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

项目摘要/摘要 听力损失是顺铂的常见副作用，顺铂是一种常用的癌症治疗药物。数量有限 的全基因组关联研究(GWAS)已经在几百名患者身上进行，并且只有一 已经确定了几个变种。ACYP2中只有一个变异体(Rs1872328)被证明是健壮的 与顺铂相关性听力损失(CAHL)相关。虽然这个变种有如此大的影响，但它仍然 不清楚潜在的生物学机制是什么，以及如何利用它们作为一种治疗选择。 这样的发现很少见的一个原因是，GWAS使用的样本量很小 如果患者还没有接受顺铂治疗，很难招募他们进行研究。在这里，我们计划 进一步测试和确定对CAHL的遗传贡献，但采用不同的方法。我们不是CAHL，而是 建议测试来自人类个体的外周血祖细胞对顺铂诱导的细胞毒性并使用该方法 作为我们的表型。我们建议测试这些细胞对顺铂毒性的敏感性。 同时从特定个体提取生物样本(PBMC、成纤维细胞等)，对其进行重新编程 分化为多能干细胞(IPSCs)，将IPSCs分化为耳源性祖细胞，并测试它们的顺铂 对于一个人来说，诱导毒性可能确实是可以实现的，对数百个不同来源的细胞这样做 捐赠者将是乏味的。因此，我们建议采用我们新发表的方法(Chan et.美国，基因组 医学，2018)，将能够通过汇集来自许多不同捐赠者的多个测试细胞表型 这些细胞聚集在一起，但仍然能够从每个捐赠者身上提取单独的水平表型。有了这个方法 将极大地减少进行实验所需的成本和时间，因为 分别在数千个不同的供体细胞上进行实验，我们只需在一个细胞池上做一次 来自许多不同的捐赠者。此外，我们不是直接从个人那里获得细胞，而是建议购买 来自科里尔医学研究所(CIMR)的淋巴母细胞，在那里它们有生物库细胞 从许多不同的捐赠者那里获得，例如1000基因组计划和个人基因组计划和整体- 每个捐赠者的基因组基因数据都是可用的。我们的目标是确定来自捐赠者的祖细胞是否 携带rs1872328风险等位基因的人比非携带者对顺铂更敏感。这样做将证明 这种体外表型可以有效地作为CAHL的替代。接下来，我们的目标是确定在多大程度上 顺铂引起的耳毒性是通过使用供体细胞的遗传性来确定的吗 相关的个人。最后，我们的目标是通过有效地发现与CAHL相关的其他变异 以顺铂所致耳毒性为表型进行GWA术。这将允许发现新的 治疗CAHL的途径和潜在的更好的靶点。我们的提案为CAHL提供了一种不同的方法 通过对捐赠者的细胞进行实验来确定表型的研究，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.