Although mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitate the treatment of diseases associated with aberrant gene upregulation and downregulation, we sought to co-formulate siRNA and mRNA in a single lipidoid nanoparticle (LNP) formulation. We accommodated the distinct molecular characteristics of mRNA and siRNA in a formulation consisting of an ionizable and biodegradable amine-containing lipidoid, cholesterol, DSPC, DOPE, and PEG-lipid. Surprisingly, the co-formulation of siRNA and mRNA in the same LNP enhanced the efficacy of both drugs in vitro and in vivo. Compared to LNPs encapsulating siRNA only, co-formulated LNPs improved Factor VII gene silencing in mice from 44 to 87% at an siRNA dose of 0.03 mg/kg. Co-formulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA co-formulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included. As not all gene therapy applications require both RNA drugs, we sought to extend the benefit of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the "helper" RNA with a negatively charged polymer, polystyrenesulfonate (PSS). LNPs containing PSS mediated the same level of protein silencing or expression as standard LNPs using 2-3-fold less RNA. For example, LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, respectively. Together, these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a negatively charged "helper polymer" enhances the efficacy of LNP delivery systems.
Keywords: Lipid nanoparticle; lipidoid; mRNA delivery; nanoparticle formulation; polystyrenesulfonate; siRNA delivery.