CTCF peptides - a new therapeutic alternative to target HIV in the brain.

CTCF 肽 - 一种针对大脑中 HIV 的新治疗替代方案。

基本信息

批准号：
7621223
负责人：
Malgorzata Simm
金额：
$ 40.65万
依托单位：
ST. LUKE'S-ROOSEVELT INST FOR HLTH SCIS
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-30 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7621223
关键词：
AIDS Dementia Complex Acquired Immunodeficiency Syndrome Affinity Amino Acid Sequence Antiviral Agents Antiviral Response Antiviral resistance Applications Grants Astrocytes Binding Binding Sites Biological Biological Assay Biological Response Modifier Therapy Brain CCCTC-binding factor CD4 Positive T Lymphocytes Cell Line Cell Surface Receptors Cells Complex Cultured Cells DNA Binding DNA Probes Data Dose Evaluation Extracellular Space Facility Construction Funding Category Foundations Future Genetic Transcription Genome Goals HIV HIV-1 Highly Active Antiretroviral Therapy Human In Vitro Incidence Infection Kinetics Length Ligation Maintenance Mediating Messenger RNA Methods Mus N-terminal NF-kappa B Names Neurotoxins Nitric Oxide Nuclear Nuclear Extract Nucleotides Oligonucleotides Organ Pathway interactions Patients Penetration Peptides Pharmaceutical Preparations Phase Phenotype Process Proteins Protocols documentation Public Health Range Reaction Recombinant Proteins Recombinants Refractory Reporter Genes Resistance Small Interfering RNA TNFRSF5 gene Testing Therapeutic Agents Time Toxic effect Transmembrane Transport Virus Virus Replication base brain tissue cytokine deletion library dimer extracellular fetal in vivo macrophage mutant novel novel therapeutics prevent promoter research study resistance factors response size therapeutic target tool uptake

AIDS Dementia Complex Acquired Immunodeficiency Syndrome Affinity Amino Acid Sequence Antiviral Agents Antiviral Response Antiviral resistance Applications Grants Astrocytes Binding Binding Sites Biological Biological Assay Biological Response Modifier Therapy Brain CCCTC-binding factor CD4 Positive T Lymphocytes Cell Line Cell Surface Receptors Cells Complex Cultured Cells DNA Binding DNA Probes Data Dose Evaluation Extracellular Space Facility Construction Funding Category Foundations Future Genetic Transcription Genome Goals HIV HIV-1 Highly Active Antiretroviral Therapy Human In Vitro Incidence Infection Kinetics Length Ligation Maintenance Mediating Messenger RNA Methods Mus N-terminal NF-kappa B Names Neurotoxins Nitric Oxide Nuclear Nuclear Extract Nucleotides Oligonucleotides Organ Pathway interactions Patients Penetration Peptides Pharmaceutical Preparations Phase Phenotype Process Proteins Protocols documentation Public Health Range Reaction Recombinant Proteins Recombinants Refractory Reporter Genes Resistance Small Interfering RNA TNFRSF5 gene Testing Therapeutic Agents Time Toxic effect Transmembrane Transport Virus Virus Replication base brain tissue cytokine deletion library dimer extracellular fetal in vivo macrophage mutant novel novel therapeutics prevent promoter research study resistance factors response size therapeutic target tool uptake

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项目摘要

DESCRIPTION (provided by applicant): Virus-infected macrophages are the major therapeutic target in the brain tissue, even though the productive virus replication in these cells is necessary but not sufficient to cause HIV Associated Dementia (HAD). HAART is still generally recommended for patients with AIDS and in general this treatment has been effective in decreasing the incidence of HAD; however a significant group of patients remains refractory to HAART even when its CNS penetration is enhanced by supplementing drugs. For this reason alone, the activation of native cellular mechanisms inducing anti-viral resistance in cells migrating to various organs including CNS should be considered a viable alternative to add to the existing antiviral approaches for HAD. We have induced such responses mediated through soluble molecule [previously named HRF (HIV-1 Resistance Factor)] able to prevent virus replication in CD4+ T cells. Recently we identified the HRF as a truncated form of a potent transcription suppressor - CTCF (tCTCF). The biological activity of tCTCF was confirmed by several experimental methods: (1) the siRNA silencing of CTCF mRNA in HRF(+) cells reversed the HIV-1 resistance phenotype and reduced the biological activity of their cell culture supernatants; (2) depletion of tCTCF from HRF(+) cell culture supernatant abolished its antiviral activity; while (3) the affinity purified tCTCF induced antiviral activity in human macrophages; and (4) the affinity purified tCTCF inhibited the transcription of HIV-1 LTR promoted expression of reporter gene. Our preliminary data showed that upon HIV-1 exposure, CTCF from nuclear extracts of HRF(+) but not HRF(-) control cells bound to DNA probe comprising NF-:B, Sp1 and YB-1 binding sequences, possibly through its interaction with YB-1 protein. We hypothesize that since NF-:B and YB-1 binding sites are separated by only 6 nucleotides the CTCF/YB1 complex bound to the virus promoter might obstruct the formation of NF- :B/DNA binding, thus halting transcription of virus genome. Taken together we discovered a novel form of CTCF protein that mediates innate responses to HIV-1 in human macrophages. The main goal of this application is to prepare foundation for future delivery of tCTCF as a biotherapeutic for treatment of HIV-1 in CNS. In this grant application we propose: (1) To study the mechanism of tCTCF mediated cellular responses to HIV-1; (2) To determine the minimum tCTCF amino-acid sequence required for the induction of antiviral responses and efficacy assessment of tCTCF treatment in primary human macrophages; and (3) To determine the minimal CTCF toxicity and brain entry in vivo. PUBLIC HEALTH RELEVANCE: Recently we identified the novel anti-HIV-1 protein called truncated form of a potent transcription suppressor - CTCF (tCTCF). tCTCF is a product of posttranslational processing of the full size CTCF protein and is secreted into extracellular space by HIV-1 resistant - tCTCF producing cells. The biological activity of tCTCF was confirmed by several experimental methods: (1) the siRNA silencing of full length CTCF mRNA in tCTCF producing cells reversed the HIV-1 resistance phenotype and reduced the biological activity of their cell culture supernatants; (2) depletion of tCTCF from tCTCF(+) cell culture supernatant abolished its antiviral activity; while (3) the affinity purified tCTCF induced antiviral activity in human macrophages; and (4) the affinity purified tCTCF inhibited the transcription of HIV-1 LTR promoted expression of reporter gene. Our preliminary data showed that upon HIV-1 exposure, CTCF from nuclear extracts of tCTCF producing but not control cells bound to DNA probe comprising NF-kB, Sp1 and YB-1 binding sequences, possibly through its interaction with YB-1 protein. We hypothesize that since NF-kB and YB-1 binding sites are separated by only 6 nucleotides the CTCF/YB1 complex bound to the virus promoter might obstruct the formation of NF-kB/DNA binding, thus halting transcription of virus genome. Taken together we discovered a novel form of CTCF protein that mediates innate responses to HIV-1 in human macrophages. The main goal of this application is to prepare foundation for future delivery of tCTCF as a biotherapeutic for treatment of HIV-1 in CNS.

描述（由申请人提供）：即使这些细胞中的生产性病毒复制是必要但不足以引起HIV相关痴呆症（HAT），但感染病毒的巨噬细胞是脑组织中的主要治疗靶标。通常，对于艾滋病患者，HAART通常仍建议使用HAART，总的来说，这种治疗方法已有效降低HAD的发生率。然而，即使通过补充药物增强了CNS的渗透率，也有很大一部分患者仍然难以忍受HAART。仅出于这个原因，应将迁移到包括CN在内的各种器官的细胞中诱导抗病毒抗性的天然细胞机制的激活视为可行的替代方法，可以添加到现有的抗病毒方法中。我们诱导了通过可溶性分子[以前命名为HRF（HIV-1抗性因子）]介导的这种反应，以防止CD4+ T细胞中的病毒复制。最近，我们将HRF确定为有效转录抑制器-CTCF（TCTCF）的截断形式。通过几种实验方法证实了TCTCF的生物学活性：（1）HRF（+）细胞中CTCF mRNA的siRNA沉默逆转了HIV-1耐药表型，并降低了其细胞培养上期诊所的生物学活性；（2）HRF（+）细胞培养上清液中TCTCF的耗竭消除了其抗病毒活性；（3）亲和力纯化的TCTCF诱导人类巨噬细胞中的抗病毒活性；（4）亲和力纯化的TCTCF抑制了HIV-1 LTR的转录促进了报告基因的表达。我们的初步数据表明，HIV-1暴露后，来自HRF（+）的核提取物的CTCF，但不是HRF（ - ）对照细胞结合到包含NF-：B，SP1和YB-1结合序列的DNA探针，可能是通过与YB-1蛋白质相互作用的。我们假设，由于NF-：B和Yb-1结合位点仅被6个核苷酸分离，因此与病毒启动子结合的CTCF/YB1复合物可能会阻碍NF-：B/DNA结合的形成，因此停止了病毒基因组的转录。综上所述，我们发现了一种新型的CTCF蛋白质形式，它介导了人类巨噬细胞中对HIV-1的先天反应。该应用的主要目标是为将来将TCTCF作为CNS中HIV-1治疗的生物治疗作品做准备。在此赠款应用中，我们建议：（1）研究TCTCF介导的细胞对HIV-1的反应的机理；（2）确定诱导抗病毒反应所需的最小TCTCF氨基酸序列以及对原代人巨噬细胞中TCTCF治疗的疗效评估；（3）确定在体内最小的CTCF毒性和大脑进入。公共卫生相关性：最近，我们确定了一种新型的抗HIV-1蛋白，称为有效转录抑制器-CTCF（TCTCF）的截断形式。 TCTCF是全尺寸CTCF蛋白翻译后加工的产物，并通过HIV -1抗性-TCTCF产生细胞分泌到细胞外空间中。通过几种实验方法证实了TCTCF的生物学活性：（1）产生TCTCF的全长CTCF mRNA的siRNA沉默逆转了HIV-1耐药表型，并降低了其细胞培养上清液的生物学活性；（2）TCTCF从TCTCF（+）细胞培养上清液中耗尽了其抗病毒活性；（3）亲和力纯化的TCTCF诱导人类巨噬细胞中的抗病毒活性；（4）亲和力纯化的TCTCF抑制了HIV-1 LTR的转录促进了报告基因的表达。我们的初步数据表明，在HIV-1暴露后，来自TCTCF产生的核提取物的CTCF，但不能通过与YB-1蛋白质相互作用的NF-KB，SP1和YB-1结合序列结合的DNA探针结合。我们假设，由于NF-KB和YB-1结合位点仅被6个核苷酸分离，因此与病毒启动子结合的CTCF/YB1复合物可能会阻碍NF-KB/DNA结合的形成，从而停止了病毒基因组的转录。综上所述，我们发现了一种新型的CTCF蛋白质形式，它介导了人类巨噬细胞中对HIV-1的先天反应。该应用的主要目标是为将来将TCTCF作为CNS中HIV-1治疗的生物治疗作品做准备。