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

摘要 囊性纤维化是一种致命的遗传性疾病，目前全球约有100,000人受到影响。Cf是由 通过一系列功能缺失突变损害囊性纤维化的生物发生和/或功能 跨膜电导调节器(CFTR)离子通道，其中大多数增强其错误折叠和 退化。最近的药物发现工作已经产生了一套经过批准的小分子“校正剂” 增强错误折叠的cftr变异体和恢复cftr电导的“增强子”的表达 门控有缺陷的变种。这些分子的组合最近已经彻底改变了治疗 ~90%的CF患者至少携带一份经过充分研究的ΔF508 CFTR型变种，这是一种高度 高加索人中的渗透者。然而，目前联合疗法的疗效在不同的人群中差异很大。 约10%的患者携带罕见的、没有特征性的CF变异的不同组合，并存在分歧 药理特性(“热型”)。努力扩大现有疗法的标签，并最大限度地 可治疗的CF型的数量，特别是在非白人人群中，受到大量 Cf变种的数量和当前方法的有限吞吐量。识别响应的罕见的CF变体 随着新的矫正剂和/或增效剂的获得，治疗鸡尾酒可能会变得更具挑战性 批准。应对这一挑战需要新的技术来实现高效的生化和/或 罕见的CF变异体的药理图谱。在下文中，我们建议通过一种独特的 融合了新兴的遗传、生物化学和计算方法。我们展示了深度突变扫描 (DMS)可以用来比较校正子对数百个变体并行表达的影响。 我们的初步发现提供了一个史无前例的关于CF变异体不同热型的一瞥，而 识别具有独特生化和/或药理学特性的众多变体。我们首先提议 对这些调查进行扩展，以衡量完整的CFTR2错义集合的反应 一组结构不同的校正剂分子的变体。然后，我们将描述 具有不同校正响应的变体，以确定对抗这些小分子相互作用的CFTR相互作用 分子。然后，我们将CRISPR/Cas9技术与DMS融合，以确定这些交互如何影响 Cf变异热型的谱带。使用最先进的结构建模方法，我们将 确定与形成拮抗相互作用相关的CFTR蛋白的结构缺陷 Cftr变异热型的偏差。然后，我们将利用机器学习来根据以下条件对CF变体进行分类 它们的观察到的药理特性。最后，我们将评估经批准的校正员对 使用行业标准短路电流分析的先前未表征的变体的功能特性 Fischer大鼠甲状腺和人呼吸道上皮细胞。总而言之，这些调查将有助于扩大名单 可治疗的卡介苗基因分型，并提供新的工具来优化卡介苗药物的靶向。

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.