Real-Time Spliced-RNA Detection to Quantify Latent HIV-Infected Cells in HAART Patients

实时剪接 RNA 检测可量化 HAART 患者中潜伏的 HIV 感染细胞

• 批准号: 9238535
• 负责人: Janet L Huie
• 金额: $ 4万
• 依托单位: JAN BIOTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-13 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9238535
• 关键词: 13 year old; Acquired Immunodeficiency Syndrome; Adherence; Anti-Retroviral Agents; Biological Assay; Biotechnology; Blood specimen; CD4 Positive T Lymphocytes; Calibration; Cell Line; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chemicals; Chronic; Clinical; Clinical Trials; Collaborations; Consultations; Detection; Development; Diagnostic; Disease remission; Engineering; Funding; HIV; HIV Infections; HIV-1; Highly Active Antiretroviral Therapy; Howard Temin Award; Individual; Investigation; Laboratories; Laboratory Research; Length; Life; Ligation; Marketing; Measures; Messenger RNA; Methods; Molecular; Nucleic Acid Probes; Oligonucleotide Probes; Patient Monitoring; Patients; Performance; Phase; Procedures; Production; RNA; RNA Sequences; RNA Splicing; Reaction; Reagent; Regimen; Research; Research Personnel; Resources; Rest; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Site; Small Business Technology Transfer Research; Technology; Testing; Time; Translations; United States National Institutes of Health; Universities; Validation; Viral; Viral Genome; Viral Load result; Virion; Virus; Work; assay development; clinical application; collaboratory; cost; design; high throughput screening; internal control; meetings; molecular diagnostics; neuroAIDS; neurocognitive disorder; novel; novel therapeutics; prevent; product development; programs; public health relevance; research facility; viral DNA; viral RNA

 DESCRIPTION (provided by applicant): 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 (CDC, 2013). Strict adherence to highly active/combination anti-retroviral therapy (HAART/cART), prevents full-blown AIDS. However, HAART/cART fails to cure HIV infection as it has little effect on CD4+ cells infected with latent forms of the virus. If a patient no longer adheres to their prescribed regimen of HAART/cART, the latent pool quickly rebounds into full-blown HIV infection. Thus, HIV-AIDS is still far from eradicated. Issues with Current Solutions & How Product Meets Unmet Needs Current methods of quantifying the latent reservoirs include the quantitative viral outgrowth assay (Q-VOA), PCR and RT-PCR. Q-VOA is accepted as the most accurate method, but is a time and resource intensive procedure. PCR grossly overestimates the latent pool through detection of unintegrated as well as nonfunctional virus DNA. 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 latent HIV-infected cells. Q-VOA, the accepted quantitation standard, is currently available only at relativel few AIDS research facilities, due to its intensive resource and labor requirements. This product will introduce a real-time molecular assay to detect transcriptionally-competent HIV mRNA directly from latently infected cells isolated from HAART/cART patients. With validation against the Q-VOA standard, this assay has the potential to provide high-throughput, real-time, and lower-cost quantitation of the latent HIV- infected reservoirs in the body and significantly accelerate testing and discovery of a cure for HIV infection. Summary of Approach The product proposed is a real-time quantitative autoligation detection reaction (qLDR), which uses fluorogenic probes for chemical ligation in a thermocycling amplification reaction. qLDR allows for real-time and accurate quantification of the level of HIV mRNA present in CD4+ latent HIV-infected cells. The proposed assay will employ modified fluorogenic nucleic acid probes for superior stability and highly specific HIV RNA detection for quantifying latent HIV-infected reservoirs. Collaborators and Unique Resources Jan Biotech, Inc., with expertise in molecular diagnostic development, will collaborate with Dr. David Putnam, a chemist in the Department of Chemical and Biomolecular Engineering of Cornell University. Dr. Harris Gelbard, investigating the phenomenon of latent reservoir-induced neuroAIDS at the University of Rochester Center for AIDS Research (CFAR), and CFAR will provide consultation and HAART/cART CD4+ samples. Cell lines will be provided by the NIH AIDS Reagent Program; Q-VOA validation testing will be performed by CARE. Phase I Specific Aims Specific Aim 1: Develop spliced-RNA detection assay for quantitation of latent HIV-1 infected cells Specific Aim 2: Test qLDR with HAART patient CD4+ cells and validate against Q-VOA How Anticipated Results will Justify Phase II and Further Product Development Superior performance of qLDR is expected compared to Q-VOA and Q-VOA with RT-PCR, with real-time, sensitive and specific detection of spliced HIV mRNA directly from latent HIV-infected CD4+ cells from HAART/ cART patients. Successful Phase I validation against the Q-VOA standard will justify Phase II full validation and product development to produce a high-throughput commercial ready laboratory research assay platform. Additional Time and Funding Necessary to Bring Product to Market after Phase I Completion It is anticipated that a high-throughput laboratory research product can be brought to market as a laboratory assay kit for research purposes at the completion of the Phase II, two years after the Phase I work has been completed. It is anticipated that an additional 2-3 years and funding through a Phase II bridge award will be needed to perform the clinical trials required for FDA approval as a clinical diagnostic.

 描述（由申请人提供）：CDC 估计，在美国，1,144,500 名 13 岁及以上的人感染 HIV，其中约 180,900 人（15.8%）已感染但未确诊（CDC，2013）。 /联合抗逆转录病毒疗法（HAART/cART）可预防全面的艾滋病。 HAART/cART 无法治愈 HIV 感染，因为它对感染潜伏病毒的 CD4+ 细胞几乎没有影响。如果患者不再遵守规定的 HAART/cART 治疗方案，潜伏池很快就会反弹为全面的 HIV 感染。因此，当前解决方案的问题以及产品如何满足未满足的需求，HIV-AIDS 仍远未被根除。 目前量化潜在病毒库的方法包括病毒定量生长测定 (Q-VOA)、PCR 和病毒定量检测。 RT-PCR 被认为是最准确的方法，但它是一种时间和资源密集型程序，通过检测未整合的和非功能性的病毒 DNA，严重高估了潜伏池。 RNA 至每毫升 20-50 个病毒颗粒，从而缩短了 QVOA 得出结果的时间，通常适用于测量病毒载量，但不能直接检测具有复制能力的潜伏 HIV 感染细胞。公认的定量标准，由于其密集的资源和劳动力需求，目前仅在相对较少的艾滋病研究机构中可用。该产品将引入实时分子测定，以直接从从 HAART/分离的潜伏感染细胞中检测具有转录能力的 HIV mRNA。通过根据 Q-VOA 标准进行验证，这有可能对体内潜在的 HIV 感染库提供高通量分析、实时且低成本的定量，并显着加速测试和发现。方法摘要 所提出的产品是一种实时定量自连接检测反应 (qLDR)，它在热循环扩增反应中使用荧光探针进行化学连接，可以实时、准确地定量 HIV 感染的水平。存在于 CD4+ 潜伏 HIV 感染细胞中的 HIV mRNA 拟议的测定将采用改良的荧光核酸探针，以实现卓越的稳定性和高度特异性的 HIV RNA 检测，以量化潜伏 HIV 感染。合作者和独特资源 Jan Biotech, Inc. 拥有分子诊断开发方面的专业知识，将与康奈尔大学化学和生物分子工程系的化学家 Harris Gelbard 博士合作，研究这一现象。罗切斯特大学艾滋病研究中心 (CFAR) 提供潜在储存库诱发的神经艾滋病，CFAR 将提供咨询，HAART/cART CD4+ 细胞系将​​由 NIH AIDS 提供。试剂计划；Q-VOA 验证测试将由 CARE 进行。 第一阶段 特定目标 特定目标 1：开发用于定量潜伏 HIV-1 感染细胞的剪接 RNA 检测方法 特定目标 2：使用 HAART 患者 CD4+ 细胞测试 qLDR 并进行验证与 Q-VOA 相比 预期结果将如何证明 II 期和进一步产品开发的合理性 与 Q-VOA 和采用 RT-PCR 的 Q-VOA 相比，预计 qLDR 具有卓越的性能，直接从 HAART/cART 患者的潜伏 HIV 感染的 CD4+ 细胞中实时、灵敏和特异性地检测剪接的 HIV mRNA，根据 Q-VOA 标准成功进行 I 期验证将证明 II 期全面验证和产品开发是合理的，以生产高通量商业化实验室研究分析平台 第一阶段完成后将产品推向市场所需的额外时间和资金 预计高通量实验室研究产品可作为用于研究目的的实验室分析套件推向市场。完成在第一阶段工作完成两年后，预计将需要额外的 2-3 年时间和通过第二阶段过渡资助来进行 FDA 批准作为临床诊断所需的临床试验。 。