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: 8607892
• 负责人: CHRISTOPHER H WOELK
• 金额: $ 14.85万
• 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 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8607892
• 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; Histones; 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; inhibitor/antagonist; innovation; memory CD4 T lymphocyte; next generation sequencing; novel; novel therapeutic intervention; promoter; public health relevance; sensor; suicide gene; synthetic biology; transcription factor; transcriptome sequencing; vector

DESCRIPTION (provided by applicant): 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 wil 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 abilty 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 lea 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.

描述(由申请人提供)：目前尚无方法可用于根除艾滋病毒感染患者的潜伏蓄积物。这项拟议研究的长期目标是开发一种基于合成分子传感器的新方法，能够针对艾滋病毒感染患者的潜伏感染细胞进行破坏。这一特殊应用的目标是设计被称为“用于根除艾滋病毒的合成分子传感器(SMASD)”的结构，并确定它们从艾滋病毒感染患者或体外潜伏模型中根除潜伏感染细胞的能力。中心假设是宿主基因的启动子区域和miRNAs的结合位点可以作为传感器在潜在感染的细胞中特异性地表达自杀基因，这些区域的表达可以区分潜伏感染和未感染的细胞。这项拟议研究的基本原理是，需要专门针对潜伏感染细胞的新方法来根除潜伏库，而不是直接针对可变的艾滋病毒前病毒，需要激活艾滋病毒复制，或依赖宿主免疫系统来根除激活的细胞。在强大的初步数据的指导下，我们的假设将通过追求以下特定目标来检验：(1)识别其表达可以区分潜伏感染的原代CD4T细胞和未感染细胞的基因和miRNAs，以及(3)确定粉碎的构建体诱导潜伏感染的原代CD4T细胞死亡的能力。目标1和2中的分析将利用RNA-Seq和Small RNA-Seq分别识别潜伏感染和未感染的初级记忆CD4T细胞之间差异表达的基因和miRNAs，这些细胞来自我们的合作者Vincente Planelle博士的体外模型。我们的最终目标是利用合成生物学领域成熟的程序来设计粉碎的结构，分别包含基因的启动子和miRNA结合位点(Aim 1)和miRNAs(Aim 2)，它们专门感知潜伏感染细胞中的环境。这些构建体在潜伏感染的原代CD4T细胞中诱导特定自杀基因表达的能力将在从Planelle博士和O‘Doherty博士的体外原代CD4T细胞潜伏模型以及来自HIV感染患者的潜伏感染和未感染的细胞的混合细胞中转染或转导后进行评估。在我们看来，这项申请中提出的研究是创新的，因为它将利用合成生物学的最新进展和从下一代测序平台(即RNA-Seq和Small RNA-Seq)获得的信息来开发一种根除潜伏感染细胞的新方法。这将是意义重大的，因为这是一系列研究的第一步，预计这些研究将有助于制定新的战略，消除艾滋病毒感染患者的潜伏病原体。最终，粉碎的构建体将被开发用于与适当的载体(即腺相关病毒)一起用于治疗艾滋病毒感染的患者，与基因治疗不同，它们将只需要瞬时表达来诱导潜在感染细胞的破坏。