BACKGROUND Several small molecule corrector and potentiator drugs have recently been licensed for Cystic Fibrosis (CF) therapy. However, other aspects of the disease, especially inflammation, are less effectively treated by these drugs. We hypothesized that small molecule drugs could function either alone or as an adjuvant to licensed therapies to treat these aspects of the disease, perhaps emulating the effects of gene therapy in CF cells. The cardiac glycoside digitoxin, which has been shown to inhibit TNFα/NFκB signaling in CF lung epithelial cells, may serve as such a therapy. METHODS IB3-1 CF lung epithelial cells were treated with different Vertex (VX) drugs, digitoxin, and various drug mixtures, and ELISA assays were used to assess suppression of baseline and TNFα-activated secretion of cytokines and chemokines. Transcriptional responses to these drugs were assessed by RNA-seq and compared with gene expression in AAV-[wildtype]CFTR-treated IB3-1 (S9) cells. We also compared in vitro gene expression signatures with in vivo data from biopsied nasal epithelial cells from digitoxin-treated CF patients. RESULTS CF cells exposed to digitoxin exhibited significant suppression of both TNFα/NFκB signaling and downstream secretion of IL-8, IL-6 and GM-CSF, with or without co-treatment with VX drugs. No evidence of drug-drug interference was observed. RNA-seq analysis showed that gene therapy-treated CF lung cells induced changes in 3134 genes. Among these, 32.6% were altered by digitoxin treatment in the same direction. Shared functional gene ontology themes for genes suppressed by both digitoxin and gene therapy included inflammation (84 gene signature), and cell-cell interactions and fibrosis (49 gene signature), while genes elevated by both were enriched for epithelial differentiation (82 gene signature). A new analysis of mRNA data from digitoxin-treated CF patients showed consistent trends in expression for genes in these signatures. CONCLUSIONS Adjuvant gene therapy-emulating activities of digitoxin may contribute to enhancing the efficacy of currently licensed correctors and potentiators in CF patients.

中文翻译：

---

基因疗法模拟小分子疗法在囊性纤维化气道上皮细胞和患者中的作用。

背景技术近来，几种小分子校正剂和增强剂药物已被许可用于囊性纤维化（CF）治疗。然而，这些药物不能有效地治疗疾病的其他方面，尤其是炎症。我们假设小分子药物可以单独发挥作用，也可以作为许可的疗法的佐剂来治疗疾病的这些方面，也许可以模拟基因疗法在CF细胞中的作用。心脏糖苷洋地黄毒苷已被证明可抑制CF肺上皮细胞中的TNFα/NFκB信号传导，可作为此类疗法。方法用不同的Vertex（VX）药物，洋地黄毒杆菌毒素和各种药物混合物处理IB3-1 CF肺上皮细胞，并使用ELISA分析评估基线和TNFα激活的细胞因子和趋化因子分泌的抑制作用。通过RNA-seq评估对这些药物的转录反应，并将其与AAV- [野生型] CFTR处理的IB3-1（S9）细胞中的基因表达进行比较。我们还比较了体外基因表达特征与来自洋地黄毒治疗的CF患者的活检鼻上皮细胞的体内数据。结果暴露于洋地黄毒素的CF细胞对TNFα/NFκB信号传导和IL-8，IL-6和GM-CSF的下游分泌均具有显着抑制作用，无论是否与VX药物共同治疗。没有观察到药物干扰的证据。RNA-seq分析显示，经基因治疗的CF肺细胞可诱导3134个基因发生变化。其中，通过洋地黄毒苷处理，有32.6％的细胞向同一方向改变。受洋地黄毒苷和基因治疗抑制的基因共有的功能基因本体论主题包括炎症（84个基因签名），以及细胞间相互作用和纤维化（49个基因特征），而两者共同升高的基因则丰富了上皮分化（82个基因特征）。对洋地黄毒治疗的CF患者的mRNA数据进行的一项新分析显示，这些签名中基因的表达趋势一致。结论洋地黄毒苷的辅助基因疗法模拟活性可能有助于增强CF患者当前许可的校正剂和增强剂的功效。