Human cytomegalovirus (HCMV) causes one of the most common opportunistic infections in patients with AIDS. Disseminated HCMV infection in these patients is usually associated with gastroenteritis, pneumonia, and sight-threatening retinitis. The emergence of drug-resistant HCMV strains to currently available drugs (e.g. ganciclovir) has posed a need to develop new drugs and novel strategies to combat HCMV infections. The objective of this study is to develop Salmonella as a novel class of gene transfer vectors for targeted delivery of anti-HCMV RNase P ribozymes for blocking viral infection. Attenuated strains of Salmonella can function as a carrier system for the delivery of eukaryotic expression vectors and have been shown to deliver therapeutic agents, including nucleic acids-based vaccines and anti-tumor molecules (e.g. small interfering RNAs) for the treatment and prevention of human diseases. We have recently shown that attenuated Salmonella efficiently delivered an anti-HCMV RNase P ribozyme sequence to human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. Furthermore, we have generated novel attenuated Salmonella strains that efficiently delivered RNase P ribozyme sequence for expression in cells. However, little is currently known about the mechanism of how Salmonella achieves efficient gene transfer for delivery of nucleic acids-based agents (e.g. RNase P ribozymes) in human cells. It has not been reported if Salmonella-mediated delivery of RNase P ribozymes is specific and effective in inhibiting viral infection and pathogenesis in animal models. To address these issues, we propose to first study how the generated Salmonella strains achieve efficient gene transfer. We will then investigate whether targeted delivery of RNase P ribozyme by the generated Salmonella abolishes HCMV gene expression and growth in cultured human cells. Using murine cytomegalovirus (MCMV) infection of mice as a model system, we will determine whether the generated Salmonella strains are highly efficient for targeted delivery of RNase P ribozyme in animals and whether the targeted delivery of RNase P ribozymes mediated by Salmonella is highly effective in blocking MCMV infection and pathogenesis in vivo. The potential immune/inflammatory responses and cytotoxicity associated with the generated Salmonella strains will also be investigated. Our research will generate novel Salmonella strains with efficient gene transfer activity that can be used in gene-targeting clinical applications. Furthermore, these results will provide insight into the mechanism of how Salmonella achieves efficient gene delivery in mammalian cells, and will determine whether Salmonella-mediated delivery of RNase P ribozymes is specific and effective in shutting down gene expression in cultured cells and in animals. This study will facilitate the development of Salmonella-mediated delivery of RNase P ribozymes as a novel therapeutic approach for treatment of infections by HCMV and other AIDS-associated viruses.

Public Health Relevance

The proposed research is to generate novel agents for targeted gene delivery of antiviral molecules in treating infections caused by AIDS-associated viruses, including human cytomegalovirus (HCMV), which causes one of the most common opportunistic infections in AIDS patients. Our study will facilitate the development of a new method that can be used as a research tool and a therapeutic approach for studies and treatment of infections associated with human viruses such as HCMV.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI091536-03
Application #
8489102
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Dempsey, Walla L
Project Start
2011-07-03
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
3
Fiscal Year
2013
Total Cost
$345,066
Indirect Cost
$110,066
Name
University of California Berkeley
Department
Internal Medicine/Medicine
Type
Schools of Public Health
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704