Polyethylenimine-modified Pluronics (PCMs) Improve Morpholino Oligomer Delivery in Cell Culture and Dystrophic mdx Mice

Abstract

We investigated a series of small-sized polyethylenimine (PEI, 0.8/1.2 k)-conjugated pluronic copolymers (PCMs) for their potential to enhance delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in dystrophic mdx mice. PCM polymers containing pluronics of molecular weight (Mw) ranging 2-6 k, with hydrophilic-lipophilic balance (HLB) 7-23, significantly enhanced PMO-induced exon-skipping in a green fluorescent protein (GFP) reporter-based myoblast culture system. Application of optimized formulations of PCMs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. Consistent with our observations in vitro, optimization of molecular size and the HLB of pluronics are important factors for PCMs to achieve enhanced PMO delivery in vivo. Observed cytotoxicity of the PCMs was lower than Endo-porter and PEI 25 k. Tissue toxicity of PCMs in muscle was not clearly detected with the concentrations used, indicating the potential of the PCMs as effective and safe PMO carriers for treating diseases such as muscular dystrophy.

Evaluation of Tris[2-(Acryloyloxy)Ethyl]Isocyanurate Cross-Linked Polyethylenimine as Antisense Morpholino Oligomer Delivery Vehicle in Cell Culture and Dystrophic mdx Mice

“…However, 5 lg PEI 25k induced large areas of muscle damage indicated by the presence of necrotic fibers and focal infiltrations. Collectively, these data further confirm the importance of charge balance and molecular size in vector microstructure for effective gene/AO delivery with reduced toxicity (Wang et al, 2012b(Wang et al, , 2013.…”

“…All other chemicals were of reagent grade. PMO targeting human dystrophin gene exon 50 (PMOE50, 5¢-AACTTCCTCTTTAACAGAAAAGCATAC-3¢) (Sazani et al, 2001;Hu et al, 2010) and PMO targeting mouse dystrophin gene exon 23 (PMOE23, 5¢-GGCCAAA CCTCGGCTTACCTGAAAT-3¢) (Gebski, et al, 2003) were purchased from GeneTools), and the specific sequences were selected as per reported (Wu et al, 2008(Wu et al, , 2009b(Wu et al, , 2012Hu et al, 2010;Wang et al, 2013).…”

Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

“…3,6 Furthermore, the complicated synthesis and purification in modification increases the costs significantly, and potential immune responses to the targeting peptide could prevent repeated administration. The nonvirus-mediated delivery strategy remains attractive because of the vector's structural flexibility chosen from synthetic or natural compounds, capacity for delivery of larger therapeutic agents, ease of handling, greater safety, and less expense than viral vectors, [18][19][20] especially for those commercial available compounds that have been widely applied in the field of environmental biology and biotechnology.…”

Poly(ester amine) constructed from polyethylenimine and pluronic for gene delivery in vitro and in vivo

“…The results indicate potential of these PCMs as delivery vectors both in pDNA and antisense oligonucleotide (AOs) because of the flexibility in structure composition and hydrophilic-lipophilic balance (HLB) (Wang et al, 2012(Wang et al, , 2013(Wang et al, , 2014. Modification with different linker is an option to improve the transfection efficiency and reduce cytotoxicity of delivery carrier properties of polymer or liposome for gene delivery (Jin et al, 2014).…”