Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency.

Arginase deficiency is caused by biallelic mutations in arginase 1 (ARG1), the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of guanidino compounds, while it is clinically notable for developmental delays, spastic diplegia, psychomotor function loss, and (uncommonly) death. There is currently no completely effective medical treatment available. While preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division. Recent advances in messenger RNA (mRNA) codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of safer, albeit temporary, treatments to restore liver metabolic function in patients with urea cycle disorders, including ARG1 deficiency. In this study, we applied such technologies to successfully treat an ARG1-deficient murine model. Mice were administered LNPs encapsulating human codon-optimized ARG1 mRNA every 3 d. Mice demonstrated 100% survival with no signs of hyperammonemia or weight loss to beyond 11 wk, compared with controls that perished by day 22. Plasma ammonia, arginine, and glutamine demonstrated good control without elevation of guanidinoacetic acid, a guanidino compound. Evidence of urea cycle activity restoration was demonstrated by the ability to fully metabolize an ammonium challenge and by achieving near-normal ureagenesis; liver arginase activity achieved 54% of wild type. Biochemical and microscopic data showed no evidence of hepatotoxicity. These results suggest that delivery of ARG1 mRNA by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency.

An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

mRNA vaccine trafficking and resulting protein expression after intramuscular administration.

The mRNA vaccine route from injection site to critical immunologic tissues, as well as the localization of protein antigen following intramuscular (i.m.) administration, is crucial to generating an effective immune response. Here, we quantified mRNA at the injection site, lymph nodes, and in select tissues. mRNA was primarily present 24 h after administration and then rapidly degraded from local and systemic tissues. Histological analyses of mRNA and expressed protein at the site of administration and in the lymph nodes following i.m. administration of our vaccine in rodents and nonhuman primates (NHPs) were completed, and mRNA and protein expression were detected in tissue resident and infiltrating immune cells at the injection site. In addition, high levels of protein expression were observed within subcapsular and medullary sinus macrophages in draining lymph nodes. More important, results were similar between rodents and NHPs, indicating cross-species similarities.

Intermittent lipid nanoparticle mRNA administration prevents cortical dysmyelination associated with arginase deficiency.

Arginase deficiency is associated with prominent neuromotor features, including spastic diplegia, clonus, and hyperreflexia; intellectual disability and progressive neurological decline are other signs. In a constitutive murine model, we recently described leukodystrophy as a significant component of the central nervous system features of arginase deficiency. In the present studies, we sought to examine if the administration of a lipid nanoparticle carrying human ARG1 mRNA to constitutive knockout mice could prevent abnormalities in myelination associated with arginase deficiency. Imaging of the cingulum, striatum, and cervical segments of the corticospinal tract revealed a drastic reduction of myelinated axons; signs of degenerating axons were also present with thin myelin layers. Lipid nanoparticle/ARG1 mRNA administration resulted in both light and electron microscopic evidence of a dramatic recovery of myelin density compared with age-matched controls; oligodendrocytes were seen to be extending processes to wrap many axons. Abnormally thin myelin layers, when myelination was present, were resolved with intermittent mRNA administration, indicative of not only a greater density of myelinated axons but also an increase in the thickness of the myelin sheath. In conclusion, lipid nanoparticle/ARG1 mRNA administration in arginase deficiency prevents the associated leukodystrophy and restores normal oligodendrocyte function.

MicroRNAs Enable mRNA Therapeutics to Selectively Program Cancer Cells to Self-Destruct.

The advent of therapeutic mRNAs significantly increases the possibilities of protein-based biologics beyond those that can be synthesized by recombinant technologies (eg, monoclonal antibodies, extracellular enzymes, and cytokines). In addition to their application in the areas of vaccine development, immune-oncology, and protein replacement therapies, one exciting possibility is to use therapeutic mRNAs to program undesired, diseased cells to synthesize a toxic intracellular protein, causing cells to self-destruct. For this approach to work, however, methods are needed to limit toxic protein expression to the intended cell type. Here, we show that inclusion of microRNA target sites in therapeutic mRNAs encoding apoptotic proteins, Caspase or PUMA, can prevent their expression in healthy hepatocytes while triggering apoptosis in hepatocellular carcinoma cells.

Pharmacological characterization of guinea pig chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2).

Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a G protein-coupled receptor activated by prostaglandin D(2) (PGD(2)), has been identified as a receptor expressed on cell types critical to the pathogenesis of asthma. The cDNA encoding guinea pig CRTH2 was cloned and mRNA expression examined in selected tissues. Transcript profiling of guinea pig CRTH2 indicated relatively high levels of expression in bone marrow, intermediate levels in brain and relatively low levels in lung, spleen, thymus, lymph node, etc. Characterization of the molecular pharmacology of guinea pig CRTH2 revealed that guinea pig CRTH2 exhibited a greater affinity for Delta(12)-PGJ(2), a stable PGD(2) metabolite relative to human CRTH2. The CRTH2 selective agonists 13,14-dihydro-15-keto PGD(2) and Delta(12)-PGJ(2) induced the recruitment of eosinophils following intradermal administration of these ligands in guinea pigs. Chemotaxis of guinea pig eosinophils was elicited by either PGD(2) or Delta(12)-PGJ(2), and was abolished by a CRTH2-specific antagonist. These results indicate that PGD(2) and the stable metabolite, Delta(12)-PGJ(2), play important roles in CRTH2 activation in the guinea pig.