Safety Evaluation of Lipid Nanoparticle–Formulated Modified mRNA in the Sprague-Dawley Rat and Cynomolgus Monkey

Abstract

The pharmacology, pharmacokinetics, and safety of modified mRNA formulated in lipid nanoparticles (LNPs) were evaluated after repeat intravenous infusion to rats and monkeys. In both species, modified mRNA encoding the protein for human erythropoietin (hEPO) had predictable and consistent pharmacologic and toxicologic effects. Pharmacokinetic analysis conducted following the first dose showed that measured hEPO levels were maximal at 6 hours after the end of intravenous infusion and in excess of 100-fold the anticipated efficacious exposure (17.6 ng/ml) at the highest dose tested. hEPO was pharmacologically active in both the rat and the monkey, as indicated by a significant increase in red blood cell mass parameters. The primary safety-related findings were caused by the exaggerated pharmacology of hEPO and included increased hematopoiesis in the liver, spleen, and bone marrow (rats) and minimal hemorrhage in the heart (monkeys). Additional primary safety-related findings in the rat included mildly increased white blood cell counts, changes in the coagulation parameters at all doses, as well as liver injury and release of interferon γ-inducible protein 10 in high-dose groups only. In the monkey, as seen with the parenteral administration of cationic LNPs, splenic necrosis and lymphocyte depletion were observed, accompanied with mild and reversible complement activation. These findings defined a well-tolerated dose level above the anticipated efficacious dose. Overall, these combined studies indicate that LNP-formulated modified mRNA can be administered by intravenous infusion in 2 toxicologically relevant test species and generate supratherapeutic levels of protein (hEPO) in vivo.

Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery

“…31 Inspired by the success of LNPs, various groups showed that MC3 can also be used to safely transfect mRNA in order to express therapeutic proteins. 32,33 Researchers at Intellia Therapeutics have reported an ionizable lipid named LP-01 ( Figure 2) as part of the LNP formulation for the co-delivery of Cas9 mRNA and single guide RNA (sgRNA) for transthyretin gene, enabling successful editing of the mouse transthyretin gene in the liver. 34 In another example, Ramaswamy et al 35 reported successful protein replacement with human recombinant factor IX mRNA in a mouse model of hemophilia B, using an LNP formulation that utilized a proprietary ionizable lipid (representative example shown as ATX-100 in Figure 2) developed by Arcturus Therapeutics for their lipid-enabled and unlocked nucleic acid-modified RNA (LUNAR) delivery platform.…”

“…Most mRNA protein therapies use liver as a biological factory for the production and secretion of therapeutic proteins. 33,40,44 Expression of hEPO could be sustained for over a month when dosed weekly, with a peak expression between 6 and 12 h after LNP administration. 40 In all of the above cases, mRNA-containing LNPs were tolerated, and they did not cause liver injury or show signs of inflammation or complement activation in NHPs at the doses tested.…”

Self-Amplifying RNA Vaccines for Venezuelan Equine Encephalitis Virus Induce Robust Protective Immunogenicity in Mice

“…Alternatively, we speculate that the lower potency of a highly attenuated or inactivated VEE RNA vaccine could be overcome by further improvement of the synthetic non-viral delivery system, for example, by optimizing CNE specifically for VEEV SAM vaccine or utilizing alternative delivery strategies such as LNPs. 8,10,42,43 On a related note, the attenuated LAV m -CNE vaccine was significantly less immunogenic than the parental LAV-CNE vaccine, whereas LAV m -VRPs was similar to LAV-VRPs. It is possible that the VRP presents glycoproteins authentically to allow for more efficient uptake of the particles as compared to CNE so that both LAV m -VRPs and LAV-VRPs could efficiently launch to induce robust immune responses.…”

Delivery of mRNA Therapeutics for the Treatment of Hepatic Diseases

“…55 These carriers are highly effective, and commercially available transfection reagents like Lipofectamine messengerMAX and TransIT are widely used in vitro. 39,[55][56][57][58][59][60][61][62][63] Unfortunately, the majority of the commercially available reagents either don't work in vivo or result in significant toxicity like liver damage and immunogenicity. 64,65 One of the underlying reasons behind this loss of activity in vivo could be positively charged character of the resulting mRNA-carrier complexes.…”

“…doses of 0.5 mg/kg mRNA induced an increase in hepatic PBGD activity of roughly 80% at 24 h post-infusion. 71 Moreover, protein replacement of angiotensin-converting enzyme 2 in the liver and lungs of mice using 1 mg/kg mRNA-LNPs was shown by Schrom et al 72 Toxicity concerns might be alleviated by a study from Sedic et al, 60 who evaluated the safety of modified mRNA encoding for human erythropoietin packaged in LNPs. Up to 0.3 mg/kg mRNA-LNP was applied two times weekly to rats and monkeys in repeated i.v.…”