Safe, Site-specific Gene Delivery using Ultrasound and Microbubble Technology
Author(s)
Alete, Julia Alice
Type
Thesis
Abstract
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.
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.
Date Issued
2008
Date Awarded
2008-11
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Wells, Dominic
Creator
Alete, Julia Alice
Publisher Department
Imaging Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)