Improved therapeutic efficacy in two mouse models of methylmalonic acidemia (MMA) using a second-generation mRNA therapy.

Isolated methylmalonic acidemia/aciduria (MMA) due to MMUT enzyme deficiency is an ultra-rare pediatric disease with high morbidity and mortality, with no approved disease-altering therapies. Previous publications showed that systemic treatment with a codon-optimized mRNA encoding wild-type human MMUT (MMUT) is a promising strategy for treatment of MMA. We developed a second-generation drug product, mRNA-3705, comprised of an mRNA encoding the MMUT enzyme formulated in a lipid nanoparticle (LNP) with incorporation of enhancements over the previous clinical candidate mRNA-3704.

Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells.

Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy.

Author Correction: Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA.

Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to -OH group is required to maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure.

Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines.

mRNA vaccines have the potential to tackle many unmet medical needs that are unable to be addressed with conventional vaccine technologies. A potent and well-tolerated delivery technology is integral to fully realizing the potential of mRNA vaccines. Pre-clinical and clinical studies have demonstrated that mRNA delivered intramuscularly (IM) with first-generation lipid nanoparticles (LNPs) generates robust immune responses.

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.

Endoglin Mediates Vascular Maturation by Promoting Vascular Smooth Muscle Cell Migration and Spreading.

Objective: Endoglin, a transforming growth factor-β superfamily coreceptor, is predominantly expressed in endothelial cells and has essential roles in vascular development. However, whether endoglin is also expressed in vascular smooth muscle cells (VSMCs), especially in vivo, remains controversial. Furthermore, the roles of endoglin in VSMC biology remain largely unknown.

Shed urinary ALCAM is an independent prognostic biomarker of three-year overall survival after cystectomy in patients with bladder cancer.

Proteins involved in tumor cell migration can potentially serve as markers of invasive disease. Activated Leukocyte Cell Adhesion Molecule (ALCAM) promotes adhesion, while shedding of its extracellular domain is associated with migration. We hypothesized that shed ALCAM in biofluids could be predictive of progressive disease.

Urinary oncofetal ED-A fibronectin correlates with poor prognosis in patients with bladder cancer.

The extracellular matrix protein fibronectin (FN) contributes to the structural integrity of tissues as well as the adhesive and migratory functions of cells. While FN is abundantly expressed in adult tissues, the expression of several alternatively spliced FN isoforms is restricted to embryonic development, tissue remodeling and cancer. These FN isoforms, designated ED-A and ED-B, are frequently expressed by cancer cells, tumor-associated fibroblasts and newly forming blood vessels.

Directional cell movement through tissues is controlled by exosome secretion.

Directional cell movement through tissues is critical for multiple biological processes and requires maintenance of polarity in the face of complex environmental cues. Here we use intravital imaging to demonstrate that secretion of exosomes from late endosomes is required for directionally persistent and efficient in vivo movement of cancer cells. Inhibiting exosome secretion or biogenesis leads to defective tumour cell migration associated with increased formation of unstable protrusions and excessive directional switching.

ALCAM/CD166 is a TGF-β-responsive marker and functional regulator of prostate cancer metastasis to bone.

The dissemination of prostate cancer to bone is a common, incurable aspect of advanced disease. Prevention and treatment of this terminal phase of prostate cancer requires improved molecular understanding of the process as well as markers indicative of molecular progression. Through biochemical analyses and loss-of-function in vivo studies, we demonstrate that the cell adhesion molecule, activated leukocyte cell adhesion molecule (ALCAM), is actively shed from metastatic prostate cancer cells by the sheddase ADAM17 in response to TGF-β.

The Purkinje neuron acts as a central regulator of spatially and functionally distinct cerebellar precursors.

The prospective white matter (PWM) in the nascent cerebellum contains a transient germinal compartment that produces all postnatally born GABAergic inhibitory interneurons and astrocytes. However, little is known about the molecular identity and developmental potential of resident progenitors or key regulatory niche signals. Here, we show that neural stem-cell-like primary progenitors (Tnc(YFP-low) CD133(+)) generate intermediate astrocyte (Tnc(YFP-low) CD15(+)) precursors and GABAergic transient amplifying (Ptf1a(+)) cells.

Integrin-free tetraspanin CD151 can inhibit tumor cell motility upon clustering and is a clinical indicator of prostate cancer progression.

Normal physiology relies on the organization of transmembrane proteins by molecular scaffolds, such as tetraspanins. Oncogenesis frequently involves changes in their organization or expression. The tetraspanin CD151 is thought to contribute to cancer progression through direct interaction with the laminin-binding integrins α3β1 and α6β1.

Widespread contribution of Gdf7 lineage to cerebellar cell types and implications for hedgehog-driven medulloblastoma formation.

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma.

Intracranial orthotopic allografting of medulloblastoma cells in immunocompromised mice.

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered.

Isolation, enrichment, and maintenance of medulloblastoma stem cells.

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.

Transventricular delivery of Sonic hedgehog is essential to cerebellar ventricular zone development.

Cerebellar neurons are generated from two germinal neuroepithelia: the ventricular zone (VZ) and rhombic lip. Signaling mechanisms that maintain the proliferative capacity of VZ resident progenitors remain elusive. We reveal that Sonic hedgehog (Shh) signaling is active in the cerebellar VZ and essential to radial glial cell proliferation and expansion of GABAergic interneurons.

Sonic hedgehog signaling regulates a novel epithelial progenitor domain of the hindbrain choroid plexus.

Choroid plexuses (ChPs) are vascularized secretory organs involved in the regulation of brain homeostasis, and function as the blood-cerebrospinal fluid (CSF) barrier. Despite their crucial roles, there is limited understanding of the regulatory mechanism driving ChP development. Sonic hedgehog (Shh), a secreted signal crucial for embryonic development and cancer, is strongly expressed in the differentiated hindbrain ChP epithelium (hChPe).

Gli3-deficient mice exhibit cleft palate associated with abnormal tongue development.

Palatogenesis depends on appropriate growth, elevation, and fusion of the palatal shelves and aberration in these processes can lead to palatal clefting. We observed a high incidence of palate clefting in mice deficient in Gli3, known for its role as a repressor in the absence of Shh signaling. In contrast with several current mouse models of cleft palate, Meckel's cartilage extension, cranial neural crest migration, palatal shelf proliferation, apoptosis, and key signaling components mediated by Shh, Bmp, Fgf, and Tgfbeta, appeared unaffected in Gli3-/- mice.