The goal of this project is to develop small non-coding RNA directed transcriptional gene silencing as a therapeutic modality for the treatment of HIV-1 infection. We have learned that small non-coding RNAs targeted to specific loci in the HIV-1 or CCR5 promoters can result in long-term stable epigenetic silencing of HIV-1 or CCR5. Notably, this form of silencing in the context of HIV-1 is refractory to viral mutation. We have also recently developed and humanized the Pddip DNA excision machinery from Tetrahymina thermophila and found that this system can be used to excise those loci targeted for transcriptional silencing by the small non-coding RNA. The work proposed here will mechanistically validate several different complimentary approaches that may result in a novel therapeutic capable of regulating transcription or excision of HIV-1 or CCR5 in a long-term manner. These approaches center around 3 methods of targeted delivery: (1) conditionally replicating HIV-2 vectors, (2) CCR5 or gp120 binding aptamers and (3) CXCR4, CCR5 binding nanoparticles, in order to introduce non-coding RNAs capable of transcriptionally silencing and excising HIV-1 or CCR5 in HIV-1 infected and relevant cell types. These approaches will be developed and mechanistically validated in vitro and in vivo as well as critically assessed for unintended secondary off-target effects. This proposal will be the first stage of validating severa targeted delivery approaches to be used as a cell specific delivery strategy for non-coding RNA directed transcriptional gene silencing and gene excision, a mechanism that has the potential to result in long-term stable silencing of viral expression in infected individuals in the absence of viral resistance. Such an outcome could be considered a functional cure.

Public Health Relevance

This project will develop and characterize methods to deliver non-coding RNAs capable of transcriptional regulation and excision of HIV-1 or CCR5 in a cell targeted manner in vivo. Such a methodology has the potential to result in long-term stable silencing or excision of HIV-1 from infected individuals.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Program Projects (P01)
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Special Emphasis Panel (ZAI1)
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Voulgaropoulou, Frosso
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Scripps Research Institute
La Jolla
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Astakhova, Kira; Ray, Roslyn; Taskova, Maria et al. (2018) ""Clicking"" Gene Therapeutics: A Successful Union of Chemistry and Biomedicine for New Solutions. Mol Pharm 15:2892-2899
Shevchenko, Galina; Morris, Kevin V (2018) All I's on the RADAR: role of ADAR in gene regulation. FEBS Lett 592:2860-2873
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Lister, Nicholas; Shevchenko, Galina; Walshe, James L et al. (2017) The molecular dynamics of long noncoding RNA control of transcription in PTEN and its pseudogene. Proc Natl Acad Sci U S A 114:9942-9947
Hewson, Chris; Morris, Kevin V (2016) Form and Function of Exosome-Associated Long Non-coding RNAs in Cancer. Curr Top Microbiol Immunol 394:41-56
Lazar, Daniel C; Morris, Kevin V; Saayman, Sheena M (2016) The emerging role of long non-coding RNAs in HIV infection. Virus Res 212:114-26
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Weinberg, Marc S; Morris, Kevin V (2016) Transcriptional gene silencing in humans. Nucleic Acids Res 44:6505-17

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