Targeted delivery of therapeutic RNAs for the treatment of ischemic stroke

Abstract

Ischemic stroke is caused by a reduction in blood flow to the brain due to narrowed cerebral arteries. Thrombolytic agents have been used to induce reperfusion of occluded cerebral arteries. However, brain damage continues to progress after reperfusion and induces ischemia-reperfusion (I/R) injury. Therefore, additional treatment is required to protect brain from I/R injury. In this study, various therapeutic RNAs was applied to alleviate I/R injury and treat ischemic stroke. For efficient gene therapy of I/R injury, replicon system equipped HO1 gene (HO1-repRNA) was delivered into ischemic brain, using deoxycholic acid conjugated PEI2k (DOCA-PEI). Since the I/R injury is long lasting damage, HO1-repRNA was utilized for expression persistence of gene. HO1-repRNA/DOCA-PEI complex was formed by charge interaction. In vitro studies showed that the expression of HO1-repRNA persisted longer than that of HO1-mRNA in Neuro2A cells. In vivo expression efficiency and therapeutic effects of HO1-repRNA/DOCA-PEI were evaluated in a middle cerebral artery occlusion (MCAO) model. In vivo studies showed that HO1 expression by HO1-repRNA/DOCA-PEI persisted until 7 days after injection. In addition, tumor necrosis factor-α (TNF-α) and apoptosis levels in the brain were reduced in the HO1-repRNA/DOCA-PEI group more efficiently than HO1-mRNA/DOCA-PEI at 7 days after injection. As a result, HO1-repRNA/DOCA-PEI significantly suppressed infarct size compared to HO1-mRNA/DOCA-PEI at 7 days after injection. For brain-specific delivery of therapeutic gene, HMGB1 small interfering RNA (siRNA) was delivered into ischemic brains by intravenous administration using rabies virus glycoprotein (RVG) peptide-decorated exosomes. A fusion protein of RVG and Lamp2b was expressed in 293T cells. Since Lamp2b is an exosome membrane-integral protein, RVG-Lamp2b is integrated into the exosomes, producing RVG-decorated exosomes (RVG-Exo). HMGB1-siRNA was loaded into RVG-Exo and unmodified exosomes (Unmod-Exo) by electroporation. In vitro studies demonstrated that RVG-Exo has higher uptake efficiency to Neuro2A cells than Unmod-Exo. Also, HMGB1 levels were reduced more effectively by RVG-Exo/HMGB1-siRNA. In vivo delivery efficiency and therapeutic effects of RVG-Exo/HMGB1-siRNA were evaluated in a MCAO model. RVG-Exo/HMGB1-siRNA, Unmod-Exo/HMGB1-siRNA, and PEI25k/HMGB1-siRNA were administrated into the MCAO model intravenously through the tail vein. The results showed that HMGB1, TNF-α, and apoptosis levels in the brain were reduced in the RVG-Exo/HMGB1-siRNA group more efficiently than the other groups. In addition, the infarct size was decreased in the RVG-Exo/HMGB1-siRNA group more effectively than the other groups. For nose-to-brain and hypoxia-specific delivery of anti-microRNA oligonucleotide (AMO), an exosome linked to RAGE-binding-peptide (RBP-Exo) was developed. In vitro studies showed that RBP-Exo delivered AMO181a to Neuro2A cells more efficiently than Unmod-Exo. In addition, RAGE was down-regulated by RBP-Exo, suggesting that RBP moiety of RBP-Exo reduced RAGE-mediated signal pathway. MicroRNA-181a (miR-181a) is one of the up-regulated miRNAs in the ischemic brain and its down-regulation can reduce the damages in the ischemic brain. Cholesterol-modified AMO181a (AMO181a-chol) was loaded onto the RBP-Exo by hydrophobic interaction. The AMO181a-chol-loaded RBP-Exo (RBP-Exo/AMO181a-chol) was administered to a MCAO model through nose-to-brain pathway. MiR-181a was knocked down and Bcl-2 was up-regulated by intranasal administration of RBP-Exo/AMO181a-chol. In addition, TNF-α expression and apoptosis were reduced by RBP-Exo/AMO181a-chol. As a result, RBP-Exo/AMO181a-chol significantly suppressed infarct size compared with the controls In conclusion, self-amplifying replicon system contributed to efficient therapeutic effect in the ischemic brain. As biocompatible carriers for therapeutic genes, RVG-Exo and RBP-Exo have been demonstrated to be brain and hypoxia-specific carriers, respectively. Especially, the combined effects of RBP and AMO181a exerted neuroprotective effect in the ischemic brain. Taken together, targeted delivery of therapeutic RNAs may be useful as a therapeutic system for the treatment of ischemic stroke.