ABSTRACT
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. Both drug products produced functional MMUT in rat livers when dosed IV, and showed long-term safety and efficacy in two mouse models of MMA. mRNA-3705 produced 2.1-3.4-fold higher levels of hepatic MMUT protein expression than the first-generation drug product mRNA-3704 when given at an identical dose level, which resulted in greater and more sustained reductions in plasma methylmalonic acid. The data presented herein provide comprehensive preclinical pharmacology to support the clinical development of mRNA-3705.
ABSTRACT
Argininosuccinate lyase (ASL) is integral to the urea cycle detoxifying neurotoxic ammonia and the nitric oxide (NO) biosynthesis cycle. Inherited ASL deficiency causes argininosuccinic aciduria (ASA), a rare disease with hyperammonemia and NO deficiency. Patients present with developmental delay, epilepsy and movement disorder, associated with NO-mediated downregulation of central catecholamine biosynthesis. A neurodegenerative phenotype has been proposed in ASA. To better characterise this neurodegenerative phenotype in ASA, we conducted a retrospective study in six paediatric and adult metabolic centres in the UK in 2022. We identified 60 patients and specifically looked for neurodegeneration-related symptoms: movement disorder such as ataxia, tremor and dystonia, hypotonia/fatigue and abnormal behaviour. We analysed neuroimaging with diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) in an individual with ASA with movement disorders. We assessed conventional and DTI MRI alongside single photon emission computer tomography (SPECT) with dopamine analogue radionuclide 123 I-ioflupane, in Asl-deficient mice treated by hASL mRNA with normalised ureagenesis. Movement disorders in ASA appear in the second and third decades of life, becoming more prevalent with ageing and independent from the age of onset of hyperammonemia. Neuroimaging can show abnormal DTI features affecting both grey and white matter, preferentially basal ganglia. ASA mouse model with normalised ureagenesis did not recapitulate these DTI findings and showed normal 123 I-ioflupane SPECT and cerebral dopamine metabolomics. Altogether these findings support the pathophysiology of a late-onset movement disorder with cell-autonomous functional central catecholamine dysregulation but without or limited neurodegeneration of dopaminergic neurons, making these symptoms amenable to targeted therapy.
ABSTRACT
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.
Subject(s)
Fabry Disease/genetics , Fabry Disease/therapy , RNA, Messenger/therapeutic use , alpha-Galactosidase/genetics , Animals , Disease Models, Animal , Endocytosis , Enzyme Replacement Therapy , Genetic Therapy , Humans , Lipids/chemistry , Lysosomes/metabolism , Macaca fascicularis , Male , Mice , Mice, Knockout , RNA, Messenger/pharmacokinetics , Tissue Distribution , Trihexosylceramides/metabolismABSTRACT
BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation. METHODS: We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4-/- mice. RESULTS: We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4-/- mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure. CONCLUSIONS: Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3. LAY SUMMARY: This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/deficiency , Cholestasis, Intrahepatic/drug therapy , Cholestasis, Intrahepatic/genetics , Gene Deletion , Liposomes/chemistry , Nanoparticle Drug Delivery System/chemistry , Nanoparticles/chemistry , Phenotype , RNA, Messenger/administration & dosage , ATP Binding Cassette Transporter, Subfamily B/administration & dosage , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Animals , Cholestasis, Intrahepatic/metabolism , Disease Models, Animal , HEK293 Cells , Homozygote , Humans , Liver/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , RNA, Messenger/genetics , Transfection , Treatment Outcome , ATP-Binding Cassette Sub-Family B Member 4ABSTRACT
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.
Subject(s)
Citrullinemia/drug therapy , Citrullinemia/metabolism , Drug Delivery Systems/methods , Genetic Therapy/methods , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , RNA, Messenger/therapeutic use , Animals , Behavior, Animal/drug effects , Disease Models, Animal , Gene Knockout Techniques , Glucosephosphate Dehydrogenase/genetics , HeLa Cells , Hep G2 Cells , Humans , Lipids/chemistry , Loss of Function Mutation , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/metabolism , Nanoparticles/chemistry , Open Reading Frames/genetics , RNA, Messenger/chemical synthesis , RNA, Messenger/chemistry , RNA, Messenger/genetics , Transfection , Treatment OutcomeABSTRACT
PURPOSE: To evaluate the effect of cervical margin relocation (CMR) for crowns designed using CAD/CAM technology, and made of pre-cured resin or lithium disilicate, before and after thermomechanical loading. The test hypothesis was that the marginal quality of the crowns would not be influenced by the CMR with composite resins before or after thermomechanical loading. MATERIALS AND METHODS: Standard crown preparations were created in 40 human molars. The margins were located in enamel, except for the mesial proximal box, where the cervical margin was 2.0 mm below the cementoenamel junction, with 4.0 mm in the buccolingual and 2.0 mm in the mesiodistal dimension. For the CMR technique, a 2-mm layer of conventional or flowable composite resin was applied to the mesial box. Using the Cerec CAD/CAM system, 40 standard crowns were prepared, and restorations were luted using a dual-curing adhesive cement. SEM analysis was performed using epoxy resin replicas before and after loading to assess the marginal quality of interfaces of the mesial proximal box with CMR/crown and the distal face of the tooth without CMR. Statistical differences between groups were analyzed using the Kruskal-Wallis test and Bonferroni's post-hoc test. RESULTS: The null hypothesis was accepted, since no statistically significant differences were found in marginal quality before and after thermomechanical cycling (p > 0.05). CONCLUSION: The implementation of CMR before and after thermomechanical cycling had no effect on the quality of cervical margins. To establish whether CMR is a suitable procedure for the adhesive luting of composite resin crowns in deep proximal boxes, additional studies are required.
Subject(s)
Composite Resins/chemistry , Computer-Aided Design , Crowns , Dental Marginal Adaptation , Dental Materials/chemistry , Tooth Preparation, Prosthodontic/methods , Dental Enamel/anatomy & histology , Dental Porcelain/chemistry , Dental Prosthesis Design , Humans , In Vitro Techniques , Materials Testing , Microscopy, Electron, Scanning , Nanocomposites/chemistry , Nanoparticles/chemistry , Replica Techniques , Resin Cements/chemistry , Self-Curing of Dental Resins/methods , Silicon Dioxide/chemistry , Stress, Mechanical , Temperature , Time Factors , Tooth Cervix/anatomy & histology , Water/chemistryABSTRACT
Purpose: Lipid nanoparticles (LNPs) show promise in their ability to introduce mRNA to drive protein expression in specific cell types of the mammalian eye. Here, we examined the ability of mRNA encapsulated in LNPs with two distinct formulations to drive gene expression in mouse and human retina and other ocular tissues. Methods: We introduced mRNA-carrying LNPs into two biological systems. Intravitreal injections were tested to deliver LNPs into the mouse eye. Human retinal pigment epithelium (RPE) and retinal explants were used to assess mRNA expression in human tissue. We analyzed specificity of expression using histology, immunofluorescence, and imaging. Results: In mice, mRNAs encoding GFP and ciliary neurotrophic factor (CNTF) were specifically expressed by Müller glia and RPE. Acute inflammatory changes measured by microglia distribution (Iba-1) or interleukin-6 (IL-6) expression were not observed 6 hours post-injection. Human RPE also expressed high levels of GFP. Human retinal explants expressed GFP in cells with apical and basal processes consistent with Müller glia and in perivascular cells consistent with macrophages. Conclusions: We demonstrated the ability to reliably transfect subpopulations of retinal cells in mouse eye tissues in vivo and in human ocular tissues. Of significance, intravitreal injections were sufficient to transfect the RPE in mice. To our knowledge, we demonstrate delivery of mRNA using LNPs in human ocular tissues for the first time. Translational Relevance: Ocular gene-replacement therapies using non-viral vector methods are a promising alternative to adeno-associated virus (AAV) vectors. Our studies show that mRNA LNP delivery can be used to transfect retinal cells in both mouse and human tissues without inducing significant inflammation. This methodology could be used to transfect retinal cell lines, tissue explants, mice, or potentially as gene-replacement therapy in a clinical setting in the future.
Subject(s)
Intravitreal Injections , Nanoparticles , RNA, Messenger , Retinal Pigment Epithelium , Animals , Humans , RNA, Messenger/administration & dosage , RNA, Messenger/metabolism , Mice , Retinal Pigment Epithelium/metabolism , Nanoparticles/chemistry , Mice, Inbred C57BL , Ciliary Neurotrophic Factor/genetics , Ciliary Neurotrophic Factor/metabolism , Ciliary Neurotrophic Factor/administration & dosage , Retina/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Ependymoglial Cells/metabolism , Gene Transfer Techniques , LiposomesABSTRACT
Messenger RNA (mRNA) therapeutics delivered via lipid nanoparticles hold the potential to treat metabolic diseases caused by protein deficiency, including propionic acidemia (PA), methylmalonic acidemia (MMA), and phenylketonuria (PKU). Herein we report results from multiple independent preclinical studies of mRNA-3927 (an investigational treatment for PA), mRNA-3705 (an investigational treatment for MMA), and mRNA-3210 (an investigational treatment for PKU) in murine models of each disease. All 3 mRNA therapeutics exhibited pharmacokinetic/pharmacodynamic (PK/PD) responses in their respective murine model by driving mRNA, protein, and/or protein activity responses, as well as by decreasing levels of the relevant biomarker(s) when compared to control-treated animals. These preclinical data were then used to develop translational PK/PD models, which were scaled allometrically to humans to predict starting doses for first-in-human clinical studies for each disease. The predicted first-in-human doses for mRNA-3927, mRNA-3705, and mRNA-3210 were determined to be 0.3, 0.1, and 0.4 mg/kg, respectively.
Subject(s)
Amino Acid Metabolism, Inborn Errors , Disease Models, Animal , Phenylketonurias , Propionic Acidemia , RNA, Messenger , Propionic Acidemia/genetics , Propionic Acidemia/therapy , Propionic Acidemia/drug therapy , Animals , Phenylketonurias/genetics , Phenylketonurias/drug therapy , Phenylketonurias/therapy , RNA, Messenger/genetics , RNA, Messenger/metabolism , Amino Acid Metabolism, Inborn Errors/genetics , Amino Acid Metabolism, Inborn Errors/therapy , Amino Acid Metabolism, Inborn Errors/drug therapy , Mice , Humans , Male , Female , Nanoparticles/chemistry , Mice, Inbred C57BL , LiposomesABSTRACT
The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [18F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.
Subject(s)
Argininosuccinic Aciduria , Liver Diseases , Adult , Humans , Animals , Mice , Argininosuccinic Aciduria/genetics , Argininosuccinic Aciduria/therapy , Cysteine , Glutathione , MetabolomicsABSTRACT
The aim of this study was to evaluate microtensile bond strength (µTBS) to dentine, interfacial nanoleakage expression, and stability after ageing, of two-step vs. one-step self-etch adhesives. Human molars were cut to expose middle/deep dentine, assigned to groups (n = 15), and treated with the following bonding systems: (i) Optibond XTR (a two-step self-etch adhesive; Kerr), (ii) Clearfil SE Bond (a two-step self-etch adhesive; Kuraray), (iii) Adper Easy Bond (a one-step self-etch adhesive; 3M ESPE), and (iv) Bond Force (a one-step self-etch adhesive; Tokuyama). Specimens were processed for µTBS testing after 24 h, 6 months, or 1 yr of storage in artificial saliva at 37°C. Nanoleakage expression was examined in similarly processed additional specimens. At baseline the µTBS results ranked in the following order: Adper Easy Bond = Optibond XTR ≥Clearfil SE = Bond Force, and interfacial nanoleakage analysis showed Clearfil SE Bond = Adper Easy Bond = Optibond XTR> Bond Force. After 1 yr of storage, Optibond XTR, Clearfil SE Bond, and Adper Easy Bond showed higher µTBS and lower interfacial nanoleakage expression compared with Bond Force. In conclusion, immediate bond strength, nanoleakage expression, and stability over time were not related to the number of steps of the bonding systems, but to their chemical formulations.
Subject(s)
Acid Etching, Dental/methods , Adhesives/chemistry , Dentin-Bonding Agents/chemistry , Dentin/chemistry , Resin Cements/chemistry , Analysis of Variance , Dental Leakage , Humans , Microscopy, Electron, Scanning , Molar, Third , Silver/chemistry , Statistics, Nonparametric , Tensile Strength , Time FactorsABSTRACT
Purpose: Lipid nanoparticles (LNPs) show promise in their ability to introduce mRNA to drive protein expression in specific cell types of the mammalian eye. Here, we examined the ability of mRNA encapsulated in lipid nanoparticles (LNPs) with two distinct formulations to drive gene expression in mouse and human retina and other ocular tissues. Methods: We introduced mRNA carrying LNPs into two biological systems. Intravitreal injections were tested to deliver LNPs into the mouse eye. Human retinal pigment epithelium (RPE) and retinal explants were used to assess mRNA expression in human tissue. We analyzed specificity of expression using histology, immunofluorescence, and imaging. Results: In mice, mRNAs encoding GFP and ciliary neurotrophic factor (CNTF) were specifically expressed by Müller glia and retinal pigment epithelium (RPE). Acute inflammatory changes measured by microglia distribution (Iba-1) or interleukin-6 (IL-6) expression were not observed 6 hours post-injection. Human RPE also expressed high levels of GFP. Human retinal explants expressed GFP in cells with apical and basal processes consistent with Müller glia and in perivascular cells consistent with macrophages. Conclusions: We demonstrated the ability to reliably transfect subpopulations of retinal cells in mice eye tissues in vivo and in human ocular tissues. Of significance, intravitreal injections were sufficient to transfect the RPE in mice. To our knowledge we demonstrate delivery of mRNA using LNPs in human ocular tissues for the first time.
ABSTRACT
Background: In academic research and the pharmaceutical industry, in vitro cell lines and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have intrinsic limitations, whilst the use of precision-cut tissue slices can bridge the gap between these mainstream models. Precision-cut tissue slices combine the advantage of high reproducibility, studying all cell sub-types whilst preserving the tissue matrix and extracellular architecture, thereby closely mimicking a mini-organ. This approach can be used to replicate the biological phenotype of liver monogenic diseases using mouse models. Methods: Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers, enabling its use as a reliable ex-vivo model to assess the therapeutic screening of inherited metabolic diseases. Results: We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects such as citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively. Conclusions: Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases.
Subject(s)
Liver Diseases , Metabolic Diseases , Animals , Mice , Reproducibility of Results , Liver Diseases/genetics , Liver Diseases/therapy , PhenotypeABSTRACT
PURPOSE: To assess the effects of grade IV titanium ultrasonic tip instrumentation on different grade IV titanium implant surfaces and compare the decontamination of different implant surfaces using chlorhexidine, blue laser, or ozone. MATERIALS AND METHODS: Profilometry and energy-dispersive x-ray spectroscopy (EDS) analyses were performed on smooth, laser-micropatterned, and sandblasted grade IV titanium sample disks before (t0) and after (t1) ultrasonic instrumentation with an ultrasonic grade IV titanium tip. Samples were also incubated with a Streptococcus sanguinis culture. Each surface type was then treated with chlorhexidine, blue laser, or ozone (three test groups + control group). Scanning electron microscopy (SEM) images were taken after bacterial growth and after decontamination. RESULTS: After ultrasonic instrumentation, surface roughness (Ra) decreased on sandblasted and micropatterned surfaces, whereas it remained substantially unvaried on the smooth surface. SEM images revealed that the laser-micropatterned structure remained substantially unvaried after instrumentation. EDS revealed a minimal quantity of carbon and iron, found in the laser-treated and sandblasted group at t0. A minimal quantity of aluminum and oxygen was found on the sandblasted surface at t0 and t1. Ozone therapy achieved the highest decontaminating effect, regardless of implant surface topography. CONCLUSION: Among the alternative therapies to ultrasonic instrumentation with titanium tips, ozone appears to be effective regardless of the type of implant surface; it can be used for the decontamination treatment of implants without altering the surface structure.
Subject(s)
Dental Implants , Dental Scaling , Microscopy, Electron, Scanning , Spectrum Analysis , Surface Properties , Titanium , Ultrasonics , X-RaysABSTRACT
Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.
Subject(s)
Disease Models, Animal , Genetic Therapy/methods , Glucose-6-Phosphatase/genetics , Glycogen Storage Disease/therapy , RNA, Messenger/genetics , Animals , Cell Line, Tumor , Cytokines/blood , Cytokines/metabolism , Glucose-6-Phosphatase/metabolism , Glycogen/metabolism , Glycogen Storage Disease/genetics , Glycogen Storage Disease/pathology , HeLa Cells , Humans , Liver/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Nanoparticles/administration & dosage , Nanoparticles/chemistry , RNA, Messenger/administration & dosage , RNA, Messenger/chemistry , Treatment Outcome , Triglycerides/metabolismABSTRACT
Alpha 1-antitrypsin (AAT) deficiency arises from an inherited mutation in the SERPINA1 gene. The disease causes damage in the liver where the majority of the AAT protein is produced. Lack of functioning circulating AAT protein also causes uninhibited elastolytic activity in the lungs leading to AAT deficiency-related emphysema. The only therapy apart from liver transplantation is augmentation with human AAT protein pooled from sera, which is only reserved for patients with advanced lung disease caused by severe AAT deficiency. We tested modified mRNA encoding human AAT in primary human hepatocytes in culture, including hepatocytes from AAT deficient patients. Both expression and functional activity were investigated. Secreted AAT protein increased from 1,14 to 3,43 µg/ml in media from primary human hepatocytes following mRNA treatment as investigated by ELISA and western blot. The translated protein showed activity and protease inhibitory function as measured by elastase activity assay. Also, mRNA formulation in lipid nanoparticles was assessed for systemic delivery in both wild type mice and the NSG-PiZ transgenic mouse model of AAT deficiency. Systemic intravenous delivery of modified mRNA led to hepatic uptake and translation into a functioning protein in mice. These data support the use of systemic mRNA therapy as a potential treatment for AAT deficiency.
Subject(s)
RNA, Messenger/metabolism , alpha 1-Antitrypsin Deficiency/genetics , alpha 1-Antitrypsin Deficiency/therapy , Animals , Blotting, Western , Cells, Cultured , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Humans , Nanoparticles/chemistry , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/physiologyABSTRACT
Propionic acidemia/aciduria (PA) is an ultra-rare, life-threatening, inherited metabolic disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC) composed of six alpha (PCCA) and six beta (PCCB) subunits. We herein report an enzyme replacement approach to treat PA using a combination of two messenger RNAs (mRNAs) (dual mRNAs) encoding both human PCCA (hPCCA) and PCCB (hPCCB) encapsulated in biodegradable lipid nanoparticles (LNPs) to produce functional PCC enzyme in liver. In patient fibroblasts, dual mRNAs encoded proteins localize in mitochondria and produce higher PCC enzyme activity vs. single (PCCA or PCCB) mRNA alone. In a hypomorphic murine model of PA, dual mRNAs normalize ammonia similarly to carglumic acid, a drug approved in Europe for the treatment of hyperammonemia due to PA. Dual mRNAs additionally restore functional PCC enzyme in liver and thus reduce primary disease-associated toxins in a dose-dependent manner in long-term 3- and 6-month repeat-dose studies in PA mice. Dual mRNAs are well-tolerated in these studies with no adverse findings. These studies demonstrate the potential of mRNA technology to chronically administer multiple mRNAs to produce large complex enzymes, with applicability to other genetic disorders.
Subject(s)
Enzyme Replacement Therapy/methods , Propionic Acidemia/therapy , RNA, Messenger/therapeutic use , Animals , Disease Models, Animal , Glutamates/therapeutic use , Humans , Kinetics , Lipids/chemistry , Liver/enzymology , Methylmalonyl-CoA Decarboxylase/chemistry , Methylmalonyl-CoA Decarboxylase/genetics , Methylmalonyl-CoA Decarboxylase/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Mitochondria/enzymology , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Propionic Acidemia/genetics , Propionic Acidemia/metabolism , Protein Subunits/chemistry , Protein Subunits/genetics , RNA, Messenger/administration & dosage , RNA, Messenger/geneticsABSTRACT
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.
Subject(s)
Amino Acid Metabolism, Inborn Errors/therapy , Liver/metabolism , Methylmalonyl-CoA Mutase/pharmacology , RNA, Messenger/pharmacology , Amino Acid Metabolism, Inborn Errors/genetics , Amino Acid Metabolism, Inborn Errors/pathology , Animals , Child , Disease Models, Animal , Humans , Lipids/genetics , Liver/drug effects , Liver/pathology , Methylmalonyl-CoA Mutase/genetics , Mice , RNA, Messenger/geneticsABSTRACT
Selective inhibitors of biogenic amine (e.g., serotonin, norepinephrine, and dopamine) uptake exhibit varying degrees of safety and efficacy as antiobesity agents. Moreover, preclinical findings suggest that the combined inhibition of monoamine neurotransmitter transporters synergistically enhances antiobesity activity. (1R,5S)-(+)-1-(3,4-Dichlorophenyl)-3-azabicyclo-[3.1.0] hexane hydrochloride (DOV 21947) inhibits norepinephrine, 5-hydroxytryptamine, and dopamine uptake, and it reduces body weight in rodent models of diet-induced obesity (DIO). DIO rats treated orally with DOV 21947 for 1 to 24 days showed significantly lower body weights than vehicle-treated DIO rats. The decrease in body weight resulted specifically from a loss of retroperitoneal and mesenteric depots of white adipose tissue. DOV 21947 also reduced daily food intake in DIO rats, but consumption returned to control levels after 11 days of treatment. With the exception of a decrease in triglyceride levels, blood chemistry was unaltered after 24 days of DOV 21947 treatments. DOV 21947 had no effect on motor activity. Although DOV 21947 increased respiratory rate and decreased the tidal volume of normal rats, it did not alter the minute volume. In addition, DOV 21947 did not significantly affect blood pressure, heart rate, electrocardiographic indices or body temperature in telemeterized dogs. However, it caused a sustained, but reversible reduction in the rate of body weight gain for as long as 6 months in normal rats, and for up to 1 year in normal dogs. In summary, DOV 21947 is effective in causing a sustained and selective reduction in fat content and triglyceride levels in animal models of obesity without significantly altering vital organ function.
Subject(s)
Aza Compounds/therapeutic use , Body Weight/drug effects , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Dietary Fats/blood , Disease Models, Animal , Obesity/drug therapy , Triglycerides/blood , Animals , Anti-Obesity Agents/pharmacology , Anti-Obesity Agents/therapeutic use , Aza Compounds/pharmacology , Body Weight/physiology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cardiovascular System/drug effects , Dietary Fats/adverse effects , Dogs , Dose-Response Relationship, Drug , Female , Male , Mice , Mice, Inbred C57BL , Neurotransmitter Uptake Inhibitors/pharmacology , Neurotransmitter Uptake Inhibitors/therapeutic use , Obesity/blood , Rats , Rats, Sprague-Dawley , Weight Gain/drug effects , Weight Gain/physiologyABSTRACT
We investigated the feeding behavior of mice carrying a non-functional 5-hydroxytryptamine-6 receptor (5-HT6). Homozygous mutant mice on C57BL/6 background were grossly normal and showed normal growth when fed a low-fat chow diet. When fed a high-fat diet, the mutant mice consumed approximately 8% less food while gaining approximately 35% less weight over an 11-week study period than did the wild-type controls. Body composition analysis of mice on high-fat feeding showed that the reduced weight gain in the mutant mice was mostly due to reduced fat accumulation. Given the documented role of the serotonin systems in human feeding, our results provide an interesting piece of evidence supporting the development of 5-HT6 receptor antagonists for treating obesity.
Subject(s)
Dietary Fats/administration & dosage , Mutation/genetics , Obesity/physiopathology , Receptors, Serotonin/genetics , Animals , Body Composition/genetics , Body Weight/genetics , Eating/physiology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Obesity/etiology , Obesity/genetics , Weight Gain/geneticsABSTRACT
LH-21 (5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole) was previously reported as a neutral antagonist at the cannabinoid CB1 receptor which, despite its reported poor ability to penetrate into the brain, suppressed food intake and body weight in rats by intraperitoneal administration. In the present study, we studied the mechanism of action of LH-21 by characterizing its in vitro pharmacological properties and in vivo efficacy. LH-21 inhibited the binding of [3H]CP55940 to cloned human and rat CB1 receptors with IC50 values of 631+/-98 nM, and 690+/-41 nM, respectively, and acted as an inverse agonist in a cAMP functional assay using cultured cells expressing human, rat or mouse CB1 receptor. The compound was shown to be brain-penetrant in rats by intravenous administration. Importantly, a single dose of LH-21 (60 mg/kg, i.p.) caused a similar suppression of overnight food intake and body weight gain in wild-type and CB1 receptor knockout mice. Our results suggest that LH-21 is a low affinity inverse agonist for the CB1 receptor and does not act on the CB1 receptor to inhibit food intake in mice.