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

Wang M, Wu B, Tucker JD, Lu P, Lu Q - Int J Nanomedicine (2015)

Bottom Line: The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro.The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone.Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, McColl-Lockwood Laboratory for Muscular Dystrophy Research, Cannon Research Center, Carolinas Medical Center, Charlotte, NC, USA.

ABSTRACT
In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases.

No MeSH data available.


Related in: MedlinePlus

Negatively stained transmission electron micrographs of PE (5 µg) with and without PMO (5 µg) complexes and PMO only (scale bar, 100 nm).Abbreviations: PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
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f7-ijn-10-5635: Negatively stained transmission electron micrographs of PE (5 µg) with and without PMO (5 µg) complexes and PMO only (scale bar, 100 nm).Abbreviations: PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.

Mentions: The affinity between a polymer and an oligonucleotide is an important parameter for their efficient delivery into cells. Here, we chose the most effective PE-4 from the PDDAC series and PE-5 for PE/PMO polyplex examination under transmission electron microscopy. As shown in Figure 7, the PE-4 polymer alone formed particles of different sizes, likely because of aggregation, whereas the PMO oligonucleotides alone formed particles with a size below 50 nm, likely resulting from hydrophobic interactions and hydrogen-bond among the PMO molecules. At a weight ratio of 5/5, the PE-4/PMO polyplex formed spherical particles with an average diameter around 20–40 nm. The PE-5/PMO polyplex formed larger particles than did the PE-4/PMO polyplex. This is probably the result of aggregation related to the uncharged hydrophobic polyacrylamide fragments within PE-5. The mechanisms of interaction between PMO and the PE molecules are not clear, but the chemical nature of PMO likely creates a hydrophobic interaction with the PEs, and a possible hydrogen-bond interaction between them. The positively charged groups within the PEs are unlikely to play a key role in the interaction with PMO; nonetheless, the surface charges of the polyplex may stabilize it in a biological environment for a longer period than PMO alone.


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

Wang M, Wu B, Tucker JD, Lu P, Lu Q - Int J Nanomedicine (2015)

Negatively stained transmission electron micrographs of PE (5 µg) with and without PMO (5 µg) complexes and PMO only (scale bar, 100 nm).Abbreviations: PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562748&req=5

f7-ijn-10-5635: Negatively stained transmission electron micrographs of PE (5 µg) with and without PMO (5 µg) complexes and PMO only (scale bar, 100 nm).Abbreviations: PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
Mentions: The affinity between a polymer and an oligonucleotide is an important parameter for their efficient delivery into cells. Here, we chose the most effective PE-4 from the PDDAC series and PE-5 for PE/PMO polyplex examination under transmission electron microscopy. As shown in Figure 7, the PE-4 polymer alone formed particles of different sizes, likely because of aggregation, whereas the PMO oligonucleotides alone formed particles with a size below 50 nm, likely resulting from hydrophobic interactions and hydrogen-bond among the PMO molecules. At a weight ratio of 5/5, the PE-4/PMO polyplex formed spherical particles with an average diameter around 20–40 nm. The PE-5/PMO polyplex formed larger particles than did the PE-4/PMO polyplex. This is probably the result of aggregation related to the uncharged hydrophobic polyacrylamide fragments within PE-5. The mechanisms of interaction between PMO and the PE molecules are not clear, but the chemical nature of PMO likely creates a hydrophobic interaction with the PEs, and a possible hydrogen-bond interaction between them. The positively charged groups within the PEs are unlikely to play a key role in the interaction with PMO; nonetheless, the surface charges of the polyplex may stabilize it in a biological environment for a longer period than PMO alone.

Bottom Line: The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro.The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone.Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, McColl-Lockwood Laboratory for Muscular Dystrophy Research, Cannon Research Center, Carolinas Medical Center, Charlotte, NC, USA.

ABSTRACT
In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases.

No MeSH data available.


Related in: MedlinePlus