Assessing the ability of hiCMs to recapitulate patient-specific doxorubicin-induced cardiotoxicity

评估 hiCM 重现患者特异性阿霉素引起的心脏毒性的能力

• 批准号: 10274149
• 负责人: Paul W. Burridge
• 金额: $ 7.65万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-13 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10274149
• 关键词: Affect; Attenuated; CRISPR/Cas technology; Cancer Patient; Candidate Disease Gene; Cardiac Myocytes; Cardiomyopathies; Cardiotoxicity; Clinical; Clinical Data; Clustered Regularly Interspaced Short Palindromic Repeats; Complication; Doxorubicin; Etiology; Exposure to; Genes; Genetic; Genetic Markers; Genetic study; Genome; Genomics; Heart Transplantation; Heart failure; Human; In Vitro; Knock-out; Knowledge; Lead; Malignant Childhood Neoplasm; Mediating; Metabolism; Methodology; Modality; Molecular; Pathway interactions; Patients; Pharmaceutical Preparations; Predisposition; Quantitative Trait Loci; Reproducibility; Research; Risk; Role; Single Nucleotide Polymorphism; Solid Neoplasm; Time; Validation; Variant; Work; base; cardioprotection; chemotherapy; clinical application; cohort; differential expression; experience; gene discovery; genetic variant; genome editing; genome wide association study; genomic tools; induced pluripotent stem cell; innovation; interest; knockout gene; leukemia/lymphoma; novel; patient response; pediatric patients; prevent; recruit; response; sarcoma; screening; side effect; tool; whole genome

PROJECT SUMMARY Doxorubicin is a highly effective chemotherapy drug commonly used in approximately 60% of pediatric patients with metastatic solid tumors (sarcomas), leukemia, and lymphoma. Treatments using doxorubicin are complicated by its well-established cardiotoxic side effect, which affects approximately 16% of pediatric patients, can lead to heart failure requiring heart transplant, and limits doxorubicin’s clinical utilization. Despite more than 50 years of research in this field, there is still, at present, little potential for either predicting or preventing cardiotoxicity. There is an obvious need for novel and innovative approaches to overcome this hurdle. Candidate gene association studies and genome–wide association studies (GWAS) have identified many single nucleotide polymorphisms (SNPs) that are statistically correlated with doxorubicin–induced cardiotoxicity (DIC), yet experimental validation of these SNPs has not been feasible due to the difficulty in isolating and culturing human cardiomyocytes in vitro. In our recent work, we showed that patient–specific human induced pluripotent stem cell–derived cardiomyocytes (hiPSC–CM) are efficient predictors of a patient’s likelihood of developing DIC, confirming for the first time that there is a genomic basis to DIC. Although GWAS has proven to be a powerful methodology for informing such genomic bases, it detects correlation rather than causation, and identified SNPs commonly fail to be replicated in subsequent studies. Here, we hypothesize that hiPSC-CMs can be utilized in three different modalities to study genetic variants associated with DIC: firstly, to discover novel predictive SNPs; secondly, to validate SNPs; and thirdly, to examine the modulated pathways and determine cardioprotective methodologies. In Aim 1, we will recruit 100 pediatric cancer patients who were exposed to doxorubicin (50 who experienced cardiotoxicity and 50 who did not), assess the response of patient-derived hiPSC-CM to doxorubicin to validate our previous findings in a large cohort and confirm this as powerful tool in the field. In Aim 2, we will use these 100 patient-specific lines to identify drug response differential expression quantitative trait loci (deQTL), validate these variants with genome editing, and mechanistically examine pathways causative to DIC susceptibility concentrating on genes with known roles in cardiomyopathy, cardioprotection, and doxorubicin metabolism. In Aim 3, we will interrogate the rigor and reproducibility of 40 existing DIC SNP studies, using CRISPR/Cas9 to knockout the gene of interest in a control isogenic hiPSC line then assess the response of hiPSC-CM to doxorubicin. We will also perform lentiviral-based pooled and arrayed whole genome CRISPR-mediated knockout screening to discover genes which modulate the risk of and protection from DIC. Finally, we will use the discoveries above to discover/repurpose genome-informed cardioprotective drugs to prevent DIC. In summary, this work will deliver us the genetic rationale for why patients experience DIC and provide 1, fully human validated SNP data for clinical application, and 2, novel cardioprotective drugs to attenuate DIC.

项目摘要 多柔比星是一种高效的化疗药物，约60%的儿科患者常用 转移性实体瘤（肉瘤）、白血病和淋巴瘤。使用多柔比星的治疗是 复杂的是其公认的心脏毒性副作用，影响了大约16%的儿童 患者，可导致需要心脏移植的心力衰竭，并限制了阿霉素的临床应用。尽管 在这一领域的研究超过50年，目前仍然没有什么潜力可以预测或 防止心脏毒性。显然需要新颖和创新的方法来克服这一点 障碍物候选基因关联研究和全基因组关联研究（GWAS）已经确定了 许多单核苷酸多态性（SNP）与阿霉素诱导的 心脏毒性（DIC），但这些SNP的实验验证还不可行，因为难以确定这些SNP是否具有心脏毒性。 体外分离培养人心肌细胞。在我们最近的工作中，我们表明， 人诱导多能干细胞衍生的心肌细胞（hiPSC-CM）是患者心肌细胞损伤的有效预测因子。 发展DIC的可能性，首次证实DIC存在基因基础。虽然GWAS 已被证明是一个强大的方法，为通知这样的基因组基地，它检测相关性，而不是 因果关系，并确定SNP通常不能在随后的研究中复制。我们假设 hiPSC-CM可以以三种不同的方式用于研究与DIC相关的遗传变异： 首先，发现新的预测SNP;其次，验证SNP;第三，检查调制的SNP。 途径和确定心脏保护方法。在目标1中，我们将招募100名儿童癌症患者， 暴露于多柔比星的患者（50例发生心脏毒性，50例未发生心脏毒性）， 患者来源的hiPSC-CM对多柔比星的反应，以验证我们先前在大型队列中的发现， 证实这是一个强大的工具。在目标2中，我们将使用这100个患者特异性线来识别药物 响应差异表达数量性状基因座（deQTL），用基因组编辑验证这些变体， 并机械地检查引起DIC易感性的途径，集中于已知的 在心肌病、心脏保护和阿霉素代谢中的作用。在目标3中，我们将询问 40个现有DIC SNP研究的可重复性，使用CRISPR/Cas9敲除感兴趣的基因， 对照等基因hiPSC系，然后评估hiPSC-CM对阿霉素的反应。我们还将表演 基于慢病毒的混合和阵列全基因组CRISPR介导的敲除筛选以发现基因 其调节DIC的风险和保护。最后，我们将利用上述发现， 发现/重新利用基因组信息的心脏保护药物来预防DIC。总之，这项工作将提供 使用遗传学原理解释患者发生DIC的原因，并提供1个完全人类验证的SNP数据， 临床应用; 2.新型心脏保护药物减轻DIC。