Reversion of metabolic dysfunction-associated steatohepatitis by skeletal muscle-directed FGF21 gene therapy.

The highly prevalent metabolic dysfunction-associated steatohepatitis (MASH) is associated with liver steatosis, inflammation and hepatocyte injury that can lead to fibrosis and may progress to hepatocellular carcinoma and death. New treatment modalities such as gene therapy may be transformative for MASH patients. Here, we describe that one-time intramuscular administration of adeno-associated viral vectors of serotype 1 (AAV1) encoding native fibroblast growth factor 21 (FGF21), a key metabolic regulator, resulted in sustained increased circulating levels of the factor, which mediated long-term (>1 year) MASH and hepatic fibrosis reversion and halted development of liver tumors in obese male and female mouse models. AAV1-FGF21 treatment also counteracted obesity, adiposity, and insulin resistance, which are significant drivers of MASH. Scale-up to large animals successfully resulted in safe skeletal muscle biodistribution and biological activity in key metabolic tissues. Moreover, as a step towards the clinic, circulating FGF21 levels were characterized in obese, insulin resistant and MASH patients. Overall, these results underscore the potential of the muscle-directed AAV1-FGF21 gene therapy to treat MASH and support its clinical translation.

A Comprehensive Study on the Effect of Plasticizers on the Characteristics of Polymer Inclusion Membranes (PIMs): Exploring Butyl Stearate as a Promising Alternative.

This study investigated the influence of various plasticizers commonly used in the manufacture of polymer inclusion membranes (PIMs), such as 2-nitrophenyl octyl ether (NPOE), phthalates, adipates, and sebacates on the mechanical, thermal, and transport properties of membranes. Additionally, butyl stearate (BTS), chosen for its non-toxic nature compared to phthalates and its cost-effectiveness relative to adipates and sebacates, was evaluated as a plasticizer in PIMs for the first time. All plasticizers were incorporated in PIMs made of either cellulose triacetate (CTA) or poly(vinyl chloride) (PVC) as the base polymers and the task-specific ionic liquid trioctylmethylammonium thiosalicylate (TOMATS) as the carrier. The plasticizers were found to significantly affect the characteristics of membrane hydrophilicity, mechanical flexibility, and thermal stability. Transport experiments using Hg(II) as a model target ion revealed that, for CTA-based PIMs, the plasticizer did not significantly affect transport efficiency. However, for PVC-based PIMs, BTS exhibited better efficiency when compared to NPOE. These findings highlight the potential of BTS as an attractive alternative to currently used plasticizers in PVC-based PIM formulations.

Seven-year follow-up of durability and safety of AAV CNS gene therapy for a lysosomal storage disorder in a large animal.

Delivery of adeno-associated viral vectors (AAVs) to cerebrospinal fluid (CSF) has emerged as a promising approach to achieve widespread transduction of the central nervous system (CNS) and peripheral nervous system (PNS), with direct applicability to the treatment of a wide range of neurological diseases, particularly lysosomal storage diseases. Although studies in small animal models have provided proof of concept and experiments in large animals demonstrated feasibility in bigger brains, there is not much information on long-term safety or durability of the effect. Here, we report a 7-year study in healthy beagle dogs after intra-CSF delivery of a single, clinically relevant dose (2 × 1013 vg/dog) of AAV9 vectors carrying the canine sulfamidase, the enzyme deficient in mucopolysaccharidosis type IIIA. Periodic monitoring of CSF and blood, clinical and neurological evaluations, and magnetic resonance and ultrasound imaging of target organs demonstrated no toxicity related to treatment. AAV9-mediated gene transfer resulted in detection of sulfamidase activity in CSF throughout the study. Analysis at tissue level showed widespread sulfamidase expression and activity in the absence of histological findings in any region of encephalon, spinal cord, or dorsal root ganglia. Altogether, these results provide proof of durability of expression and long-term safety for intra-CSF delivery of AAV-based gene transfer vectors encoding therapeutic proteins to the CNS.

Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy.

Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.

Vitamin D Receptor Overexpression in ß-Cells Ameliorates Diabetes in Mice.

Vitamin D deficiency has been associated with increased incidence of diabetes, both in humans and in animal models. In addition, an association between vitamin D receptor (VDR) gene polymorphisms and diabetes has also been described. However, the involvement of VDR in the development of diabetes, specifically in pancreatic ß-cells, has not been elucidated yet. Here, we aimed to study the role of VDR in ß-cells in the pathophysiology of diabetes. Our results indicate that Vdr expression was modulated by glucose in healthy islets and decreased in islets from both type 1 diabetes and type 2 diabetes mouse models. In addition, transgenic mice overexpressing VDR in ß-cells were protected against streptozotocin-induced diabetes and presented a preserved ß-cell mass and a reduction in islet inflammation. Altogether, these results suggest that sustained VDR levels in ß-cells may preserve ß-cell mass and ß-cell function and protect against diabetes.

Development of a new binding phase for the diffusive gradients in thin films technique based on an ionic liquid for mercury determination.

Determining bioavailable trace concentrations of mercury (Hg) in water is still a challenging analytical task. In this study, we report a methodology for determining labile Hg in natural waters using newly developed sorbents. Silicon dioxide at a nanoparticle range (Si-np) and cellulose powder at a microparticle range (Cel-p), both modified with the ionic liquid trioctylmethylammonium thiosalicylate (TOMATS), have been tested as sorbents (sorb-TOMATS) for Hg(II) uptake from solution. These novel sorb-TOMATS materials were characterized, and parameters affecting the uptake were examined. A similar Hg(II) uptake efficiency (97%) and binding capacity (9 mg Hg/g) was obtained for both sorb-TOMATS, while only a 25% of Hg(II) was taken up using non-impregnated materials. Moreover, these sorb-TOMATS were effectively embedded in agarose gel and were tested as a novel binding phase for the Diffusive Gradients in Thin Films (DGT) technique. Research revealed Si(np)-TOMATS sorbent as a suitable binding phase in the DGT technique for Hg(II) measurements, since it also allowed the efficient elution of the bound Hg(II). This new binding phase showed strong linear correlation between the accumulated Hg(II) mass and deployment time, which is in agreement with the DGT principle. In summary, this novel sorbent has a great potential to improve Hg monitoring in natural waters when integrated it in the DGT design.

System for mercury preconcentration in natural waters based on a polymer inclusion membrane incorporating an ionic liquid.

In this study, we have evaluated two different ionic liquids (IL) as extractants based on the same cation (trioctylmethylammonium) but bearing the anion thiosalicylate (TOMATS) or salicylate (TOMAS). Both IL have been incorporated as carriers in polymer inclusion membranes (PIMs), and mercury (Hg) has been preconcentrated using a special device. Results show that among the tested IL, TOMATS has given better results. A PIM made of 50% cellulose triacetate, 30% TOMATS and 20% nitrophenyl octyl ether as a plasticizer enabled the effective transport of Hg to a 10-3M cysteine solution used as a stripping phase. This novel and simple PIM-device system allows the transport of Hg at low concentration levels in different types of natural waters such as rivers, groundwater and seawater without any previous treatment. Since no matrix effect was observed on Hg transport efficiency with different waters, this newly developed PIM-system could be used as a global detection system for this metal. The effect of biofilm growth on the surface of PIMs has been investigated for the first time, and no significant differences on Hg transport have been found when using a fresh PIM and a PIM deployed for 7 days in a pond.

FGF21 gene therapy as treatment for obesity and insulin resistance.

Prevalence of type 2 diabetes (T2D) and obesity is increasing worldwide. Currently available therapies are not suited for all patients in the heterogeneous obese/T2D population, hence the need for novel treatments. Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic agent for T2D/obesity. Native FGF21 has, however, poor pharmacokinetic properties, making gene therapy an attractive strategy to achieve sustained circulating levels of this protein. Here, adeno-associated viral vectors (AAV) were used to genetically engineer liver, adipose tissue, or skeletal muscle to secrete FGF21. Treatment of animals under long-term high-fat diet feeding or of ob/ob mice resulted in marked reductions in body weight, adipose tissue hypertrophy and inflammation, hepatic steatosis, inflammation and fibrosis, and insulin resistance for > 1 year. This therapeutic effect was achieved in the absence of side effects despite continuously elevated serum FGF21. Furthermore, FGF21 overproduction in healthy animals fed a standard diet prevented the increase in weight and insulin resistance associated with aging. Our study underscores the potential of FGF21 gene therapy to treat obesity, insulin resistance, and T2D.

Polymer Inclusion Membrane as an Effective Sorbent To Facilitate Mercury Storage and Detection by X-ray Fluorescence in Natural Waters.

A novel and simple method is presented for the preconcentration and determination of mercury (Hg) from natural waters through its extraction into a polymer inclusion membrane (PIM) containing the task-specific ionic liquid trioctylmethylammonium thiosalicylate (TOMATS) followed by Energy Dispersive X-ray fluorescence (EDXRF) analysis. The determination was made directly on the membrane without any treatment or elution step, and due to the characteristics of the PIM no matrix or thickness corrections were required in EDXRF analysis. Under the best extracting and EDXRF operating conditions, a Hg limit of detection of 0.2 µg Hg L-1 was obtained. Moreover, no water matrix effect was observed when Hg was extracted from different types of water such as river, seawater, groundwater, and tap water, showing this extraction system as a global solution when dealing with natural waters. Interestingly, this Hg collected in the PIM has shown to be stable for at least 6 months without the use of any preservative. This fact is of prime importance taking into account the usual stability problems of Hg during sample storage.

Exploring new DGT samplers containing a polymer inclusion membrane for mercury monitoring.

A polymer inclusion membrane (PIM) made of cellulose triacetate as a polymer and the task specific ionic liquid (IL) trioctylmethylammonium thiosalicylate (TOMATS) was assembled as a new Diffusive Gradients in Thin film (DGT) device to test its efficiency as a binding phase for mercury (Hg) monitoring. The effect of IL content was assessed, showing that higher TOMATS percentage is better for short deployment studies (up to hours), whereas for long-term exposure (up to days), a lower content can be more suitable. Different configurations of PIM-DGT samplers have been tested under controlled conditions and compared with in-house DGT conventional ones, manufactured with thiol groups as resin layer, for the determination of labile Hg. According to our results, a nonlinear accumulation profile of Hg with deployment time for the different designs of PIM-DGT was observed, limiting the range of applicability of the DGT technique. Promising results for the efficient removal of Hg from aqueous solutions and/or environmental monitoring studies were obtained with TOMATS.