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

基本信息

批准号：
10538547
负责人：
Mary Bedard
金额：
$ 5.05万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
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/10538547
关键词：
3-Dimensional Address Adherent Culture Adjuvant Adjuvant Therapy Adult Aerodigestive Tract Automobile Driving Benign Biological Markers Biological Models Biopsy Caring Cell model Cells Characteristics Child Childhood Clinical Collaborations Data Development Diagnosis Disease Embryo Endoderm Engineering Epidermis Epithelial Excision FDA approved Face Gene Expression Genome Geography Goals Growth HPV-High Risk Healthcare Systems Histopathology Human Papillomavirus Human papillomavirus 6 Hyperplasia Infection K-18 conjugate Label Laboratories Laryngeal neoplasm Length Life Cycle Stages Literature Low risk HPV Malignant Conversion Maps Medical Methodology Modeling Molecular Target Morbidity - disease rate Mucous Membrane Ontology Open Reading Frames Operative Surgical Procedures Otolaryngology Papilloma Pathogenesis Pathology Patients Pharmaceutical Preparations Phenotype Population Procedures Production Public Health Quality of life Recording of previous events Recurrent respiratory papillomatosis Reporting Respiratory System Risk Role Sampling Scientist Severities Signal Transduction Site Specimen Stratified Epithelium Stratum Basale Surface Ectoderm System Testing Therapeutic Tissues Treatment Protocols Validation Viral Viral Genes Viral Load result Work bench to bedside chemical genetics clinical phenotype cohort disease phenotype epithelial to mesenchymal transition guided inquiry in vitro Model keratinocyte monolayer particle predictive marker predictive test premature progenitor regenerative screening single-cell RNA sequencing statistics stem stem cells therapeutically effective three-dimensional modeling time use transcriptome transcriptome sequencing transcriptomics translational study treatment response young adult

项目摘要

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/摘要拟议的研究重点是复发性呼吸道乳头状瘤病（RRP），这是一种持续性人乳头瘤病毒（HPV）驱动的疾病具有显着发病率但尚无 FDA 批准的治疗方案的疾病，是最常见的良性肿瘤。在儿童的喉部，RRP 是由低危 HPV 毒株（最常见的是 HPV6）引起的，并表现为复发性上皮性病变威胁气道的呼吸道乳头状瘤平均无法治愈。仅第一年就需要进行 4-5 次切除肿块的手术，一生中可能会面临数百次 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，并且当它发展到其他方面时具有恶性转化的风险尽管使用了一系列标签外辅助治疗的尝试，但没有任何一种药物能够有效治疗呼吸消化道中的部位。能够有效消除儿科 RRP，我们无法预测哪些患者会对任何特定药物或药物产生反应因此，更深入地了解疾病的病毒和细胞驱动因素对于更好地确定治疗方案至关重要。为此，对来自 RRP-N(ormal) 匹配的样本进行了单细胞 RNA 测序 (scRNAseq)。未接受治疗的患者并进行分析以选择候选病毒（HPV6 E5）和细胞（NOTCH 信号）驱动因素用于机械询问。由于缺乏可靠的模型系统来定义和测试预测，RRP 领域的临床进展受到阻碍来自成人而非儿童的原代单层 RRP 细胞是 RRP 发育的生物标志物和关键调节因子。然而，单层培养不利于HPV驱动疾病的研究，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内部控制的管道。患者特异性 RRP 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构建了 23 名患者的 3D 器官型上皮筏。已生成用于拟议研究的初步转录组和 scRNAseq 数据的验证。器官型筏支持使用这些模型进行拟议的转化研究来识别疾病的可行性 RRP 的生物标志物、驱动因素和分子靶点该项目得到了具有丰富经验的科学家团队的支持。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，以及照顾美国最大的 RRP 儿童和年轻人群体之一的新移民。