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: 9731244
• 负责人: David James Looney
• 金额: $ 52.45万
• 依托单位: VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
• 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 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9731244
• 关键词: 3' Untranslated Regions; Apoptosis; Binding Sites; Biological Assay; CASP3 gene; 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; National Institute of Allergy and Infectious Disease; Patients; Pattern; Phase; Procedures; Promoter Regions; Proteins; Proviruses; Public Health; RNA; Research; Sensitivity and Specificity; Source; Specificity; Testing; Transfection; Variant; Viral reservoir; Virus; Virus Activation; Virus Latency; adeno-associated viral vector; base; design; design and construction; differential expression; 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; sequencing platform; 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（由申请方提供）：目前尚无方法可用于根除HIV感染患者的潜伏性储库。这项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，该方法能够靶向HIV感染患者体内潜伏感染的细胞进行破坏。该特定应用的目的是设计被称为“用于HIV根除的合成分子传感器（SMaSHEd）”的构建体，并确定它们从HIV感染患者或从体外潜伏模型中根除潜伏感染细胞的能力。核心假设是，来自宿主基因的启动子区域和其表达可以区分潜伏感染细胞与未感染细胞的miRNA的结合位点可以用作SMaSHEd构建体中的传感器，以在潜伏感染细胞中特异性表达自杀基因。拟议研究的基本原理是，需要新的方法来消除潜伏的水库，专门针对潜伏感染的细胞，而不是直接针对可变的HIV前病毒，需要激活HIV复制，或依赖于宿主免疫系统来消除激活的细胞。在强有力的初步数据的指导下，我们的假设将通过追求以下具体目标来测试：（1和2）鉴定其表达可以区分潜伏感染的原代CD 4 T细胞与未感染的细胞的基因和miRNA，以及（3）确定SMaSHEd构建体诱导潜伏感染的原代CD 4 T细胞中的细胞死亡的能力。目标1和2中的分析将利用RNA-Seq和smallRNA-Seq来识别基因和miRNA，分别在来自我们的合作者Vincente Planelles博士的体外模型的潜伏感染和未感染的初级记忆CD 4 T细胞之间差异表达。我们的最终目标是利用合成生物学领域中建立的程序来设计分别含有基因（Aim 1）和miRNA（Aim 2）的启动子和miRNA结合位点的SMaSHEd构建体，其特异性地感测潜伏感染细胞中的环境。这些构建体特异性诱导自杀基因在潜伏感染的原代CD 4 T细胞中表达的能力将在转染或转导来自Planelles博士和O ′ Doherty博士的体外原代CD 4 T细胞潜伏模型以及来自HIV感染患者的潜伏感染和未感染细胞的混合物后进行评估。在我们看来，本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smallRNA-Seq），以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，预计这些研究将莱亚发展一种新的战略，以根除艾滋病毒感染患者的潜伏储存库。最终，SMaSHEd构建体将被开发用于适当的载体（即，腺相关病毒）用于治疗HIV感染的患者，与基因治疗相反，它们仅需要瞬时表达来诱导潜在感染细胞的破坏。