Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes

Abstract

In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA comprising codon-optimized firefly luciferase into stable LNPs. Mice were injected with 0.005–0.250 mg/kg doses of mRNA-LNPs by 6 different routes and high levels of protein translation could be measured using in vivo imaging. Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10 days. For several days, high levels of protein production could be achieved in the lung from the intratracheal administration of mRNA. Intravenous and intraperitoneal and to a lesser extent intramuscular and intratracheal deliveries led to trafficking of mRNA-LNPs systemically resulting in active translation of the mRNA in the liver for 1–4 days. Our results demonstrate that LNPs are appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA encoding therapeutic proteins.

Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA

“…For example in the study by Zangi et al, protein expression driven by exogenously administered mRNA returned to baseline within 48–72 hours post-injection [3]. Similar findings were reported with intravenously administered mRNA [4]. Unlike small RNAs, such as siRNA, that can be synthesized to have extended half-lives, in vitro transcribed mRNA is not as amenable to stability conferring chemical modifications.…”

“…For example, Zangi et al used intra-myocardial injection of exogenous luciferase or VEGF-A mRNA to increase local protein production in the heart and improved survival in a preclinical myocardial infarction model [3]. Moreover, lipid nanoparticle formulated mRNA has been successfully delivered to the liver, spleen and lung in preclinical models [4–6]. One major drawback of mRNA based therapy is the relatively short half-life of exogenously delivered mRNA in vivo.…”

mRNA vaccines — a new era in vaccinology

“…Engineering of the RNA sequence has rendered synthetic mRNA more translatable than ever before. Highly efficient and non-toxic RNA carriers have been developed that in some cases 21,22 allow prolonged antigen expression in vivo (TABLE 1). Some vaccine formulations contain novel adjuvants, while others elicit potent responses in the absence of known adjuvants.…”

“…81), but it has only recently been shown that LNPs are potent tools for in vivo delivery of self-amplifying RNA 19 and conventional, non-replicating mRNA 21 . Systemically delivered mRNA–LNP complexes mainly target the liver owing to binding of apolipoprotein E and subsequent receptor-mediated uptake by hepatocytes 82 , and intradermal, intramuscular and subcutaneous administration have been shown to produce prolonged protein expression at the site of the injection 21,22 . The mechanisms of mRNA escape into the cytoplasm are incompletely understood, not only for artificial liposomes but also for naturally occurring exosomes 83 .…”

Polyamine‐Mediated Stoichiometric Assembly of Ribonucleoproteins for Enhanced mRNA Delivery

“…On the other hand, the synthetic mRNA utilized in this study contains pseudouridine-and 5-methylcytidinesubstituted nucleotides along with a poly A tail at the 3′ end (120 adenosine residues) for optimal mRNA expression, which has been demonstrated in previous studies. [1][2]20] Since each PABP protein is known to bind a continuous stretch of 12 adenosine residues, each mRNA molecule is theoretically able to bind a maximum of ten PABP proteins. [21] Therefore, we first tested three different molar ratios of PABP to mRNA (2:1, 10:1 and 50:1) and subsequently encapsulated them with seven different polyamines.…”