Williams Jason H, Schray Rebecca C, Sirsi Shashank R, Lutz Gordon J
Drexel University College of Medicine, Department of Pharmacology and Physiology, Philadelphia, Pennsylvania 19102, USA.
BMC Biotechnol. 2008 Apr 2;8:35. doi: 10.1186/1472-6750-8-35.
Exon skipping oligonucleotides (ESOs) of 2'O-Methyl (2'OMe) and morpholino chemistry have been shown to restore dystrophin expression in muscle fibers from the mdx mouse, and are currently being tested in phase I clinical trials for Duchenne Muscular Dystrophy (DMD). However, ESOs remain limited in their effectiveness because of an inadequate delivery profile. Synthetic cationic copolymers of poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) are regarded as effective agents for enhanced delivery of nucleic acids in various applications.
We examined whether PEG-PEI copolymers can facilitate ESO-mediated dystrophin expression after intramuscular injections into tibialis anterior (TA) muscles of mdx mice. We utilized a set of PEG-PEI copolymers containing 2 kDa PEI and either 550 Da or 5 kDa PEG, both of which bind 2'OMe ESOs with high affinity and form stable nanoparticulates with a relatively low surface charge. Three weekly intramuscular injections of 5 microg of ESO complexed with PEI2K-PEG550 copolymers resulted in about 500 dystrophin-positive fibers and about 12% of normal levels of dystrophin expression at 3 weeks after the initial injection, which is significantly greater than for injections of ESO alone, which are known to be almost completely ineffective. In an effort to enhance biocompatibility and cellular uptake, the PEI2K-PEG550 and PEI2K-PEG5K copolymers were functionalized by covalent conjugation with nanogold (NG) or adsorbtion of colloidal gold (CG), respectively. Surprisingly, using the same injection and dosing regimen, we found no significant difference in dystrophin expression by Western blot between the NG-PEI2K-PEG550, CG-PEI2K-PEG5K, and non-functionalized PEI2K-PEG550 copolymers. Dose-response experiments using the CG-PEI2K-PEG5K copolymer with total ESO ranging from 3-60 microg yielded a maximum of about 15% dystrophin expression. Further improvements in dystrophin expression up to 20% of normal levels were found at 6 weeks after 10 twice-weekly injections of the NG-PEI2K-PEG550 copolymer complexed with 5 microg of ESO per injection. This injection and dosing regimen showed over 1000 dystrophin-positive fibers. H&E staining of all treated muscle groups revealed no overt signs of cytotoxicity.
We conclude that PEGylated PEI2K copolymers are efficient carriers for local delivery of 2'OMe ESOs and warrant further development as potential therapeutics for treatment of DMD.
2'-O-甲基（2'OMe）和吗啉代化学修饰的外显子跳跃寡核苷酸（ESO）已被证明可恢复mdx小鼠肌肉纤维中的肌营养不良蛋白表达，目前正在进行杜氏肌营养不良症（DMD）的I期临床试验。然而，由于递送情况不佳，ESO的有效性仍然有限。聚（乙烯亚胺）（PEI）和聚（乙二醇）（PEG）的合成阳离子共聚物被认为是在各种应用中增强核酸递送的有效试剂。
我们研究了PEG-PEI共聚物在向mdx小鼠胫前肌（TA）肌肉进行肌肉注射后是否能促进ESO介导的肌营养不良蛋白表达。我们使用了一组含有2 kDa PEI和550 Da或5 kDa PEG的PEG-PEI共聚物，它们都能与2'OMe ESO高亲和力结合，并形成表面电荷相对较低的稳定纳米颗粒。每周三次肌肉注射5 μg与PEI2K-PEG550共聚物复合的ESO，在初次注射后3周时产生了约500条肌营养不良蛋白阳性纤维，肌营养不良蛋白表达水平约为正常水平的12%，这明显高于单独注射ESO的情况，已知单独注射ESO几乎完全无效。为了提高生物相容性和细胞摄取，PEI2K-PEG550和PEI2K-PEG5K共聚物分别通过与纳米金（NG）共价结合或吸附胶体金（CG）进行功能化。令人惊讶的是，使用相同的注射和给药方案，我们发现通过蛋白质免疫印迹法检测，NG-PEI2K-PEG550、CG-PEI2K-PEG5K和未功能化的PEI2K-PEG550共聚物在肌营养不良蛋白表达方面没有显著差异。使用CG-PEI2K-PEG5K共聚物与总量为3 - 60 μg的ESO进行剂量反应实验，产生的肌营养不良蛋白表达最高约为15%。在每周两次注射10次与每次5 μg ESO复合的NG-PEI2K-PEG550共聚物后6周，发现肌营养不良蛋白表达进一步提高至正常水平的20%。这种注射和给药方案显示有超过1000条肌营养不良蛋白阳性纤维。对所有处理过的肌肉组进行苏木精-伊红染色显示没有明显的细胞毒性迹象。
我们得出结论，聚乙二醇化的PEI2K共聚物是局部递送2'OMe ESO的有效载体，值得作为治疗DMD的潜在疗法进一步开发。
