Schissel Carly K, Farquhar Charlotte E, Malmberg Annika B, Loas Andrei, Pentelute Bradley L
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
Sarepta Therapeutics, 215 First Street, Cambridge, Massachusetts 02142, United States.
ACS Bio Med Chem Au. 2022 Feb 16;2(2):150-160. doi: 10.1021/acsbiomedchemau.1c00053. eCollection 2022 Apr 20.
Cell-penetrating peptides (CPPs) can cross the cell membrane to enter the cytosol and deliver otherwise nonpenetrant macromolecules such as proteins and oligonucleotides. For example, recent clinical trials have shown that a CPP attached to phosphorodiamidate morpholino oligomers (PMOs) resulted in higher muscle concentration, increased exon skipping, and dystrophin production relative to another study of the PMO alone in patients of Duchenne muscular dystrophy. Therefore, effective design and the study of CPPs could help enhance therapies for difficult-to-treat diseases. So far, the study of CPPs for PMO delivery has been restricted to predominantly canonical l-peptides. We hypothesized that mirror-image d-peptides could have similar PMO delivery activity as well as enhanced proteolytic stability, facilitating their characterization and quantification from biological milieu. We found that several enantiomeric peptide sequences could deliver a PMO-biotin cargo with similar activities while remaining stable against serum proteolysis. The biotin label allowed for affinity capture of fully intact PMO-peptide conjugates from whole-cell and cytosolic lysates. By profiling a mixture of these constructs in cells, we determined their relative intracellular concentrations. When combined with PMO activity, these concentrations provide a new metric for delivery efficiency, which may be useful for determining which peptide sequence to pursue in further preclinical studies.
细胞穿透肽（CPPs）能够穿过细胞膜进入胞质溶胶，并递送诸如蛋白质和寡核苷酸等原本无法穿透的大分子。例如，最近的临床试验表明，与另一项仅对杜氏肌营养不良症患者使用磷二酰胺吗啉代寡聚物（PMO）的研究相比，与磷二酰胺吗啉代寡聚物相连的CPPs能使肌肉中的浓度更高，外显子跳跃增加，肌营养不良蛋白产量提高。因此，有效的CPPs设计和研究有助于增强对难治性疾病的治疗效果。到目前为止，用于递送PMO的CPPs研究主要局限于典型的L-肽。我们推测镜像D-肽可能具有类似的PMO递送活性以及更高的蛋白水解稳定性，便于从生物环境中对其进行表征和定量分析。我们发现，几个对映体肽序列能够递送具有相似活性的PMO-生物素货物，同时对血清蛋白水解保持稳定。生物素标签允许从全细胞和胞质裂解物中亲和捕获完全完整的PMO-肽缀合物。通过在细胞中分析这些构建体的混合物，我们确定了它们相对的细胞内浓度。当与PMO活性相结合时，这些浓度提供了一种新的递送效率指标，这可能有助于确定在进一步的临床前研究中应采用哪种肽序列。
