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.
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| Schroers, R; Davis, C M; Wagner, H-J et al. (2002) Lentiviral transduction of human T-lymphocytes with a RANTES intrakine inhibits human immunodeficiency virus type 1 infection. Gene Ther 9:889-97 |
