PDF(Pubmed)
Abstract：
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.
摘要：
p47phox缺陷型慢性肉芽肿病（p47-CGD）是由中性粒细胞胞质因子1（NCF1）基因突变引起的原发性免疫缺陷，导致吞噬细胞中NADPH氧化酶功能缺陷。由于其复杂的基因组背景，NCF1基因座不适合使用当前的基因组编辑技术进行安全的基因编辑。因此,我们通过CRISPR-Cas9核糖核蛋白和病毒载体模板递送开发了靶向NCF1编码序列敲入，在内源性NCF2基因座的控制下恢复p47phox表达。NCF2编码p67phox，一种与p47phox紧密相互作用并主要在骨髓细胞中表达的NADPH氧化酶亚基。这种方法恢复了p47-CGD患者造血干细胞和祖细胞（HSPCs）和p47phox缺陷型小鼠HSPCs中p47phox的表达和NADPH氧化酶的功能，转基因表达遵循髓样分化模式。腺相关病毒载体在模板递送方面优于整合缺陷的慢病毒载体，在HSPC中具有更少的脱靶整合和更高的校正功效。这种针对骨髓的基因编辑有望用于临床CGD基因治疗。因为它导致p47phox和p67phox的共表达，确保骨髓细胞中的时空和近生理转基因表达。
