Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

[Correlation study of NCF2 in chronic rhinosinusitis with nasal polyps].

Objective:After selecting NCF2 based on bioinformatics, clinical experiments were conducted to verify the expression of NCF2 in chronic rhinosinusitis with nasal polyps to study its correlation. Methods:The differentially expressed genes（DEGs） between CRSwNP and non-CRS patients were explored using the CRS-related dataset from the gene expression omnibus GEO database. The weighted gene co-expression network（WGCNA） was used for cluster analysis. The expression and cell distribution of NCF2 in the tissues were determined by single gene enrichment analysis（GSEA）, immune inflammatory infiltration analysis, and principal component（PCA） analysis. The expression degree of NCF2 in the tissues of the subjects was determined by immunohistochemistry, and the percentage of EOS in the peripheral blood of the subjects was detected and the correlation was analyzed. EOS in the tissues of the subjects were counted under a microscope and compared. Results:①The Venn diagram was obtained by crossing the module with the highest correlation between DEGs and WGCNA to determine the core gene NCF2. ②GSEA analysis showed that NCF2 was significantly related to the immunological processes such as allogeneic rejection and asthma. ③The area under the ROC curve was 1, indicating that NCF2 had diagnostic value for CRSwNP. ④NCF2 was highly expressed in nasal polyps, mainly distributed in monocytes and eosinophils. ⑤HE staining showed that the number of EOS in ECRSwNP tissues and the percentage of eosinophils in peripheral blood were higher than those in nonECRSwNP and control groups. ⑥The immunohistochemistry results showed that NCF2 was significantly expressed in the nasal polyps of ECRSwNP patients, which was higher than that in the nasal mucosa of nonECRSwNP group and control group. ⑦The expression of NCF2 in tissues was positively correlated with EOS count in ECRSwNP group and EOS expression in peripheral blood. Conclusion:The expression of NCF2 is increased in eosinophilic chronic rhinosinusitis with nasal polyps, and it is significantly correlated with the expression of eosinophils in peripheral blood and tissues, suggesting that NCF2 may be used as a basis for the intrinsic classification of ECRSwNP and a reference index for clinical diagnosis and treatment.

Clinical and molecular significance of flow cytometric analysis for reactive oxygen species production and residual p67phox expression in p67phox-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by molecular defects in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. p67phox-CGD is an autosomal recessive CGD, which is caused by a defect in the cytosolic components of NADPH oxidase, p67phox, encoded by NCF2. We previously established a flow cytometric analysis for p67phox expression, which allows accurate assessment of residual protein expression in p67phox-CGD. We evaluated the correlation between oxidase function and p67phox expression, and assessed the relevancy to genotypes and clinical phenotypes in 11 patients with p67phox-CGD. Reactive oxygen species (ROS) production by granulocytes was evaluated using dihydrorhodamine-1,2,3 (DHR) assays. p67phox expression was evaluated in the monocyte population. DHR activity and p67phox expression were significantly correlated (r = 0.718, p < 0.0162). Additionally, DHR activity and p67phox expression were significantly higher in patients carrying one missense variant in combination with one nonsense or frameshift variant in the NCF2 gene than in patients with only null variants. The available clinical parameters of our patients (i.e., age at disease onset, number of infectious episodes, and each infection complication) were not linked with DHR activity or p67phox expression levels. In summary, our flow cytometric analysis revealed a significant correlation between residual ROS production and p67phox expression. More deleterious NCF2 genotypes were associated with lower levels of DHR activity and p67phox expression. DHR assays and protein expression analysis by using flow cytometry may be relevant strategies for predicting the genotypes of p67phox-CGD.