Harnessing Otic Progenitor Cells Derived From Many Donors to Test for Cisplatin Associated Hearing Loss

项目总结/摘要 听力损失是顺铂的常见副作用，顺铂是一种常用于癌症治疗的药物。有限数量 全基因组关联研究（GWAS）已经在几百名患者中进行， 已经确定了一些变体。ACYP 2中只有1个变体（rs 1872328）已被证明是稳健的。 与顺铂相关的听力损失（CAHL）有关。虽然这种变体具有如此大的影响，但它仍然存在。 目前还不清楚潜在的生物学机制是什么，以及如何将其作为一种治疗选择。 开始很少有这样的发现的一个原因是，GWAS使用的样本量很小，事实上 如果患者尚未接受顺铂治疗，则难以招募患者进行研究。在这里，我们计划 进一步测试和鉴定CAHL的遗传贡献，但采用不同的方法。而不是CAHL，我们 提出测试源自人类个体的耳祖细胞的顺铂诱导的细胞毒性，并使用该细胞， 作为我们的表型。我们建议测试这些细胞对顺铂诱导的毒性的敏感性。 当从给定个体提取生物样品（PBMC、成纤维细胞等）时， 多能干细胞（iPSC），将iPSC分化为耳祖细胞并测试它们的顺铂 诱导的毒性可能是现实的实现1个人，这样做的细胞从数百个不同的 捐赠者会很无聊因此，我们建议采用我们新发表的方法（Chan et.例如，基因组 Medicine，2018），能够通过汇集来自许多不同供体的细胞表型进行多重检测 这些细胞在一起，还能够从每个供体中提取个体水平的表型。用这种方法 将极大地减少执行实验所需的成本和时间， 在数千个不同的捐赠细胞上分别做实验，我们只需要在一个细胞池上做一次， 来自不同的捐赠者。此外，我们建议购买，而不是直接从个人获得细胞， 来自Coriell医学研究所（CIMR）的淋巴母细胞，他们在那里拥有生物库细胞 从许多不同的捐赠者获得，例如1000个基因组计划和个人基因组计划和全基因组计划。 每个捐赠者的基因组基因型数据都是可用的。我们的目标是确定是否从捐赠者的耳祖细胞 携带rs 1872328危险等位基因的患者比非携带者更易受顺铂的影响。这样做将证明， 这种体外表型可以有效地用作CAHL的代表。接下来，我们的目标是确定在多大程度上 顺铂诱导的耳毒性是通过使用来自以下的供体细胞测量遗传性来遗传确定的： 相关的个人。最后，我们的目标是通过有效地发现与CAHL相关的其他变体， 以顺铂诱导的耳毒性作为表型进行GWAS。这将有助于发现新的 可能是治疗CAHL的更好靶点。我们的提案为CAHL提供了一种不同的方法 通过对从供体获得的细胞进行的实验确定表型的研究，而不是 而不是在人类患者中测量的听力损失的临床结果。

PROJECT SUMMARY / ABSTRACT Hearing loss is a common side effect of cisplatin, a drug commonly use for cancer treatment. Limited number of genome-wide association studies (GWAS) have been performed on a few hundred patients and only a handful of variants have been identified. Only 1 variant (rs1872328), in ACYP2 have been shown to be robustly associated with cisplatin associated hearing loss (CAHL). While this variant has such a large effect, it remains unclear what is the underlying biological mechanisms are and how they can be exploited as a treatment option. One reason why such findings are rare to begin with is that the sample size used for GWAS is small, as it is difficult to recruit patients for research if they are not already undergoing cisplatin treatment. Here, we plan to further test and identify genetic contributions to CAHL but with a different approach. Instead of CAHL, we propose to test otic progenitor cells derived from human individuals for cisplatin induced cytotoxity and use that as our phenotype instead. We propose to test these cells for their susceptibility to cisplatin induced toxicity. While extracting biological samples (PBMCs, Fibroblasts, etc) from a given individual, reprogramming them into pluripotent stem cells (iPSCs), differentiating the iPSCs into otic progenitors and testing them for cisplatin induced toxicity might be realistically attainable for 1 individual, doing so on cells from hundreds of different donors would be tedious. As such, we propose to employ our newly published method (Chan et. al., Genome Medicine, 2018) that would be able to multiplex test cellular phenotypes from many different donors by pooling the cells together, yet be able to extract individual level phenotypes from each donor. Having this method would tremendously reduce the cost and time needed to perform the experiment as instead of doing the experiments separately on thousands of different donor cells, we just have to do them once on a pool of cells from many different donors. Also, instead of obtaining cells directly from individuals, we propose to purchase lymphoblastoid cells from the Coriell Institute for Medical Research (CIMR), where they have biobanked cells obtained from many different donors, e.g. 1000 Genomes Project and Personal Genome Project and whole- genome genotype data are available for every donor. We aim to determine if otic progenitor cells from donors carrying the rs1872328 risk allele are more susceptible to cisplatin than non-carriers. Doing so will prove that this in-vitro phenotype can be effecitively used as a proxy for CAHL. Next, we aim to determine to what extent is cisplatin-induced ototoxicity is genetically determined by measuring the heritability using donor cells from related individuals. Finally, we aim to discover additional variants associated with CAHL by effectively performing a GWAS with cisplatin-induced ototoxcitiy as the phenotype. This will allow for the discovery of new pathways and potentially better target for treatment of CAHL. Our proposal offers a different approach to CAHL research where the phenotype is determined from experiments performed on cells obtained from donors rather than a clinical outcome of hearing loss as measured in human patients.