Rational optimization of combinatorial therapies for the treatment of rare cystic fibrosis variants

摘要 囊性纤维化（CF）是一种致命的遗传性疾病，目前影响全球约10万人。CF是由 一系列功能丧失突变损害了囊性纤维化的生物发生和/或功能， 跨膜传导调节因子（CFTR）离子通道，其中大部分增强其错误折叠， 降解最近的药物发现工作已经产生了一套批准的小分子“校正剂”， 增强错误折叠的CFTR变体和恢复CFTR电导的“增强剂”的表达 门控缺陷的变体。这些分子的组合最近彻底改变了对 约90%的CF患者携带至少一个经过充分研究的ΔF508 CFTR变异体拷贝，这是高度相关的。 在高加索人中的渗透。然而，目前的组合疗法的功效在不同人群中差异很大。 约10%的患者携带罕见、未表征的CF变体的不同组合， 药理学性质（“治疗型”）。努力扩大目前治疗的标签， 可治疗的CF基因型的数量，特别是在非白人群体中，受到大的 CF变体的数量和当前方法的有限通量。识别响应的罕见CF变体 随着新的校正剂和/或增效剂的获得， 批准应对这一挑战需要新的技术，使有效的生物化学和/或生物技术成为可能。 罕见CF变体的药理学分析。在下文中，我们建议以一种独特的 融合了新兴的遗传、生化和计算方法。我们展示了深度突变扫描 (DMS)可用于平行比较校正子对数百种变体表达的影响。 我们的初步研究结果提供了一个前所未有的CF变体的不同治疗方法的一瞥， 鉴定具有独特生物化学和/或药理学性质的多种变体。我们首先建议 扩大这些调查，以衡量的反应，完整的CFTR 2错义 一组结构多样的校正剂分子的变体。然后，我们将描述以下相互作用组的特征： 具有不同校正反应的变体，以鉴定拮抗这些小分子的影响的CFTR相互作用。 分子。然后，我们将CRISPR/ Cas9技术与DMS融合，以确定这些相互作用如何影响 CF变体治疗谱。使用最先进的结构建模方法，我们将 鉴定CFTR蛋白中与拮抗相互作用形成相关的结构缺陷 CFTR变异型的变异。然后，我们将利用机器学习对CF变体进行分类， 观察到的药理学特性。最后，我们将评估经批准的矫正剂对 使用行业标准短路电流分析的以前未表征的变体的功能特性 在Fischer大鼠甲状腺和人气道上皮细胞中。这些调查将有助于扩大名单 的可治疗的CF基因型，并提供新的工具，以优化CF药物的靶向。

Abstract Cystic fibrosis (CF) is a lethal genetic disease that currently affects ~100,000 people worldwide. CF is caused by a spectrum of loss-of-function mutations that compromise the biogenesis and/ or function of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel, most of which enhance its misfolding and degradation. Recent drug discovery efforts have yielded a suite of approved small molecule “correctors” that enhance the expression of misfolded CFTR variants and “potentiators” that restore conductance to CFTR variants with defective gating. Combinations of these molecules have recently revolutionized the treatment of the ~90% of CF patients bearing at least one copy of the well-studied ΔF508 CFTR variant, which is highly penetrant among Caucasians. However, the efficacy of current combinatorial therapies varies widely among the ~10% of patients bearing diverse combinations of rare, uncharacterized CF variants with divergent pharmacological properties (“theratype”). Efforts to expand the labels of current therapeutics and maximize the number of treatable CF genotypes, in particular amongst non-white populations, are constrained by the large number of CF variants and the limited throughput of current methods. Identifying rare CF variants that respond to therapeutic cocktails is likely to become even more challenging as new correctors and/ or potentiators gain approval. Addressing this challenge requires new techniques that enable efficient biochemical and/ or pharmacological profiling of rare CF variants. In the following, we propose to address this challenge with a unique fusion of emerging genetic, biochemical, and computational methods. We show how deep mutational scanning (DMS) can be used to compare the effects of correctors on the expression of hundreds of variants in parallel. Our preliminary findings provide an unprecedented glimpse of the divergent theratypes of CF variants while identifying numerous variants with unique biochemical and/ or pharmacological properties. We first propose to expand on these investigations in order to measure the response of the complete set of CFTR2 missense variants to a panel of structurally diverse corrector molecules. We will then characterize the interactomes of variants with distinct corrector responses to identify CFTR interactions that antagonize the effects of these small molecules. We will then fuse CRISPR/ Cas9 technology with DMS to determine how these interactions impact the spectrum of CF variant theratypes. Using state-of-the-art structural modeling approaches, we will then identify structural defects in the CFTR protein that are associated with the formation of antagonistic interactions and deviations in CFTR variant theratype. We will then utilize machine learning to classify CF variants based on their observed pharmacological properties. Finally, we will assess the effects of approved correctors on the functional properties of previously uncharacterized variants using industry-standard short-circuit current analysis in Fischer Rat Thyroid and human airway epithelial cells. Together, these investigations will help expand the list of treatable CF genotypes and provide new tools to optimize the targeting of CF drugs.