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

项目总结摘要 公共卫生问题 在美国，1,144,500名13岁及以上的人感染艾滋病毒，约有180,900人 (15.8%)其他感染者但未确诊。对于大多数艾滋病毒阳性的人来说，可用的治疗方法只能 控制艾滋病毒感染，延缓向艾滋病的进展。HAART有很大的副作用，未能预防 至少50%的患者患有HIV-1相关的神经认知障碍。暴露前预防 (PREP)在减少艾滋病毒感染风险和传播方面取得了令人兴奋的进展，但它仍然 尚不清楚何时停止PrEP治疗是安全的。 SBIR第二阶段项目的最终价值将证明我们的技术预测时间的能力 病毒载量在治疗中断后反弹，这在艾滋病毒治疗领域仍然是一个关键的未得到满足的需求。 当前解决方案存在的问题以及产品如何满足未满足的需求 目前量化潜在储备的方法包括定量病毒生长试验(Q-VOA)、聚合酶链式反应 和RT-PCR法。Q-VOA是一个耗费时间和资源的过程，而RT-PCR可以用来检测病毒 RNA减少到每毫升20-50个病毒颗粒，从而缩短了QVOA产生的时间，并且普遍适用 用于测量病毒载量，但不直接检测具有复制能力的潜伏感染艾滋病毒的细胞。一月 生物技术公司的艾滋病毒qLDR提供了明显的优势，包括：1)检测血液中的潜在存储细胞，而不需要 活化法；2)直接检测核糖核酸以提高定量能力，这是其他分析方法所不具备的；3)非酶 扩增，这减轻了对RNA纯化的需要和随之而来的RNA的损失；4)检测短 RNA片段，即使在存在核糖核酸酶降解酶的情况下也能提供有效的检测；5)1月 Biotech的qLDR PNA探针能够使用与高度保守的区域互补的短序列 HIV-1基因组，它允许对高度遗传多样性的HIV-1亚型进行广泛的检测。 方法概述 第一阶段qLDR探针组区分外周血中潜伏的和激活的HIV感染细胞， 这对准确测量具有复制能力的潜在储集层的水平具有很大的希望。 第二阶段的特定目标将展示该检测方法预测病毒载量在以下时间反弹的能力 使用体外和体内样本中断治疗。测试将评估化验作为一种 临床终点，以指导治疗中断决定，并促进艾滋病毒缓解和治愈发现。 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Jonathan Li，MD，MMSc进行咨询 哈佛医学院医学助理教授，参与艾滋病临床试验小组(ACTG)的研究。朱迪思 ACTG网络副主席Currier，MD，MSc将监督治疗过程中的样本收集 中断研究，A5345；Q-VOA交叉验证测试将由南方研究院进行。 第二阶段的具体目标 具体目标1：对具有复制能力的潜在储存库进行艾滋病毒qLDR预测 任务1：优化qLDR探针组 任务2：治疗中断后的体外反弹(TI) 具体目标2：分析治疗中断期间艾滋病毒反弹的QLDR预测 任务1：分析治疗中断(ATI)后病毒反弹的艾滋病毒qLDR预测 任务2：HIV qLDR与ATI研究中使用的其他检测方法的比较 具体目标3：艾滋病毒qLDR的临床前验证 任务1：验证qLDR性能特征 任务2：QVOA验证 第二期完成后的市场 Gilead和Bionor都对这项技术有商业兴趣，并期待着产生的结果 来自第二阶段和ACTG研究。商业化将寻求监管部门的批准。

PROJECT SUMMARY ABSTRACT Public Health Problem 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 treatments can only control HIV infection and delay progression to AIDS. HAART has substantial side effects and fails to prevent at least 50% of patients from developing HIV-1 associated neurocognitive disorders. Pre-exposure prophylaxis (PrEP) has been an exciting development for reduction of HIV acquisition risk and transmission, but it remains unclear when it is safe to discontinue PrEP treatment. The ultimate value of the SBIR Phase II project will demonstrate the ability of our technology to predict time to viral load rebound after treatment interruption, which remains a critical unmet need in the HIV cure field. Issues with Current Solutions & How Product Meets Unmet Needs Current methods of quantifying the latent reserve include the quantitative viral outgrowth assay (Q-VOA), PCR and RT-PCR. Q-VOA 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 latent HIV-infected cells. Jan Biotech’s HIV qLDR offers clear advantages including: 1) detection of latent reservoir cells in the blood without activation; 2) direct detection of RNA to improve quantitative ability that is lost in other assays; 3) nonenzymatic amplification, which relieves the need for RNA purification and its attendant loss of RNA; 4) detection of short segments of RNA, providing effective detection even in the presence of RNase degradative enzymes; 5) Jan Biotech’s qLDR PNA probes enable the use of short sequences complementary to highly conserved regions of the HIV-1 genome, which allows for breadth of detection across the highly genetically diverse HIV-1 subtypes. Summary of Approach The Phase I qLDR probe sets differentiated between latent and activated HIV-infected cells in peripheral blood, which has great promise for accurate measurement of the level of the replication-competent latent reservoir. The Phase II Specific Aims will demonstrate the ability of the assay to predict time to viral load rebound after treatment interruption using ex vivo and in vivo samples. Testing will evaluate the utility of the assay as a clinical endpoint to guide treatment interruption decisions and facilitate HIV remission and cure discovery. Collaborators and Unique Resources Jan Biotech, Inc., with expertise in molecular diagnostic development, will consult with Jonathan Li, MD, MMSc Assistant Professor of Medicine, Harvard Medical School, in a AIDS Clinical Trials Group (ACTG) study. Judith Currier, MD, MSc, Vice Chair of the ACTG Network will oversee sample collection from the treatment interruption study, A5345; Q-VOA cross-validation testing will be performed by Southern Research Institute. Phase II Specific Aims Specific Aim 1: HIV qLDR prediction of replication-competent latent reservoir Task 1: Optimization of qLDR probe sets Task 2: Ex vivo rebound after treatment interruption (TI) Specific Aim 2: HIV qLDR prediction of HIV rebound during Analytical Treatment Interruption Task 1: HIV qLDR prediction of Viral Rebound after Analytical Treatment Interruption (ATI) Task 2: Comparison of HIV qLDR to other assays used in ATI study Specific Aim 3: Preclinical Validation of HIV qLDR Task 1: Validation of qLDR performance characteristics Task 2: QVOA validation Market after Phase II Completion Both Gilead and Bionor are commercially interested in the technology and look forward to results generated from the Phase II and the ACTG study. Regulatory approval will be sought for commercialization.