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ELANE knockout restores granulopoiesis in congenital neutropenia
PMN, compared to cells carrying mutated ELANE. As expected, ELANE mRNA levels were severely diminished in ELANE KO cells (Online Supplementary Figure S11).
Discussion
The majority of patients suffering from congenital neu- tropenia respond well to daily treatment with rhG-CSF leading to a normal quality of life. However, in the last 25 years, we learned that CN is a preleukemic syndrome and that approximately 15 % of patients do not respond to
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even ultra-high dosages (>50 mg/kg/d) of rhG-CSF. Therefore, we are searching for other treatment modali- ties for CN patients that may prevent leukemic transfor- mation and may be useful for those who are requiring high dosages of rhG-CSF or not responding at all to rhG-CSF. For these patients, the only available treatment is stem cell transplantation with the risk of transplant- associated adverse events such as acute or chronic GvHD.
In the present study, we described for the first time the establishment of an in vitro cellular model of CRISPR/Cas9 mediated gene therapy of CN associated with autosomal dominant ELANE mutations, the most frequent cause of
Figure 6. ELANE KO restored granulocytic differ- entiation of ELANE-CN primary HSPC. (A) Differentiation capacity of ELANE knockout CD34+ cells was assessed by liquid culture differ- entiation after 14 days by investigating neu- trophilic surface marker expression. Data repre- sent means ± standard deviation (SD) from tripli- cates. *P<0.05, **P<0.01, ****P<0.0001. (B) Wright-Giemsa staining of differentiated cells was conducted on day 14 allowing morphologic discrimination of the cells. Representative images are depicted. (C-D) Electron micrographs of neutrophils generated on day 14 of liquid cul- ture analyzed by scanning electron microscopy (SEM) (C) and transmission electron microscopy (TEM) (D). Representative SEM and TEM images are depicted. Typical neutrophil morphology is observed in all studied samples.
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haematologica | 2020; 105(3)
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