Design and development of lipid nanoparticle formulations for brain gene therapy

Many genetic neurological diseases are caused by toxic gain-of-function of a mutant protein or loss-of-function of a wild-type protein. Treatment of these disorders may be feasible using gene therapy, which requires delivering therapeutic agents to affected brain regions and cells. Lipid nanoparticles (LNPs) have significant potential for this purpose: the clinical safety and efficacy of LNP systems is well-established, and neurons are amenable to LNP-mediated transfection. To adapt LNP technology for brain gene therapy applications, we iteratively designed LNPs to deliver nucleic acids to ex vivo primary neurons, and evaluated optimized formulations in vivo in the brain. Our approach improved ex vivo LNP potency over 1.7-fold for siRNA-containing systems and over 22-fold for mRNA-containing systems, and identified distinct compositions optimal for siRNA and mRNA delivery. We show that the activity of ex vivo-optimized LNPs is not always correlated with in vivo activity in murine striatum and establish the apparent pKa of ionizable cationic lipids as an important contributor to LNP efficacy in both model systems. Collectively, we demonstrate robust delivery of multiple macromolecular payloads to primary neurons ex vivo and to the brain in vivo and validate the utility of LNP systems for gene knockdown and protein replacement brain gene therapies.