Controlled Delivery of Antisense Oligonucleotides Based on PEG-fibrinogen Hydrogel in the Treatment of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a muscular disorder, caused by almost complete absence of dystrophin protein due to out-of-frame mutations in the DMD gene. The loss of dystrophin characterized by progressive muscle weakness that causes degeneration of skeletal and cardiac muscle. Currently, there are no effective cures available for DMD; therefore, the therapy is limited to management of symptoms. Antisense oligonucleotides (AONs) exon-skipping is one of the most promising therapeutic strategies for DMD, designed to skip a specific DMD exon, producing a shortened transcript but a functional dystrophin protein. In order to increase AONs stability and enhance cellular uptake, carriers can be used as an efficient approach for AON delivery. Here we designed a biomaterial strategy for encapsulation of therapeutic AONs in PEG-fibrinogen (PF) hydrogel-based microspheres as a controlled-release system that can improve the AONs pharmacokinetic properties, increase their stability and greatly decrease the overall administered dosage. PF microspheres loaded with 6-FAM labeled AONs that were complexed with polyethyleneimine (PEI), were fabricated by dual photo-initiator emulsion method. The microspheres were spherical with a homogenous distribution of AONs encapsulated within the PF material. Additionally, an in vitro study revealed that AONs polyplexes, released from the microspheres, penetrated into isolated satellite cells from mdx mice, after several incubation time points. For evaluation of dystrophin expression, isolated mdx mice myoblasts treated with PF microspheres and immune-stained with dystrophin antibodies showed positive dystrophin expression, suggesting that the use of microsphere for the controlled release of AONs can be a useful modality for treating DMD.