High throughput analysis of latency/reactivation with barcoded proviruses

 产品说明：清除所有可能重新引发感染的携带前病毒的细胞可能导致许多患者的功能性治愈，并减少病毒在人群中的传播。因此，更好地了解潜在的前病毒库是一个关键的公共卫生优先事项。一些HIV-1整合事件导致感染性子代或可重新激活的潜伏前病毒。换句 在某些情况下，逆转录过程中的突变或表观遗传沉默使整合成为功能性死胡同。已经使用有限数量的克隆细胞系或合并细胞的聚集表型对潜伏期和再活化进行了广泛研究。然而，更好地了解个体未表达的前病毒的再活化表型的全谱应大大推进减少钝性治疗以追求功能性治愈的长期目标。本申请将分子病毒学、T细胞发育和人类基因组学方法应用于一种新的高通量方法，用于更好地定义潜伏感染细胞的类别及其再活化特征。为R21探索性阶段提出的研究将开发一种新的条形码化前病毒技术，并验证其在培养细胞中的用途，建立使该系统在原代T细胞中发挥作用的技术，并开发生物信息学，用于在测试案例中对大量单个整合体进行高通量分析，该测试案例解决了T细胞系中沉默的程度由整合位置与随机效应决定。在R33期概念验证研究中，条形码化前病毒系统将被进一步完善并应用于定义T细胞系和原代CD4 + T细胞中响应于细胞分化或治疗性再活化的再活化和持续性的分子和基因组特征，以研究CD4效应T细胞的潜伏期和功能多样性之间的关系，并评估一种新的分子遗传学方法用于追踪残留病毒血症的克隆性的潜力，该方法将来可能应用于患者样本。

 DESCRIPTION: Purging all cells harboring proviruses that are likely to reinitiate infection could lead to a functional cure for many patients and would diminish viral spread in populations. Thus, better understanding of the latent proviral reservoir is a key public health priority. Some HIV-1 integration events lead to infectious progeny or to latent proviruses that can reactivate. In other cases, mutation during reverse transcription or epigenetic silencing makes integration a functional dead end. Latency and reactivation have been studied extensively using limited numbers of clonal cell lines or aggregate phenotypes of pooled cells. However, a better understanding of the full spectrum of reactivation phenotypes of individual unexpressed proviruses should greatly advance the long term goal of reducing blunt treatments in pursuit of a functional cure. The current application applies molecular virology, T cell development, and human genomics approaches to a novel high-throughput method for better defining classes of latently infected cells and their signatures of reactivation. Studies proposed for the R21 exploratory phase will develop a novel barcoded provirus technology and validate its use in cultured cells, establish technologies to enable this system in primary T-cells, and develop the bioinformatics for high throughput analysis of large numbers of individual integrants in a test case that addresses the extent to which silencing in a T cell line is determined by integration location vs. stochastic effects. In R33 phase proof-of- concept studies, the barcoded provirus system will be further refined and applied to defining molecular and genomic signatures of reactivation and persistence in response to cellular differentiation or therapeutic reactivation inT cell lines and primary CD4+ T-cells, to studying the relationship between latency and functional diversity of CD4 effector T cells, and to evaluating the potential of a novel molecular genetic approach for tracking the clonality of residual viremia, which may in the future be applied to patient samples.