mRNA Therapy Improves Metabolic and Behavioral Abnormalities in a Murine Model of Citrin Deficiency.

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.

A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates.

The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option. However, there are concerns whether an acceptable tolerability profile for chronic dosing can be achieved. The efficiency and tolerability of lipid nanoparticles has been attributed to the amino lipid. Therefore, we developed a new series of amino lipids that address this concern. Clear structure-activity relationships were developed that resulted in a new amino lipid that affords efficient mRNA delivery in rodent and primate models with optimal pharmacokinetics. A 1-month toxicology evaluation in rat and non-human primate demonstrated no adverse events with the new lipid nanoparticle system. Mechanistic studies demonstrate that the improved efficiency can be attributed to increased endosomal escape. This effort has resulted in the first example of the ability to safely repeat dose mRNA-containing lipid nanoparticles in non-human primate at therapeutically relevant levels.

Macrocyclic MEK1/2 inhibitor with efficacy in a mouse model of cardiomyopathy caused by lamin A/C gene mutation.

Signaling mediated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) is involved in numerous cellular processes. Mitogen-activated protein kinase kinases (MEK1/2) catalyze the phosphorylation of ERK1/2, converting it into an active kinase that regulates the expression of numerous genes and cellular processes. Inhibitors of MEK1/2 have demonstrated preclinical and clinical efficacy in certain cancers and types of cardiomyopathy. We report the synthesis of a novel, allosteric, macrocyclic MEK1/2 inhibitor that potently inhibits ERK1/2 activity in cultured cells and tissues of mice after systemic administration. Mice with dilated cardiomyopathy caused by a lamin A/C gene mutation have abnormally increased cardiac ERK1/2 activity. In these mice, this novel MEK1/2 inhibitor is well tolerated, improves left ventricular systolic function, decreases left ventricular fibrosis, has beneficial effects on skeletal muscle structure and pathology and prolongs survival. The novel MEK1/2 inhibitor described herein may therefore find clinical utility in the treatment of this rare cardiomyopathy, other types of cardiomyopathy and cancers in humans.

Systemic messenger RNA as an etiological treatment for acute intermittent porphyria.

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP.

Systemic Messenger RNA Therapy as a Treatment for Methylmalonic Acidemia.

Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs). Intravenous (i.v.) administration of hMUT mRNA in two different mouse models of MMA resulted in a 75%-85% reduction in plasma methylmalonic acid and was associated with increased hMUT protein expression and activity in liver. Repeat dosing of hMUT mRNA reduced circulating metabolites and dramatically improved survival and weight gain. Additionally, repeat i.v. dosing did not increase markers of liver toxicity or inflammation in heterozygote MMA mice.