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Tween 85-Modified Low Molecular Weight PEI Enhances Exon-Skipping of Antisense Morpholino Oligomer In Vitro and in mdx Mice.

ABSTRACT: We investigated a series of Tween 85 modified low molecular weight polyethylenimine (LPEI, 0.8k/1.2k/2.0k)-copolymers (Zs) through simple formulation and covalent conjugation with phosphorodiamidate morpholino oligomer (PMO) for their potential to enhance delivery in vitro and in dystrophic mdx mice. Z polymers significantly enhanced PMO-induced exon-skipping in a GFP reporter-based cell culture system. Application of optimized formulations of Zs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in mdx mice. Consistent with our observations in vitro, optimization of molecular size and hydropholic-lipopholic balance (HLB) of polymers are important factors to achieve enhanced PMO delivery in vivo. The best formulation of Zs enhanced PMO delivery with 20- and 6-fold over PMO alone in vitro and in vivo, respectively. Further, chemical conjugation of the polymer and PMO exhibits greater benefit than polymer/PMO simple formulation in PMO delivery efficiency. Observed cytotoxicity of the Zs was lower than Endo-porter and PEI 25k in vitro, and no tissue toxicity was clearly detected with the Zs at the dosage tested. These results indicate the potential of the Zs as effective and safe PMO delivery carriers for treating diseases such as muscular dystrophy.

SUBMITTER: Wang M

PROVIDER: S-EPMC5633364 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Tween 85-Modified Low Molecular Weight PEI Enhances Exon-Skipping of Antisense Morpholino Oligomer In Vitro and in mdx Mice.

Wang Mingxing M Wu Bo B Tucker Jason D JD Shah Sapana N SN Lu Peijuan P Bollinger Lauren E LE Lu Qilong Q

Molecular therapy. Nucleic acids 20170920

We investigated a series of Tween 85 modified low molecular weight polyethylenimine (LPEI, 0.8k/1.2k/2.0k)-copolymers (Zs) through simple formulation and covalent conjugation with phosphorodiamidate morpholino oligomer (PMO) for their potential to enhance delivery in vitro and in dystrophic mdx mice. Z polymers significantly enhanced PMO-induced exon-skipping in a GFP reporter-based cell culture system. Application of optimized formulations of Zs with PMO targeted to dystrophin exon 23 demonstra ...[more]

PMID: 29246291

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Project description:Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded 'revertant' dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dataset Information

Tween 85-Modified Low Molecular Weight PEI Enhances Exon-Skipping of Antisense Morpholino Oligomer In Vitro and in mdx Mice.

Publications

Tween 85-Modified Low Molecular Weight PEI Enhances Exon-Skipping of Antisense Morpholino Oligomer In Vitro and in mdx Mice.

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