Synthetic Molecular Sensors to Target Latently Infected Cells for HIV Eradication

合成分子传感器瞄准潜在感染细胞以根除艾滋病毒

• 批准号: 8657164
• 负责人: CHRISTOPHER H WOELK
• 金额: $ 14.29万
• 依托单位: UNIVERSITY OF SOUTHAMPTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8657164
• 关键词: 3' Untranslated Regions; Apoptosis; Binding Sites; Biological Assay; CD4 Positive T Lymphocytes; Cell Death; Cell Line; Cells; DNA; Data; Dependovirus; Derivation procedure; Development; Environment; Genes; Genome; Goals; HIV; Hela Cells; Histone Deacetylase Inhibitor; Homing; Immune response; Immune system; In Vitro; Individual; Knowledge; Lead; Letters; Machine Learning; Malignant neoplasm of cervix uteri; Methods; MicroRNAs; Mission; Modeling; Molecular; Patients; Pattern; Phase; Procedures; Promoter Regions; Proteins; Proviruses; Public Health; RNA; Research; Sensitivity and Specificity; Source; Specificity; Testing; Transfection; Variant; Viral; Virus; Virus Activation; adeno-associated viral vector; base; caspase-3; design; design and construction; endonuclease; gene therapy; in vitro Model; infectious disease treatment; innovation; memory CD4 T lymphocyte; next generation sequencing; novel; novel therapeutic intervention; promoter; sensor; suicide gene; synthetic biology; transcription factor; transcriptome sequencing; vector

PROJECT SUMMARY There are no methods currently available for the eradication of the latent reservoir in HIV-infected patients. The long-term goal of the proposed research is to develop a new method based on synthetic molecular sensors that is capable of targeting latently infected cells in HIV-infected patients for destruction. The objective of this particular application is to design constructs known as "Synthetic Molecular Sensors for HIV Eradication (SMaSHEd)" and determine their ability to eradicate latently infected cells from HIV-infected patients or from in vitro latency models. The central hypothesis is that the promoter regions from host genes and the binding sites for miRNAs whose expression can discriminate latently infected from uninfected cells can be used as sensors in SMaSHEd constructs to specifically express a suicide gene in latently infected cells. The rationale for the proposed research is that new methods for eradicating the latent reservoir are required that specifically target latently infected cells, and do not directly target the variable HIV provirus, require activation of HIV replication, or rely on the host immune system to eradicate activated cells. Guided by strong preliminary data, our hypothesis will be tested by pursuing the following specific aims: (1 & 2) Identify genes and miRNAs whose expression can discriminate latently infected primary CD4 T cells from uninfected cells, and (3) Determine the ability of SMaSHEd constructs to induce cell death in latently infected primary CD4 T cells. The analyses in Aim 1 & 2 will utilize RNA-Seq and smallRNA-Seq to identify the genes and miRNAs, respectively, that are differentially expressed between latently infected and uninfected primary memory CD4 T cells derived from the in vitro model of our collaborator Dr. Vincente Planelles. Our final aim will utilize procedures well established in the realm of synthetic biology to design SMaSHEd constructs containing the promoters and miRNA binding sites of genes (Aim 1) and miRNAs (Aim 2), respectively, that specifically sense the environment in latently infected cells. The ability of these constructs to induce the expression of a suicide gene specifically in latently infected primary CD4 T cells will be assessed following transfection or transduction of mixtures of latently infected and uninfected cells from the in vitro primary CD4 T cell latency model of Dr. Planelles and of Dr. O'Doherty, as well as from HIV-infected patients. The research proposed in this application is innovative, in our opinion, because it will utilize the latest advances in synthetic biology and information gained from next generation sequencing platforms (i.e., RNA-Seq and smallRNA-Seq) to develop a novel method for eradicating latently infected cells. This will be significant because it is the first step in a continuum of research that is expected to lead to the development of a new strategy for the eradication of the latent reservoir in HIV-infected patients. Ultimately, SMaSHEd constructs will be developed for use with appropriate vectors (i.e., adeno-associated virus) for the treatment of HIV-infected patients, where, in contrast to gene therapy, they will only require transient expression to induce the destruction of latently infected cells.

项目总结 目前还没有方法可用于根除艾滋病毒感染者的潜伏蓄水池 病人。提出的研究的长期目标是开发一种新的方法，基于合成 分子传感器，能够针对艾滋病毒感染患者的潜伏感染细胞进行破坏。 这一特定应用的目标是设计被称为用于 根除艾滋病毒(粉碎)“，并确定它们从艾滋病毒感染者中清除潜伏感染细胞的能力 患者或来自体外潜伏期模型。中心假设是宿主基因的启动子区域 而miRNAs的结合部位可以区分潜伏感染的细胞和未感染的细胞 在被粉碎的结构中用作传感器，在潜伏感染的细胞中特异性地表达自杀基因。 建议进行这项研究的理由是需要新的方法来根除潜伏的油气藏。 它们专门针对潜伏感染的细胞，而不直接针对可变的艾滋病毒前病毒，需要 激活艾滋病毒复制，或依靠宿主免疫系统根除激活的细胞。以Strong为指导 初步数据，我们的假设将通过追求以下具体目标来验证：(1)识别基因 以及其表达可以区分潜伏感染的原代CD4T细胞和未感染细胞的miRNAs， 以及(3)确定破碎的构建体在潜伏感染的原代CD4T细胞中诱导细胞死亡的能力 细胞。目标1和2中的分析将利用RNA-Seq和Small RNA-Seq来识别基因和miRNAs， 分别在潜伏感染和未感染的初级记忆CD4T之间有差异表达 细胞来源于我们的合作者Vincente Planelle博士的体外模型。我们的最终目标将利用 在合成生物学领域建立了一套程序来设计包含 基因的启动子和miRNA结合位点(Aim 1)和miRNAs(Aim 2) 潜伏感染细胞中的环境。这些结构诱导自杀表达的能力 潜伏感染的原代CD4T细胞中的特异性基因将在转基因或转导后进行评估 来自DR体外原代CD4T细胞潜伏期模型的潜伏感染和未感染细胞的混合。 Planelle和O‘Doherty博士的，以及艾滋病毒感染患者的。这项研究中提出的 在我们看来，应用是创新的，因为它将利用合成生物学和 从下一代测序平台(即RNA-Seq和SmallRNA-Seq)获得的信息，以开发 根除潜伏感染细胞的新方法。这将是意义重大的，因为这是 预计将导致制定根除艾滋病的新战略的研究连续体 HIV感染者体内的潜伏蓄水池。最终，粉碎的构造将被开发用于 用于治疗艾滋病毒感染者的适当载体(即腺相关病毒)，与之相反 对于基因治疗，它们只需要瞬时表达就可以诱导对潜伏感染细胞的破坏。