Gene Therapy

Gene therapy involves the delivery of DNA or RNA to diseased organ or cells to correct defective genes implicated in disease.


Gene therapy may be achieved through a number of different approaches. If the condition is a consequence of an absent or non-functional gene product, a functional copy of the gene may be delivered to the disease loci. Alternatively, gene expression may be controlled using RNA interference technologies such as small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA (miRNA). RNA interference (RNAi), a natural cell process by which specific mRNAs are targeted for degradation by complementary small interfering RNAs (siRNAs), enables the specific silencing of a single gene at the cell level. A variety of biomedical and clinical research have showed that RNAi has a great potential as an efficient therapeutic approach. Typically RNA interference is used to down-regulate expression of a pathogenic gene, however, up-regulation of genes is possible by targeting regulatory regions in gene promoters.

Despite the tremendous therapeutic potential of gene therapy, and the large number of disorders identified as good candidates, the field has so far been unsuccessful. These failures are from complications associated with gene delivery. Delivery efficiency is high for viral vectors, the most common delivery method, however these have limited therapeutic potential because of problems observed in clinical trials including toxicity, immune and inflammatory responses, difficulties in targeting and controlling dose. In addition there is justifiable concern that the vectors will integrate into the genome, with unknown long-term effects, and the possibility that the virus may recover its ability to cause disease.

Much effort has therefore been directed towards non-viral vector systems, such as plasmid DNA. These vectors are attractive because they are simple to produce and store, can stably persist in cells, however they contain bacterial DNA sequences that may trigger immunotoxic responses. Some human cells, including dendritic cells and T-cells, cannot be efficiently transfected with current plasmid vectors. Short, linear DNA vectors and small RNAs such as short hairpin RNA (shRNA) and micro-RNA (miRNA) are more easily introduced into cells than plasmids. Linear DNA and RNA ends, however, trigger rapid degradation by cell, requiring continuous replenishment. Furthermore, the DNA ends can signal repair and recombination pathways to cause apoptosis. For these reasons, RNAi- and miRNA-based technologies have not yet been highly successful in the clinic. There is clearly a desperate need for a new and efficient way to therapeutically deliver gene therapy vectors to cells.

Plasmid DNA vectors have some utility in basic research because they are straightforward to generate and isolate. They are propagated in bacterial strains and recovered from the bacterial cells. As mentioned above, however, this requires them to contain bacterial DNA sequences notably a prokaryotic origin of replication and an antibiotic resistance marker for maintenance of the plasmid. The presence of these bacterial sequences has a number of very serious and deleterious consequences. Most notably it limits how small the plasmids can be made. Large plasmids, of several kbp, are transfected at very low efficiency. Their large size also makes them susceptible to hydrodynamic shearing forces associated with delivery (e.g., through aersolisation) or in the bloodstream. Shear-induced degradation leads to a loss of biological activity that is at least partially responsible for the current lack of success in using non-vivral vectors for gene therapy. Various cationic and liposomal transfection reagents have been designed to try and alleviate these problems with transfection but these suffer from problems with cytotoxicity. In addition, the bacterial sequences on plasmids can induce silencing of the gene carried on the plasmid leading to loss of efficacy even if the plasmid is successfully transfected. The CpG motifs that are more common in bacterial than eukaryotic DNA sequences also elicit immune responses in mammalian cells. Reducing the size of DNA vectors appears to be a reasonable approach to increase cell transfection efficiency . One may envision that the bacterial sequences on the plasmid could be physically removed and resultant short linear DNA fragments that contain only the therapeutic sequences more easily introduced into cells than conventional plasmid vectors. Unfortunately, the ends of linear DNA are highly bioreactive in vivo, triggering cellular DNA repair and recombination processes as well as apoptosis. Thus, there is a need for gene targeting therapies that are stable in biological environments and that allow for greater cell transfection and transgene expression.