Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency.
Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency.
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嵌合肽吗啉代缀合物的背景依赖性效应有助于肌营养不良蛋白外显子跳跃效率
DOI：
10.1038/mtna.2013.51
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发表时间：
2013-09-24
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影响因子：
--
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中科院分区：
文献类型：
--
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我们最近报道，细胞穿透肽（CPP）和新的嵌合肽含有CPP（称为B肽）和肌肉靶向肽（称为MSP）基序显着提高系统性外显子跳跃活性的吗啉代磷酸二酰胺寡聚体（PMO）在dystrophin缺陷mdx小鼠。在本研究中，研究了嵌合肽构型对肽缀合的PM 0在体内的活性和组织摄取的一般机制意义。在mdx小鼠中测试了四种另外的嵌合肽-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比其对应物（9-B-PMO）诱导显著更高水平的外显子跳跃和肌营养不良蛋白恢复，进一步证实了嵌合肽-PMO缀合物的活性取决于嵌合肽内组织靶向肽基序相对于PMO的相对位置的观点。随后的机制研究表明，与MSP-B-PMO相比，B-MSP-PMO进入肌肉细胞的增强的细胞摄取导致外显子跳跃活性增加。令人惊讶的是，进一步的证据表明，两种取向的嵌合肽-PMO缀合物（B-MSP-PMO和MSP-B-PMO）的摄取是ATP依赖性和温度依赖性的，并且还部分地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。总的来说，我们的数据表明，嵌合肽中的肽取向是一个重要的参数，决定细胞的摄取和活性时，直接缀合到寡核苷酸。这些观察结果为未来的治疗研究提供了设计改进的细胞靶向化合物的见解。
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-deficientmdxmice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOsin vivowas 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 inmdxmice. 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.
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影响因子：
14.9
作者：
Wu RP;Youngblood DS;Hassinger JN;Lovejoy CE;Nelson MH;Iversen PL;Moulton HM
通讯作者：
Moulton HM
影响因子：
3
作者：
Seow, Yiqi;Yin, Haifang;Wood, Matthew J. A.
通讯作者：
Wood, Matthew J. A.
影响因子：
5.1
作者：
Wu, B.;Lu, P.;Lu, Q. L.
通讯作者：
Lu, Q. L.
影响因子：
3.5
作者：
Yin, HaiFang;Moulton, Hong M.;Wood, Matthew J. A.
通讯作者：
Wood, Matthew J. A.
影响因子：
2.9
作者：
Ziegler, A;Nervi, P;Seelig, J
通讯作者：
Seelig, J