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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

Confocal microscopic images of C2C12 cells treated with FITC-PMO (2 µg) without and with PE-4 (5 µg). Nuclear staining with Hoechst 33258. The images were obtained under a magnification of 63×. Labeled PMO can be observed at lower levels throughout the treated cells; however, with the addition of PE-4, greater levels of labeled PMO can be observed with greater concentrations in the perinuclear space.Abbreviations: PE, polyelectrolyte; FITC, fluorescein isothiocyanate; PMO, phosphorodiamidate morpholino oligomer.
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f6-ijn-10-5635: Confocal microscopic images of C2C12 cells treated with FITC-PMO (2 µg) without and with PE-4 (5 µg). Nuclear staining with Hoechst 33258. The images were obtained under a magnification of 63×. Labeled PMO can be observed at lower levels throughout the treated cells; however, with the addition of PE-4, greater levels of labeled PMO can be observed with greater concentrations in the perinuclear space.Abbreviations: PE, polyelectrolyte; FITC, fluorescein isothiocyanate; PMO, phosphorodiamidate morpholino oligomer.

Mentions: To examine whether PEs improve the cell uptake of PMO, we examined the intracellular localization of the PE/PMO polyplex. PE was complexed with 3′-carboxyfluorescein-labeled PMO at a weight ratio of 5/2. The presence of PE appeared to affect the pathway of PMO uptake, as demonstrated by confocal microscopy analysis. PMO alone distributed evenly within the cytoplasm of the cells, in agreement with a reported passive diffusion model.16 Signals for PMO were considerably stronger in cells treated with PE-4 and visualized as punctuates within the cytosol and especially around the nucleus (Figure 6). These results suggest that the presence of PEs alters the route of PMO internalization, probably eliciting endocytosis through formation of a complex.


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)

Confocal microscopic images of C2C12 cells treated with FITC-PMO (2 µg) without and with PE-4 (5 µg). Nuclear staining with Hoechst 33258. The images were obtained under a magnification of 63×. Labeled PMO can be observed at lower levels throughout the treated cells; however, with the addition of PE-4, greater levels of labeled PMO can be observed with greater concentrations in the perinuclear space.Abbreviations: PE, polyelectrolyte; FITC, fluorescein isothiocyanate; PMO, phosphorodiamidate morpholino oligomer.
© Copyright Policy
Related In: Results  -  Collection

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

f6-ijn-10-5635: Confocal microscopic images of C2C12 cells treated with FITC-PMO (2 µg) without and with PE-4 (5 µg). Nuclear staining with Hoechst 33258. The images were obtained under a magnification of 63×. Labeled PMO can be observed at lower levels throughout the treated cells; however, with the addition of PE-4, greater levels of labeled PMO can be observed with greater concentrations in the perinuclear space.Abbreviations: PE, polyelectrolyte; FITC, fluorescein isothiocyanate; PMO, phosphorodiamidate morpholino oligomer.
Mentions: To examine whether PEs improve the cell uptake of PMO, we examined the intracellular localization of the PE/PMO polyplex. PE was complexed with 3′-carboxyfluorescein-labeled PMO at a weight ratio of 5/2. The presence of PE appeared to affect the pathway of PMO uptake, as demonstrated by confocal microscopy analysis. PMO alone distributed evenly within the cytoplasm of the cells, in agreement with a reported passive diffusion model.16 Signals for PMO were considerably stronger in cells treated with PE-4 and visualized as punctuates within the cytosol and especially around the nucleus (Figure 6). These results suggest that the presence of PEs alters the route of PMO internalization, probably eliciting endocytosis through formation of a complex.

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