a-positioning-device-for-placement-mice-during-intranasal-sirna

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Hanyang University

Yale University School of Medicine

Adipose tissue macrophage (ATM)-mediated inflammation is a key feature contributing to the adverse metabolic outcomes of dietary obesity. Recruitment of macrophages to obese adipose tissues (AT) can occur through the engagement of CCR2, the receptor for MCP-1 (monocyte chemoattractant protein-1), which is expressed on peripheral monocytes/macrophages. Here, we show that i.p. administration of a rabies virus glycoprotein-derived acetylcholine receptor-binding peptide effectively delivers complexed siRNA into peritoneal macrophages and ATMs in a mouse model of high-fat diet-induced obesity. Treatment with siRNA against CCR2 inhibited macrophage infiltration and accumulation in AT and, therefore, proinflammatory cytokines produced by macrophages. Consequently, the treatment significantly improved glucose tolerance and insulin sensitivity profiles, and also alleviated the associated symptoms of hepatic steatosis and reduced hepatic triglyceride production. These results demonstrate that disruption of macrophage chemotaxis to the AT through cell-targeted gene knockdown strategies can provide a therapeutic intervention for obesity-related metabolic diseases. The study also highlights a siRNA delivery approach for targeting specific monocyte subsets that contribute to obesity-associated inflammation without affecting the function of other tissue-resident macrophages that are essential for host homeostasis and survival. 

Silencing CCR2 in Macrophages Alleviates Adipose Tissue Inflammation and the Associated Metabolic Syndrome in Dietary Obese Mice.

siRNA entrapment within endosomes is a significant problem encountered with siRNA delivery platforms that co-opt receptor-mediated entry pathways. Attachment of a cell-penetrating peptide (CPP), such as nona-arginine (9R) to a cell receptor-binding ligand like the Rabies virus glycoprotein, RVG, allows effective siRNA delivery to the cytoplasm by non-endocytic pathways, but a significant amount of siRNA complexes also enters the cell by ligand-induced receptor endocytosis and remain localized in endosomes. Here, we report that the incorporation of trileucine (3 Leu) residues as an endo-osmolytic moiety in the peptide improves endosomal escape and intracellular delivery of siRNA. The trileucine motif did not affect early non-endosomal mechanism of cytoplasmic siRNA delivery but enhanced target gene silencing by >20% only beyond 24 h of transfection when siRNA delivery is mostly through the endocytic route and siRNA trapped in the endosomes at later stages were subject to release into cytoplasm. The mechanism may involve endosomal membrane disruption as trileucine residues lysed RBCs selectively under endosomal pH conditions. Interestingly <3 Leu or >3 Leu residues were not as effective, suggesting that 3 Leu residues are useful for enhancing cytoplasmic delivery of siRNA routed through endosomes.

Trileucine residues in a ligand-CPP-based siRNA delivery platform improve endosomal escape of siRNA.

No vaccines or therapeutics are licensed for West Nile virus (WNV), a mosquito-transmitted neuroencephalitic flavivirus. The small interfering RNA siFvE targets a conserved sequence within the WNV E protein and limits virus infection. Using a rabies virus-derived neuron-targeting peptide (RVG9R) and an intranasal route for delivering siFvE to the CNS, we demonstrate that treatment of WNV-infected mice at late stages of neuroinvasive disease results in recovery. Selectively targeting virus in the CNS lowers viral burdens in the brain, reduces neuropathology, and results in a 90% survival rate at 5-6 days post-infection (when viral titers peak in the CNS), while placebo-treated mice succumb by days 9-10. Importantly, CNS virus clearance is achieved by humoral and cell-mediated immune responses to WNV infection in peripheral tissues, which also engender sterilizing immunity against subsequent WNV infection. These results indicate that intranasal RVG9R-siRNA treatment offers efficient late-stage therapy and facilitates natural long-term immunity against neuroinvasive flaviviruses.

Irfan Ullah

Department of Bioengineering and Institute of Nanoscience and Technology

Hanyang University

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Irfan Ullah

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Bioengineering and Institute of Nanoscience and Technology

Hanyang University

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Department of Bioengineering and Institute of Nanoscience and Technology