Non-Viral Gene Therapy
Original project description:
We are pursuing two gene therapy approaches. One approach involves direct delivery of plasmid DNA into muscle fibers by using hydrodynamic injection. The other approach employs transfection of mesoangioblast stem cells with plasmid DNA, followed by in vivo transplantation of the corrected stem cells. In either strategy, the phiC31 integrase, a site-specific phage recombinase, will be used to carry out integration of the dysferlin gene into the chromosomes of target cells. This process should produce robust, long-term expression of dysferlin at a set of safe integration sites. The initial experiments seek to establish whether hydrodynamic DNA delivery is effective and safe in dysferlin-deficient A/J mice. In addition, transfection, integration, and stable expression of plasmid DNA in mesoangioblast stem cells will be demonstrated, along with differentiation of the transfected stem cells into skeletal muscle fibers in vitro. If justified by the initial studies, the next phase of the work will evaluate and compare the levels of dysferlin production that result from the direct DNA delivery and stem cell approaches in the muscles of A/J mice in vivo.
Hydrodynamic delivery of plasmid DNA was not successful due to technical issues with the small size of the veins in mice. Therefore, this delivery method was not pursued further, even though it may still be a viable option in human. The use of mesoangioblast stem cells for the delivery of the dysferlin gene to the muscle also had technical issues, so we switched to the use of adipose derived mesenchymal stem cells (AD-MSC) instead. However, due to the AD-MSC's poor ability to grow after transfection, the inability to utilize a clone, and poor ability to differentiate into muscle in vitro and in vivo, we believe that AD-MSC are not an appropriate stem cell choice for dysferlin gene therapy/cell therapy approach in muscle. We believe that induced pluripotent stem cells may be a better choice. Click here for a complete summary of the AD-MSC results.