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: 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
• 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

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.

项目摘要/摘要 拟议的研究重点是复发性呼吸乳头状瘤病（RRP），持续的人乳头瘤病毒（HPV）驱动 疾病具有明显的发病率，但没有FDA批准的治疗选择。作为最常见的良性肿瘤 RRP的儿童喉部是由HPV的低风险菌株（最常见的HPV6）引起的 沿呼吸道威胁气道的乳头瘤。手术是非疗法的；孩子平均要 仅在第一年就可以去除群众的4-5个程序，并且一生就可以面对数百个。 RRP的临床课程 因此，由于无法预测，因此将其定位为严重的燃烧 气化道中的位置。尽管使用了一系列尝试以外的标签调整疗法，但没有一个代理 我们有效地消除了小儿RRP，我们无法预测哪些患者会对任何特定药物做出反应或 治疗方案。因此，对疾病的病毒和细胞驱动因素的更深入了解对于更好地识别 疗法。为此，在RRP-N（Ormal）匹配的标本上进行了单细胞RNA测序（SCRNASEQ） 对幼稚的患者进行治疗和分析，以选择候选病毒（HPV6 E5）和细胞（Notch信号传导）驱动因素 用于机械审讯。 没有真正的模型系统来定义和测试预测，RRP领域的临床进展受到了阻碍 RRP开发的生物标志物和关键调节剂。来自成年人但不是儿童的主要单层RRP细胞是 在文献中报道。然而，单层培养并未进行HPV驱动疾病的研究，因为HPV病毒 生命周期需要3D区分粘膜。我们实验室的初步工作建立了一条内部控制的管道， 由RRP-N组成的RRP的患者特异性模型匹配的患者组织培养为2D主要细胞，这些细胞已经 成功地设计成3D有机上皮筏。来自23例患者的组织标本和衍生作用细胞 已在拟议的研究中生成。 3D中初步转录组和SCRNASEQ数据的验证 有机筏支持将这些模型用于拟议的翻译研究的可行性 RRP的生物标志物，驱动因素和分子靶标。该项目得到了一个科学家团队的支持 上皮模型，SCRNASEQ，OMICS方法，统计和病理学的协作和完整专业知识， 以及关心美国RRP最大的儿童和年轻人队列之一的临床医生。