An Efficacy and Mechanism Driven Study on the Impact of Hypoxia on Lipid Nanoparticle Mediated mRNA Delivery

Abstract

Hypoxia is a common hallmark of human disease that is characterized by abnormally low oxygen levels in the body. While the effects of hypoxia on many small molecule-based drugs are known, its effects on several classes of next-generation medications including messenger RNA therapies warrant further study. Here, we provide an efficacy- and mechanism-driven study that details how hypoxia impacts the cellular response to mRNA therapies delivered using 4 different chemistries of lipid nanoparticles (LNPs, the frontrunner class of drug delivery vehicles for translational mRNA therapy utilized in the Moderna and Pfizer/BioNTech COVID-19 vaccines). Specifically, our work provides a comparative analysis as to how various states of oxygenation impact LNP-delivered mRNA expression, cellular association, endosomal escape, and intracellular ATP concentrations following treatment with 4 different LNPs across 3 different cell lines. In brief, we first identify that hypoxic cells express less LNP-delivered mRNA into protein than normoxic cells. Next, we identify generalizable cellular reoxygenation protocols that can reverse the negative effects that hypoxia imparts on LNP-delivered mRNA expression. Finally, mechanistic studies that utilize fluorescence-activated cell sorting, confocal microscopy, and enzyme inhibition reveal that decreases in mRNA expression correlate with decreases in intracellular ATP (rather than with differences in mRNA LNP uptake pathways). In presenting this data, we hope that our work provides a comprehensive efficacy and mechanism-driven study that explores the impact of differential oxygenation on LNP-delivered mRNA expression while simultaneously establishing fundamental criteria that may one day be useful for the development of mRNA drugs to treat hypoxia-associated disease.

A Unified Strategy to Improve Lipid Nanoparticle Mediated mRNA Delivery Using Adenosine Triphosphate

“…In brief, our strategy involves co-delivery of adenosine triphosphate (ATP, the cellular currency used to translate mRNA into protein) within mRNA LNPs. 31,32 Specifically, we reveal that our ATP co-delivery strategy results in greater amounts of protein expression across eight different LNP chemistries, three different cell lines, two different oxygenation protocols, and for mRNA encoding for intracellular and secreted proteins. studies are then performed to confirm that our approach is able to increase both intracellular and secreted protein expression levels in mice without impacting the innate biodistribution properties of parent mRNA LNPs (i.e., analogous mRNA LNP formulations without ATP).…”

“…Here, we present a unified strategy to increase the amount of mRNA-encoded protein expressed by target cells in vitro and in vivo following treatment with mRNA LNPs (Figure ). In brief, our strategy involves co-delivery of adenosine triphosphate (ATP, the cellular currency used to translate mRNA into protein) within mRNA LNPs. , Specifically, we reveal that our ATP co-delivery strategy results in greater amounts of protein expression across eight different LNP chemistries, three different cell lines, two different oxygenation protocols, and for mRNA encoding for intracellular and secreted proteins. Mechanistic studies on intracellular biochemical pathways, cellular association properties, and endosomal escape parameters are then performed to better understand how and why our ATP co-delivery strategy increases the amount of LNP-delivered mRNA into protein.…”

Messenger RNA Therapy for Female Reproductive Health

“…As such, optimization of the chemistries and molar ratios of each of these components is a large area of research in the field of mRNA delivery. 95,96…”

“…Each of these components is tunable, and modulation of each can cause changes in the size, polydispersity index, charge, encapsulation efficiency, biodistribution, and overall efficacy of the LNPs. As such, optimization of the chemistries and molar ratios of each of these components is a large area of research in the field of mRNA delivery. , …”

Bioinspired Spatiotemporal Management toward RNA Therapies

“…In additional examples, adding additional excipients like tannic acid (TA) could also facilitate the endosomal escape of the LNPs in MDCK cells . They also reported that hypoxia decreased the efficacy of LNPs due to differences in ATP in normoxic vs hypoxic cells rather than in how cells respond to mRNA LNPs . A follow up work is to develop a unified strategy by codelivering mRNA and ATP inside LNPs, which may have the potential for treating hypoxic diseases …”