Single cell RNAseq guides discovery of viral and cellular drivers of RRP pathologies

单细胞 RNAseq 指导发现 RRP 病理的病毒和细胞驱动因素

• 批准号: 10315369
• 负责人: Mary Bedard
• 金额: $ 4.98万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10315369
• 关键词: Single; cell; RNAseq; guides; discovery

Project summary/Abstract The proposed studies focus on recurrent respiratory papillomatosis (RRP), a persistent human papillomavirus (HPV)-driven disease that has significant morbidity yet no FDA-approved treatment options. As the most common benign neoplasm of the larynx in children, RRP is caused by low risk strains of HPV, most commonly HPV6, and presents as recurring epithelial papillomas along the respiratory tract that threaten the airway. Surgeries are non-curative; children undergo an average of 4-5 procedures to remove masses in the first year alone and can face hundreds in their lifetime. The clinical course of RRP thus poses a severe burden as it is unpredictable and carries a risk of malignant conversion when it progresses to other sites in the aerodigestive tract. Despite the use of an array of attempts at off-label adjuvant therapies, no single agent has been effective at eliminating pediatric RRP, and we cannot predict which patients will respond to any particular drug or treatment regimen. A deeper understanding of the viral and cellular drivers of disease is thus essential to identify better therapies. To this end, single cell RNA sequencing (scRNAseq) was performed on RRP-N(ormal) matched specimens from a treatment naïve patient and analysis performed to select candidate viral (HPV6 E5) and cellular (NOTCH signaling) drivers for mechanistic interrogation. Clinical progress in the RRP field has been hindered by the absence of authentic model systems to define and test predictive biomarkers and key regulators of RRP development. Primary monolayer RRP cells from adults, but not children, are reported in the literature. However, monolayer culture is not conducive to the study of HPV-driven disease as the HPV viral life cycle requires 3D differentiated mucosa. Preliminary work in our laboratory establishes a pipeline of internally controlled, patient-specific models of RRP consisting of RRP-N matched patient tissue cultured into 2D primary cells that have been successfully engineered into 3D organotypic epithelial rafts. Tissue specimens and derivative primary cells from 23 patients have been generated for use in the proposed studies. Validation of preliminary transcriptomic and scRNAseq data in 3D organotypic rafts support the feasibility of using these models for the proposed translational studies to identify disease biomarkers, drivers, and molecular targets of RRP. The project is supported out by a team of scientists with a history of collaboration and complementary expertise in epithelial models, scRNAseq, omics methodologies, statistics and pathology, as well as clinicians who care for one of the largest cohort of children and young adults with RRP in the USA.

项目概要/摘要 拟议的研究集中在复发性呼吸道乳头状瘤病（RRP），一种持续性人乳头状瘤病毒（HPV）驱动的 具有显著发病率但没有FDA批准的治疗方案的疾病。作为最常见的良性肿瘤， 在儿童喉部，RRP是由HPV的低风险菌株引起的，最常见的是HPV 6，并表现为复发的上皮细胞， 乳头状瘤沿着呼吸道威胁气道。surgery是非治愈性的;儿童平均接受 4-5仅在第一年就需要办理迁移群众的手续，而且在他们的一生中可能要面对数百人。RRP的临床过程 因此造成了严重的负担，因为它是不可预测的，并且当它进展到其他疾病时具有恶性转化的风险。 在呼吸消化道的位置。尽管使用了一系列的尝试标签外辅助治疗，没有单一的药物， 有效地消除了儿科RRP，我们无法预测哪些患者会对任何特定药物产生反应， 治疗方案。因此，更深入地了解疾病的病毒和细胞驱动因素对于更好地识别 治疗为此，对来自美国的RRP-N（正常）匹配标本进行单细胞RNA测序（scRNAseq）。 未经治疗的患者，进行分析以选择候选病毒（HPV 6 E5）和细胞（NOTCH信号传导）驱动因子 进行机械审讯 RRP领域的临床进展受到缺乏真实模型系统来定义和测试预测性的阻碍。 生物标志物和RRP发展的关键调控因子。来自成人而非儿童的原代单层RRP细胞， 在文献中报道。然而，单层培养不利于研究HPV驱动的疾病，因为HPV病毒 生命周期需要3D分化的粘膜。我们实验室的初步工作建立了一个内部控制的管道， RRP的患者特异性模型由培养成2D原代细胞的RRP-N匹配患者组织组成， 成功地被改造成3D器官型上皮筏。23例患者的组织标本和衍生原代细胞 已生成用于拟议研究。初步转录组学和scRNAseq数据在3D中的验证 器官型筏支持使用这些模型进行翻译研究以鉴定疾病的可行性 RRP的生物标志物、驱动因子和分子靶标。该项目得到了一个科学家团队的支持， 在上皮模型、scRNAseq、组学方法、统计学和病理学方面的合作和互补专业知识， 以及临床医生谁照顾一个最大的队列的儿童和年轻人与RRP在美国。