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道乳头状瘤病(RRP)，这是一种由人乳头瘤病毒(HPV)驱动的持续性疾病 发病率很高，但没有FDA批准的治疗方案的疾病。最常见的良性肿瘤 在儿童的喉部，RRP是由低风险的HPV株引起的，最常见的是HPV6，表现为复发的上皮细胞 呼吸道乳头状瘤，威胁呼吸道。手术是不能治愈的；儿童平均要经历 仅在第一年就需要4-5次手术来切除肿块，在他们的一生中可能会面临数百次。RRP的临床病程 因此造成了严重的负担，因为它是不可预测的，并有恶性转化的风险，当它进展到 在空气中的消化道的位置。尽管使用了一系列非标签辅助疗法的尝试，但没有一种药物 在消除儿科RRP方面是有效的，我们无法预测哪些患者会对任何特定的药物或 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RRP-N(正常)匹配的样本进行了单细胞RNA测序(ScRNAseq)。 一名治疗幼稚的患者和分析，以选择候选病毒(HPV6 E5)和细胞(Noch信号)驱动因素 进行机械式审讯。 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了阻碍 RRP发展的生物标志物和关键调节因子。来自成人而不是儿童的原代单层RRP细胞是 在文献中有报道。然而，单层培养不利于作为HPV病毒的HPV引起的疾病的研究。 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条内部控制的管道， 由RRP-N匹配的患者组织培养成2D原代细胞的特定患者模型 成功地设计成了3D器官型上皮筏。23例患者的组织标本及其衍生的原代细胞 已生成供拟议研究使用的数据。3D中初步转录和scRNAseq数据的验证 器官型木筏支持将这些模型用于拟议的转译研究以识别疾病的可行性 RRP的生物标记物、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，他们拥有 在上皮模型、scRNAseq、组学方法、统计学和病理学方面的协作和互补专业知识， 以及护理美国最大的RRP儿童和年轻人队列之一的临床医生。