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.

项目概要/摘要 公共卫生问题 CDC 估计，美国有 1,144,500 名 13 岁及以上的人 感染艾滋病毒的人，还有大约 180,900 人（15.8%）已感染但未确诊。 对于个人而言，现有药物只能控制艾滋病毒感染并延缓艾滋病毒治疗的进展。 正在积极开发中，需要经过验证的生物标志物来清除感染者体内的艾滋病毒。 当前解决方案的问题以及产品如何满足当前量化 HIV 的方法。 病毒库包括定量病毒生长测定（QVOA）、PCR 和 RT-PCR QVOA 是一种时间和资源。 密集程序，而 RT-PCR 可用于检测每毫升 20-50 个病毒颗粒的病毒 RNA，从而 缩短了 QVOA 得出结果的时间，通常适用于测量病毒载量，但不直接 使用组合检测具有复制能力的 HIV 感染细胞。 在 Jan Biotech 的检测中，抗体和探针杂交需要多个步骤，并且信号会显着丢失。 所有 RNA 分子都会被计数，而 q 和 ddPCR 则受到反转录效率的限制。 Jan Biotech 基于序列的潜伏 HIV 检测能够检测到单个 HIV 感染细胞水平 将提供监测潜在艾滋病毒储存库所急需的高通量、可扩展的检测方法。 方法摘要 Jan Biotech 的检测采用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，将评估其与 RT-ddPCR 的相关性。 转录分析和 QVOA 与两种比较分析的统计相关性将大于 0.6。 迈向 II 期验证测试和评估该检测的预测价值的里程碑 治疗中断后病毒反弹的时间将被开发并开源共享。 拥有分子诊断开发专业知识的合作者和独特资源。 与 Steven Yukl 博士（医学博士、UCSF/SFVAMC）合作进行转录分析，并与 Michael Busch 医学博士合作， 博士和 Mars Stone 博士，使用 RAVEN 样本面板，并与医学博士、理学硕士 Jonathan Li 合作 哈佛医学院和布莱根妇女医院医学副教授 Harris A. 罗彻斯特大学医学院的 Gelbard 医学博士、哲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 具体目标 1（第一阶段）：与转录分析和 QVOA 正交测定的测定相关性 将在 Jan Biotech 的 RNAamp 检测和 Yukl 博士的转录检测之间进行比较测试 RT-ddPCR 分析和 QVOA 的关键阶段里程碑将是 0.6 或 0.6 的统计相关性。 RNAamp 优于 RT-ddPCR 转录分析或 QVOA 值。 具体目标 2（第二阶段）：使用 RAVEN 评估小组和 QVOA 样品进行测定验证 成功完成 RNAamp 和 RT-ddPCR 转录的 I 期比较测试 在目标 2 中，第一阶段指标的分析和实现，Jan Biotech 将使用 Reservoir 验证 RNAamp 检测验证和评估网络 (RAVEN) 评估小组。 具体目标 3（第二阶段）：评估 HIV 病毒库激活的临床相关性 该目标将评估这些新型生物标志物，以预测艾滋病毒反弹的时间，这对于确定优先顺序至关重要 治疗以确定那些可能受益于新的艾滋病毒缓解策略的参与者。 具体目标 4（第二阶段）：分析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 HIV 病毒库 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基于经过验证的序列的定量结果。 HIV 储存库诊断分析和计算软件将使我们能够获得 FDA 的批准 流程，包括导致 510(k) 批准的额外临床前和临床评估、临床试验、 以及准确、高通量的 HIV 储存库检测的商业化。