Brolin Camilla, Lim Ernest Wee Kiat, Grizot Sylvestre, Olsen Caroline Holkmann, Yavari Niloofar, Krag Thomas O, Nielsen Peter E
Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
MedinCell, Jacou, France.
Nucleic Acid Ther. 2021 Jun;31(3):208-219. doi: 10.1089/nat.2020.0856. Epub 2020 Jul 15.
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 forming depot technology (BEPO) and PNA-oligonucleotide formulation was studied. fluorescence imaging analysis showed fast renal/bladder excretion of the PNA (t ∼ 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 ∼ 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.
反义介导的外显子跳跃是治疗杜氏肌营养不良症（DMD）的一种有前景的新方法，DMD由基因突变引起，这些突变通常在肌营养不良蛋白基因中引入翻译终止密码子，从而导致功能性肌营养不良蛋白的产生被阻断。外显子去除可恢复翻译，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，此前已在DMD的mdx小鼠模型中通过剪接调节恢复了功能性肌营养不良蛋白的表达。在本研究中，我们比较了在mdx小鼠中通过静脉内（i.v.）和皮下（s.c.）途径全身给药20聚体剪接转换反义PNA寡聚物的情况。此外，还研究了形成长效注射技术（BEPO）和PNA-寡核苷酸制剂的效果。荧光成像分析显示，静脉内给药时PNA在肾脏/膀胱的排泄速度很快（t约20分钟），而皮下给药的排泄速度慢两到三倍。BEPO长效注射剂的释放呈现双相动力学特征，剂量的50%缓慢释放（t约10天）。在所有情况下，均可检测到在肾脏和肝脏中有一定的蓄积。将PNA与互补的硫代磷酸酯寡核苷酸配制成双链杂交复合物可提高PNA的溶解度。然而，这些替代给药方法均未显著提高反义活性。因此，必须采用更复杂的制剂，如设计的纳米颗粒或与递送配体偶联，以改善药代动力学以及组织靶向性和可及性。另一方面，结果表明，PNA的皮下和BEPO长效注射剂给药是可行的，且给药更简便、剂量更高、频率更低，以及释放更可控，这在动物模型研究以及最终临床给药优化方面均具有应用价值。
