Ribozymes as Antivirals for Respiratory Syncytial Virus

Michelle Meyer (2011). Ribozymes as Antivirals for Respiratory Syncytial Virus PhD Thesis, School of Chemistry & Molecular Bioscience, The University of Queensland.

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S33431796_Thesis_final.pdf Final thesis application/pdf 5.76MB 12
Author Michelle Meyer
Thesis Title Ribozymes as Antivirals for Respiratory Syncytial Virus
School, Centre or Institute School of Chemistry & Molecular Bioscience
Institution The University of Queensland
Publication date 2011-06
Thesis type PhD Thesis
Supervisor Prof. Paul Young
Total pages 327
Total colour pages 30
Total black and white pages 297
Language eng
Subjects 03 Chemical Sciences
Abstract/Summary Respiratory syncytial virus (RSV) remains the leading cause of lower respiratory tract infections in infants and young children worldwide. Despite the widespread incidence of RSV, the search for an effective treatment is ongoing as an effective vaccine or therapeutic remains elusive. Ribozymes are catalytic RNAs which have been employed as gene therapeutics. Ribozymes were designed to target two conserved sites within the L polymerase mRNA of RSV A and B-subtypes. Algorithm-based secondary structure modelling of the L mRNA highlights potential constraints with chosen target sites. Ribozyme-mediated cleavage of transcripts was examined in an in vitro, cell free system. Transient and stable clonal expression systems using plasmid vectors to deliver subtype A-specific ribozymes were generated and challenged with RSV. An inducible RSV minigenome replicon system, transiently expressing the antiviral ribozymes only in the presence of an infection, was developed and evaluated. The minigenome system allowed for the generation of defective RSV particles carrying the therapeutic ribozyme, highlighting the potential use of pseudovirus particles as delivery vehicles. The level of virus reduction achieved by the ribozyme was examined by measuring the abundance of virus RNA and/or protein and the formation of virus particles/progeny. In addition to ribozyme design and target site properties, the antiviral effect of the ribozyme was influenced by the strain and dose of RSV, transfection efficiency, the timing of therapeutic application and key features of the mammalian expression vector and minigenome delivery cassettes. While a direct comparison between the minigenome and plasmid vector delivery vehicles was not achieved, both systems inhibited the replication of RSV at the RNA, protein and progeny level. A mutant, inactive version of the double ribozyme demonstrated the potential involvement of antisense activity. This study emphasizes the possible application of ribozymes targeting RSV and serves as a basis for its further development thus contributing to the field of RSV therapeutics. Moreover, the minigenome system has the potential to be developed into an effective strategy for RSV therapeutic delivery.
Keyword Respiratory Syncytial Virus
Reverse genetics
Additional Notes Colour Pages: 17-22, 29, 67-68, 97-99, 101, 103, 108, 111, 116-117, 123, 141, 144, 151-153, 165, 186, 190, 200, 218, 326.

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Created: Fri, 04 Nov 2011, 11:42:20 EST by Miss Michelle Meyer on behalf of Library - Information Access Service