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Functional correction of episomal mutations with short DNA fragments and RNA-DNA oligonucleotides
P H Thorpe1, B J Stevenson, D J Porteous
1Medical Genetics Section, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK. peter.thorpe@ed.ac.uk
Background:
Gene correction is an alternative approach to replacement gene therapy. By correcting mutations within the genome, some of the barriers to effective gene therapy are avoided. Homologous nucleic acid sequences can correct mutations by inducing recombination or mismatch repair. Recently, encouraging data have been presented using both short DNA fragments (SDFs) and RNA-DNA oligonucleotides (RDOs) in experimental strategies to realize clinical gene correction.
Methods:
The delivery of labelled SDFs and RDOs to a variety of cell lines was tested using both FACS analysis and confocal microscopy. A GFP-based reporter system was constructed, containing a nonsense mutation, to allow quantitation of gene correction in living cells. This reporter was used to compare efficiencies of functional gene correction using SDFs and RDOs in arange of mammalian cell lines.
Results:
The delivery experiments highlight the inefficient delivery of SDFs and RDOs to the nucleus using polyethylenimine (PEI) transfection. This study compared the episomal correction efficiency of the reporter plasmid mediated by SDFs and RDOs within different cell types; low levels of functional correction were detected in cell culture.
Conclusions:
Whilst delivery of PEI-complexed SDFs or RDOs to the cell is highly effective, nuclear entry appears to be a limiting factor. SDFs elicited episomal GFP correction across a range of cell lines, whereas RDOs only corrected the reporter in a cell line that overexpresses RAD51.