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：
7692168
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/7692168
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 Culture Techniques Cell Line Cell Surface Receptors Cells Complex DNA Binding DNA Probes Data Dose Evaluation Extracellular Space 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 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 vitro activity in vivo macrophage mutant novel novel therapeutics prevent promoter protein complex public health relevance research study resistance factors response therapeutic target tool uptake

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）：病毒感染的巨噬细胞是脑组织中的主要治疗靶点，尽管这些细胞中有效的病毒复制是必要的，但不足以引起 HIV 相关痴呆（HAD）。 HAART 仍被普遍推荐用于 AIDS 患者，总体而言，这种治疗方法可有效降低 HAD 的发病率；然而，即使通过补充药物增强了 HAART 的中枢神经系统渗透性，仍有相当一部分患者对 HAART 仍难治。仅出于这个原因，激活天然细胞机制，诱导迁移到包括中枢神经系统在内的各种器官的细胞产生抗病毒耐药性，就应该被认为是 HAD 现有抗病毒方法的可行替代方案。我们已经通过可溶性分子 [以前称为 HRF（HIV-1 抵抗因子）] 诱导了这种反应，能够阻止病毒在 CD4+ T 细胞中复制。最近我们发现 HRF 是一种有效的转录抑制因子 CTCF (tCTCF) 的截短形式。 tCTCF的生物活性通过多种实验方法得到证实：（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清液的生物活性； (2)从HRF(+)细胞培养上清液中去除tCTCF，消除了其抗病毒活性； (3)亲和纯化的tCTCF在人巨噬细胞中诱导抗病毒活性； (4)亲和纯化的tCTCF抑制HIV-1 LTR的转录，促进报告基因的表达。我们的初步数据表明，在暴露于 HIV-1 后，来自 HRF(+) 而不是 HRF(-) 对照细胞的核提取物的 CTCF 可能通过其相互作用与包含 NF-:B、Sp1 和 YB-1 结合序列的 DNA 探针结合与 YB-1 蛋白。我们假设，由于 NF-:B 和 YB-1 结合位点仅相隔 6 个核苷酸，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，从而停止病毒基因组的转录。总之，我们发现了一种新形式的 CTCF 蛋白，可介导人类巨噬细胞对 HIV-1 的先天反应。该应用的主要目标是为未来将 tCTCF 作为治疗 CNS 中 HIV-1 的生物疗法奠定基础。在本次资助申请中，我们建议：（1）研究tCTCF介导的HIV-1细胞反应机制； (2) 确定在原代人巨噬细胞中诱导抗病毒反应和评估 tCTCF 治疗功效所需的最小 tCTCF 氨基酸序列； (3)确定CTCF的最小体内毒性和脑进入。公共健康相关性：最近我们发现了一种新型抗 HIV-1 蛋白，称为截短形式的有效转录抑制因子 - CTCF (tCTCF)。 tCTCF 是全尺寸 CTCF 蛋白翻译后加工的产物，由 HIV-1 抗性 - tCTCF 产生细胞分泌到细胞外空间。 tCTCF的生物活性通过多种实验方法得到证实：（1）tCTCF产生细胞中全长CTCF mRNA的siRNA沉默可逆转HIV-1耐药表型并降低其细胞培养上清液的生物活性； (2)从tCTCF(+)细胞培养上清液中去除tCTCF，消除了其抗病毒活性； (3)亲和纯化的tCTCF在人巨噬细胞中诱导抗病毒活性； (4)亲和纯化的tCTCF抑制HIV-1 LTR的转录，促进报告基因的表达。我们的初步数据表明，在暴露于 HIV-1 后，来自 tCTCF 产生细胞（而非对照细胞）的核提取物中的 CTCF 与包含 NF-kB、Sp1 和 YB-1 结合序列的 DNA 探针结合，可能是通过其与 YB-1 蛋白的相互作用。我们假设，由于 NF-kB 和 YB-1 结合位点仅相隔 6 个核苷酸，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，从而停止病毒基因组的转录。总之，我们发现了一种新形式的 CTCF 蛋白，可介导人类巨噬细胞对 HIV-1 的先天反应。该应用的主要目标是为未来将 tCTCF 作为治疗 CNS 中 HIV-1 的生物疗法奠定基础。