Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The development of gene therapy for clinical use continues to face many hurdles. A major issue is the limitation of gene delivery technology. This body of work describes strategies for improving the selectivity and efficacy of gene delivery to vascular endothelium, with emphasis on delivery to pulmonary vasculature in vivo. Several important principles were established which continue to be of relevance to the field. The work progresses from vector development through to the use of new vector strategies in the application of novel gene delivery approaches in disease models. Work in gene therapy and vector development began in the Division of Human Gene Therapy, University of Alabama at Birmingham, under the mentorship of Prof David T Curiel and has continued through international collaborations and the establishment of my own laboratory in the Hanson Institute with affiliate links to the University of Adelaide. The work presented in this thesis consists entirely of published material, either as book chapters (three) or peer reviewed journal articles (twelve). The sequence of material progresses from a broad introduction to the field on Gene Therapy, more specific chapters dealing with pulmonary gene delivery including a detailed methodology chapter. The peer reviewed works contain an evolution of work dealing with the development of strategies to target adenoviral gene delivery vectors to the pulmonary vascular endothelium. This work encompasses the use of bi-specific conjugates, genetic modification of viral capsid (outer coat) proteins and the use of cell-specific promoters. The work progresses to a demonstration of the therapeutic gains achieved with the use of targeted over non-targeted vectors in animal models and culminates with a highly novel application of modulation of the bone morphogenetic protein pathway in pulmonary hypertension. A component of the work focuses on enhanced gene delivery to vein grafts exVIVO. There are many key original contributions encompassed within the work, including 1) first use of conjugate-based retargeting to vascular cells, 2) first demonstration that tropism modification could alter in vivo biodistribution of virus, 3) first demonstration of cell-specific retargeting of adenoviral vector after systemic vascular injection in vivo (a technique still unsurpassed in the field), 4) first demonstration of the in vivo selectivity gains achieved by combined cell-specific promoters with viral retargeting, 5) first demonstration of therapeutic gains achieved by targeting in a vascular context and 6) first demonstration that modulation of the BMPR2 pathway can have a therapeutic impact in pulmonary hypertension. Importantly, the targeting work I have developed has been adapted and used by others and laid a foundation for further vector improvements. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1349837 / Thesis (M.D.) -- University of Adelaide, School of Medicine, 2009

Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The development of gene therapy for clinical use continues to face many hurdles. A major issue is the limitation of gene delivery technology. This body of work describes strategies for improving the selectivity and efficacy of gene delivery to vascular endothelium, with emphasis on delivery to pulmonary vasculature in vivo. Several important principles were established which continue to be of relevance to the field. The work progresses from vector development through to the use of new vector strategies in the application of novel gene delivery approaches in disease models. Work in gene therapy and vector development began in the Division of Human Gene Therapy, University of Alabama at Birmingham, under the mentorship of Prof David T Curiel and has continued through international collaborations and the establishment of my own laboratory in the Hanson Institute with affiliate links to the University of Adelaide. The work presented in this thesis consists entirely of published material, either as book chapters (three) or peer reviewed journal articles (twelve). The sequence of material progresses from a broad introduction to the field on Gene Therapy, more specific chapters dealing with pulmonary gene delivery including a detailed methodology chapter. The peer reviewed works contain an evolution of work dealing with the development of strategies to target adenoviral gene delivery vectors to the pulmonary vascular endothelium. This work encompasses the use of bi-specific conjugates, genetic modification of viral capsid (outer coat) proteins and the use of cell-specific promoters. The work progresses to a demonstration of the therapeutic gains achieved with the use of targeted over non-targeted vectors in animal models and culminates with a highly novel application of modulation of the bone morphogenetic protein pathway in pulmonary hypertension. A component of the work focuses on enhanced gene delivery to vein grafts exVIVO. There are many key original contributions encompassed within the work, including 1) first use of conjugate-based retargeting to vascular cells, 2) first demonstration that tropism modification could alter in vivo biodistribution of virus, 3) first demonstration of cell-specific retargeting of adenoviral vector after systemic vascular injection in vivo (a technique still unsurpassed in the field), 4) first demonstration of the in vivo selectivity gains achieved by combined cell-specific promoters with viral retargeting, 5) first demonstration of therapeutic gains achieved by targeting in a vascular context and 6) first demonstration that modulation of the BMPR2 pathway can have a therapeutic impact in pulmonary hypertension. Importantly, the targeting work I have developed has been adapted and used by others and laid a foundation for further vector improvements. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1349837 / Thesis (M.D.) -- University of Adelaide, School of Medicine, 2009

Intrapulmonary arteriovenous anastomoses (IPAVA) have been known to exist as part of the normal pulmonary vasculature for over 50 years but have been underappreciated by physiologists and clinicians. Using a technique called saline contrast echocardiography we and others have demonstrated that during exercise or when breathing low oxygen gas mixtures IPAVA open, but breathing 100% oxygen during exercise prevents them from opening. However, the mechanism(s) for this dynamic regulation and the role IPAVA play in affecting pulmonary gas exchange efficiency remain unknown. In Chapter IV the infusion of epinephrine and dopamine into resting subjects opened IPAVA. While it is possible this opening was due to the direct vasoactive action of these catecholamines, the opening may simply be due to increases in cardiac output and pulmonary artery systolic pressure secondary to the cardiac effects of these drugs. In Chapter V I used Technetium-99m labeled macroaggregated albumin (99mTc-MAA) to quantify blood flow through IPAVA in exercising healthy humans. Initial attempts to correct for attenuation of the emitted signal were unsuccessful due to the time necessary for data acquisition and the resulting accumulation of free-99mTc. However, I used a blood sample to calculate freely circulating 99mTc which could be subtracted from the shunt fraction. Using this procedure I demonstrated for the first time using filtered solid particles that breathing 100% oxygen reduces blood flow through IPAVA during exercise. Finally, in Chapter VI I tackled the elephant in the room surrounding IPAVA in healthy humans: do these vessels play a role in pulmonary gas exchange efficiency? Our data suggest that the efficiency of pulmonary gas exchange is dependent on the driving pressure gradient for oxygen and the distance to blood flowing through the core of IPAVA. As such, with increases in exercise intensity the diffusion distance and transit time of blood at the core of IPAVA prevent complete gas exchange, thus blood flow through IPAVA acts as a shunt. This dissertation includes previously unpublished co-authored material.

Yes / Aberrant transforming growth factor-β (TGF-β) signaling activation is linked to pulmonary arterial hypertension (PAH). BMPR2 mutations perturb the balance between bone morphogenetic protein (BMP) and TGF-β pathways, leading to vascular remodeling, narrowing of the lumen of pulmonary vasculature, and clinical symptoms. This forum highlights the association of the TGF-β pathway with pathogenesis and therapeutic approaches. / Research carried out at Nasim laboratories is funded by GrowMedtech, the Royal Society, the Commonwealth Scholarship Commission (CSC) and the University of Bradford (UoB). N.S. is funded by the CSC and C.C.N. is partly funded by the UoB.