Engineered tRNAs suppress nonsense mutations in cells and in vivo.

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2-7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into  efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP-sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.

GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells.

In response to liver injury, hepatic stellate cells activate and acquire proliferative and contractile features. The regression of liver fibrosis appears to involve the clearance of activated hepatic stellate cells, either by apoptosis or by reversion toward a quiescent-like state, a process called deactivation. Thus, deactivation of active hepatic stellate cells has emerged as a novel and promising therapeutic approach for liver fibrosis. However, our knowledge of the master regulators involved in the deactivation and/or activation of fibrotic hepatic stellate cells is still limited. The transcription factor GATA4 has been previously shown to play an important role in embryonic hepatic stellate cell quiescence. In this work, we show that lack of GATA4 in adult mice caused hepatic stellate cell activation and, consequently, liver fibrosis. During regression of liver fibrosis, Gata4 was reexpressed in deactivated hepatic stellate cells. Overexpression of Gata4 in hepatic stellate cells promoted liver fibrosis regression in CCl4-treated mice. GATA4 induced changes in the expression of fibrogenic and antifibrogenic genes, promoting hepatic stellate cell deactivation. Finally, we show that GATA4 directly repressed EPAS1 transcription in hepatic stellate cells and that stabilization of the HIF2α protein in hepatic stellate cells leads to liver fibrosis.

Viral gene delivery vectors: the next generation medicines for immune-related diseases.

Viruses have evolved to efficiently express their genes in host cells, which makes them ideally suited as gene delivery vectors for gene and immunotherapies. Replication competent (RC) viral vectors encoding foreign or self-proteins induce strong T-cell responses that can be used for the development of effective cancer treatments. Replication-defective (RD) viral vectors encoding self-proteins are non-immunogenic when introduced in a host naïve for the cognate virus. RD viral vectors can be used to develop gene replacement therapies for genetic disorders and tolerization therapies for autoimmune diseases and allergies. Degenerative/inflammatory diseases are associated with chronic inflammation and immune responses that damage the tissues involved. These diseases therefore strongly resemble autoimmune diseases. This review deals with the use of RC and RD viral vectors for unraveling the pathogenesis of immune-related diseases and their application to the development of the next generation prophylactics and therapeutics for todays' major diseases.

Supercapacitor/biofuel cell hybrid device employing biomolecules for energy conversion and charge storage.

We report on a hybrid bioelectrochemical system that integrates an energy converting part, viz. a glucose/oxygen enzymatic fuel cell, with a charge-storing component, in which the redox features of the immobilized redox protein cytochrome c (cyt c) were utilized. Bilirubin oxidase and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) were employed as the biocatalysts for dioxygen reduction and glucose oxidation, respectively. A bi-protein PQQ-GDH/cyt c signal chain was created that facilitates electron transfer between the enzyme and the electrode surface. The assembled supercapacitor/biofuel cell hybrid biodevice displays a 15 times higher power density tested in the pulse mode compared to the performance achieved from the continuously operating regime (4.5 and 0.3 µW cm-2, respectively) with an 80% residual activity after 50 charge/discharge pulses. This can be considered as a notable step forward in the field of glucose/oxygen membrane-free, biocompatible hybrid power sources.

Two-dimensional graphene paper supported flexible enzymatic fuel cells.

Application of enzymatic biofuel cells (EBFCs) in wearable or implantable biomedical devices requires flexible and biocompatible electrode materials. To this end, freestanding and low-cost graphene paper is emerging among the most promising support materials. In this work, we have exploited the potential of using graphene paper with a two-dimensional active surface (2D-GP) as a carrier for enzyme immobilization to fabricate EBFCs, representing the first case of flexible graphene papers directly used in EBFCs. The 2D-GP electrodes were prepared via the assembly of graphene oxide (GO) nanosheets into a paper-like architecture, followed by reduction to form layered and cross-linked networks with good mechanical strength, high conductivity and little dependence on the degree of mechanical bending. 2D-GP electrodes served as both a current collector and an enzyme loading substrate that can be used directly as a bioanode and biocathode. Pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) and bilirubin oxidase (BOx) adsorbed on the 2D-GP electrodes both retain their biocatalytic activities. Electron transfer (ET) at the bioanode required Meldola blue (MB) as an ET mediator to shuttle electrons between PQQ-GDH and the electrode, but direct electron transfer (DET) at the biocathode was achieved. The resulting glucose/oxygen EBFC displayed a notable mechanical flexibility, with a wide open circuit voltage range up to 0.665 V and a maximum power density of approximately 4 µW cm-2 both fully competitive with reported values for related EBFCs, and with mechanical flexibility and facile enzyme immobilization as novel merits.

Transient PAX8 Expression in Islets During Pregnancy Correlates With ß-Cell Survival, Revealing a Novel Candidate Gene in Gestational Diabetes Mellitus.

Transient Pax8 expression was reported in mouse islets during gestation, whereas a genome-wide linkage and admixture mapping study highlighted PAX8 as a candidate gene for diabetes mellitus (DM). We sought the significance of PAX8 expression in mouse and human islet biology. PAX8 was induced in gestating mouse islets and in human islets treated with recombinant prolactin. Global gene expression profiling of human and mouse islets overexpressing the corresponding species-specific PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets. These findings support that PAX8 could be a candidate gene for the study of gestational DM (GDM). PAX8 was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8 A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identified in a female diagnosed with GDM and GTD as well as in her father with type 2 DM but was absent in control patients. The p.T356M variant did not alter protein stability or cellular localization, whereas its transactivation activity was hindered. In parallel, a retrospective clinical analysis uncovered that a pregnant female harboring a second PAX8 mutation (p.P25R, c.74C>G) previously reported to cause congenital hypothyroidism also developed GDM. These data indicate that increased expression of PAX8 affects islet viability and that PAX8 could be considered as a candidate gene for the study of GDM.

Fuel-independent and membrane-less self-charging biosupercapacitor.

We present a fuel-independent self-charging biosupercapacitor comprising an oxygen reducing enzymatic biocathode and an opposing bioelectrode, in which the supercapacitive properties of immobilised protein were utilised. Our findings disclose a novel hybrid type of bioelectrochemical systems, which can potentially be employed as an autonomous power supplier under substrate-deficient conditions.

How Simian Virus 40 Hijacks the Intracellular Protein Trafficking Pathway to Its Own Benefit and Ours.

Viruses efficiently transfer and express their genes in host cells and evolve to evade the host's defense responses. These properties render them highly attractive for use as gene delivery vectors in vaccines, gene, and immunotherapies. Among the viruses used as gene delivery vectors, the macaque polyomavirus Simian Virus 40 (SV40) is unique in its capacity to evade intracellular antiviral defense responses upon cell entry. We here describe the unique way by which SV40 particles deliver their genomes in the nucleus of permissive cells and how they prevent presentation of viral antigens to the host's immune system. The non-immunogenicity in its natural host is not only of benefit to the virus but also to us in developing effective SV40 vector-based treatments for today's major human diseases.

LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.

Rechargeable, flexible and mediator-free biosupercapacitor based on transparent ITO nanoparticle modified electrodes acting in µM glucose containing buffers.

We present a transparent and flexible self-charging biosupercapacitor based on an optimised mediator- and membrane-free enzymatic glucose/oxygen biofuel cell. Indium tin oxide (ITO) nanoparticles were spray-coated on transparent conducting ITO supports resulting in a flocculent, porous and nanostructured electrode surface. By this, high capacitive currents caused by an increased electrochemical double layer as well as enhanced catalytic currents due to a higher number of immobilised enzyme molecules were obtained. After a chemical pre-treatment with a silane derivative, bilirubin oxidase from Myrothecium verrucaria was immobilized onto the ITO nanostructured electrode surface under formation of a biocathode, while bioanodes were obtained by either immobilisation of cellobiose dehydrogenase from Corynascus thermophilus or soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus. The latter showed a lower apparent KM value for glucose conversion and higher catalytic currents at µM glucose concentrations. Applying the optimised device as a biosupercapacitor in a discontinuous charge/discharge mode led to a generated power output of 0.030mW/cm2 at 50µM glucose, simulating the glucose concentration in human tears. This represents an enhancement by a factor of 350 compared to the power density obtained from the continuously operating biofuel cell with a maximum power output of 0.086µW/cm2 under the same conditions. After 17h of charging/discharging cycles a remarkable current enhancement was still measured. The entire device was transferred to flexible materials and applied for powering a flexible display showing its potential applicability as an intermittent power source in smart contact lenses.

Generation of a Vero-Based Packaging Cell Line to Produce SV40 Gene Delivery Vectors for Use in Clinical Gene Therapy Studies.

Replication-defective (RD) recombinant simian virus 40 (SV40)-based gene delivery vectors hold a great potential for clinical applications because of their presumed non-immunogenicity and capacity to induce immune tolerance to the transgene products in humans. However, the clinical use of SV40 vectors has been hampered by the lack of a packaging cell line that produces replication-competent (RC) free SV40 particles in the vector production process. To solve this problem, we have adapted the current SV40 vector genome used for the production of vector particles and generated a novel Vero-based packaging cell line named SuperVero that exclusively expresses the SV40 large T antigen. SuperVero cells produce similar numbers of SV40 vector particles compared to the currently used packaging cell lines, albeit in the absence of contaminating RC SV40 particles. Our unique SV40 vector platform named SVac paves the way to clinically test a whole new generation of SV40-based therapeutics for a broad range of important diseases.

Transparent, mediator- and membrane-free enzymatic fuel cell based on nanostructured chemically modified indium tin oxide electrodes.

We detail a mediator- and membrane-free enzymatic glucose/oxygen biofuel cell based on transparent and nanostructured conducting supports. Chemically modified indium tin oxide nanoparticle modified electrodes were used to substantially increase the active surface area without significantly compromising transparency. Two different procedures for surface nanostructuring were employed, viz. spray-coating and drop-coating. The spray-coated biodevice showed superior characteristics as compared to the drop-coated enzymatic fuel cell, as a result of the higher nanostructured surface area as confirmed by electrochemical characterisation, as well as scanning electron and atomic force microscopy. Subsequent chemical modification with silanes, followed by the immobilisation of either cellobiose dehydrogenase from Corynascus thermophiles or bilirubin oxidase from Myrothecium verrucaria, were performed to obtain the bioanodes and biocathodes, respectively. The optimised biodevice exhibited an OCV of 0.67V and power output of up to 1.4µW/cm2 at an operating voltage of 0.35V. This is considered a significant step forward in the field of glucose/oxygen membrane- and mediator-free, transparent enzymatic fuel cells.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

The 'Omics' of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease that primarily affects motor neurons and is accompanied by sustained unregulated immune responses, but without clear indications of the ultimate causative mechanisms. The identification of a diverse array of ALS phenotypes, a series of recently discovered mutations, and the links between ALS and frontotemporal degeneration have significantly increased our knowledge of the disease. In this review we discuss the main features involved in ALS pathophysiology in the context of recent advances in 'omics' approaches, including genomics, proteomics, and others. We emphasize the pressing need to combine clinical imaging with various different parameters taken from omics fields to facilitate early, accurate diagnosis and rational drug design in the treatment of ALS.

SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

Mesenchymal stromal cells express GARP/LRRC32 on their surface: effects on their biology and immunomodulatory capacity.

Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-ß1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-ß1 to the cell surface of activated Foxp3(+) regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-ß1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-ß1 and reduced their proliferative capacity in a TGF-ß1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker.

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission.

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases.

A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39-0.40s(-1)) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.

Oxygen biosensor based on bilirubin oxidase immobilized on a nanostructured gold electrode.

Gold disk electrodes modified with gold nanoparticles have been used as a scaffold for the covalent immobilization of bilirubin oxidase. The nanostructured bioelectrodes were tested as mediator-less biosensors for oxygen in a buffer that mimics the content and the composition of human physiological fluids. Chronoamperometry measurements showed a detection limit towards oxygen of 6 ± 1 µM with a linear range of 6-300 µM, i.e. exceeding usual physiological ranges of oxygen in human tissues and fluids. The biosensor presented is the first ever-reported oxygen amperometric biosensor based on direct electron transfer of bilirubin oxidase.