RNAi-based therapeutics is in various stages of study for treating a number of different diseases, including viral infections (e.g., hepatitis, HIV, influenza, RSV), cancer, hyperlipidemia, Huntington's disease, and age-related macular degeneration, to name a few. Our previous studies have provided important information on the rate- limiting processes in primary cells in tissues that limit RNAi effectiveness due to toxicity. The first section of the application is designed to further dissect the molecular limitations of RNAi. We propose studies that examine the limitations of RNAi delivery via a transcriptional template or direct double-stranded delivery, while still developing a robust RNAi delivery system that can safely knock down transgene expression by 3 to 4 orders of magnitude. This level of knockdown will likely be important for a number of the diseases under consideration for preclinical study and clinical efficacy. The second part of the application deals with new discoveries that we uncovered while examining the RNAi pathway in mammals. First, we discovered new mechanisms on how microRNAs are loaded into the RISC complex. Second, we found the biologic basis for why miRNA targets in the 3'UTR are heavily favored over those in the coding regions of mRNAs. A third and forth discovery are related to identifying two new classes of small RNAs. One group of these RNAs may be involved in RNA-directed RNA transcription, a process known to be robust in invertebrates and plants but not previously believed to exist in mammals. The second class of small RNAs is derived from tRNAs. We discovered two different types of abundant tRNA- derived small RNAs. We have established in part how these small RNAs are generated from the tRNAs. While we have established that these RNAs are localized to the cytoplasm, our preliminary data suggest that these RNAs are not microRNA-like, even though they bind to specific argonaute proteins. We hypothesize that these RNAs serve as a buffer to regulate the number of miRNAs that can be taken up into an active RISC complex. We propose studies to attempt to further elucidate their function. Moreover, we have discovered that for at least the type II tRNA-derived small RNAs, there is the potential to use them in combination with an antisense oligonucleotide for regulated gene silencing in a process we call Sense-Induced-Trans Silencing or SITS. We propose studies to determine the likelihood of developing this approach into a bona-fide therapeutic.

Public Health Relevance

The ultimate goal of our studies is to use small RNAs as therapeutics to treat diseases like viral infections, cancer, and some genetic disorders. We are studying some of the molecular mechanisms that limit RNA interference effectiveness in order to develop more robust treatment approaches. In addition, we have discovered other classes of small RNAs for which there is no defined biological function. We are studying the role of that these RNAs might play in gene regulation and designing new strategies to develop these into novel therapeutic approaches to treat disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK078424-09
Application #
8667424
Study Section
Therapeutic Approaches to Genetic Diseases (TAG)
Program Officer
Serrano, Jose
Project Start
2006-09-30
Project End
2015-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
9
Fiscal Year
2014
Total Cost
$515,649
Indirect Cost
$169,507
Name
Stanford University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Valdmanis, Paul N; Gu, Shuo; Chu, Kirk et al. (2016) RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice. Nat Med 22:557-62
Valdmanis, P N; Roy-Chaudhuri, B; Kim, H K et al. (2015) Upregulation of the microRNA cluster at the Dlk1-Dio3 locus in lung adenocarcinoma. Oncogene 34:94-103
Gu, Shuo; Zhang, Yue; Jin, Lan et al. (2014) Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs. Nucleic Acids Res 42:12169-76
Chung, Janet; Scherer, Lisa J; Gu, Angel et al. (2014) Optimized lentiviral vectors for HIV gene therapy: multiplexed expression of small RNAs and inclusion of MGMT(P140K) drug resistance gene. Mol Ther 22:952-63
Roy-Chaudhuri, Biswajoy; Valdmanis, Paul N; Zhang, Yue et al. (2014) Regulation of microRNA-mediated gene silencing by microRNA precursors. Nat Struct Mol Biol 21:825-32
Gu, Shuo; Jin, Lan; Huang, Yong et al. (2012) Slicing-independent RISC activation requires the argonaute PAZ domain. Curr Biol 22:1536-42
Valdmanis, Paul N; Gu, Shuo; Schuermann, Nina et al. (2012) Expression determinants of mammalian argonaute proteins in mediating gene silencing. Nucleic Acids Res 40:3704-13
Gu, Shuo; Jin, Lan; Zhang, Yue et al. (2012) The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo. Cell 151:900-11
Haussecker, Dirk; Huang, Yong; Lau, Ashley et al. (2010) Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 16:673-95
Beer, Shelly; Bellovin, David I; Lee, Joyce S et al. (2010) Low-level shRNA cytotoxicity can contribute to MYC-induced hepatocellular carcinoma in adult mice. Mol Ther 18:161-70

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