| Summary: | The following study investigates the use of diagnostic ultrasound in combination with microbubbles (ultrasound contrast agents) as a physical enhancer for non-viral gene delivery. The aim of this work was firstly, to demonstrate that ultrasound exposure using settings within the range of diagnostic ultrasound, in combination with microbubbles can improve gene delivery, and secondly, to show that it is a safe, site-specific technique which mitigates the risk of tissue damage often seen with other physical enhancers of gene delivery such as, electroporation. Initially, a feasibility study was carried out to test the efficiency and safety of microbubble ultrasound (MBUS) in a reporter gene setting. Experiments using intravenous injections of a luciferase reporter gene established that MBUS is a safe, site-specific technique which improved levels of the luciferase expression in the organ targeted by MBUS. Luciferase was successfully delivered to the liver and heart, showing significantly higher levels compared to injections without MBUS, and with no detectable expression in other non-target organs. A therapeutic application of MBUS was tested using the mdx mouse, an animal model for Duchenne Muscular Dystrophy (DMD), a genetic disorder caused by the lack of functional dystrophin in muscle fibres due to premature termination of translation. The most successful treatment approach in the mdx mouse thus far had been the injection of Phosphorodiamidate Morpholino Oligomers (PMOs), which by inducing exon skipping, re-introduced dystrophin expression in most muscles in the body, with the exception of the heart. Injections of PMOs with MBUS to the heart successfully re-introduced dystrophin expression in cardiomyocytes. Furthermore, treatment parameters were investigated in more detail in order to optimize PMO delivery to the heart. Finally, an investigation into different types of commercially available microbubbles compared the efficiencies (with respect to gene delivery) of the different bubbles, in order to understand why different microbubbles show different results when used for MBUS, potentially enabling the design of microbubbles specifically for gene delivery.
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