The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option. However, there are concerns whether an acceptable tolerability profile for chronic dosing can be achieved. The efficiency and tolerability of lipid nanoparticles has been attributed to the amino lipid. Therefore, we developed a new series of amino lipids that address this concern. Clear structure-activity relationships were developed that resulted in a new amino lipid that affords efficient mRNA delivery in rodent and primate models with optimal pharmacokinetics. A 1-month toxicology evaluation in rat and non-human primate demonstrated no adverse events with the new lipid nanoparticle system. Mechanistic studies demonstrate that the improved efficiency can be attributed to increased endosomal escape. This effort has resulted in the first example of the ability to safely repeat dose mRNA-containing lipid nanoparticles in non-human primate at therapeutically relevant levels.

mRNA vaccines have the potential to tackle many unmet medical needs that are unable to be addressed with conventional vaccine technologies. A potent and well-tolerated delivery technology is integral to fully realizing the potential of mRNA vaccines. Pre-clinical and clinical studies have demonstrated that mRNA delivered intramuscularly (IM) with first-generation lipid nanoparticles (LNPs) generates robust immune responses. Despite progress made over the past several years, there remains significant opportunity for improvement, as the most advanced LNPs were designed for intravenous (IV) delivery of siRNA to the liver. Here, we screened a panel of proprietary biodegradable ionizable lipids for both expression and immunogenicity in a rodent model when administered IM. A subset of compounds was selected and further evaluated for tolerability, immunogenicity, and expression in rodents and non-human primates (NHPs). A lead formulation was identified that yielded a robust immune response with improved tolerability. More importantly for vaccines, increased innate immune stimulation driven by LNPs does not equate to increased immunogenicity, illustrating that mRNA vaccine tolerability can be improved without affecting potency.

Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to-OH group is required to maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit higher cellular uptake and retention, potentially leading to a steady release from the endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape. Our findings show the importance of cholesterol in subcellular transport of LNPs carrying mRNA and emphasize the need for greater insights into surface composition and structural properties of nanoparticles, and their subcellular interactions which enable designs to improve endosomal escape.

The discoidin domain receptors (DDRs) are receptor tyrosine kinases that recognize collagens as their ligands. DDRs display unique structural features and distinctive activation kinetics, which set them apart from other members of the kinase superfamily. DDRs regulate cell-collagen interactions in normal and pathological conditions and thus are emerging as major sensors of collagen matrices and potential novel therapeutic targets. New structural and biological information has shed light on the molecular mechanisms that regulate DDR signaling, turnover, and function. This minireview provides an overview of these areas of DDR research with the goal of fostering further investigation of these intriguing and unique receptors. The discoidin domain receptor (DDR)2 family comprises two distinct members, DDR1 and DDR2, which were initially discovered in the early 1990s and characterized as receptor tyrosine kinases (RTKs) based on the presence of a catalytic kinase domain (KD) (1-7). Subsequently, collagens were identified as ligands for DDRs (8), thus establishing the unique characteristic of these receptors among other members of the RTK superfamily. Upon collagen binding, DDRs undergo tyrosine autophosphorylation with distinctive activation kinetics, which elicits genetic and cellular programs that regulate a variety of cell-collagen interactions. Despite their unique characteristics, the biochemical and cellular mechanisms by which DDRs mediate their multiple biological effects remain poorly defined. This minireview provides an overview of current information on DDR structure, regulation, and signaling. For information on specific DDR biological functions in processes such as cell adhesion, migration, and invasion over collagen matrices and their role in normal and pathological processes, the reader is directed to the following recent reviews (9 -11) DDR StructureThe DDR1 subfamily is composed of five membrane-anchored isoforms, and the DDR2 subfamily is represented by a single protein. The five DDR1 isoforms are generated by alternative splicing. DDR1a, DDR1b, and DDR1c are full-length functional receptors, and DDR1d and DDR1e are truncated or kinase-inactive receptors (10, 12). Two additional secreted splice variants of DDR1 have also been identified (13). DDR1b and DDR1c contain an additional 37 residues within the intracellular juxtamembrane (IJXM) region. With the exception of the two secreted DDR1 isoforms, all DDRs are single-pass type I transmembrane glycoproteins that are characterized by the presence of six distinct protein domains: a discoidin (DS) domain, a DS-like domain, an extracellular juxtamembrane (EJXM) region, a transmembrane (TM) segment, a long IJXM region, and an intracellular KD (Fig. 1A). The presence of the N-terminal DS and DS-like domains is the defining feature of the DDR RTK subfamily. The DS domain exhibits high homology to a protein module originally identified in proteins from Dictyostelium discoideum (14). In this organism, the DS domain functions as a galactose-binding lectin, whic...

Intracellular delivery of mRNA holds great potential for vaccine1–3 and therapeutic4 discovery and development. Despite increasing recognition of the utility of lipid-based nanoparticles (LNPs) for intracellular delivery of mRNA, particle engineering is hindered by insufficient understanding of endosomal escape, which is believed to be a main limiter of cytosolic availability and activity of the nucleic acid inside the cell. Using a series of CRISPR-based genetic perturbations of the lysosomal pathway, we have identified that late endosome/lysosome (LE/Ly) formation is essential for functional delivery of exogenously presented mRNA. Lysosomes provide a spatio-temporal hub to orchestrate mTOR signaling and are known to control cell proliferation, nutrient sensing, ribosomal biogenesis, and mRNA translation. Through modulation of the mTOR pathway we were able to enhance or inhibit LNP-mediated mRNA delivery. To further boost intracellular delivery of mRNA we screened 212 bioactive lipid-like molecules that are either enriched in vesicular compartments or modulate cell signaling. Surprisingly, we have discovered that leukotriene-antagonists, clinically approved for treatment of asthma and other lung diseases, enhance intracellular mRNA delivery in vitro (over 3-fold, p<0.005) and in vivo (over 2-fold, p<0.005). Understanding LNP-mediated intracellular delivery will inspire the next generation of RNA therapeutics that have high potency and. limited toxicity.