Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs

Abstract

Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.

Systematic optimization of an engineered hydrogel allows for selective control of human neural stem cell survival and differentiation after transplantation in the stroke brain

“…Recently, studies have addressed transplant survival along with differentiation and repair of the recipient tissue with a data-driven statistical approach to optimize the gel composition to promote tissue repair. Many of these investigations have transitioned from in vitro to in vivo in the tissue-engineering field of organs such as retina [46], skin [47], liver [48], digestive tract [49], ear [50], pancreas [51], lungs [52] and skeletal muscle [53]. However the systematic development of a bioengineered scaffold for the brain with DOE studies has been rarely pursued past the in vitro stage [21, 22].…”

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

“…To date, naked, chemically modified, or protamine-complexed mRNA have shown promise in phase I/II cancer trials (24)(25)(26). Recently, preclinical development of materials specific for mRNA delivery has resulted in cationic polymers such as polymethacrylates (27)(28)(29), poly(aspartamides) (30,31), and polypeptides (32), as well as multicomponent cationic lipid or lipid-like formulations (21,(33)(34)(35)(36). In many of these examples, however, transfection efficiencies can be quite low, ranging 20-80% in cells (18), with likely much lower efficiencies in vivo, which requires either high mRNA doses or hydrodynamic injections (32,37).…”

Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery

“…[4] To circumvent this barrier, our group and others have developed and implemented an array of lipid nanoparticles (LNPs) for the entrapment and subsequent delivery of nucleic acids in vivo . [1] Although these LNPs have been largely optimized for siRNA sequences, both Schlake’s group [5] and our research team [6] have recently employed LNPs derived from previously described components to deliver mRNA in vivo . Successful delivery was confirmed by quantifying serum protein levels, thereby establishing LNPs as viable delivery vehicles for mRNA.…”