interest, as well as cause complications for the patient themselves when used clinically, especially in those who already have a serious genetic illness. Another difficulty is the possibility that some viruses will randomly integrate their nucleic acids into the genome, which can interrupt gene function and generate new mutations. This is a significant concern when considering germline gene therapy, due to the potential to generate new mutations in the embryo or offspring. Non-viral vectors Non-viral methods of nucleic acid transfection involved injecting a naked DNA plasmid into cell for incorporation into the genome. This method used to be relatively ineffective with low frequency of integration, however, efficiency has since greatly improved, using methods to enhance the delivery of the gene of interest into cells. Furthermore, non-viral vectors are simple to produce on a large scale and are not highly immunogenic. Some non-viral methods are detailed below: Electroporation is a technique in which high voltage pulses are used to carry DNA into the target cell across the membrane. The method is believed to function due to the formation of pores across the membrane, but although these are temporary, electroporation results in a high rate of cell death which has limited its use. An improved version of this technology, electron-avalanche transfection, has since been developed, which involves shorter (microsecond) high voltage pulses which result in more effective DNA integration and less cellular damage. The gene gun is a physical method of DNA transfection, where a DNA plasmid is loaded onto a particle of heavy metal (usually gold) and loaded onto the 'gun'. The device generates a force to penetrate the cell membrane, allowing the DNA to enter whilst retaining the metal particle. Oligonucleotides are used as chemical vectors for gene therapy, often used to disrupt mutated DNA sequences to prevent their expression. Disruption in this way can be achieved by introduction of small RNA molecules, called siRNA, which signal cellular machinery to cleave the unwanted mRNA sequences to prevent their transcription. Another method utilises double-stranded oligonucleotides, which bind transcription factors required for transcription of the target gene. By competitively binding these transcription factors, the oligonucleotides can prevent the gene's expression. ZFNs Zinc-finger nucleases (ZFNs) are enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger recognizes between 9 and 18 bases of sequence. Thus by mixing those modules, it becomes easier to target any sequence researchers wish