INACTIVATION OF CHEMOKINE RECEPTORS BY INTRAKINES

通过内因子使趋化因子受体失活

基本信息

批准号：
6696571
负责人：
Si-Yi Chen
金额：
$ 30万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-02-01 至 2006-01-31
项目状态：
已结题

项目摘要

Our recent studies have demonstrated that intrakines can efficiently down-regulate surface chemokine receptors such as CCR5 and CXCR4 on T-cells to inhibit HIV-1 infection, while having no apparent detrimental effects on T-cell functions (PNAS. 94:11567-11572, 1997; Nature Medicine 3:1110-1118, 1997). Thus, the unique features and therapeutic potential of intrakines merit the further development of this strategy for HIV-l gene therapy. In this, competing continuation application, we propose to improve the specificity of intrakines and to determine their efficiency and mechanism in down-regulating chemokine coreceptors and inhibiting HIV-1 infection. The hypothesis of this study is that intrakines able to specifically down-regulate CCR5 can be generated by modifying RANTES-intrakine or using a chemokine MIP-1B which specifically binds to CCR5, and transduced into human lymphocytes to down-regulate surface chemokine receptors even on highly activated T-cells to a level sufficient to block HIV-1 entry. We further hypothesize that intrakines may be superior to chemokines for blocking HIV-1 infection of macrophages by intracellular trapping of newly synthesized C CR5. The goal of this study is to refine this novel intrakine strategy, so that it becomes an attractive form of HIV- 1 therapy and a generic means for inactivating other pathogen's receptors. In this study, we will enhance the specificity of intrakines in down-regulating CCR5 by generating RANTES-intrakine mutants and an intrakine derived from MIP-1B that specifically binds to CCR5. We will quantitatively determine the efficiency of intrakines or their mutants in down-regulating chemokine receptors on T-cells under various stimulating conditions. We will then determine the ability of intrakines to prevent virus-to-cell as well as cell-to-cell transmission in highly activated T-cells. We will further assess the correlation of receptor surface levels with the cell susceptibility to HIV- 1 infection by quantitatively determining HIV- 1 load and surface receptor levels. Moreover, the anti-HIV activity of intrakines in macrophages will be evaluated by using lentiviral vectors. To determine if intrakines can compromise T-cell functions, we will first evaluate the effects of intrakines on T-cell function in vitro. We will then evaluate potential adverse effects of intrakines in vivo by generating and evaluating tissue-specific transgenic mice expressing intrakines in hematopoietic stem cells, as well as T-lymphoid and myeloid cells. We will also evaluate in vivo anti-HIV activity of intrakine in hu-PBL SCID mouse models. By accomplishing the goals of this study, we will lay the foundation to use intrakines targeting chemokine receptors for potential HIV gene therapy. Ultimately, intrakines may afford a novel class of therapeutic molecules targeting HIV- 1 entry to be used with chemotherapy for the long-term control of HIV- 1 infection. Moreover, this study will further establish the intrakine approach as a generic means for inactivating other viruses' and pathogens' receptors.

我们最近的研究表明，肌内因子可以有效下调表面趋化因子受体，如 CCR5 和 CXCR4 T细胞抑制HIV-1感染，同时没有明显的有害作用对 T 细胞功能的影响 (PNAS. 94:11567-11572, 1997; Nature Medicine 3：1110-1118，1997）。因此，其独特的功能和治疗潜力内因子值得进一步开发这种针对 HIV-1 基因的策略治疗。在这个竞争性的延续应用中，我们建议改进内因子的特异性并确定其效率和机制下调趋化因子辅助受体并抑制 HIV-1 感染。这这项研究的假设是内因子能够特异性下调 CCR5 可以通过修饰 RANTES-intrakin 或使用趋化因子 MIP-1B 来生成特异性结合 CCR5，并转入人淋巴细胞即使在高度活化的 T 细胞上，也能下调表面趋化因子受体水平足以阻止 HIV-1 进入。我们进一步假设内因子可能比趋化因子更能阻断巨噬细胞的 HIV-1 感染新合成的 C CR5 的细胞内捕获。这项研究的目标是完善这种新颖的内因子策略，使其成为一种有吸引力的形式 HIV-1 疗法和灭活其他病原体受体的通用方法。在这项研究中，我们将增强肌内因子下调的特异性 CCR5通过产生RANTES-内因子突变体和衍生自的内因子 MIP-1B 与 CCR5 特异性结合。我们将定量确定内因子或其突变体下调趋化因子的效率各种刺激条件下 T 细胞上的受体。我们随后将确定内因子阻止病毒传播至细胞的能力以及高度活化的 T 细胞中的细胞间传播。我们将进一步评估受体表面水平与细胞对 HIV 易感性的相关性通过定量测定HIV-1载量和表面受体1感染水平。此外，巨噬细胞中的肌内因子的抗 HIV 活性将通过使用慢病毒载体进行评估。确定内因子是否可以损害T细胞功能，我们首先评估intrakines的影响体外 T 细胞功能。然后我们将评估潜在的不利影响通过生成和评估组织特异性转基因小鼠体内因子在造血干细胞以及 T 淋巴细胞中表达内因子骨髓细胞。我们还将评估 Intrakine 的体内抗 HIV 活性 hu-PBL SCID 小鼠模型。通过实现本研究的目标，我们将奠定为使用靶向趋化因子受体的内因子潜在的基础 HIV基因治疗。最终，内因子可能提供一类新型的针对 HIV-1 进入的治疗分子与化疗一起使用长期控制HIV-1感染。此外，本研究将进一步建立 Intrakine 方法作为灭活其他物质的通用方法病毒和病原体的受体。