Transcript – Delivery of CRISPR/Cas-9 Using Cationic Liposome

Duchenne muscular dystrophy is a genetic disease that occurs in one in 5,000 boys that are born. Mutations in the DMD gene lead to absolute dystrophin evident in both skeletal and cardiac muscle. Without dystrophin, the muscle cells tear during contraction and eventually die. DMD boys ultimately become so weak that they lose the ability to walk by seven years will then lose the ability to use their arms and will sadly succumb to respiratory or cardiac muscle failure. Gene therapies have the potential to lessen the severity of the disease. For boys with DMD, gene editing is a tool in which we can hone in on specific regions of the DNA gene with targeted arnas. Once there, we can cut out mutated DNA regions with nucleases such as caspase nine. These techniques can lead to a smaller but functional dystrophin protein. As a result, skeletal and cardiac muscle can be preserved. Affected boys will improve upon their daily functions, such as breathing and walking unassisted, eating and drinking. One of the main concerns of using these types of gene therapies is the potential immune response to the various viral vectors. As an alternative, we propose to encapsulate our gene editing plasmids with liposomal nanoparticles. We hypothesize that this will improve delivery of the plasmids to the proper location in the cell and will also reduce the immune response, observed with viral vectors. We developed several gene editing RNA specific for the mutated area in the mouse model for DMD. We then transformed, grew and purified plasmids in our laboratory, resulting in several replicates. Ribosomes were then prepared by a film hydration method followed by extrusion cycles, and particle size was determined by the dynamic light scattering method.

Each plasmid replicate was then encapsulated with liposomal nanoparticles. Next, we plan to test which plasmids are best encapsulated with the nanoparticle. Once the nanoparticles delivered in vivo to the next mouse model for DMD, DNA and protein will be examined, and function of various muscles in arterials throughout the body will be examined.