Bouncers block everyday people from entering trendy nightclubs. Similarly, the blood-brain barrier blocks molecules in the periphery from entering neural tissue. How can therapeutic molecules, like gene therapies, sneak through? Kumar et al. report a new transvascular delivery system based on the rabies virus in a recent article in Nature.
Neurotropic viruses, like rabies, cross the blood-brain barrier to infect brain cells. Rabies virus glycoprotein (RVG) enters neurons at acetylcholine receptors. The authors proposed that intravenous RVG would shuttle attached short interfering RNA into neurons.
A 29-amino acid peptide from RVG bound acetylcholine receptors and displaced 
-bungarotoxin, the acetylcholine receptor inhibitor active in snake venom. This peptide also bound mouse neuroblastoma Neuro2a cells but not human cervical cancer HeLa cells, which do not express acetylcholine receptors. Similarly, the peptide targeted cells isolated from mouse brain but not mouse spleen. Flow cytometry isolated neurons positive for rabies protein in mice injected intravenously with the RVG peptide, suggesting that it crosses the blood-brain barrier.
The RVG peptide does not normally bind nucleic acids. Because charge interaction binds negatively charged nucleic acids to positively charged peptides, the authors added a string of 9 arginine residues (9R) to RVG. RVG-9R bound short interfering RNA (siRNA) and infected Neuro2a but not HeLa cells. RVG-9R attached to green fluorescent protein (GFP) siRNA reduced GFP expression in Neuro2a cells stably expressing GFP, suggesting that siRNA bound to the RVG peptide is functional.
RVG-9R-siRNA silenced gene expression in the brain. The authors detected siRNA in multiple brain regions in mice injected with RVG-9R-siRNA. GFP transgenic mice injected with GFP siRNA associated with RVG-9R showed reduced GFP expression in brain but not liver. Superoxide dismutase 1 (SOD1) is associated with the familial form of amyotrophic lateral sclerosis (ALS). Intravenous injection of RVG-9R-SOD1 siRNA reduced SOD1 expression in brain by approximately 5-fold but did not reduce SOD1 expression in liver or spleen relative to control, suggesting that the RVG peptide is specific for brain in vivo.
Antiviral siRNAs protects mice from flaviviral encephalitis (FvE), which is fatal. The authors exposed mice to Japanese encephalitis virus (JEV) and treated them intravenously 4 hours later with RVG-9R-siFvE. All of the mice treated with siRNA attached to the rabies viral matrix protein died within 10 days. In contrast, 80% of mice treated with the RVG-9R-siFvE survived at least 30 days.
Therefore, intravenous injection of RVG-9R can deliver therapeutic agents to the brain in vivo, potentially reducing the need for invasive brain surgery to target therapies for diseases, like Parkinson's and epilepsy.