Lithotripter shock wave induced RNA-based gene therapy

Gene therapy is the process of introducing genes to augment or minimise the expression of absent or defective genes, respectively. Non-viral gene delivery systems for the treatment of genetic diseases, have evolved into highly appreciable nucleic acid-based therapies due to considerably less risk of host immunogenicity and induction of inﬂammatory responses. However, they are challenged by limited delivery. Thus, more eﬃcient strategies are continually being sought. Lithotripter shock waves (LSW) are powerful acoustic waves that are an attractive choice of delivery system, as they oﬀer a non-invasive, targeted and safe approach. Furthermore, the delivery of messenger ribonucleic acid (mRNA) possesses several advantages over commonly delivered plasmid deoxyribonucleic acids (pDNA), because it does not require opening of the nuclear envelope, thereby reducing the level of cell injury necessary for transfection.
This work presents the ﬁrst investigation on the eﬃcacy of LSW mediated mRNA delivery, based on optimised SW parameters that balance the desired enhanced permeability of cell membranes against undesired cytotoxicity, and maintain the structural and biological stability of the RNA. A transfectability measure that deﬁnes the ability of SWs to permeabilise a cell whilst keeping it alive was established for dissimilar cell types, as a function of the acoustic pressure and number of SWs. Statistically signiﬁcant RNA uptake was recorded in a tissue mimicking system, and using RNA analogues at various concentrations, the SW induced bio-distribution was characterised. In addition to LSW induced gene augmentation using mRNA, it was shown that LSWs could be used to eﬀect gene inhibition through the delivery of siRNA.
Kinetic experiments were carried out to measure mRNA uptake during shock wave exposure and indicated that rate of delivery was highest at the start of the SW dose and decreased during treatment. The results also suggested that the enhancement of cell permeability was signiﬁcantly transient, and that mRNA was highly susceptible to degradation in its naked state. Furthermore, mRNA-based gene expression was shown to be predictive but quantal.
The in vitro tissue model was improved from a gel-based system, to one that incorporated multi-cellular spheroids which capture aspects of 3-D tumours. Static overpressure was applied during SW exposure in order to suppress cavitation eﬀects and isolate eﬀects that could be attributed to shear due to cell-to-cell coupling. The results showed that mild overpressure improved RNA uptake the most, but that at higher overpressure, the level of increase in RNA uptake relative to controls, was dependent on the type of RNA nucleotide being delivered. This suggested that a complex interaction between LSW cavitation and direct stress dominates delivery.
A ﬁnal report was on the signiﬁcant improvement of gene delivery when mRNA was encapsulated within a lipid nanoparticle vector, and SW exposure was assisted by cavitation agents. Also, by exposure to another acoustic stimulus – focused ultrasound (US), direct comparisons were made between SWs and US on the eﬃciency of delivery and tissue penetration. In conclusion, this thesis has shown that by choosing parameters appropriately, shock waves can be a promising strategy for the delivery of genes to cells.

Advisor: Cleveland, R
Carlisle, R