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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

Viability of C2C12E50 cells after treatment with PEs at three doses (4, 10, 20 µg/mL from left to right for each polymer; PEI 25,000 was used as the comparison) determined by MTS assay. The cells were seeded in 96-well plates at an initial density of 1×104 cells/well in 0.2 mL of growth medium. The results are presented as the mean ± standard deviation (n=3, Student’s t-test, *P≤0.05 compared with untreated cells).Abbreviations: PEs, polyelectrolytes; PEI, polyethylenimine; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].
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f2-ijn-10-5635: Viability of C2C12E50 cells after treatment with PEs at three doses (4, 10, 20 µg/mL from left to right for each polymer; PEI 25,000 was used as the comparison) determined by MTS assay. The cells were seeded in 96-well plates at an initial density of 1×104 cells/well in 0.2 mL of growth medium. The results are presented as the mean ± standard deviation (n=3, Student’s t-test, *P≤0.05 compared with untreated cells).Abbreviations: PEs, polyelectrolytes; PEI, polyethylenimine; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].

Mentions: The C2C12E50 cell line was used to evaluate the efficacy of PEs for the delivery of PMO.18,28 This cell line expresses a GFP reporter, but its expression is disrupted by the insertion of the hDysE50. The expression of GFP in the reporter cells relies on the targeted removal of exon 50 by antisense oligonucleotides. First, we examined the cytotoxicity of the PEs using an MTS-based assay as shown in Figure 2. The toxicity of the PDDAC series including PE-1, PE-2, PE-3, and PE-4 was clearly size-dependent, being higher with increasing molecular weight of PDDAC. PE-5 (PADAC) and PE-6 (PBEBP) are structurally different from PDDAC. PE-5 contains insertion of around 55% polyacrylamide in composition; while PE-6 has a dispersed charged distribution due to the propyl urea spacer as compared with the PDDAC series. Protamine sulfate (PE-7) is a biocompatible and arginine-rich cell-penetrating polypeptide composed of only 50–110 amino acids, thus has a lower molecular weight and less toxicity than the PDDAC series.29–31


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)

Viability of C2C12E50 cells after treatment with PEs at three doses (4, 10, 20 µg/mL from left to right for each polymer; PEI 25,000 was used as the comparison) determined by MTS assay. The cells were seeded in 96-well plates at an initial density of 1×104 cells/well in 0.2 mL of growth medium. The results are presented as the mean ± standard deviation (n=3, Student’s t-test, *P≤0.05 compared with untreated cells).Abbreviations: PEs, polyelectrolytes; PEI, polyethylenimine; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].
© Copyright Policy
Related In: Results  -  Collection

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

f2-ijn-10-5635: Viability of C2C12E50 cells after treatment with PEs at three doses (4, 10, 20 µg/mL from left to right for each polymer; PEI 25,000 was used as the comparison) determined by MTS assay. The cells were seeded in 96-well plates at an initial density of 1×104 cells/well in 0.2 mL of growth medium. The results are presented as the mean ± standard deviation (n=3, Student’s t-test, *P≤0.05 compared with untreated cells).Abbreviations: PEs, polyelectrolytes; PEI, polyethylenimine; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].
Mentions: The C2C12E50 cell line was used to evaluate the efficacy of PEs for the delivery of PMO.18,28 This cell line expresses a GFP reporter, but its expression is disrupted by the insertion of the hDysE50. The expression of GFP in the reporter cells relies on the targeted removal of exon 50 by antisense oligonucleotides. First, we examined the cytotoxicity of the PEs using an MTS-based assay as shown in Figure 2. The toxicity of the PDDAC series including PE-1, PE-2, PE-3, and PE-4 was clearly size-dependent, being higher with increasing molecular weight of PDDAC. PE-5 (PADAC) and PE-6 (PBEBP) are structurally different from PDDAC. PE-5 contains insertion of around 55% polyacrylamide in composition; while PE-6 has a dispersed charged distribution due to the propyl urea spacer as compared with the PDDAC series. Protamine sulfate (PE-7) is a biocompatible and arginine-rich cell-penetrating polypeptide composed of only 50–110 amino acids, thus has a lower molecular weight and less toxicity than the PDDAC series.29–31

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