Abstracts Category : Other

Optimization and analysis of lipid nanoparticles for invivo mRNA delivery

by Kevin John Kauffman

Messenger RNA (mRNA) therapeutics have the potentialto treat a diverse array of diseases requiring protein expression,with applications in protein replacement therapies,immunotherapies, and genome engineering. However, the intracellulardelivery of mRNA is challenging and necessitates a safe andeffective delivery vector. Lipid nanoparticles (LNPs) have shownconsiderable promise for the delivery of small interfering RNAs(siRNA) to the liver but their utility as agents for mRNA deliveryhave only been recently investigated. New delivery materials formRNA delivery are also being developed which have the potential totransfect nonliver targets, but the screening of these vectors invivo is low-throughput and it is difficult to determine transfectedcell types. There is a need both for efficacious,well-characterized mRNA delivery materials and for methods tofacilitate in vivo screening of novel materials. We first developeda generalized strategy to optimize LNP formulations for mRNAdelivery to the liver using Design of Experiment methodologies. Bysimultaneously varying lipid ratios and structures, we developed anoptimized formulation which increased the potency oferyrthopoietin-mRNA-loaded LNPs in vivo 7-fold relative toformulations previously used for siRNA delivery. Next, we exploredthe immune response and activity of base-modified LNPformulatedmRNA administered systemically in vivo. We observed indications ofa previously uncharacterized transient, extracellular innate immuneresponse to mRNA-LNPs, including neutrophilia, myeloid cellactivation, and up-regulation of four serum cytokines. Although wehave developed a more efficacious liver-targeting LNP, many mRNAtherapies will require delivery to non-liver tissues. Usingtrial-and-error approaches, we discover novel formulations capableof inducing mRNA expression in vivo in the spleen, lung, and fat.To increase the throughput of in vivo screening, we report a newbarcoding-based approach capable of evaluating the biodistributionand pharmacokinetics of many LNP formulations in a single mouse.Then, we develop a method that can identify mRNA expressiondelivered from LNPs in both bulk tissues and with single cellresolution. Together, the work reported here contributes to thedevelopment of mRNA therapeutics by increasing mRNA-LNP potency andcharacterizing their immunogenicity in vivo. Furthermore, we hopethe multiple in vivo screening methods described in this Thesiswill accelerate the discovery of new delivery vectors capable oftransfecting desired tissues and cell types.Advisors/Committee Members: Daniel G. Anderson (advisor).