Page 94 - Haematologica March 2020

M. Nasri et al.
including also cathepsin G (CG), proteinase 3 (PR3) and azurocidin (AZU1). NSP are stored in cytoplasmic gran- ules, can be secreted into the extra- and peri-cellular space upon cellular activation and considered to be crucially involved in bacterial defense. NE and PR3 are very similar in their substrate specificity supporting a potentially redundant function for these two enzymes. Elane-/- mice have normal neutrophil counts, but there are conflicting results regarding the effect of NE-deficiency on neutrophil extravasation to sites of inflammation, phagocytosis, and neutrophil extracellular traps in mice. NE may or may not be essential for these processes.29-33 Papillon-Lefevre Syndrome (PLS) is the only human disorder known to cause NE deficiency. This rare autosomal recessive disease is due to loss-of-function mutations in the DPPI gene locus with the loss of the lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI). The activation of NSP, including NE, depends on the N-terminal processing activ- ity of DPPI. Therefore, PLS patients exhibit a severe reduc- tion in the activity and stability of all three NSP including NE. Intriguingly, patients with PLS have no defects in their ability to kill bacteria e.g. Staphylococcus aureus or E.coli, suggesting that redundancies in the neutrophil’s bacterici- dal mechanisms negate the necessity for serine proteases for killing common bacteria.34 Moreover, since the other serine proteases including CG, PR3 and neutrophil serine protease 4 remain intact, we do not expect for the result- ant cells to develop any neutrophil-specific functional anti-bacterial or immunodeficiency phenotype in ELANE KO cells. Based on these observations, at this juncture, we believe that CRISPR/Cas9 based knockout of ELANE in HSPC of CN patients may restore defective granulopoiesis in CN patients without seriously impairing neutrophil functions. Further studies, including gene expression analysis to understand which pathways are affected by ELANE mutations and verifying that these pathways are indeed restored by ELANE KO, are essential to justify the therapeutic applications of ELANE KO technology in the future. It will also contribute to the understanding of the pathophysiology of the CN caused by ELANE mutations. Our first attempts to investigate intracellular signaling pathways affected by mutated ELANE revealed the restoration of mRNA expression of anti-apoptotic Bcl-xl factor that is downregulated in CN myeloid progenitor cells.35 Moreover, we found downregulation of mRNA lev- els of the key UPR player BiP, normally upregulated in HSPC of CN patients harboring ELANE mutations.8-10
CRISPR/Cas9 technology also allowed correction of the specific gene mutations. We selected the ELANE knockout approach since ELANE mutations are heterozygous gain- of-function gene defects that are distributed throughout all five exons and two introns of ELANE and specific ELANE mutations correction would require specific set- tings for each patient based on the mutation position. In addition, the requirement of the introduction of the donor repair template DNA in a gene correction approach
requires the activation of HDR making it difficult to achieve efficient correction in primary HSPC. We and other investigators have reported that ELANE mutations induce UPR and ER stress.8-11 We also described deregulat- ed signaling pathways in HSPC of CN patients down- stream of ELANE mutations, such as diminished expres- sion of transcription factors LEF-1, and C/EBPα,36−39 abro- gated expression and phosphorylation of the adaptor pro- tein HCLS1,40 elevated apoptosis35 and hyperactivated NAMPT/sirtuins.41 These intracellular defects may lead to the elevated fragility of HSPC during ex vivo gene manipu- lations and may affect gene correction efficiency. Moreover, gene editing strategy directed to the correction of ELANE mutations may lead to the creation of novel missense or frameshift mutations that may result in the novel mutant NE protein with damaging functions and potential generation of the pre-leukemic HSPC clones with proliferative advantage and possible leukemic trans- formation. Adeno-associated virus (AAV)-based vector may be used for the delivery of the donor repair template and is considered safer than retroviral constructs. Two groups recently published successful gene deletions as a gene therapy approach to cure sickle cell disease, a com- mon inherited blood disorder.17,18 It should be noted that AAV-based expression constructs may induce anti-viral host immune responses and may non-specifically inte- grate into the host genome.
In summary, we report here for the first time a method of CRISPR/Cas9 mediated ELANE gene deletion in hematopoietic stem cells and iPSC from CN patients har- boring ELANE mutations. The ELANE gene deletion resulted in the increase of granulocytic differentiation to functional normal mature neutrophils in these patients in vitro. Therefore, CRISPR/Cas9 based gene knockout of ELANE in CN patients harboring ELANE mutation might be a useful treatment option especially in patients requir- ing high G-CSF dosages or do not responding to G-CSF at all. In addition, it remains to be investigated in subsequent clinical studies whether, in CN patients harboring ELANE mutations, the ELANE gene knockout would also abrogate the leukemogenesis.
Acknowledgments
We would like to thank the FACS core facility of the UKT especially Stella Autenrieth for the assistance in flow cytometry; Michael Schindler and Esther Lehmann for their support in con- focal microscopy. This work was supported by the Excellence Initiative of the Faculty of Medicine, University of Tübingen (JS), Jose Carreras Leukemia Foundation (JS, PM, MR), Madeleine Schickedanz Kinderkrebsstiftung (JS, MK, MN), DFG (JS, MK), intramural Fortüne program of the Medical Faculty of the Tübingen University (MK, DA, YX), German-Israeli Foundation for Scientific Research and Development (GIF) (KW, BD), German Cancer Consortium (PM), Else Kröner-Fresenius Stiftung (MK, BD), Fritz Thyssen Foundation (MR and BD), NIH R24 AI 049393 (VM, BF, CZ, DCD).
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