Schiroli Davide, Gómara María J, Maurizi Eleonora, Atkinson Sarah D, Mairs Laura, Christie Kathleen A, Cobice Diego F, McCrudden Cian M, Nesbit M Andrew, Haro Isabel, Moore Tara
Biomedical Sciences Research Institute, University of Ulster, Coleraine BT52 1SA, Northern Ireland.
Unit of Synthesis and Biomedical Applications of Peptides, Department of Biomedical Chemistry, Institute for Advanced Chemistry of Catalonia, Consejo Superior de Investigaciones Científicas (IQAC-CSIC), Barcelona, Spain.
Mol Ther Nucleic Acids. 2019 Sep 6;17:891-906. doi: 10.1016/j.omtn.2019.07.017. Epub 2019 Aug 1.
Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations.
常染色体显性遗传的遗传性疾病，如角膜营养不良，可通过小干扰RNA（siRNA）进行等位基因特异性基因沉默。通过注射将siRNA递送至角膜，虽然有效，但不适用于频繁的长期治疗方案，而将siRNA局部递送至角膜则受到眼表保护机制的阻碍。在此，我们描述了一种有吸引力的创新替代方法，即使用能够与siRNA非共价结合并将其递送至角膜的细胞穿透肽衍生物进行局部应用。通过合理的设计方法，我们修饰了一种细胞穿透肽——眼用递送肽（POD）的衍生物，该肽已被证明可扩散至角膜各层。这些POD衍生物能够形成大小和ζ电位与报道的能够进行细胞内化的复合物相似的siRNA-肽复合物（多聚体）。当加入内体破坏剂氯喹时，在体外成功实现了细胞质释放和基因沉默。一种具有最佳递送特性的棕榈酰化-POD与氯喹类似物三氟甲基喹啉进行了共价功能化。这种修饰后的POD，名为三氟甲基喹啉-棕榈酰-POD（QN-Palm-POD），当与siRNA复合并局部应用于体内眼部时，导致角膜上皮中荧光素酶报告基因表达降低了30%。本文开发的方法代表了一种有效的标准化方法，非常适合筛选一系列递送制剂。
