Rare mutations in Cystic Fibrosis: Overcoming barriers to personalized medicine

该项目将侧重于必须克服的知识差距，以提高精确度，基因型特异性 囊性纤维化（CF）的治疗。首先，我们将描述疾病相关的等位基因，其中许多是 非常罕见，并已被分配到一个不正确或不完整的机械类别。其他变体 代表之前未评估的新缺陷。我们的目标将是阐明传统的限制， 疾病相关CF等位基因的诊断类别，并解决调节剂药物反应性（即， “治疗型”），用于许多以前未以这种方式测试的罕见CFTR突变。我们将 还提供了最近解决的CFTR cryo-EM结构可以帮助定义疾病的方法的例子- 相关突变及其对CFTR折叠的影响（目的1）。我们的研究将提供细胞系统， 代表数千名患者的等位基因的分子表型，应该对科学研究有价值。 社区、临床医生和患者。第二，需要新的模型系统， 使用药物如ivacaftor或Orkambi（其含有ivacaftor和lumacaftor两者）的体内益处。作为测试 为了这个目的，我们将产生对应于20个CFTR变体的iPS系 目标1下的配置文件。这样的实验将提供以下初步评估：1）iPS在多大程度上 根据最好的可用的和创新的方案 2）这些模型系统是否表现出可检测的调制器 3）iPS细胞是否在分子表型上与广泛使用的（FRT）细胞模型相似 编码相同的突变，4）尖端基因编辑技术在iPS中的有用性 细胞生产一线系统，以及5）iPS技术的实用功能维斯个性化 治疗，包括个体患者样本可以有效扩增的程度，耐久性 CFTR的表达，体外功能，以这种方式开发的单层的稳定性（目的2）等。最后，我们 我们将迎接挑战，通过促进新的干预措施， 携带P67 L/F508 del基因型的CF患者，一项象征数百种其他CFTR的病例研究 变体。我们有令人信服的数据表明，P67 L患者将从药物中显著获益。 例如Orkambi。Aim 3将在临床环境中通过直接测试人类 有这种罕见等位基因的人如果成功的预测，一组病人谁目前没有有效的 调节剂治疗将直接受益。此外，因为我们相信iPS细胞很可能会重演 临床疗效，这些数据旨在帮助建立一个体外替代品，将改善药物的获取， 用于携带P67 L以外的罕见变体的CF患者群体的精确治疗。

This project will focus on gaps in knowledge that must be overcome to advance precision, genotype-specific intervention for cystic fibrosis (CF). First, we will characterize disease-associated alleles, many of which are extremely rare, and have been assigned to an incorrect or incomplete mechanistic category. Other variants represent novel defects not evaluated previously. Our goal will be to elucidate limitations of the conventional diagnostic categories for disease-associated CF alleles, and to address modulator drug responsiveness (i.e., ‘theratype’) for a number of rare CFTR mutations that have not been previously tested in this manner. We will also provide examples of ways the recently solved CFTR cryo-EM structure can help define disease- associated mutations and their effects on CFTR folding (Aim 1). Our studies will provide cell systems for molecular phenotyping of alleles representing thousands of patients, and should be of value to the scientific community, clinicians, and patients with the disease. Second, new model systems are needed that predict in vivo benefit using drugs such as ivacaftor or Orkambi (which contains both ivacaftor and lumacaftor). As a test of emerging cell systems for this purpose, we will generate iPS lines corresponding to 20 of the CFTR variants profiled under Aim 1. Experiments such as these will provide a first evaluation of: 1) the extent to which iPS cells can be used effectively as polarized monolayers according to the best available and innovative protocols developed by our laboratory and others, 2) whether these model systems exhibit detectable modulator response in vitro, 3) whether iPS cells are similar in molecular phenotype to widely used (FRT) cell models encoding the same mutations for this purpose, 4) usefulness of leading edge gene editing technologies in iPS cells to produce first-line systems, and 5) practical features of iPS technology vis-à-vis personalized therapeutics, including the extent to which an individual patient sample can be expanded effectively, durability of CFTR expression, function in vitro, stability of monolayers developed in this manner (Aim 2), etc. Finally, we will take up the challenge to establish the practical usefulness of our work by facilitating a new intervention in CF patients carrying the P67L/F508del genotype, a case study emblematic of hundreds of other CFTR variants. We have a compelling body of data indicating that P67L patients will benefit significantly from a drug such as Orkambi. Aim 3 will formally pursue this assertion in a clinical setting by directly testing human subjects with this very rare allele. If successful as predicted, a group of patients who currently have no effective modulator therapy will directly benefit. Moreover, because we believe it is likely that iPS cells will recapitulate clinical efficacy, the data is intended to help establish an in vitro surrogate that will improve drug access and precision therapeutics for a population of CF patients carrying rare variants other than P67L.