Recombinant Adeno-associated Viral Vector Mediated Gene Transfer of hTBX18: Advancing the Development of a Biological Pacemaker

Abstract

Recombinant adeno-associated viral (rAAV) vectors, have been developed and are being clinically trialled for treatment of cardiovascular diseases. With an improved understanding of the molecular mechanisms involved in pacemaker dysfunction, gene therapy is being used to advance viable treatment options that can potentially replace the need for electronic devices. Currently, the only viable management option for pacemaker dysfunction is the insertion of an electronic pacemaker. We however proposed an alternative gene therapy approach using rAAV vectors to deliver the gene human T-box 18 (hTBX18), to advance the creation of a biological pacemaker. The work in this thesis aimed to address the limitations with current approaches to biological pacemaker development, to set up a relevant animal model to assess this gene transfer approach and to assess rAAV vector biosafety in this novel model. In the first phase of work, we successfully generated an original rAAV construct expressing hTBX18 and showed that rAAV6-hTBX18 gene transfer to ventricular cardiomyocytes resulted in molecular, physiological, morphological and functional changes, recapitulating the pacemaker phenotype in an in vitro setting. In the second body of work, we successfully developed, characterised and validated a large animal model of atrioventricular block that is stable and technically feasible in adult sheep. In the third body of work, we successfully performed rAAV cardiac infusions in sheep and analysed vector shedding in excreta samples from urine, nasal mucus, saliva and faeces. We concluded that rAAV-mediated gene transfer into sheep hearts results in low-grade shedding of non-functional vector particles following vector delivery. This thesis therefore lays the groundwork for the next phase of pre-clinical development of biological pacemakers using clinically relevant rAAV vectors in a previously non-existent sheep animal model.