Biodistribution of lipid nanoparticle, eGFP mRNA and translated protein following subcutaneous administration in mouse.

Aim: Increased knowledge of biodistribution and pharmacokinetics of lipid nanoparticle (LNP)-encapsulated mRNA drug components may aid efficacy and safety evaluation. Methods: Mice were subcutaneously administrated LNP encapsulated enhanced green fluorescent protein mRNA and sampled up to 72 h after dosing. LNP, mRNA and translated protein were quantified by LC-MS, branched DNA and ELISA. Results: Highest levels of LNP and mRNA were detected in skin, followed by spleen, but also rapidly distributed to circulation. Translated protein showed high concentration in skin and spleen, but also in liver and kidney across 24 h where the LNP was cleared at 4 h. Conclusion: Subcutaneously dosing LNP encapsulated mRNA in mice resulted in a nonlinear relationship of LNP, mRNA and protein concentration across multiple tissues.

[Box: see text].

Spray dried lipid nanoparticle formulations enable intratracheal delivery of mRNA.

RNA therapies have recently taken a giant leap forward with the approval of Onpattro™, a siRNA therapy delivered using a lipid nanoparticle (LNP), and the LNP-enabled mRNA vaccines against COVID-19, which are the first mRNA drugs to reach the marketplace. The latter medicines have illustrated that stability is a significant challenge in the distribution of RNA drugs using non-viral delivery systems, particularly in areas without cold chain storage. Here, we describe a proof-of-concept study on the engineering of an LNP mRNA formulation suitable for spray drying. This process produced a dry powder formulation that maintained stability and preserved mRNA functionality with increased performance compared to liquid formulations stored two weeks at 4 °C. Intratracheal delivery of spray dried LNPs loaded with eGFP mRNA to rats resulted in the production of the eGFP protein in a range of cell types including bronchiolar epithelial cells, macrophages and type II pneumocytes; cell types involved in adaptive immunity and which would be valuable targets for inhaled vaccines against respiratory pathogens. Together, these data show that spray drying of LNPs enhances their stability and may enable RNA delivery to the lung for protein replacement therapy, gene editing, vaccination, and beyond.

Functionalized lipid nanoparticles for subcutaneous administration of mRNA to achieve systemic exposures of a therapeutic protein.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required.

Overcoming analytical challenges to generate data critical to understanding lipid nanoparticle-delivered modified mRNA biodistribution.

Aim: Chemically modified mRNA offers a novel approach to treat disease. Due to susceptibility to extracellular nucleases in vivo, dosed modified mRNA therapeutics can benefit from encapsulation within novel delivery systems, such as lipid nanoparticles (LNPs). To understand the holistic effect of dosing LNP-encapsulated modified mRNA therapeutics can require bioanalysis of several components including the mRNA, protein and LNP. Methodology: These components can require bespoke preanalytical strategies to preserve analyte integrity to achieve successful analysis. Here we describe the sample collection, processing steps and bioanalytical technologies that can be used to overcome these challenges. Discussion: Understanding the biodistribution and holistic effects of the different components allow the pharmaceutical industry to evaluate safety and efficacy of modified mRNA therapeutics.

Synthesis of di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate in unlabelled and C-13 labelled forms for use as a biomarker of drug induced phospholipidosis.

Di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) has been identified as a promising biomarker for drug-induced phospholipidosis (DIPL). Both unlabelled and stable isotope labelled versions of BMP were desired for use as internal standards. Isopropylideneglycerol was converted to 4-methoxyphenyldiphenylmethyl-3-PMB-glycerol in three steps. Initially, the 2-postion of the glycerol was protected as a t-butyldiphenylsilyl ether, which proved to be a mistake; deprotection of the ether resulted in the decomposition of the compound. A switch to a t-butyldimethylsilyl ether protecting group resulted in an intermediate that could be deprotected to the alcohol to give the target compound after salt exchange. The same procedure was used to prepare [13 C6 ]BMP from [13 C3 ]glycerol.