Mesenchymal stem cell therapy for the treatment of myocardial infarction

Mesenchymal stem cells (MSCs) are currently under investigation as repair agents in the preservation of cardiac function following myocardial infarction (MI). However concerns have emerged regarding the safety of acute intracoronary (IC) MSC delivery specifically related to mortality, micro-infarction and microvascular flow restriction post cell therapy in animal models. This thesis aimed to firstly identify an optimal dose of MSC that could be tolerated when delivered via the coronary artery in a porcine model of acute MI (AMI). Initial dosing studies identified 25x106 MSC to be a safe MSC cell dose, however, angiographic observations from these studies recognised that on delivery of MSC there was a significant adverse decrease in distal blood flow within the artery. This observation along with additional supportive data in the literature (published during the course of this thesis) suggested MSC may be contributing to such adverse events through the propagation of thrombosis. Therefore further studies aimed to investigate the innate prothrombotic activity of MSC. Expression of the initiator of the coagulation cascade initiator tissue factor (TF) on MSC was detected in high levels on the surface of these cells. MSC-derived TF antigen was catalytically active, capable of supporting thrombin generation in vitro and enhancing platelet-driven thrombus deposition on collagen under flow. Infusion of MSC via IC route was associated with a decreased coronary flow reserve when delivered but not when coadministered with an antithrombin agent heparin. Heparin also reduced MSC-associated in situ thrombosis incorporating platelets and VWF in the microvasculature. Heparin-assisted MSC delivery reduced acute apoptosis and significantly improved infarct size, left ventricular ejection fraction, LV volumes, wall motion and scar formation at 6 weeks post AMI. In addition, this thesis investigated the paracrine factors secreted by MSC, in particular focusing on the effect on cardiac repair of a novel MSC-paracrine factor SPARCL1. In summary this work provides new insight into the mechanism by which MSC may be deleterious when delivered by an IC route and a means of abrogating this effect. Moreover we present new data on the MSC secretome with elucidation of the challenges encountered using a single paracrine factor cardiac repair strategy.

Ex vivo culture of patient tissue and examination of gene delivery

Gene therapy has emerged as a realistic prospect for the treatment of cancer due to its potential for selective tumour cell targeting. The greatest challenge gene delivery vectors face is the ability to safely and efficiently deliver genes into target cells. The overall objectives of this thesis are to evaluate the efficacy of various gene delivery methods in a clinically relevant tumour model and to also investigate potential strategies for tumour selective delivery. We began with the development of a tumour slice model system using patient waste tissue. This model involves the use of fresh human tumour tissue, cut into thin slices and maintained ex vivo and is universally applicable to gene delivery methods, using a real-time luminescence detection method to assess gene delivery. The nature of the ex vivo culture system permitted examination of specific physiological variables, the influence of intratumoural factors and tissue specific effects on vector expression. Adenoviral vectors under the control of the human CXCR4 promoter demonstrated a 'tumour on' and 'normal off' expression profile when compared with the ubiquitously active CMV promoter when tested in patient tumour tissue. In addition, we developed an ex vivo system of changing oxygenation using the hypoxia inducer, cobalt, to mimic the transient hypoxic conditions found in solid tumours. We found that Adenoviral transgene expression was robust in the cycling hypoxic conditions relevant to solid tumours and re-oxygenation of chronically hypoxic tissue enhanced transgene expression. Finally, we demonstrated an AAV-based tumour targeting strategy using a tumour-selective promoter allowing for the efficient targeting of AAV vectors to cancer cells and the sparing of normal tissue in both murine metastatic liver tumours models and patient tissue. The thesis highlights the importance of indepth preclinical assessment of novel therapeutics and may serve as a platform for further testing of novel gene delivery approaches.

Development of tumour imaging and therapy strategies utilising unengineered bacteria

The ability of systemically administered bacteria to target and replicate to high numbers within solid tumours is well established. Tumour localising bacteria can be exploited as biological vehicles for the delivery of nucleic acid, protein or therapeutic payloads to tumour sites and present researchers with a highly targeted and safe vehicle for tumour imaging and cancer therapy. This work aimed to utilise bacteria to activate imaging probes or prodrugs specifically within target tissue in order to facilitate the development of novel imaging and therapeutic strategies. The vast majority of existing bacterial-mediated cancer therapy strategies rely on the use of bacteria that have been genetically modified (GM) to express genes of interest. While these approaches have been shown to be effective in a preclinical setting, GM presents extra regulatory hurdles in a clinical context. Also, many strains of bacteria are not genetically tractably and hence cannot currently be engineered to express genes of interest. For this reason, the development of imaging and therapeutic systems that utilise unengineered bacteria for the activation of probes or drugs represents a significant improvement on the current gold standard. Endogenously expressed bacterial enzymes that are not found in mammalian cells can be used for the targeted activation of imaging probes or prodrugs whose activation is only achieved in the presence of these enzymes. Exploitation of the intrinsic enzymatic activity of bacteria allows the use of a wider range of bacteria and presents a more clinically relevant system than those that are currently in use. The nitroreductase (NTR) enzymes, found only in bacteria, represent one such option. Chapter 2 introduces the novel concept of utilising native bacterial NTRs for the targeted activation of the fluorophore CytoCy5S. Bacterial-mediated probe activation allowed for non-invasive fluorescence imaging of in vivo bacteria in models of infection and cancer. Chapter 3 extends the concept of using native bacterial enzymes to activate a novel luminescent, NTR activated probe. The use of luminescence based imaging improved the sensitivity of the system and provides researchers with a more accessible modality for preclinical imaging. It also represents an improvement over existing caged luciferin probe systems described to date. Chapter 4 focuses on the employment of endogenous bacterial enzymes for use in a therapeutic setting. Native bacterial enzymatic activity (including NTR enzymes) was shown to be capable of activating multiple prodrugs, in isolation and in combination, and eliciting therapeutic responses in murine models of cancer. Overall, the data presented in this thesis advance the fields of bacterial therapy and imaging and introduce novel strategies for disease diagnosis and treatment. These preclinical studies demonstrate potential for clinical translation in multiple fields of research and medicine.