1] Research Centre of Basic Medical Science, Tianjin Medical University, Qixiangtai Road, Heping District, Tianjin, China [2] Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, UK.
Mol Ther Nucleic Acids. 2013 Sep 24;2(9):e124. doi: 10.1038/mtna.2013.51.
We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.Molecular Therapy-Nucleic Acids (2013) 2, e124; doi:10.1038/mtna.2013.51; published online 24 September 2013.
我们最近报道称，细胞穿透肽（CPPs）和新型嵌合肽（称为 B 肽）和肌肉靶向肽（称为 MSP）基序可显著提高肌营养不良症缺失型 mdx 小鼠中多聚体（PMOs）的系统外显子跳跃活性。在本研究中，研究了嵌合肽结构对体内肽缀合 PMO 的活性和组织摄取的一般机制意义。在 mdx 小鼠中测试了另外四种嵌合肽-PMO 缀合物，包括新鉴定的肽 9（B-9-PMO 和 9-B-PMO）和对照肽 3（B-3-PMO 和 3-B-PMO）。免疫组织化学染色、RT-PCR 和 Western blot 结果表明，B-9-PMO 诱导的外显子跳跃和肌营养不良蛋白恢复水平明显高于其对应物（9-B-PMO），进一步证实了嵌合肽-PMO 缀合物的活性取决于组织靶向肽基序在嵌合肽中相对于 PMO 的相对位置。随后的机制研究表明，与 MSP-B-PMO 相比，B-MSP-PMO 进入肌肉细胞的摄取增强导致外显子跳跃活性增强。令人惊讶的是，进一步的证据表明，两种取向（B-MSP-PMO 和 MSP-B-PMO）的嵌合肽-PMO 缀合物的摄取均依赖于 ATP 和温度，并且部分由硫酸乙酰肝素蛋白聚糖（HSPG）介导，表明内吞作用可能是两种嵌合肽-PMO 缀合物的主要摄取途径。总之，我们的数据表明，当直接缀合到寡核苷酸时，嵌合肽中的肽取向是决定细胞摄取和活性的重要参数。这些观察结果为未来治疗学研究中改进的细胞靶向化合物的设计提供了思路。分子治疗-核酸（2013 年）2，e124；doi:10.1038/mtna.2013.51；在线发布 2013 年 9 月 24 日。
