Antiviral RNAi inhalation inhibits human parainfluenza infection ex vivo and in vivo

Human parainfluenza virus 3 (HPIV3) infection can cause severe clinical outcomes in high-risk patients, with no approved antiviral therapy available. RNA interference (RNAi)-based antiviral treatment is a promising new therapy approach. We report the co-development of an siRNA targeting HPIV3 and a lipid nanoparticle (LNP)-based formulation that is suitable for inhaled delivery. We investigated compound safety and efficacy in a series of in vitro, ex vivo, and in vivo experiments, and developed an LNP formulations for the in vivo application of antiviral RNAi. 56 in silico predicted RNAi candidates were screened using fluorescent reporter cells, LLCMK2, Calu3, and human precision-cut lung slice (hPCLS) infection models. 12 different LNP compositions were screened for their stability after nebulization and initial LNP pharmacokinetics were monitored using an isolated perfused rat lung (IPL) model. Prophylactic inhalation treatment of the optimized LNP-formulation and lead siRNA compound (300 µg/kg) were finally tested in an in vivo cotton rat infection model. The best siRNA reached IC₅₀ values from 0.7 to 127 nM, depending on the test system, and did not show signs of cytotoxicity. LNP-formulated siRNA was successfully delivered to the airway epithelial cells in the IPL lungs. In vivo, the siRNA was primarily found in the lungs of cotton rats but not in blood or any other organs. HPIV3 viral load could be reduced in the lungs after prophylactic treatment, and infection-induced neutrophilia could be reverted. We could successfully demonstrate efficacy and delivery of antiviral RNAi using LNP-formulated siRNA against HPIV3 infection, with further toxicological and pharmacological assessment to come.