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

 描述(由申请人提供)：美国疾病控制与预防中心估计，在美国，13岁及以上的人中有1,144,500人感染了艾滋病毒，还有大约180,900人(15.8%)感染了艾滋病毒，但未确诊(CDC，2013)。严格遵守高效/联合抗逆转录病毒疗法(HAART/CART)，防止艾滋病全面爆发。然而，HAART/CART未能治愈艾滋病毒感染，因为它对感染潜伏型病毒的CD4+细胞几乎没有影响。如果患者不再坚持HAART/CART的处方方案，潜伏池很快就会反弹为全面的艾滋病毒感染。因此，艾滋病毒-艾滋病仍远未根除。当前解决方案的问题和产品如何满足未满足的需求目前量化潜在储存库的方法包括定量病毒生长分析(Q-VOA)、聚合酶链式反应和逆转录-聚合酶链式反应。Q-VOA被认为是最准确的方法，但它是一个耗费时间和资源的过程。通过检测整合型和非功能性病毒DNA，聚合酶链式反应严重高估了潜伏池。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QVOA结果的时间，并且通常适用于病毒载量的测量，但不直接检测具有复制能力的潜伏HIV感染细胞。Q-VOA是公认的定量标准，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可用。该产品将推出一种实时分子分析方法，直接从HAART/CART患者的潜伏感染细胞中检测具有转录能力的HIV mRNA。通过Q-VOA标准的验证，这种检测方法有可能提供高通量、实时和低成本的体内潜在艾滋病毒感染宿主的量化，并显著加快艾滋病毒感染治疗方法的测试和发现。该产品提出的方法概述是实时定量自配基检测反应(QLDR)，它使用荧光探针在热循环扩增反应中进行化学连接。QLDR允许实时和准确地量化存在于CD4+潜伏的HIV感染细胞中的HIV mRNA水平。建议的检测将使用改进的荧光核酸探针，以实现卓越的稳定性和高度特异性的艾滋病毒RNA检测，以量化潜在的艾滋病毒感染宿主。合作者和独特资源Jan Biotech，Inc.在分子诊断开发方面拥有专业知识，将与康奈尔大学化学和生物分子工程系化学家大卫·帕特南博士合作。Harris Gelbard博士在罗切斯特大学艾滋病研究中心(CFAR)研究潜伏的水库诱发神经艾滋病现象，CFAR将提供咨询和HAART/CART CD4+样本。细胞系将由NIH艾滋病试剂计划提供；Q-VOA验证测试将由CARE进行。第一阶段具体目标具体目标1：建立剪接RNA检测方法以定量检测潜伏的HIV-1感染细胞特定目标2：用HAART患者的CD4+细胞测试qLDR并验证Q-VOA如何验证预期结果将如何证明第二阶段和进一步的产品开发与RT-PCR的Q-VOA和Q-VOA相比，qLDR预期具有更好的性能，通过实时、灵敏和特异的直接从HAART/CART患者的潜伏感染的HIV+细胞中检测剪接的HIV mRNA。针对Q-VOA标准的成功第一阶段验证将证明第二阶段全面验证和产品开发是合理的，以生产一个高通量、商业就绪的实验室研究分析平台。在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在第二阶段完成时，即第一阶段工作完成两年后，高通量实验室研究产品可以作为用于研究目的的实验室分析试剂盒推向市场。预计将需要额外的2-3年和通过第二阶段桥梁奖励的资金来执行FDA批准作为临床诊断所需的临床试验。

 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.