Targeted siRNA Delivery to Neurons for Prion Protein Silencing Using Liposomal Complexes

Begin with a small interfering RNA that suppresses prion protein expression, a protein linked to neurodegenerative prion diseases.

Add a cationic liposome suspension.

The negatively-charged siRNA binds to the positively-charged liposome surface.

Introduce RVG-9r peptide, a Rabies Virus Glycoprotein attached to a polyarginine tail.

The arginine residues facilitate RVG-9r-siRNA binding.

Inject the liposome-siRNA-peptide complexes into the tail vein of an anesthetized mouse.

LSPCs circulate through the bloodstream and reach the brain.

The RVG-9r peptide binds to nicotinic acetylcholine receptors on endothelial cells, triggering receptor-mediated transcytosis across the blood-brain barrier.

Inside the brain, RVG-9r binds to neuronal acetylcholine receptors, facilitating LSPC endocytosis and siRNA release.

The RNA-induced silencing complex binds to the siRNA.

The passenger strand dissociates, while the guide strand binds to and cleaves PrP mRNA, preventing translation and reducing PrP expression.

Add 20 microliters of the 4 nanomole liposome suspension to 200 microliters of a 20 micromolar stock siRNA solution, and incubate for 10 minutes at room temperature. Then add 80 microliters of a 500 micromolar stock of RVG-9r targeting peptide to the siRNA liposome solution, and incubate for 10 minutes at room temperature.

After anesthetizing a mouse using 2 to 3% isofluorane in oxygen, cover the eyes of the mouse with ophthalmic ointment and confirm anesthetization by performing a toe pinch. Proceed only if a reaction is absent. While maintaining anesthesia, place the mouse on its back or side to access the tail vein.

Wipe the tail with 70% ethanol, and then use a 26-gauge insulin syringe to inject 300 microliters of LSPCs or PALETS into the tail vein. Begin injecting distally and move proximally if the vein collapses or ruptures. Place the mouse in a clean cage until it maintains sternal recumbency.