabella N. , Kelley Merideth L. , Fletcher Breanna , Dale David C. 
Published: February 2020

Background : Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease expressed primarily in neutrophils. Expression of the mutant protein impairs survival and maturation of myeloid precursors in bone marrow. More than 130 different ELANE mutations have been found in patients with cyclic and congenital neutropenia, and genotype-phenotype studies suggest that specific mutations cause more severe disease. (Curr Op Hematol. 2015;22:3-11) Mutant NE is also implicated as the primary cause of ELANE associated neutropenia by studies showing that cell permeable inhibitors of NE correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143).
Hypothesis: If inhibition of NE can correct the cellular defect, deletion of ELANE will have similar effects and permit studies to see if NE is an essential neutrophil protease.
Methods: We used CRISPR/Cas9 mediated gene editing to create a cellular model of congenital neutropenia in the commercially available human promyelocytic cell line, HL60. We used CRISPR/Cas9 editing technology to create engineered HL60 cell lines with knock-in (KI) ELANE P139L, C151Y and G214R single point heterozygous mutations. Using the same technology, we then performed ELANE gene complete knock out (KO) of ELANE in each mutant cell line. All engineered cell lines as well as wild-type HL60 cells were cultured for 5-7 days in complete RPMI supplemented with 2uM all-trans retinoic acid (ATRA) to trigger myeloid differentiation. Survival of these cell lines was investigated using Annexin V-PE staining and flow cytometric analysis. Granulocytic differentiation was evaluated using CD11b surface marker staining and flow cytometry and by performing manual differential cell counts. We measured the unfolded protein response (UPR) by western blotting using UPR specific antibodies, phagocytosis with E. coli particles using fluorescence detection, IL-8 stimulated chemotaxis in trans-well system and PMA activated respiratory burst by flow cytometry.
Results: Cells expressing the P139L and C151Y mutant ELANE had increased Annexin V staining more than a 2-fold increase in apoptotic cells at 7 days in culture. Granulocytic differentiation, measured by surface CD11b expression, was significantly impaired. (p&lt;0.0001). Cytospins stained with Diff-Quik showed a typical block of myeloid differentiation and a significant deficiency of mature neutrophils in 7-day cultures. Western blot analysis using antibodies to GRP78/BiP and ATF6 showed a typical UPR signature in both ELANE mutant cell lines compared to wild type. Chemotaxis to the IL8 chemo-attractant was severely impaired in cells expressing mutant NE. Respiratory burst and phagocytosis was also altered in cells expressing mutant NE. ELANE KO corrected all these cellular and functional abnormalities, reverting these functions toward the wild phenotype. Studies are in progress for the third cell line expressing the G214R mutation.
Conclusions: CRISPR/Cas9 engineered HL60 cell lines expressing mutant NE are a highly reproducible and reliable cellular model for investigating ELANE associated neutropenia. The presence of mutant NE severely impairs neutrophil functions, and its deletion through KO of ELANE corrects the cellular abnormalities and reverses the induced abnormalities. These results suggest that CRISPR/Cas9 mediated genetic knock down of NE is a novel therapeutic approach for treatment of this disorder.
This data also suggests that total absence of NE does not alter basic functions of granulocytes.

Disclosures
Dale: Sanofi Aventis: Consultancy, Honoraria; Athelas: Equity Ownership; Amgen: Consultancy, Research Funding; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer/Ingelheim: Consultancy; Coherus: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding.