Human immunodeficiency virus type 1 (HIV-1) has been infecting humans for ~20 years now with little hope for an efficacious vaccine in the near future (Ho 2002). Alternative strategies to inhibit viral replication either alone or in combination with the currently administered therapies are tremendously desirable. One such strategy involves the use of small interfering 21-26 bp double stranded RNAs (siRNAs) which have recently been shown to be ubiquitous and specific repressers of gene expression (reviewed (Finnegan and Matzke 2003)). Of particular interest is the use of siRNAs to specifically target genes involved in diseases such as HIV-1. Importantly, the use of these siRNAs to target HIV-1 or its chemokine co-receptor at the level of promoter-controlled expression (i.e. transcriptional gene silencing, TGS) would be an immeasurable and potent therapeutic agent. Until recently there was no published data suggesting that TGS is operable in mammalian cells and the majority of siRNA activity is cytoplasmic (Zeng 2002). Recently we demonstrated in human cells that siRNAs constructed to target a promoter can effectively suppress a genes mRNA expression (Morris, Chan et al. 2004). Moreover, the preliminary data presented here adds further support to the observation that siRNAs can induce TGS in human cells only upon successful delivery of the siRNA to the nucleus and that the pathway of the observed inhibition involves histone deacetylation (HDAC). We hypothesize that the HIV-1 promoter, specifically the 5' LTR, and the promoter for the chemokine co-receptor CCR5 used for entry by HIV-1 can be specifically targeted by siRNAs (delivered directly into the nucleus) and that these promoters undergo epigenetic changes that are specific, long lasting, and significantly alter the transcriptional profile of the integrated virus or chemokine co-receptor. To test this hypothesis we propose to 1) determine the magnitude, duration, and specificity of siRNA mediated suppression by targeting a minimum of 4 sites in the HIV-1 LTR and CCR5 promoter, 2) Characterize promoter specific siRNA mediated suppression of HIV-1 viral replication (LTR specific siRNAs) or HIV-1 entry (CCR5 specific siRNAs) in transduced cells challenged with HIV-1, 3) characterize the epigenetic changes (DNA methylation, histone acetylation/ phosphorylation and methylation) associated with siRNA mediated TGS of the HIV-1 LTR and CCR5 promoter, and 4) determine the mechanism of action of the siRNA mediated TGS by knocking out DNMT-1 and HDAC's as well as determine if an interaction with Sin3 or Mi2/NuRD protein complexes is involved. The work outlined in this project should further develop our understanding of this complex molecular pathway of target specific gene regulation in human cells, specifically HIV-1 and it's chemokine co-receptor CCR5, and provide findings that will be applicable in future therapeutic interventions aimed at altering patterns of HIV-1 and co-receptor expression. ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL083473-04
Application #
7263926
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Barbosa, Luiz H
Project Start
2005-09-01
Project End
2010-08-31
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
4
Fiscal Year
2007
Total Cost
$453,828
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Turner, Anne-Marie W; Ackley, Amanda M; Matrone, Michael A et al. (2012) Characterization of an HIV-targeted transcriptional gene-silencing RNA in primary cells. Hum Gene Ther 23:473-83
Morris, Kevin V (2011) The emerging role of RNA in the regulation of gene transcription in human cells. Semin Cell Dev Biol 22:351-8
Knowling, Stuart; Morris, Kevin V (2011) Epigenetic regulation of gene expression in human cells by noncoding RNAs. Prog Mol Biol Transl Sci 102:1-10
Hawkins, Peter G; Morris, Kevin V (2010) Transcriptional regulation of Oct4 by a long non-coding RNA antisense to Oct4-pseudogene 5. Transcription 1:165-175
Morris, Kevin V; Vogt, Peter K (2010) Long antisense non-coding RNAs and their role in transcription and oncogenesis. Cell Cycle 9:2544-7
Malecova, Barbora; Morris, Kevin V (2010) Transcriptional gene silencing through epigenetic changes mediated by non-coding RNAs. Curr Opin Mol Ther 12:214-22
Turner, Anne-Marie W; Morris, Kevin V (2010) Controlling transcription with noncoding RNAs in mammalian cells. Biotechniques 48:ix-xvi
Turner, Anne-Marie W; De La Cruz, Justin; Morris, Kevin V (2009) Mobilization-competent Lentiviral Vector-mediated Sustained Transcriptional Modulation of HIV-1 Expression. Mol Ther 17:360-8
Morris, Kevin V (2009) Non-coding RNAs, epigenetic memory and the passage of information to progeny. RNA Biol 6:242-7
Morris, Kevin V (2009) RNA-directed transcriptional gene silencing and activation in human cells. Oligonucleotides 19:299-306

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