Page 92 - Haematologica March 2020

M. Nasri et al.
CN. We tested ex vivo CRISPR/Cas9 RNP-based ELANE knockout in HSPC of CN patients that may be used for autologous transplantation as a therapeutic approach for ELANE-CN patients. Virus- and DNA-free application of CRISPR/Cas9 RNP markedly increases gene editing effi- ciency and simultaneously decreases the probability and frequency of off-target effects, because CRISPR/Cas9 RNP activity is preserved in cells for only approximately 48 hours. We recently reported the establishment of the flu- orescent labeling of CRISPR/Cas9 RNP complexes for gene editing of primary hematopoietic stem cells and sub- sequent sorting of gene-modified cells for further applica- tions.26 Implementation of this method will improve the efficiency of gene knockout or gene correction in HSPC, including ELANE knockout or correction of ELANE muta- tions.
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In case of ELANE KO, different combinations of the ELANE gene editing are expected: we may generate unedited, monoallelic edited (of mutated or WT allele), or bi-allelic edited HSPC. Since we did not use any selection marker for edited HSPC, we were not able to estimate the proportion of HSPC with inactivation of the mutated ELANE allele. The fact that the proportion of ELANE KO cells was elevated upon granulocytic differentiation strongly argues for the differentiation advantage of the edited cells lacking ELANE (including loss of the mutated allele).
There are several potential unforeseen consequences of the ELANE gene knockout strategy. For example, Tidwell et al. reported the presence of two in-frame ATG codons in exon 2 and exon 4 of ELANE.27 They showed that the internal translation of NE can be initiated when the canon-
Figure 7. Unaffected functions of ELANE KO neu- trophils. (A, B, C) Reactive oxygen species (ROS) assay (A) and phagocytosis assay (B, C) of granulo- cytes generated on day 14 of liquid culture, as described in the Methods section. Data represent means ± SD from duplicates. *P<0.05, **P<0.01, ***P<0.0001. (C) Phagocytosis Kinetic using IncuCyte ZOOM System of granulocytes generated on day 14 of liquid culture as described in the Methods section. Data represent mean ± standard deviation (SD). (D) Chemotaxis depicted as fold change between fMLP-treated and untreated gran- ulocytes generated on day 14 of liquid culture. Data represents mean ± SD, healthy control (ctrl) (n=2), CN patient (n=1).
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