Treatment and prevention of hepatitis C virus (HCV) infections remains a major challenge for controlling this worldwide health problem; existing therapies are only partially effective and no vaccine is currently available. RNA interference offers the potential of a novel therapeutic approach for treating HCV infections. Toward this end, we are developing small hairpin interfering RNAs (shRNAs) and modified siRNAs targeting the conserved internal ribosome entry site (IRES) element of the HCV genome. Phase I experiments utilized a reporter gene plasmid in which firefly luciferase (fLuc) expression is dependent on the HCV IRES. Direct transfection of HCV IRES shRNAs, or alternatively shRNAs expressed from pol Ill-promoter vectors, efficiently blocked HCV IRES-mediated fLuc expression in human hepatocytes and a mouse model where nucleic acids were delivered to cells in the liver by hydrodynamic transfection via the tail vein. In vivo bioluminescent imaging revealed that HCVwt shRNA inhibited HCV IRES-dependent reporter gene expression up to 99% in the mouse model; doubly mutated or irrelevant shRNAs had little or no effect. Phase II builds on the Phase I accomplishments to develop efficient methods for inhibitor delivery to liver by 1) derivatizing siRNAs to facilitate liver targeting and uptake and 2) preparation of adeno-associated virus (AAV) serotypes that deliver shRNAs specifically to the liver. Side-by-side experiments with shRNA, siRNAs (unmodified and modified) and AAV-delivered shRNAs will be performed to determine ability to block HCV IRES-dependent gene expression. As HCV does not infect tissue culture cells, lead inhibitors will be tested in human hepatocytes stably expressing HCV replicons prior to engaging in large scale pre-clinical animal studies. The use of single photo emission computed tomography (SPECT) allows non-invasive imaging and hence optimization of delivery and si/shRNA targeting of HCV IRES-dependent somatostatin receptor-2 reporter expression in mice as well as large animals such as monkeys. Pharmacokinetic and toxicology experiments will be done in rabbits and monkeys prior to investigating the inhibitors in HCV-infected animal models, including non-human primates chronically infected with HCV and the chimeric KMT mouse model.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
Project #
2R44AI056611-03
Application #
6998667
Study Section
Special Emphasis Panel (ZRG1-IDM-B (12))
Program Officer
Koshy, Rajen
Project Start
2003-08-01
Project End
2008-08-31
Budget Start
2005-09-01
Budget End
2006-08-31
Support Year
3
Fiscal Year
2005
Total Cost
$831,879
Indirect Cost
Name
Somagenics, Inc.
Department
Type
DUNS #
013494781
City
Santa Cruz
State
CA
Country
United States
Zip Code
95060
Ma, Han; Dallas, Anne; Ilves, Heini et al. (2014) Formulated minimal-length synthetic small hairpin RNAs are potent inhibitors of hepatitis C virus in mice with humanized livers. Gastroenterology 146:63-6.e5
Dallas, Anne; Ilves, Heini; Ma, Han et al. (2014) Inhibition of hepatitis C virus in chimeric mice by short synthetic hairpin RNAs: sequence analysis of surviving virus shows added selective pressure of combination therapy. J Virol 88:4647-56
Dallas, Anne; Ilves, Heini; Ge, Qing et al. (2012) Right- and left-loop short shRNAs have distinct and unusual mechanisms of gene silencing. Nucleic Acids Res 40:9255-71
Ge, Qing; Dallas, Anne; Ilves, Heini et al. (2010) Effects of chemical modification on the potency, serum stability, and immunostimulatory properties of short shRNAs. RNA 16:118-30
Ge, Qing; Ilves, Heini; Dallas, Anne et al. (2010) Minimal-length short hairpin RNAs: the relationship of structure and RNAi activity. RNA 16:106-17
Vlassov, Alexander V; Korba, Brent; Farrar, Kristine et al. (2007) shRNAs targeting hepatitis C: effects of sequence and structural features, and comparision with siRNA. Oligonucleotides 17:223-36
Seyhan, Attila A; Alizadeh, Babak N; Lundstrom, Kenneth et al. (2007) RNA interference-mediated inhibition of Semliki Forest virus replication in mammalian cells. Oligonucleotides 17:473-84
Dallas, Anne; Vlassov, Alexander V (2006) RNAi: a novel antisense technology and its therapeutic potential. Med Sci Monit 12:RA67-74