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

 描述（由申请人提供）：CDC估计，在美国，1，144，500名13岁及以上的人感染了艾滋病毒，另有约180，900人（15.8%）感染但未确诊（CDC，2013）。严格遵守高效/联合抗逆转录病毒疗法（HAART/cART），可预防艾滋病全面爆发。然而，HAART/cART无法治愈HIV感染，因为它对感染潜伏形式病毒的CD 4+细胞几乎没有影响。如果患者不再坚持他们规定的HAART/cART方案，潜伏池迅速反弹到全面的HIV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以及产品如何满足未满足的需求当前定量潜伏储库的方法包括定量病毒生长测定（Q-VOA）、PCR和RT-PCR。Q-VOA被认为是最准确的方法，但这是一个时间和资源密集型的程序。PCR通过检测未整合的以及无功能的病毒DNA严重高估了潜伏池。RT-PCR可用于检测病毒RNA至20-50个病毒颗粒/mL，从而缩短了QVOA结果的时间，并且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。目前，公认的定量标准Q-VOA仅在相对较少的艾滋病研究机构中可用，这是由于其密集的资源和劳动力需求。该产品将引入实时分子检测，以直接从HAART/cART患者分离的潜伏感染细胞中检测具有转录能力的HIV mRNA。通过对Q-VOA标准的验证，该检测有可能提供体内潜伏的HIV感染储库的高通量、实时和低成本定量，并显着加速HIV感染治愈的测试和发现。方法概述所提出的产品是实时定量自连接检测反应（qLDR），其在热循环扩增反应中使用荧光探针进行化学连接。qLDR允许实时和准确定量存在于CD 4+潜伏HIV感染细胞中的HIV mRNA水平。拟定的检测方法将采用改良的荧光核酸探针，以获得上级稳定性和高度特异性的HIV RNA检测，用于定量潜伏的HIV感染储库。合作者和独特资源Jan Biotech，Inc.在分子诊断开发方面具有专业知识，将与康奈尔大学化学和生物分子工程系的化学家大卫普特南博士合作。Harris Gelbard博士在罗切斯特大学艾滋病研究中心（CFAR）研究潜伏性艾滋病诱导的神经艾滋病现象，CFAR将提供咨询和HAART/cART CD 4+样本。细胞系将由NIH AIDS Reagent Program提供; Q-VOA验证检测将由CARE进行。第一阶段具体目标具体目标1：开发用于定量潜伏HIV-1感染细胞的剪接RNA检测试验具体目标2：使用HAART患者CD 4+细胞测试qLDR，并根据Q-VOA验证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Q-VOA相比，qLDR的性能预期优于Q-VOA和Q-VOA与RT-PCR，实时，直接从HAART/ cART患者的潜伏HIV感染的CD 4+细胞中灵敏和特异地检测剪接的HIV mRNA。根据Q-VOA标准成功进行的第一阶段验证将证明第二阶段的全面验证和产品开发是合理的，以产生高通量商业化的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实验室测定试剂盒推向市场，用于研究目的。预计将需要额外的2-3年时间和通过II期桥梁奖提供的资金，以进行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.