Our overall hypothesis is that RNA interference (RNAi) can be developed to reduce specific gene expression over long-term without toxic effects in human lymphocytes. RNAi using siRNA to inhibit specific gene expression is a powerful and promising technology for both basic research and therapeutic intervention. However, our results and several recent studies have shown unintended nonspecific cytotoxic effects associated with this technology in mammalian cells raising a concern for the long term use of the RNAi in therapeutic applications in humans. The cytotoxic effects were attributed at least in part to apoptosis, although other mechanisms are also possible. The cytotoxicity was associated with higher levels of shRNA expression. Based upon these results, we hypothesized that that the levels of shRNA are important for determining whether a given shRNA will be cytotoxic or not and that optimization of those levels can yield shRNAs that are both effective and not or minimally cytotoxic. We will also address whether the cell localization of the shRNA and/or its pathway for processing can affect the extent of cytotoxicity within cells. Through our proposed studies we will understand the effects of shRNA expression in mature lymphoid cells in vitro as well as the effects on the differentiation of the lymphocyte populations in an in vivo setting. We will minimize the cytotoxic effect and maximize stable and effective function of RNAi in primary T-lymphocytes. The relevance of this research to public health. RNA interference is a powerful and promising technology to inhibit specific gene expression for both basic research and therapeutic intervention. The inhibition of specific gene expression needs to be effective without unintended side effects. This proposal propose to study the effects of RNAi in human T lymphocytes to maximize the RNAi effect and minimize side effects. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL086409-03
Application #
7479088
Study Section
Special Emphasis Panel (ZRG1-BST-Z (52))
Program Officer
Thomas, John
Project Start
2006-09-30
Project End
2010-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
3
Fiscal Year
2008
Total Cost
$337,544
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Shimizu, Saki; Yadav, Swati Seth; An, Dong Sung (2016) Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector. Methods Mol Biol 1364:235-48
Shimizu, Saki; Ringpis, Gene-Errol; Marsden, Matthew D et al. (2015) RNAi-Mediated CCR5 Knockdown Provides HIV-1 Resistance to Memory T Cells in Humanized BLT Mice. Mol Ther Nucleic Acids 4:e227
Pang, Shen; Pokomo, Lauren; Chen, Kevin et al. (2014) High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA. PLoS One 9:e96445
Wolstein, Orit; Boyd, Maureen; Millington, Michelle et al. (2014) Preclinical safety and efficacy of an anti-HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor. Mol Ther Methods Clin Dev 1:11
Suree, Nuttee; Koizumi, Naoya; Sahakyan, Anna et al. (2012) A novel HIV-1 reporter virus with a membrane-bound Gaussia princeps luciferase. J Virol Methods 183:49-56
Marsden, Matthew D; Kovochich, Michael; Suree, Nuttee et al. (2012) HIV latency in the humanized BLT mouse. J Virol 86:339-47
Ringpis, Gene-Errol E; Shimizu, Saki; Arokium, Hubert et al. (2012) Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice. PLoS One 7:e53492
Kitchen, Scott G; Shimizu, Saki; An, Dong Sung (2011) Stem cell-based anti-HIV gene therapy. Virology 411:260-72
Shimizu, Saki; Hong, Patrick; Arumugam, Balamurugan et al. (2010) A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model. Blood 115:1534-44
Shimizu, Saki; Kamata, Masakazu; Kittipongdaja, Panyamol et al. (2009) Characterization of a potent non-cytotoxic shRNA directed to the HIV-1 co-receptor CCR5. Genet Vaccines Ther 7:8

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