Characterization and applications in muscle of a Minicircle vector for Nonviral gene therapy

Sammanfattning: In gene therapy, the aim is to change the behaviour of a cell by introduction of genetic material, often DNA encoding a protein or a therapeutic RNA. The purpose can be to replace a malfunctioning copy of a gene, as in clinical trials for treatment of X-linked severe combined immunodeficiency, or introduce a new gene into the body to help fight a disease, as has been done in clinical trials for e.g. leukaemia and lymphoma where immune cells has been modified to recognize and destroy cancer. In order to alter the behaviour, the genetic material must be transported into the cell and reach the nucleus. The two main ways to achieve this is either using viral vectors, where engineered viruses carry the therapeutic DNA, or nonviral vectors, which are commonly based on plasmids produced in bacteria. This thesis focuses on nonviral vectors. Nonviral vectors are generally considered safer and more easily produced than viral vectors, but are less efficient in delivery and long term expression. This is thought to be partly due to the plasmid backbone, i.e. sequences needed only for propagation in the bacteria such as origin of replication and selection markers, commonly antibiotics resistance genes. Bacterially produced DNA sequences have a different methylation pattern than eukaryotic DNA. It has been shown that this can induce an immune response, especially in combination with the use of lipids for transfection. Also for naked delivery of plasmids, the expression is transient, which could be due to epigenetic phenomenon. A way to optimize the plasmid vector is to remove the bacterial backbone by recombination in the production bacteria. The resulting vector is called the minicircle (MC). In one of studies included in this thesis, we investigate how the size of the MC vector affects coiling and relate these findings to analysis of other aspects such as robustness, expression efficiency and transfection. We find that reducing the size of the MC affects the configuration of the vector, causing an increased frequency of dimer and trimer formation during production. We also find that there seems to be a lower size limit for efficient expression. However, the smaller sizes also result in a vector which is more robust than conventional plasmids when exposed to shearing forces, and shows extended expression in vivo. In the two other studies, we evaluate the vector for use in muscle. A comparison of the MC to a conventional plasmid for expression of a growth factor in heart and skeletal muscle in the mouse shows that the smaller size allows for a higher effective dose, and thus, higher gene expression. The third study demonstrates that it is possible to use the MC to express small regulatory RNAs for splice-switching, targeting Duchenne muscular dystrophy, and that treatment with these MCs in mouse muscle results in increased dystrophin levels. However, development of suitable delivery methods is required to realize the full potential of MCs in vivo. Thus, the smaller size enabling a higher dose, prolonged expression and increased robustness, and the fact that the MC construct is devoid of bacterial sequences and antibiotics resistance gene make the MC vector an attractive alternative for nonviral gene. However, for use where systemic treatment is needed, delivery must be enhanced. Consequently, the vector might be more suitable for treatments where only local expression is required, such as single organ treatment, DNA vaccination or ex vivo treatments.