Intravenous Delivery of a Dicer Substrate siRNA to the Central Nervous System via Rabies Virus Glycoprotein

Introduction: A Rabies Virus Glycoprotein (RVG) peptide was found to be an effective carrier for siRNA molecules across the selectively impermeable blood brain barrier and into neurons. This technique facilitates intravenous small interfering RNA administration, but its application remains limited to mice. Expansion into other animal species is required to broaden its research use and enhance its clinical relevance.

Methods: The RVG peptide was fused with an alternating arginine-glycine tail and separated by a glycine spacer. This peptide component was then combined with a Dicer substrate small interfering RNA (DsiRNA) designed to knock down mRNA specific to a protein, Shisa7, that associates with GABAA receptors. Two cohorts of rats implanted with femoral catheters were dosed once per day for two consecutive days and sacrificed at varying time points after administration of RVG-9r/DsiRNA. Brain tissue was analyzed by RT-PCR and Western blot analysis to verify gene silencing.

Results: The first cohort of rats was analyzed via Western blot analysis. The pons/medulla and cerebellum showed robust knockdown of Shisa7, with a mean reduction of 51% and 68%, respectively. Shisa7 in the frontal cortex was reduced by 43% and 6% at the 24-hour and 48-hour time point, respectively. The second cohort of rats produced similar results for the pons/medulla and cerebellum, while the frontal cortex showed a reduction of 17% and 49% at 24 and 48 hours. The hippocampus only showed gene silencing at 24 hours post-infusion. The RT-PCR data supported the Western blot data, in that Shisa7 mRNA levels decreased in multiple brain areas.

Conclusions: This study effectively knocked down Shisa7, an auxiliary protein of the GABAA receptor complex, in multiple brain areas after intravenous delivery of RVG-9r/DsiRNA in rats, making this non-invasive technique a viable method for studying Central Nervous System (CNS) proteins.