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

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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.