Antisense-mediated exon skipping constitutes a promising new modality for treatment of Duchenne Muscular Dystrophy (DMD), which is caused by gene mutations that typically introduce a translation stop codon in the dystrophin gene, thereby abolishing production of functional dystrophin protein. The exon removal can restore translation to produce a shortened, but still partially functional dystrophin protein. Peptide nucleic acid (PNA) as a potential antisense drug has previously been shown to restore the expression of functional dystrophin by splice modulation in the mdx mouse model of DMD. In this study, we compare systemic administration of a 20-mer splice switching antisense PNA oligomer through intravenous (i.v.) and subcutaneous (s.c.) routes in the mdx mice. Furthermore, the effect of in situ forming depot technology (BEPO^®) and PNA-oligonucleotide formulation was studied. In vivo fluorescence imaging analysis showed fast renal/bladder excretion of the PNA (t_½ ∼ 20 min) for i.v. administration, while s.c. administration showed a two to three times slower excretion. The release from the BEPO depot exhibited biphasic kinetics with a slow release (t_½ ∼ 10 days) of 50% of the dose. In all cases, some accumulation in kidneys and liver could be detected. Formulation of PNA as a duplex hybridization complex with a complementary phosphorothioate oligonucleotide increased the solubility of the PNA. However, none of these alternative administration methods resulted in significantly improved antisense activity. Therefore, either more sophisticated formulations such as designed nanoparticles or conjugation to delivery ligands must be utilized to improve both pharmacokinetics as well as tissue targeting and availability. On the other hand, the results show that s.c. and BEPO depot administration of PNA are feasible and allow easier, higher, and less frequent dosing, as well as more controlled release, which can be exploited both for animal model studies as well as eventually in the clinic in terms of dosing optimization.

中文翻译：

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在 mdx 小鼠模型中系统递送抗肌萎缩蛋白反义肽核酸的方法

反义介导的外显子跳跃是治疗杜氏肌营养不良症 (DMD) 的一种有前途的新模式，该模式是由基因突变引起的，该基因突变通常在肌营养不良蛋白基因中引入翻译终止密码子，从而消除功能性肌营养不良蛋白的产生。外显子去除可以恢复翻译以产生缩短但仍具有部分功能的肌营养不良蛋白。肽核酸 (PNA) 作为一种潜在的反义药物，先前已显示通过剪接调节在 DMD 的 mdx 小鼠模型中恢复功能性肌营养不良蛋白的表达。在这项研究中，我们比较了 mdx 小鼠通过静脉内 (iv) 和皮下 (sc) 途径对 20 聚体剪接转换反义 PNA 寡聚体的全身给药。此外，现场效果研究了形成仓库技术（BEPO ^®）和PNA-寡核苷酸配方。体内荧光成像分析显示静脉内给药的 PNA 快速肾/膀胱排泄（t _½ ∼ 20 分钟），而皮下给药显示慢 2 到 3 倍的排泄。BEPO 仓库的释放表现出缓慢释放的双相动力学（t _½∼ 10 天）的 50% 剂量。在所有情况下，都可以检测到肾脏和肝脏中的一些积聚。将 PNA 作为具有互补硫代磷酸酯寡核苷酸的双链杂交复合物制剂增加了 PNA 的溶解度。然而，这些替代给药方法均未显着提高反义活性。因此，必须使用更复杂的制剂（例如设计的纳米颗粒）或与递送配体结合来改善药代动力学以及组织靶向性和可用性。另一方面，结果表明，皮下和 BEPO 长效给药 PNA 是可行的，并且允许更容易、更高和更少的给药频率，以及更可控的释放，