Long-term efficacy and safety of mRNA therapy in two murine models of methylmalonic acidemia.

BACKGROUND: Isolated methylmalonic acidemia/aciduria (MMA) is an ultra-rare, serious, inherited metabolic disorder with significant morbidity and mortality. Exogenously delivered mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, is a potential therapy to produce functional MUT enzyme in liver. METHODS: Two 12-week repeat-dose studies were conducted to evaluate the efficacy and safety of intravenously-administered hMUT mRNA encapsulated in lipid nanoparticles in two murine models of MMA. FINDINGS: In MMA hypomorphic mice, hMUT mRNA treatment resulted in dose-dependent and reproducible biomarker responses after each dose. Enzymatically-active MUT protein was produced in liver in a dose-dependent manner. hMUT mRNA was well-tolerated with no adverse effects, as indicated by the lack of clinical observations, minimal changes in clinical chemistry parameters, and histopathology examination across all tissues. In severe MMA mice, hMUT mRNA led to substantially improved survival and growth and ameliorated biochemical abnormalities, all of which are cardinal clinical manifestations in severely affected patients. INTERPRETATION: These data demonstrate durable functional benefit of hMUT mRNA and support development of this new class of therapy for a devastating, pediatric disorder. FUND: This work was funded by Moderna, Inc.

Accelerated Blood Clearance of Lipid Nanoparticles Entails a Biphasic Humoral Response of B-1 Followed by B-2 Lymphocytes to Distinct Antigenic Moieties.

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.

Systemic mRNA Therapy for the Treatment of Fabry Disease: Preclinical Studies in Wild-Type Mice, Fabry Mouse Model, and Wild-Type Non-human Primates.

Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla-deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders.

Targeted Interleukin-10 Nanotherapeutics Developed with a Microfluidic Chip Enhance Resolution of Inflammation in Advanced Atherosclerosis.

Inflammation is an essential protective biological response involving a coordinated cascade of signals between cytokines and immune signaling molecules that facilitate return to tissue homeostasis after acute injury or infection. However, inflammation is not effectively resolved in chronic inflammatory diseases such as atherosclerosis and can lead to tissue damage and exacerbation of the underlying condition. Therapeutics that dampen inflammation and enhance resolution are currently of considerable interest, in particular those that temper inflammation with minimal host collateral damage. Here we present the development and efficacy investigations of controlled-release polymeric nanoparticles incorporating the anti-inflammatory cytokine interleukin 10 (IL-10) for targeted delivery to atherosclerotic plaques. Nanoparticles were nanoengineered via self-assembly of biodegradable polyester polymers by nanoprecipitation using a rapid micromixer chip capable of producing nanoparticles with retained IL-10 bioactivity post-exposure to organic solvent. A systematic combinatorial approach was taken to screen nanoparticles, resulting in an optimal bioactive formulation from in vitro and ex vivo studies. The most potent nanoparticle termed Col-IV IL-10 NP22 significantly tempered acute inflammation in a self-limited peritonitis model and was shown to be more potent than native IL-10. Furthermore, the Col-IV IL-10 nanoparticles prevented vulnerable plaque formation by increasing fibrous cap thickness and decreasing necrotic cores in advanced lesions of high fat-fed LDLr(-/-) mice. These results demonstrate the efficacy and pro-resolving potential of this engineered nanoparticle for controlled delivery of the potent IL-10 cytokine for the treatment of atherosclerosis.

Targeted nanoparticles containing the proresolving peptide Ac2-26 protect against advanced atherosclerosis in hypercholesterolemic mice.

Chronic, nonresolving inflammation is a critical factor in the clinical progression of advanced atherosclerotic lesions. In the normal inflammatory response, resolution is mediated by several agonists, among which is the glucocorticoid-regulated protein called annexin A1. The proresolving actions of annexin A1, which are mediated through its receptor N-formyl peptide receptor 2 (FPR2/ALX), can be mimicked by an amino-terminal peptide encompassing amino acids 2-26 (Ac2-26). Collagen IV (Col IV)-targeted nanoparticles (NPs) containing Ac2-26 were evaluated for their therapeutic effect on chronic, advanced atherosclerosis in fat-fed Ldlr(-/-) mice. When administered to mice with preexisting lesions, Col IV-Ac2-26 NPs were targeted to lesions and led to a marked improvement in key advanced plaque properties, including an increase in the protective collagen layer overlying lesions (which was associated with a decrease in lesional collagenase activity), suppression of oxidative stress, and a decrease in plaque necrosis. In mice lacking FPR2/ALX in myeloid cells, these improvements were not seen. Thus, administration of a resolution-mediating peptide in a targeted NP activates its receptor on myeloid cells to stabilize advanced atherosclerotic lesions. These findings support the concept that defective inflammation resolution plays a role in advanced atherosclerosis, and suggest a new form of therapy.