Sequence-based RNA Fluorescence Assay to Measure Latent HIV Reservoirs

基于序列的 RNA 荧光测定来测量潜在的 HIV 病毒库

• 批准号: 10081917
• 负责人: Janet L Huie
• 金额: $ 29.13万
• 依托单位: JAN BIOTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-07 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10081917
• 关键词: 13 year old; AIDS clinical trial group; Achievement; Acquired Immunodeficiency Syndrome; Aftercare; Antibodies; Base Sequence; Biological Assay; Biological Markers; Biotechnology; CD4 Positive T Lymphocytes; Calibration; Cell Count; Cells; Centers for Disease Control and Prevention (U.S.); Chemicals; Clinical; Clinical Trials; Collaborations; Combined Modality Therapy; Complementary DNA; Computer software; Consult; Correlation Studies; Development; Diagnostic; Disease remission; Doctor of Philosophy; Evaluation; Fluorescence; Fluorescence-Activated Cell Sorting; Gene Expression Profiling; HIV; HIV Infections; HIV Seropositivity; Highly Active Antiretroviral Therapy; Hospitals; Human; Individual; Interruption; Length; Leukapheresis; Measurement; Measures; Medical center; Medicine; Methods; Monitor; Participant; Patients; Pharmaceutical Preparations; Phase; Planet Mars; Predictive Value; Procedures; Process; Public Health; RNA; Resources; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Sampling; San Francisco; Shock; Signal Transduction; Testing; Time; Universities; Validation; Veterans; Viral; Viral Load result; Virion; Virus; Woman; antiretroviral therapy; base; clinically relevant; commercialization; data sharing; diagnostic assay; internal control; latent HIV reservoir; medical schools; mid-career faculty; molecular diagnostics; multiplex detection; novel; novel marker; open source; phase 2 testing; preclinical evaluation; research clinical testing; software development; therapy outcome; viral RNA; viral rebound

Project Summary/Abstract Public Health Problem. The CDC estimates that in the U.S., 1,144,500 people aged 13 years and older are living with HIV infection, with approximately 180,900 (15.8%) others infected but undiagnosed. For most HIV-positive individuals, available drugs can only control HIV infection and delay progression to AIDS. HIV cure treatments in active development require validated biomarkers for HIV clearance from infected individuals. Issues with Current Solutions & How Product Meets Unmet Needs. Current methods of quantifying the HIV reservoir include the quantitative viral outgrowth assay (QVOA), PCR and RT-PCR. QVOA is a time and resource intensive procedure while RT-PCR can be used to detect viral RNA to 20-50 virus particles per mL and thus reduces the time to result of QVOA and is generally applicable for measuring viral load, but does not directly detect replication-competent HIV-infected cells. Fluorescence-activated cell sorting (FACS) using combined antibody and probe hybridizations requires multiple steps with significant loss of signal. In Jan Biotech’s assay, all RNA molecules are counted, whereas q and ddPCR are limited by the efficiency of reverse transcription. The capability of Jan Biotech’s sequence-based latent HIV assay to detect down to the single HIV-infected cell level would provide a high throughput, scalable assay critically needed to monitor latent HIV reservoirs. Summary of Approach. Jan Biotech’s assay employs sequence-specific fluorogenic probes with a high signal amplification to directly detect HIV RNA species. It will be evaluated for correlation with RT-ddPCR Transcriptional Profiling and QVOA. A statistical correlation of >0.6 with both comparison assays will be the milestone to move forward to Phase II validation testing and assessment of the predictive value of the assay for time to viral rebound after treatment interruption. Software analytics will be developed and shared open source. Collaborators and Unique Resources. Jan Biotech, Inc., with expertise in molecular diagnostic development, will collaborate with Dr. Steven Yukl, MD, UCSF/SFVAMC, for Transcriptional Profiling, with Michael Busch, MD, PhD, and Mars Stone, PhD, for the use of the RAVEN sample panels, and with Jonathan Li, MD, MMSc Associate Professor of Medicine at Harvard Medical School and Brigham and Women’s Hospital, Harris A. Gelbard, MD, PhD, of University of Rochester Medical School, and John Mellors, MD, of University of Pittsburgh Medical School, for ATCG sample testing. Fast-Track Specific Aims Specific Aim 1 (Phase I): Assay correlation to Transcriptional Profiling and QVOA orthogonal assays Comparison testing will be performed between Jan Biotech’s RNAamp assay and Dr. Yukl’s transcriptional profiling RT-ddPCR assays and QVOA. The Critical Phase I milestone will be a statistical correlation of 0.6 or better of RNAamp to both RT-ddPCR transcriptional profiling or QVOA values. Specific Aim 2 (Phase II): Assay validation using RAVEN Evaluation Panel and QVOA Samples Given successful completion of Phase I comparison testing between RNAamp and RT-ddPCR transcriptional profiling and achievement of the Phase I metric, in Aim 2, Jan Biotech will validate RNAamp using the Reservoir Assay Validation and Evaluation Network (RAVEN) Evaluation Panel. Specific Aim 3 (Phase II): Evaluation of clinical relevance to HIV reservoir activation This Aim will evaluate these novel biomarkers for prediction of time to HIV rebound, which is crucial to prioritize promising treatments to determine those participants likely to benefit from novel HIV remission strategies. Specific Aim 4 (Phase II): Software development and integration for assay analytics In Aim 4, we will build Bayesian software to analyze and share the data from the HIV reservoir RNAamp assay. Market after Phase II Completion. The end result of the project will be a validated sequence-based, quantitative HIV reservoir diagnostic assay and computational software that will enable us to proceed to the FDA approval process, including additional preclinical and clinical evaluation leading towards 510(k) approval, clinical trials, and commercialization of an accurate, high-throughput HIV reservoir assay.

项目总结/摘要 公共卫生问题。CDC估计，在美国，1，144，500名13岁及以上的人生活在 艾滋病毒感染者，约180，900人（15.8%）感染但未确诊。对于大多数艾滋病毒阳性者来说， 对于个体而言，现有的药物只能控制艾滋病毒感染并延缓艾滋病的发展。艾滋病毒治愈治疗 在积极开发中，需要用于从感染个体清除HIV的经验证的生物标志物。 当前解决方案的问题以及产品如何满足未满足的需求。目前的艾滋病毒定量方法 库的方法包括定量病毒生长测定（QVOA）、PCR和RT-PCR。QVOA是一种时间和资源 而RT-PCR可用于检测病毒RNA至20-50个病毒颗粒/mL，因此 缩短了获得QVOA结果的时间，通常适用于测量病毒载量，但不直接 检测有复制能力的HIV感染细胞。使用组合的流式细胞仪进行流式细胞术活化细胞分选（FACS） 抗体和探针杂交需要多个步骤，信号损失显著。在Jan Biotech的检测中， 所有的RNA分子都被计数，而q和ddPCR受到逆转录效率的限制。的 Jan Biotech的基于序列的潜伏期HIV检测试剂盒能够检测到单个HIV感染细胞水平 将提供一种高通量、可扩展的检测方法，这是监测潜伏HIV库所急需的。 方法总结。Jan Biotech的检测采用了具有高信号的序列特异性荧光探针 扩增以直接检测HIV RNA种类。将评价其与RT-ddPCR的相关性 转录谱和QVOA。与两种比较测定的统计相关性>0.6将是 这是一个里程碑，可以推进到第二阶段验证测试，并评估测定的预测价值， 治疗中断后至病毒反弹的时间。软件分析将以开源方式开发和共享。 合作伙伴和独特的资源。Jan Biotech，Inc.，拥有分子诊断开发方面的专业知识， 与UCSF/SFVAMC的Steven Yukl博士合作进行转录分析，与Michael Busch博士合作， 博士和Mars Stone博士，用于RAVEN样品板的使用，以及Jonathan Li，MD，MMSC 哈佛医学院和布里格姆妇女医院的医学副教授哈里斯·A. 罗切斯特大学医学院的Gelbard医学博士和匹兹堡大学的John Mellors医学博士 医学院，用于ATCG样本测试。 快速通道具体目标 特定目标1（I期）：检测与转录谱分析和QVOA正交检测的相关性 将在Jan Biotech的RNAamp检测试剂盒和Dr. Yukl的转录检测试剂盒之间进行比较检测。 分析RT-ddPCR测定和QVOA。第一阶段关键里程碑的统计相关性为0.6或 RNAamp对RT-ddPCR转录谱分析或QVOA值都更好。 特定目标2（第II阶段）：使用RAVEN评价样本组和QVOA样本进行测定验证 鉴于成功完成RNAamp和RT-ddPCR转录之间的I期比较测试， 在目标2中，Jan Biotech将使用储库验证RNAamp， 测定验证和评价网络（RAVEN）评价小组。 具体目标3（II期）：评价与HIV储库激活的临床相关性 该目标将评估这些新的生物标志物，用于预测HIV反弹的时间，这对于优先考虑至关重要 有希望的治疗，以确定那些参与者可能受益于新的艾滋病毒缓解策略。 具体目标4（第II阶段）：用于检测分析的软件开发和集成 在目标4中，我们将构建贝叶斯软件来分析和共享来自HIV储库RNAamp检测的数据。 第二阶段完成后的市场。该项目的最终结果将是一个经过验证的基于序列的定量 HIV储库诊断分析和计算软件，使我们能够继续获得FDA批准 过程，包括导致510（k）批准的额外临床前和临床评价，临床试验， 和商业化的准确，高通量的HIV储库测定。