Tween 85-Modified Low Molecular Weight PEI Enhances Exon-Skipping of Antisense Morpholino Oligomer In Vitro and in mdx Mice

We investigated a series of Tween 85 modified low molecular weight polyethylenimine (LPEI, 0.8k/1.2k/2.0k)-copolymers (Zs) through simple formulation and covalent conjugation with phosphorodiamidate morpholino oligomer (PMO) for their potential to enhance delivery in vitro and in dystrophic mdx mice. Z polymers significantly enhanced PMO-induced exon-skipping in a GFP reporter-based cell culture system. Application of optimized formulations of Zs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in mdx mice. Consistent with our observations in vitro, optimization of molecular size and hydropholic-lipopholic balance (HLB) of polymers are important factors to achieve enhanced PMO delivery in vivo. The best formulation of Zs enhanced PMO delivery with 20- and 6-fold over PMO alone in vitro and in vivo, respectively. Further, chemical conjugation of the polymer and PMO exhibits greater benefit than polymer/PMO simple formulation in PMO delivery efficiency. Observed cytotoxicity of the Zs was lower than Endo-porter and PEI 25k in vitro, and no tissue toxicity was clearly detected with the Zs at the dosage tested. These results indicate the potential of the Zs as effective and safe PMO delivery carriers for treating diseases such as muscular dystrophy.