Abstract
The delivery of genes into cells through the transfer of ribonucleic acids (RNAs) has been found to cause a change in the level of target protein expression. RNA-based transfection is conceptually more efficient than commonly delivered plasmid DNA because it does not require division or damage of the nuclear envelope, thereby increasing the chances of the cell remaining viable. Shock waves (SWs) have been found to induce cellular uptake by transiently altering the permeability of the plasma membrane, thereby overcoming a critical step in gene therapy. However, accompanying SW bio-effects include dose-dependent irreversible cell injury and cytotoxicity. Here, the effect of SWs generated by a clinical lithotripter on the viability and permeabilisation of three different cell lines in vitro was investigated. Comparison of RNA stability before and after SW exposure revealed no statistically significant difference. Optimal SW exposure parameters were identified to minimise cell death and maximise permeabilisation, and applied to enhanced green fluorescent protein (eGFP) messenger RNA (mRNA) or anti-eGFP small interfering RNA delivery. As a result, eGFP mRNA expression levels increased up to 52-fold in CT26 cells, whereas a 2-fold decrease in GFP expression was achieved after anti-eGFP small interfering RNA delivery to MCF-7/GFP cells. These results indicate that SW parameters can be employed to achieve effective nucleotide delivery, laying the foundation for non-invasive and high-tolerability RNA-based gene therapy.
Highlights
Nucleic acid–based therapies provide a powerful approach to the treatment of genetic diseases, by introducing into target cells, healthy replacements of mutated or absent genes, or gene-specific inhibitory molecules; to reinstate typical cellular function either through the expression of normal protein or the repression of defective protein
The efficacy of sonoporation strongly depends on the acoustic parameters of the employed technology, because a trade-off exists between maintaining high cell viability and achieving nucleotide uptake
The cell line revealed reduced amenability to be efficiently transfected as the highest proportion of permeabilised cells above that of killed cells was found to be 4.1% by exposure to 134 shock waves, energy level 5.0, at 2 Hz
Summary
Nucleic acid–based therapies provide a powerful approach to the treatment of genetic diseases, by introducing into target cells, healthy replacements of mutated or absent genes, or gene-specific inhibitory molecules; to reinstate typical cellular function either through the expression of normal protein or the repression of defective protein. Physical transfection systems for the delivery of nucleic acids have attracted substantial attention in recent years, as they permit accessibility to Address correspondence to: Sandra Nwokeoha, Institute of Biomedical Engineering, Old Road Campus Research Building, University of Oxford, OX3 7DQ, Oxford, UK. Ox.ac.uk the target site and entry into the cell’s cytosol. Such methods include electroporation, the gene gun, laser irradiation, magnetofection and microinjection. Electroporation has achieved comparably high transfection levels, whereby up to 1000-fold increases in gene expression have been reported relative to the admittedly highly inefficient level achieved with standard plasmid DNA injection (Wells 2004). Sonoporation, the process of transiently permeabilising the cell membrane using ultrasound, provides the most practical and least invasive device-based option when deep access is needed (Mo et al 2012). The efficacy of sonoporation strongly depends on the acoustic parameters of the employed technology, because a trade-off exists between maintaining high cell viability and achieving nucleotide uptake
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