Moreno Pedro M D, Ferreira Ana R, Salvador Daniela, Rodrigues Maria T, Torrado Marília, Carvalho Eva D, Tedebark Ulf, Sousa Mónica M, Amaral Isabel F, Wengel Jesper, Pêgo Ana P
i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal; INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 4200-135 Porto, Portugal.
GE Healthcare Bio-Sciences AB, 75184 Uppsala, Sweden; SynMer AB, 17568 Järfälla, Sweden.
Mol Ther Nucleic Acids. 2018 Jun 1;11:393-406. doi: 10.1016/j.omtn.2018.03.009. Epub 2018 Mar 20.
After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels, allowing the regulation of cell survival and cell function, together with the availability of chemically modified nucleic acids with favorable biopharmaceutical properties, make AONs an attractive tool for novel SCI therapy developments. In this work, we explored the potential of locked nucleic acid (LNA)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration-RhoA and GSK3β. The fibrin-matrix-assisted AON delivery system mediated potent RNA knockdown in vitro in a dorsal root ganglion explant culture system and in vivo at a SCI lesion site, achieving around 75% downregulation 5 days after hydrogel injection. Our results show that local implantation of a AON-gapmer-loaded hydrogel matrix mediated efficient gene silencing in the lesioned spinal cord and is an innovative platform that can potentially combine gene regulation with regenerative permissive substrates aiming at SCI therapeutics and nerve regeneration.
脊髓损伤（SCI）后，由于损伤部位形成了高度抑制性的微环境，多种因素共同作用，神经再生受到严重阻碍。反义寡核苷酸（AONs）具有在不同水平修饰基因表达的潜力，能够调节细胞存活和细胞功能，同时具有生物药学性质良好的化学修饰核酸，这使得AONs成为新型SCI治疗开发的一个有吸引力的工具。在这项工作中，我们探索了锁核酸（LNA）修饰的AON缺口寡核苷酸与纤维蛋白水凝胶桥接材料联合使用，在SCI损伤部位原位诱导基因沉默的潜力。针对两个有望促进轴突再生的基因靶点——RhoA和GSK3β，有效地开发了LNA缺口寡核苷酸。纤维蛋白基质辅助的AON递送系统在体外背根神经节外植体培养系统和体内SCI损伤部位介导了有效的RNA敲低，水凝胶注射5天后实现了约75%的下调。我们的结果表明，局部植入负载AON缺口寡核苷酸的水凝胶基质介导了损伤脊髓中的有效基因沉默，是一个创新平台，有可能将基因调控与旨在SCI治疗和神经再生的再生允许性基质相结合。
