Adenovirus Mediated Gene Transfer of Skeletal Troponin C to Myocardium

Docherty, Andrew (2001) Adenovirus Mediated Gene Transfer of Skeletal Troponin C to Myocardium. PhD thesis, University of Glasgow.

Abstract

Ischaemic heart disease and the syndrome of congestive cardiac failure are increasing in prevalence within western societies. The limitations of present day interventions are discussed. There is a need to identify new therapeutic targets and the potential of molecular genetic methods is discussed. Experimental data from transgenic mice expressing skeletal troponin C is considered together with data from substitution of troponin isoforms in isolated preparation. The function of cardiac troponin C is significantly affected by intracellular acidosis and this is largely responsible for the early decoupling of the calcium transient and contractile dysfunction. In comparison skeletal troponin C is relatively unaffected by moderate degrees of intracellular acidosis. This led to the hypothesis that by displacing a proportion of the cardiac troponin C with the skeletal isoform, there would be the possibility of rendering cardiac muscle relatively resistant to the effects of intracellular acidosis. Perhaps such altered myocardium would be able to contract for longer in conditions of ischaemia. Vectors for gene transfer are considered and the attractive properties of adenoviral vectors detailed. Routes by which to deliver vector to the heart are discussed and particular consideration is given to the intra-coronary injection of adenoviral vector. Chapter 3 deals with the production and quality assurance of replication defective adenoviral vectors. The "supernatant rescue assay" method to detect wild type contamination is discussed but not utilised. Further experiments depend on the adenoviral vector which is free of wild type contamination. Although virus was propagated from single plaques and the 15 viral DNA tested by restriction enzyme analysis, the possibility of wild type contamination remained. This was to undermine the value of the data from subsequent experiments. Chapter 4 details the failure of the Swan-Ganz method of viral delivery, which in part may have been due to contamination of vector by replication competent wild-type virus. A selective coronary injection method of vector delivery was achieved and then compared with an aortic cross-clamp method of vector delivery. The experimental design permitted a direct comparison of the efficiency of both methods. The selective coronary injection method was found to be more efficient. Neither method achieved adequate efficiency of gene transfer to allow investigation of genetically altered contractile physiology by means of a working heart preparation. The selective coronary injection method achieved foreign gene expression in up to 30% of myocytes in the trabeculae rich basal right ventricle. Pre-injection of serotonin before injection of low dose virus resulted in more myocardial damage than expected. The lack of a control group, injected with only vehicle, limited the value of these data. Chapter 7 details the results of physiological investigation of right ventricular trabeculae, isolated from rabbits following selective coronary injection of adenoviral vector encoding either beta-galactosidase or skeletal troponin C. The results are negative and the possible reasons discussed. The vector used for these experiments encoded a skeletal troponin C mutant where the final 9 amino-acids at the C-terminal had been deleted and replaced with a 12 amino-acid sequence which included a haemagglutinin antigen motif. In retrospect, confirmation of the integration of the gene product into the sarcomere should have been undertaken prior to proceeding to in vivo investigation. The final chapter places the results of this work in the context of a rapidly advancing field.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: S M Cobbe
• Keywords: Genetics
• Date of Award: 2001
• Depositing User: Enlighten Team
• Unique ID: glathesis:2001-75726
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Dec 2019 09:15
• Last Modified: 19 Dec 2019 09:15
• URI: https://theses.gla.ac.uk/id/eprint/75726