RATIONALE AND OBJECTIVE Cystic fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators offer significant improvements, but approximately 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimized for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial. METHODS Air-liquid interface cultures of primary human bronchial epithelial cells (HBEC) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalized human lung epithelial cell line mimicking Class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay (PLA) for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies. RESULTS rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a Class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence ≤10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalization assay. CONCLUSIONS The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.

中文翻译：

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rSIV.F/HN 的药理学和临床前安全性概况，rSIV.F/HN 是一种用于囊性纤维化基因治疗的杂交慢病毒载体。


基本原理和目标 囊性纤维化 （CF） 是由 CF 跨膜传导调节因子 （CFTR） 基因突变引起的。CFTR 调节剂提供了显着的改善，但大约 10% 的患者仍然无反应或不耐受。本研究提供了对 rSIV.F/HN 的分析，rSIV.F/HN 是一种针对肺递送优化的慢病毒载体，包括 CFTR 蛋白表达、CFTR 缺陷的功能校正和基因组整合位点分析，为首次人体临床试验做准备。方法 使用 CF 患者原代人支气管上皮细胞 （HBEC） 的气液界面培养物 （F508del/F508del） 以及模拟 I 类 （CFTR-null） 纯合突变的 CFTR 缺陷永生化人肺上皮细胞系，用于评估转导效率。定量方法包括用于 CFTR 蛋白表达的新型邻位连接测定 （PLA）。为了评估 CFTR 通道活性，进行了 Ussing 室研究。使用整合位点分析和体外插入诱变研究评估安全性。结果 rSIV.F/HN 在原代 F508del/F508del HBECs 以及 I 类细胞中表达 CFTR 并将 CFTR 介导的氯电流恢复到生理水平。相比之下，后者无法通过小分子 CFTR 调节剂实现，这凸显了基因治疗对这类突变的潜力。rSIV.F/HN-CFTR 与增效剂 ivacaftor 的组合显示出大于加法的效果。基因组整合模式显示没有位点优势（发生频率 ≤10%），并且在体外永生化测定中观察到插入诱变的风险较低。结论 结果强调了 rSIV。F/HN 作为一种很有前途的 CF 基因治疗载体，提供了一种与突变无关的治疗选择。