Limits...
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

Green fluorescent protein expression induced by PMOE23 (5 µg) formulated with PEs in C2C12E23 cells (PE or Endoporter 5 µg and PMOE23 5 µg in 0.5 mL of 10% fetal bovine serum-Dulbecco’s Modified Eagle’s Medium after 48 hours treatment). Upper panel shows fluorescence detection of green fluorescent protein expression, original magnification, 100×; Lower panel shows reverse transcription-polymerase chain reaction of exon 23 skipping.Abbreviations: PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4562748&req=5

f5-ijn-10-5635: Green fluorescent protein expression induced by PMOE23 (5 µg) formulated with PEs in C2C12E23 cells (PE or Endoporter 5 µg and PMOE23 5 µg in 0.5 mL of 10% fetal bovine serum-Dulbecco’s Modified Eagle’s Medium after 48 hours treatment). Upper panel shows fluorescence detection of green fluorescent protein expression, original magnification, 100×; Lower panel shows reverse transcription-polymerase chain reaction of exon 23 skipping.Abbreviations: PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.

Mentions: In order to assess the delivery potential of the PEs for PMO exon-skipping in muscle fibers, rather than in myoblasts, the PEs were also tested in the mouse dystrophin exon 23 reporter C2C12 myoblast cell (C2C12E23). GFP reporter expression is driven by a muscle creatine kinase promoter, therefore allowed us to test the potential for PMO delivery in differentiating or differentiated myotubes.18 Cells reaching around 70% confluence were incubated in the differentiation medium for 2 days and then treated with PE-formulated PMOE23. The results showed a similar trend as that obtained in C2C12E50 cells, with PE-2, PE-3, and PE-4 achieving higher GFP expression than other PEs, as illustrated in Figure 5 by fluorescence images and RT-PCR detection of exon 23 skipping. The levels of exon 23 skipping were 56.4%, 64.1%, 59.0%, 32.6%, 24.7%, 38.5%, 1.8%, 23.7%, and 10.1% for PE-1, PE-2, PE-3, PE-4, PE-5, PE-6, PE-7, Endoporter-formulated PMO, and PMO only, respectively. The results indicate the importance of molecular size, chemical structure, and positive charge distribution in the vector microstructure for both delivery efficiency and toxicity.


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)

Green fluorescent protein expression induced by PMOE23 (5 µg) formulated with PEs in C2C12E23 cells (PE or Endoporter 5 µg and PMOE23 5 µg in 0.5 mL of 10% fetal bovine serum-Dulbecco’s Modified Eagle’s Medium after 48 hours treatment). Upper panel shows fluorescence detection of green fluorescent protein expression, original magnification, 100×; Lower panel shows reverse transcription-polymerase chain reaction of exon 23 skipping.Abbreviations: PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
© Copyright Policy
Related In: Results  -  Collection

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

f5-ijn-10-5635: Green fluorescent protein expression induced by PMOE23 (5 µg) formulated with PEs in C2C12E23 cells (PE or Endoporter 5 µg and PMOE23 5 µg in 0.5 mL of 10% fetal bovine serum-Dulbecco’s Modified Eagle’s Medium after 48 hours treatment). Upper panel shows fluorescence detection of green fluorescent protein expression, original magnification, 100×; Lower panel shows reverse transcription-polymerase chain reaction of exon 23 skipping.Abbreviations: PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
Mentions: In order to assess the delivery potential of the PEs for PMO exon-skipping in muscle fibers, rather than in myoblasts, the PEs were also tested in the mouse dystrophin exon 23 reporter C2C12 myoblast cell (C2C12E23). GFP reporter expression is driven by a muscle creatine kinase promoter, therefore allowed us to test the potential for PMO delivery in differentiating or differentiated myotubes.18 Cells reaching around 70% confluence were incubated in the differentiation medium for 2 days and then treated with PE-formulated PMOE23. The results showed a similar trend as that obtained in C2C12E50 cells, with PE-2, PE-3, and PE-4 achieving higher GFP expression than other PEs, as illustrated in Figure 5 by fluorescence images and RT-PCR detection of exon 23 skipping. The levels of exon 23 skipping were 56.4%, 64.1%, 59.0%, 32.6%, 24.7%, 38.5%, 1.8%, 23.7%, and 10.1% for PE-1, PE-2, PE-3, PE-4, PE-5, PE-6, PE-7, Endoporter-formulated PMO, and PMO only, respectively. The results indicate the importance of molecular size, chemical structure, and positive charge distribution in the vector microstructure for both delivery efficiency and toxicity.

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