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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

Restoration of dystrophin in tibialis anterior muscles of mdx mice (aged 4–5 weeks) 2 weeks after intramuscular injection.Notes: (A) Dystrophin was detected by immunohistochemistry with rabbit polyclonal antibody P7 against dystrophin. Blue nuclear staining with 4,6-diamidino-2-phenylindole. Muscles treated with PMOE23 (2 µg) only was used as controls. All other samples were from muscles treated with 2 µg polymer and 2 µg PMOE23 in 40 µL saline. Original magnification, 100×. (B) The percentage of dystrophin-positive fibers in muscles treated with 2 µg PMOE23 with and without polymers (2 µg). The maximum numbers of dystrophin-positive fibers were counted in a single cross-section (n=5, one-way analysis of variance test, #P≤0.05, there were significant difference between PE groups; Student’s t-test, *P≤0.05 compared with 2 µg PMO). (C) Detection of exon 23 skipping by reverse transcription-polymerase chain reaction. Total RNA of 100 ng from each sample was used for amplification of dystrophin mRNA from exon 20 to exon 26. The upper bands (indicated by E22 + E23 + E24) correspond to the normal mRNA, and the lower bands (indicated by E22 + E24) correspond to the mRNA with exon E23 skipped. (D) Western blots demonstrate the expression of dystrophin protein. Dystrophin detected with monoclonal antibody Dys 1. A loading control (α-actin) was used.Abbreviations: Dys, dystrophin; PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
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f8-ijn-10-5635: Restoration of dystrophin in tibialis anterior muscles of mdx mice (aged 4–5 weeks) 2 weeks after intramuscular injection.Notes: (A) Dystrophin was detected by immunohistochemistry with rabbit polyclonal antibody P7 against dystrophin. Blue nuclear staining with 4,6-diamidino-2-phenylindole. Muscles treated with PMOE23 (2 µg) only was used as controls. All other samples were from muscles treated with 2 µg polymer and 2 µg PMOE23 in 40 µL saline. Original magnification, 100×. (B) The percentage of dystrophin-positive fibers in muscles treated with 2 µg PMOE23 with and without polymers (2 µg). The maximum numbers of dystrophin-positive fibers were counted in a single cross-section (n=5, one-way analysis of variance test, #P≤0.05, there were significant difference between PE groups; Student’s t-test, *P≤0.05 compared with 2 µg PMO). (C) Detection of exon 23 skipping by reverse transcription-polymerase chain reaction. Total RNA of 100 ng from each sample was used for amplification of dystrophin mRNA from exon 20 to exon 26. The upper bands (indicated by E22 + E23 + E24) correspond to the normal mRNA, and the lower bands (indicated by E22 + E24) correspond to the mRNA with exon E23 skipped. (D) Western blots demonstrate the expression of dystrophin protein. Dystrophin detected with monoclonal antibody Dys 1. A loading control (α-actin) was used.Abbreviations: Dys, dystrophin; PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.

Mentions: Immunohistochemistry showed that the PMOE23 alone induced up to 12% maximum dystrophin-positive fibers in one cross-section of the tibialis anterior muscle. The number of dystrophin-positive fibers increased dramatically in the muscles treated with PMOE23 mediated by PEs. The PDDAC series enhanced PMO-mediated exon-skipping with increasing molecular size. PE-3 and PE-4 achieved over 40% and 50% positive fibers respectively, ie, over fourfold as compared with PMO alone at the tested dose. Meanwhile, PE-5, PE-6, and PE-7 did not dramatically change the number of dystrophin-positive fibers (Figure 8). These results correlate well with the data in muscle cell lines in vitro, suggesting that the smaller PE molecule was less able to form an optimal complex with PMO, resulting in low transfection efficiency.32,33 PE-3 or PE-4 with higher transfection efficiency is probably due to larger molecular size, thus creating higher affinity binding sites with PMO. The positively charged PE-PMO complex is likely to be more stable in biological systems as the complex is expected to interact with various cells and biomacromolecules in the tissue and circulation, maintaining a longer circulation time.34,35 The levels of exon-skipping and corresponding dystrophin expression were also quantitatively determined by RT-PCR and Western blot, respectively. PMO formulated with PE-1, PE-2, PE-3, PE-4, PE-5, PE-6, PE-7 and PMO only achieved levels of exon-skipping at 4.1%, 30.5%, 22.9%, 25.5%, 0.2%, 20.8%, 0.8%, and 2.7%, respectively. Dystrophin protein expression levels were found to be 3.5%, 23.7%, 6.2%, 51.2%, 9.5%, and 3.7% of normal levels (taking muscles from C57 mouse as 100%) for PMO formulated with PE-1, PE-2, PE-3, PE-4, PE-6 and PMO only, respectively. Both quantitative and qualitative data therefore demonstrated the best performance with the PDDAC series, especially with PE-4. The results indicate the importance of molecular size and composition for the efficacy of delivery of uncharged PMO. However, more effective PEs are associated with higher toxicity, further highlighting the difficulty and complexity of developing non-viral oligonucleotide delivery agents for clinical use.


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)

Restoration of dystrophin in tibialis anterior muscles of mdx mice (aged 4–5 weeks) 2 weeks after intramuscular injection.Notes: (A) Dystrophin was detected by immunohistochemistry with rabbit polyclonal antibody P7 against dystrophin. Blue nuclear staining with 4,6-diamidino-2-phenylindole. Muscles treated with PMOE23 (2 µg) only was used as controls. All other samples were from muscles treated with 2 µg polymer and 2 µg PMOE23 in 40 µL saline. Original magnification, 100×. (B) The percentage of dystrophin-positive fibers in muscles treated with 2 µg PMOE23 with and without polymers (2 µg). The maximum numbers of dystrophin-positive fibers were counted in a single cross-section (n=5, one-way analysis of variance test, #P≤0.05, there were significant difference between PE groups; Student’s t-test, *P≤0.05 compared with 2 µg PMO). (C) Detection of exon 23 skipping by reverse transcription-polymerase chain reaction. Total RNA of 100 ng from each sample was used for amplification of dystrophin mRNA from exon 20 to exon 26. The upper bands (indicated by E22 + E23 + E24) correspond to the normal mRNA, and the lower bands (indicated by E22 + E24) correspond to the mRNA with exon E23 skipped. (D) Western blots demonstrate the expression of dystrophin protein. Dystrophin detected with monoclonal antibody Dys 1. A loading control (α-actin) was used.Abbreviations: Dys, dystrophin; PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
© Copyright Policy
Related In: Results  -  Collection

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

f8-ijn-10-5635: Restoration of dystrophin in tibialis anterior muscles of mdx mice (aged 4–5 weeks) 2 weeks after intramuscular injection.Notes: (A) Dystrophin was detected by immunohistochemistry with rabbit polyclonal antibody P7 against dystrophin. Blue nuclear staining with 4,6-diamidino-2-phenylindole. Muscles treated with PMOE23 (2 µg) only was used as controls. All other samples were from muscles treated with 2 µg polymer and 2 µg PMOE23 in 40 µL saline. Original magnification, 100×. (B) The percentage of dystrophin-positive fibers in muscles treated with 2 µg PMOE23 with and without polymers (2 µg). The maximum numbers of dystrophin-positive fibers were counted in a single cross-section (n=5, one-way analysis of variance test, #P≤0.05, there were significant difference between PE groups; Student’s t-test, *P≤0.05 compared with 2 µg PMO). (C) Detection of exon 23 skipping by reverse transcription-polymerase chain reaction. Total RNA of 100 ng from each sample was used for amplification of dystrophin mRNA from exon 20 to exon 26. The upper bands (indicated by E22 + E23 + E24) correspond to the normal mRNA, and the lower bands (indicated by E22 + E24) correspond to the mRNA with exon E23 skipped. (D) Western blots demonstrate the expression of dystrophin protein. Dystrophin detected with monoclonal antibody Dys 1. A loading control (α-actin) was used.Abbreviations: Dys, dystrophin; PE, polyelectrolyte; PMO, phosphorodiamidate morpholino oligomer.
Mentions: Immunohistochemistry showed that the PMOE23 alone induced up to 12% maximum dystrophin-positive fibers in one cross-section of the tibialis anterior muscle. The number of dystrophin-positive fibers increased dramatically in the muscles treated with PMOE23 mediated by PEs. The PDDAC series enhanced PMO-mediated exon-skipping with increasing molecular size. PE-3 and PE-4 achieved over 40% and 50% positive fibers respectively, ie, over fourfold as compared with PMO alone at the tested dose. Meanwhile, PE-5, PE-6, and PE-7 did not dramatically change the number of dystrophin-positive fibers (Figure 8). These results correlate well with the data in muscle cell lines in vitro, suggesting that the smaller PE molecule was less able to form an optimal complex with PMO, resulting in low transfection efficiency.32,33 PE-3 or PE-4 with higher transfection efficiency is probably due to larger molecular size, thus creating higher affinity binding sites with PMO. The positively charged PE-PMO complex is likely to be more stable in biological systems as the complex is expected to interact with various cells and biomacromolecules in the tissue and circulation, maintaining a longer circulation time.34,35 The levels of exon-skipping and corresponding dystrophin expression were also quantitatively determined by RT-PCR and Western blot, respectively. PMO formulated with PE-1, PE-2, PE-3, PE-4, PE-5, PE-6, PE-7 and PMO only achieved levels of exon-skipping at 4.1%, 30.5%, 22.9%, 25.5%, 0.2%, 20.8%, 0.8%, and 2.7%, respectively. Dystrophin protein expression levels were found to be 3.5%, 23.7%, 6.2%, 51.2%, 9.5%, and 3.7% of normal levels (taking muscles from C57 mouse as 100%) for PMO formulated with PE-1, PE-2, PE-3, PE-4, PE-6 and PMO only, respectively. Both quantitative and qualitative data therefore demonstrated the best performance with the PDDAC series, especially with PE-4. The results indicate the importance of molecular size and composition for the efficacy of delivery of uncharged PMO. However, more effective PEs are associated with higher toxicity, further highlighting the difficulty and complexity of developing non-viral oligonucleotide delivery agents for clinical use.

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