Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation, here, we compared, in multiple cell and in vivo models, mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1mΨ), synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1mΨ made by the modified process, while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA.

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.

Accelerated blood clearance (ABC) is a phenomenon in which certain pharmaceutical agents are rapidly cleared from the blood upon second and subsequent administrations. ABC has been observed for many lipid-delivery vehicles, including liposomes and lipid nanoparticles (LNP). Previous studies have demonstrated a role for humoral responses against the polyethylene glycol motifs in clearance, but significant gaps remain in our understanding of the mechanism of ABC, and strategies for limiting the impact of ABC in a clinical setting have been elusive. mRNA therapeutics have great promise, but require chronic administration in encapsulating delivery systems, of which LNP are the most clinically advanced. In this study, we investigate the mechanisms of ABC for mRNA-formulated LNP in vivo and in vitro. We present evidence that ABC of mRNA-formulated LNP is dramatic and proceeds rapidly, based on a previously unrecognized ability of LNP to directly activate B-1 lymphocytes, resulting in the production of antiphosphorylcholine IgM Abs in response to initial injection. Upon repeated injections, B-2 lymphocytes also become activated and generate a classic anti-polyethylene glycol adaptive humoral response. The ABC response to phosphorylcholine/LNP-encapsulated mRNA is therefore a combination of early B-1 lymphocyte and later B-2 lymphocyte responses. ImmunoHorizons, 2019, 3: 282-293.

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Study Design: Biomechanical investigation. Objective: To compare the biomechanical performance of nitinol memory metal rods and titanium rods when used as posterior spinal instrumentation in a synthetic model. Methods: Biomechanical testing was performed using ultra-high-molecular-weight polyethylene blocks. Nineteen spinal constructs were created to allow comparison of 5.5-mm nitinol rods with 5.5-mm titanium rods. Static compression and rotational testing were performed on an Instron 8874 and Instron 4202 at 37°C to simulate body temperature. Results: The average titanium construct stiffness under static compression or bending was 47.2 ± 9.1 N/mm while nitinol’s stiffness averaged 48.9 ± 12.4 N/mm ( P = .83). During axial rotation testing, the nitinol rod system showed no torsional stiffness difference from the titanium system: 0.95 ± 0.03 Nm/deg versus 0.96 ± 0.17 Nm/deg, respectively ( P = 0.91). There was a statistically significant difference between the average torsional yield point for the titanium constructs (14.4 ± 1.6 Nm/deg) and nitinol constructs (21.3 ± 0.8 Nm/deg) ( P = .004). The torsional toughness of the nitinol constructs was also statistically greater than the titanium rods: 473 GN/m3 versus 784 GN/m3 ( P = .0006). There was no statistically significant difference between the nitinol group sustaining a higher number of fatigue cycles until failure and the titanium group (181 660 cycles vs 64 104 cycles, respectively, P = .22). Conclusions: This study provides biomechanical evidence that nitinol rods used in a posterior construct are comparable to titanium rods with regard to compression and have increased torsional failure load and torsional toughness. While nitinol trended toward superior fatigue resistance, there was no significant difference in nitinol versus titanium construct fatigue resistance.