An Investigation into Factors affecting Ultrasound and Microbubble mediated Gene Transfection

Abstract

Gene therapy is a promising tool in the treatment of genetic disorders. Viral vectors have a natural ability to genetically alter cells, however, concern over their immunogenic and mutagenic effects has led to the development of safer non-viral alternatives, such as ultrasound and microbubble mediated gene transfection (UMGT). UMGT causes cellular bioeffects leading to uptake of genetic material; however transfection efficiencies in vivo are poor. It was the aim of this project to investigate key factors that affect UMGT in vivo. A Model Protocol was created with techniques capable of characterising the acoustic response and stability of microbubbles, as well as their population statistics and ability to transfect the heart of six to eight week old, CD1, female mice with a luciferase plasmid. This was used to investigate the effect of needle gauge and microbubble formulation on transfection. It was found that the needle gauge used to systemically deliver SonoVue microbubbles affected microbubble number and transfection. 29G needles caused a decrease in microbubble concentration and transfection, whilst 25G needles caused no microbubble destruction and resulted in significantly greater transfection. Albumin microbubbles are more effective than lipid microbubbles at transfection, but are not optimised. It was found that altering the dextrose and albumin content of an albumin microbubble affected their acoustic and transfection properties. A link between the attenuation behaviour of a microbubble and its transfection efficiency was found, suggesting that optimising acoustic behaviour could improve transfection. DNA-loaded, cationic lipid microbubbles have shown greater transfection ability than neutral microbubbles due to co-localisation of DNA at the site of microbubble activation. Albumin microbubbles have not been fully investigated for this effect. A novel, potentially safe, microbubble formulation was created and fluorescence techniques used to demonstrate its loading capability. This project has identified needle gauge and microbubble acoustic behaviour as key factors affecting the effectiveness of UMGT.