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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2019)

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.

    Cover page: RNase P Catalytic RNA as a Tool for Anti-Viral Therapy
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2014)
    RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application.
      Cover page: Engineered RNase P Ribozymes Effectively Inhibit Human Cytomegalovirus Gene Expression and Replication
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2015)
      An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.
        Cover page: RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2021)
        Abstract. Long-range transport of biogenic emissions from the coast of Antarctica, precipitation scavenging, and cloud processing are the main processes that influence the observed variability in Southern Ocean (SO) marine boundary layer (MBL) condensation nuclei (CN) and cloud condensation nuclei (CCN) concentrations during the austral summer. Airborne particle measurements on the HIAPER GV from north-south transects between Hobart, Tasmania and 62° S during the Southern Ocean Clouds, Radiation Aerosol Transport Experimental Study (SOCRATES) were separated into four regimes comprising combinations of high and low concentrations of CCN and CN. In 5-day HYSPLIT back trajectories, air parcels with elevated CCN concentrations were almost always shown to have crossed the Antarctic coast, a location with elevated phytoplankton emissions relative to the rest of the SO. The presence of high CCN concentrations was also consistent with high cloud fractions over their trajectory, suggesting there was substantial growth of biogenically formed particles through cloud processing. Cases with low cloud fraction, due to the presence of cumulus clouds, had high CN concentrations, consistent with previously reported new particle formation in cumulus outflow regions. Measurements associated with elevated precipitation during the previous 1.5-days of their trajectory had low CCN concentrations indicating CCN were effectively scavenged by precipitation. A course-mode fitting algorithm was used to determine the primary marine aerosol (PMA) contribution which accounted for  0.07 µm) indicated that particle formation occurs more frequently above the MBL; however, the growth of recently formed particles typically occurs in the MBL, consistent with cloud processing and the condensation of volatile compound oxidation products. CCN measurements on the R/V Investigator as part of the second Clouds, Aerosols, Precipitation, Radiation and atmospheric Composition Over the southeRn Ocean (CAPRICORN-2) campaign were also conducted during the same period as the SOCRATES study. The R/V Investigator observed elevated CCN concentrations near Australia, likely due to continental and coastal biogenic emissions. The Antarctic coastal source of CCN from the south as well as CCN sources from the mid-latitudes create a latitudinal gradient in CCN concentration with an observed minimum in the SO between 55° S and 60° S. The SOCRATES airborne measurements are not influenced by Australian continental emissions, but still show evidence of elevated CCN concentrations to the south of 60° S, consistent with biogenic coastal emissions. In addition, a latitudinal gradient in the particle composition is observed; more hygroscopic particles to the north, consistent with a greater fraction of sea salt from PMA, and more sulfate and organic particles to the south, which are likely from biogenic sources in coastal Antarctica.
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