UC Berkeley

Engineered RNase P-based gene-targeting agents derived from the RNA subunit of E.coli RNase P, represent a promising approach for anti-viral applications. The focus of the research in this dissertation has been to develop anti-viral therapeutic agents from RNase P-based RNAs, with enhanced efficacy at cleaving targeted human cytomegalovirus (HCMV) mRNAs in cells. This dissertation is organized into six chapters that conclude my research. In Chapters 2 and 3, engineered RNase P-based ribozyme variants V661-IE2 and V718-A, which were generated using an in-vitro selection procedure, were designed to target HCMV mRNAs of the immediate-early protein (IE2), and the overlapping mRNA region of capsid assembly protein (AP) and protease (PR), respectively. Variant ribozyme V718-A showed a nearly 60-fold greater activity than wild type M1 RNA in cleaving target AP/PR mRNAs in-vitro. Additionally, a reduction in AP/PR expression of 98% – 99% and a viral growth inhibition of 50,000-fold was observed in cells expressing V718-A, while a 75% decrease in AP/PR expression and a viral growth reduction of 500-fold was detected in cells expressing M1-A. Variant V661-IE2 showed a nearly 50-fold greater in-vitro cleavage activity than wild type M1-IE2 in targeting IE2 mRNA. Likewise, A nearly 98% decrease in expression of IE2 and a 3,500-fold reduction in viral growth was observed in cells expressing variant V661-IE2, compared to a 75% decrease in IE2 expression and a 100-fold reduction in viral growth in cells expressing wild type ribozyme M1-IE2. These results indicate that the generated ribozyme variants selected to be highly active in-vitro are also more effective in targeting mRNAs in cultured cells, and are a potential effective approach for anti-viral therapeutic applications. In Chapter 4, engineered external guide sequence (EGS) molecules, which were generated from an in-vitro selection procedure, was designed to target mRNAs of the herpes simplex virus 1 (HSV-1) major transcriptional regulator (ICP4). The EGS variant induced human RNase P to cleave ICP4 mRNA in-vitro, with a 60-fold greater activity than EGSs generated from a natural ptRNA substrate. Additionally, a nearly 97% inhibition in ICP4 expression, and a 7,000-fold decrease in viral replication was seen in HSV-1-infected cells expressing the variant EGS, compared to a 75% reduction in ICP4 expression and a 500-fold decrease in replication in cells expressing the EGS derived form a natural ptRNA. These results suggest that engineered EGS variants can effectively block HSV-1 gene expression and viral growth in cells, and show the potential for engineered EGS variants to be developed as effective anti-viral therapeutic agents.

Attenuated strains of Salmonella bacteria represent promising nucleic acid delivery agents. Lastly, in Chapter 5 we constructed a novel strain of attenuated Salmonella for the delivery and expression of antigens hemagglutinin (HA) and neuraminidase (NA) from the H5N1 influenza virus. Here, we showed that the Salmonella vaccine induced a substantial production of anti-HA serum IgG and mucosal IgA, and anti-HA interferon-gamma producing T cells in orally-vaccinated mice. Additionally, we observed orally-vaccinated mice that expressed viral antigens HA and NA were completely protected from H5N1 virus in lethal amounts, while all mice treated with empty expression vector-carrying Salmonella were not protected. These results indicate that Salmonella-based vaccines induces strong humoral and cellular immune responses that are antigen-specific. Thus, Salmonella-based vectors can be designed to provide effective immune protection against multiple influenza virus strains. Moreover, this study highlights the potential in developing attenuated Salmonella strains as new and viable oral vaccine vectors or as effective gene-delivery agents for anti-viral gene-targeting applications in vivo.