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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

Flow cytometry diagram. The upper panel shows the flow cytometry dot plots (FSC-SSC) and the lower panel shows the histograms (FL).Abbreviations: PE, polyelectrolyte; PEI, polyethylenimine; PMO, phosphorodiamidate morpholino oligomer; FSC, forward scatter; SSC, side scatter; FL, flourescence.
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f4-ijn-10-5635: Flow cytometry diagram. The upper panel shows the flow cytometry dot plots (FSC-SSC) and the lower panel shows the histograms (FL).Abbreviations: PE, polyelectrolyte; PEI, polyethylenimine; PMO, phosphorodiamidate morpholino oligomer; FSC, forward scatter; SSC, side scatter; FL, flourescence.

Mentions: We next examined the effect of the PE polymers on exon-skipping of PMO. The PMO sequence, PMOE50 (5′-AACTTCCTCTTTAACAGAAAAGCATAC-3′) with previously confirmed efficacy for targeted removal of human dystrophin exon 50 was used.18–20 C2C12E50 GFP reporter cells were treated with a fixed amount (5 µg) of PMOE50 in 500 µL of 10% fetal bovine serum-DMEM medium formulated with each polymer at four different doses (1, 2, 5, and 10 µg). Transfection efficiency was examined by fluorescence microscopy. The results showed that almost all PE polymers, even at 1 µg, improved GFP expression compared with the PMOE50 alone. The highest levels of GFP expression were achieved at a dose of 2–5 µg with most PEs, especially with the PDDAC series, reaching up to 90% with PE-2/3/4 at the dose of 2 µg and comparable to or higher than Endoporter-mediated delivery (5 µg, effective dose in vitro, commercial reagent produced by GeneTools). A dose-dependent GFP expression is also illustrated by PE-3 at the doses of 1, 2, 5 µg (Figure 3). In contrast, less than 5% of the cells were GFP-positive when treated with PMOE50 alone. The exon-skipping efficiency remained higher at the 10 µg dose of PEs, but some toxicity was observed with PE-3/4. The transfection efficiency and cell viability of the PE/PMO formulation at the PE doses (1, 2, 5 µg) mixed with PMO (5 µg) in 500 µL of medium were also quantified by flow cytometry with Endoporter (5 µg) and PEI 25,000 (2 µg) as controls (Figures 3 and 4). GFP expression resulting from PDDAC-mediated PMO delivery was noted to be up to 20-fold higher when compared with PMO alone. The PDDAC series (PE-1/2/3/4) has more positive charges compared with PE-5 or PE-6 at the same weight dose. PE-5 has around 55% polyacrylamide in the main chain compared with the PDDAC series, thus certainly contains a reduced surface charge when forming a PE/PMO polyplex; PE-6 has a dispersed charged distribution as compared with the PDDAC series. The low transfection efficiency with protamine sulfate is likely due to a much lower molecular size, thus limited charge groups within the polyplex.


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)

Flow cytometry diagram. The upper panel shows the flow cytometry dot plots (FSC-SSC) and the lower panel shows the histograms (FL).Abbreviations: PE, polyelectrolyte; PEI, polyethylenimine; PMO, phosphorodiamidate morpholino oligomer; FSC, forward scatter; SSC, side scatter; FL, flourescence.
© Copyright Policy
Related In: Results  -  Collection

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

f4-ijn-10-5635: Flow cytometry diagram. The upper panel shows the flow cytometry dot plots (FSC-SSC) and the lower panel shows the histograms (FL).Abbreviations: PE, polyelectrolyte; PEI, polyethylenimine; PMO, phosphorodiamidate morpholino oligomer; FSC, forward scatter; SSC, side scatter; FL, flourescence.
Mentions: We next examined the effect of the PE polymers on exon-skipping of PMO. The PMO sequence, PMOE50 (5′-AACTTCCTCTTTAACAGAAAAGCATAC-3′) with previously confirmed efficacy for targeted removal of human dystrophin exon 50 was used.18–20 C2C12E50 GFP reporter cells were treated with a fixed amount (5 µg) of PMOE50 in 500 µL of 10% fetal bovine serum-DMEM medium formulated with each polymer at four different doses (1, 2, 5, and 10 µg). Transfection efficiency was examined by fluorescence microscopy. The results showed that almost all PE polymers, even at 1 µg, improved GFP expression compared with the PMOE50 alone. The highest levels of GFP expression were achieved at a dose of 2–5 µg with most PEs, especially with the PDDAC series, reaching up to 90% with PE-2/3/4 at the dose of 2 µg and comparable to or higher than Endoporter-mediated delivery (5 µg, effective dose in vitro, commercial reagent produced by GeneTools). A dose-dependent GFP expression is also illustrated by PE-3 at the doses of 1, 2, 5 µg (Figure 3). In contrast, less than 5% of the cells were GFP-positive when treated with PMOE50 alone. The exon-skipping efficiency remained higher at the 10 µg dose of PEs, but some toxicity was observed with PE-3/4. The transfection efficiency and cell viability of the PE/PMO formulation at the PE doses (1, 2, 5 µg) mixed with PMO (5 µg) in 500 µL of medium were also quantified by flow cytometry with Endoporter (5 µg) and PEI 25,000 (2 µg) as controls (Figures 3 and 4). GFP expression resulting from PDDAC-mediated PMO delivery was noted to be up to 20-fold higher when compared with PMO alone. The PDDAC series (PE-1/2/3/4) has more positive charges compared with PE-5 or PE-6 at the same weight dose. PE-5 has around 55% polyacrylamide in the main chain compared with the PDDAC series, thus certainly contains a reduced surface charge when forming a PE/PMO polyplex; PE-6 has a dispersed charged distribution as compared with the PDDAC series. The low transfection efficiency with protamine sulfate is likely due to a much lower molecular size, thus limited charge groups within the polyplex.

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