Department of Developmental Biology, Rangos Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Dualsystems Biotech AG, Schlieren, Switzerland.
Methods Mol Biol. 2021;2211:97-112. doi: 10.1007/978-1-0716-0943-9_8.
Cell-penetrating peptides (CPPs), also known as protein transduction domains, were first identified 25 years ago. They are small, ~6-30 amino acid long, synthetic, or naturally occurring peptides, able to carry a variety of cargoes across the cellular membranes in an intact, functional form. These cargoes can range from other small peptides, full-length proteins, nucleic acids including RNA and DNA, nanoparticles, and viral particles as well as radioisotopes and other fluorescent probes for imaging purposes. However, this ability to enter all cell types indiscriminately, and even cross the blood-brain barrier, hinders their development into viable vectors. Hence, researchers have adopted various strategies ranging from pH activatable cargoes to using phage display to identify tissue-specific CPPs. Use of this phage display strategy has led to an ever-expanding number of tissue-specific CPPs. Using phage display, we identified a 12-amino acid, non-naturally occurring peptide that targets the heart with peak uptake at 15 min after a peripheral intravenous injection, that we termed Cardiac Targeting Peptide (CTP). In this chapter, we use CTP as an example to describe techniques for validation of cell-specific transduction as well as provide details on a technology to identify binding partner(s) for these ever-increasing plethora of tissue-specific peptides. Given the myriad cargoes CTP can deliver, as well as rapid uptake after an intravenous injection, it can be applied to deliver radioisotopes, miRNA, siRNA, peptides, and proteins of therapeutic potential for acute cardiac conditions like myocardial infarction, where the window of opportunity for salvaging at-risk myocardium is limited to 6 hrs.
细胞穿透肽（CPPs），也称为蛋白转导结构域，于 25 年前首次被发现。它们是小的、约 6-30 个氨基酸长的合成或天然存在的肽，能够以完整、功能性的形式将各种货物携带穿过细胞膜。这些货物可以包括其他小肽、全长蛋白质、核酸（包括 RNA 和 DNA）、纳米颗粒和病毒颗粒以及放射性同位素和其他用于成像目的的荧光探针。然而，这种能够无差别地进入所有细胞类型甚至穿过血脑屏障的能力，阻碍了它们发展成为可行的载体。因此，研究人员采用了各种策略，从 pH 激活的货物到使用噬菌体展示来识别组织特异性 CPPs。使用噬菌体展示策略导致了组织特异性 CPPs 的数量不断增加。使用噬菌体展示，我们鉴定出一种 12 个氨基酸的非天然存在的肽，它靶向心脏，在静脉外周注射后 15 分钟达到峰值摄取，我们将其命名为心脏靶向肽（CTP）。在本章中，我们将 CTP 作为一个例子来描述验证细胞特异性转导的技术，并详细介绍一种识别这些不断增加的组织特异性肽的结合伴侣的技术。鉴于 CTP 可以输送的多种货物，以及静脉注射后迅速摄取，它可用于输送放射性同位素、miRNA、siRNA、肽和具有治疗潜力的蛋白质，用于急性心脏病况，如心肌梗死，其中挽救风险心肌的机会窗口仅限于 6 小时。
