Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Aromatic l-amino acid decarboxylase deficiency: a patient-derived neuronal model for precision therapies.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a complex inherited neurological disorder of monoamine synthesis which results in dopamine and serotonin deficiency. The majority of affected individuals have variable, though often severe cognitive and motor delay, with a complex movement disorder and high risk of premature mortality. For most, standard pharmacological treatment provides only limited clinical benefit. Promising gene therapy approaches are emerging, though may not be either suitable or easily accessible for all patients. To characterize the underlying disease pathophysiology and guide precision therapies, we generated a patient-derived midbrain dopaminergic neuronal model of AADC deficiency from induced pluripotent stem cells. The neuronal model recapitulates key disease features, including absent AADC enzyme activity and dysregulated dopamine metabolism. We observed developmental defects affecting synaptic maturation and neuronal electrical properties, which were improved by lentiviral gene therapy. Bioinformatic and biochemical analyses on recombinant AADC predicted that the activity of one variant could be improved by l-3,4-dihydroxyphenylalanine (l-DOPA) administration; this hypothesis was corroborated in the patient-derived neuronal model, where l-DOPA treatment leads to amelioration of dopamine metabolites. Our study has shown that patient-derived disease modelling provides further insight into the neurodevelopmental sequelae of AADC deficiency, as well as a robust platform to investigate and develop personalized therapeutic approaches.

Effects of Mini-Dystrophin on Dystrophin-Deficient, Human Skeletal Muscle-Derived Cells.

BACKGROUND: We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. AIMS: Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. METHODS: Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3-R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. RESULTS: Cell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. CONCLUSIONS: Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

Production of lentiviral vectors using novel, enzymatically produced, linear DNA.

The manufacture of large quantities of high-quality DNA is a major bottleneck in the production of viral vectors for gene therapy. Touchlight Genetics has developed a proprietary abiological technology that addresses the major issues in commercial DNA supply. The technology uses 'rolling-circle' amplification to produce large quantities of concatameric DNA that is then processed to create closed linear double-stranded DNA by enzymatic digestion. This novel form of DNA, Doggybone™ DNA (dbDNA™), is structurally distinct from plasmid DNA. Here we compare lentiviral vectors production from dbDNA™ and plasmid DNA. Lentiviral vectors were administered to neonatal mice via intracerebroventricular injection. Luciferase expression was quantified in conscious mice continually by whole-body bioluminescent imaging. We observed long-term luciferase expression using dbDNA™-derived vectors, which was comparable to plasmid-derived lentivirus vectors. Here we have demonstrated that functional lentiviral vectors can be produced using the novel dbDNA™ configuration for delivery in vitro and in vivo. Importantly, this could enable lentiviral vector packaging of complex DNA sequences that have previously been incompatible with bacterial propagation systems, as dbDNA™ technology could circumvent such restrictions through its phi29-based rolling-circle amplification.

Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy.

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model.

Payload-delivering engineered γδ T cells display enhanced cytotoxicity, persistence, and efficacy in preclinical models of osteosarcoma.

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous αß T cells. These approaches are limited by tonic signaling of synthetic constructs and costs associated with manufacturing. γδ T cells are an emerging alternative for cellular therapy, having innate antitumor activity, potent antibody-dependent cellular cytotoxicity, and minimal alloreactivity. We present an immunotherapeutic platform technology built around the innate properties of the Vγ9Vδ2 T cell, harnessing specific characteristics of this cell type and offering an allocompatible cellular therapy that recruits bystander immunity. We engineered γδ T cells to secrete synthetic tumor-targeting opsonins in the form of an scFv-Fc fusion protein and a mitogenic IL-15Rα-IL-15 fusion protein (stIL15). Using GD2 as a model antigen, we show that GD2-specific opsonin-secreting Vγ9Vδ2 T cells (stIL15-OPS-γδ T cells) have enhanced cytotoxicity and promote bystander activity of other lymphoid and myeloid cells. Secretion of stIL-15 abrogated the need for exogenous cytokine supplementation and further mediated activation of bystander natural killer cells. Compared with unmodified γδ T cells, stIL15-OPS-γδ T cells exhibited superior in vivo control of subcutaneous tumors and persistence in the blood. Moreover, stIL15-OPS-γδ T cells were efficacious against patient-derived osteosarcomas in animal models and in vitro, where efficacy could be boosted with the addition of zoledronic acid. Together, the data identify stIL15-OPS-γδ T cells as a candidate allogeneic cell therapy platform combining direct cytolysis with bystander activation to promote tumor control.

Expression of gain-of-function CFTR in cystic fibrosis airway cells restores epithelial function better than wild-type or codon-optimized CFTR.

Class Ia/b cystic fibrosis transmembrane regulator (CFTR) variants cause severe lung disease in 10% of cystic fibrosis (CF) patients and are untreatable with small-molecule pharmaceuticals. Genetic replacement of CFTR offers a cure, but its effectiveness is limited in vivo. We hypothesized that enhancing protein levels (using codon optimization) and/or activity (using gain-of-function variants) of CFTR would more effectively restore function to CF bronchial epithelial cells. Three different variants of the CFTR protein were tested: codon optimized (high codon adaptation index [hCAI]), a gain-of-function (GOF) variant (K978C), and a combination of both (hˆK978C). In human embryonic kidney (HEK293T) cells, initial results showed that hCAI and hˆK978C produced greater than 10-fold more CFTR protein and displayed ∼4-fold greater activity than wild-type (WT) CFTR. However, functionality was profoundly different in CF bronchial epithelial cells. Here, K978C CFTR more potently restored essential epithelial functions (anion transport, airway surface liquid height, and pH) than WT CFTR. hCAI and hˆK978C CFTRs had limited impact because of mislocalization in the cell. These data provide a proof of principle showing that GOF variants may be more effective than codon-optimized forms of CFTR for CF gene therapy.

Urokinase-type plasminogen activator (uPA) regulates invasion and matrix remodelling in colorectal cancer.

Background: Cancer cells remodel their local physical environment through processes of matrix reorganisation, deposition, stiffening and degradation. Urokinase-type plasminogen activator (uPA), which is encoded by the PLAU gene, is an extracellular proteolytic enzyme known to be involved in cancer progression and tumour microenvironment (TME) remodelling. Perturbing uPA therefore has a strong potential as a mechano-based cancer therapy. This work is a bioengineering investigation to validate whether 1) uPA is involved in matrix degradation and 2) preventing matrix degradation by targeting uPA can reduce cancer cell invasion and metastasis. Methods: To this aim, we used an engineered 3D in vitro model, termed the tumouroid, that appropriately mimics the tumour's native biophysical environment (3 kPa). A CRISPR-Cas9 mediated uPA knockout was performed to introduce a loss of function mutation in the gene coding sequence. Subsequently, to validate the translational potential of blocking uPA action, we tested a pharmacological inhibitor, UK-371,801. The changes in matrix stiffness were measured by atomic force microscopy (AFM). Invasion was quantified using images of the tumouroid, obtained after 21 days of culture. Results: We showed that uPA is highly expressed in invasive breast and colorectal cancers, and these invasive cancer cells locally degrade their TME. PLAU (uPA) gene knock-out (KO) completely stopped matrix remodelling and significantly reduced cancer invasion. Many invasive cancer gene markers were also downregulated in the PLAU KO tumouroids. Pharmacological inhibition of uPA showed similarly promising results, where matrix degradation was reduced and so was the cancer invasion. Conclusion: This work supports the role of uPA in matrix degradation. It demonstrates that the invasion of cancer cells was significantly reduced when enzymatic breakdown of the TME matrix was prevented. Collectively, this provides strong evidence of the effectiveness of targeting uPA as a mechano-based cancer therapy.

3D human induced pluripotent stem cell-derived bioengineered skeletal muscles for tissue, disease and therapy modeling.

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells and then combined within 3D hydrogels under tension to generate an aligned myofiber scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers. Importantly, bioengineered muscles provide a high-fidelity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 d, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof of principle of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.

Optimized lentiviral vector to restore full-length dystrophin via a cell-mediated approach in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the DMD gene. Restoration of full-length dystrophin protein in skeletal muscle would have therapeutic benefit, but lentivirally mediated delivery of such a large gene in vivo has been hindered by lack of tissue specificity, limited transduction, and insufficient transgene expression. To address these problems, we developed a lentiviral vector, which contains a muscle-specific promoter and sequence-optimized full-length dystrophin, to constrain dystrophin expression to differentiated myotubes/myofibers and enhance the transgene expression. We further explored the efficiency of restoration of full-length dystrophin in vivo, by grafting DMD myoblasts that had been corrected by this optimized lentiviral vector intramuscularly into an immunodeficient DMD mouse model. We show that these lentivirally corrected DMD myoblasts effectively reconstituted full-length dystrophin expression in 93.58% ± 2.17% of the myotubes in vitro. Moreover, dystrophin was restored in 64.4% ± 2.87% of the donor-derived regenerated muscle fibers in vivo, which were able to recruit members of the dystrophin-glycoprotein complex at the sarcolemma. This study represents a significant advance over existing cell-mediated gene therapy strategies for DMD that aim to restore full-length dystrophin expression in skeletal muscle.

Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications.

Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10-15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches.

Re-structuring lentiviral vectors to express genomic RNA via cap-dependent translation.

Lentiviral (LV) vectors based on human immunodeficiency virus type I (HIV-1) package two copies of their single-stranded RNA into vector particles. Normally, this RNA genome is reverse transcribed into a double-stranded DNA provirus that integrates into the cell genome, providing permanent gene transfer and long-term expression. Integration-deficient LV vectors have been developed to reduce the frequency of genomic integration and thereby limit their persistence in dividing cells. Here, we describe optimization of a reverse-transcriptase-deficient LV vector, which enables direct translation of LV RNA genomes upon cell entry, for transient expression of vector payloads as mRNA without a DNA intermediate. We have engineered a novel LV genome arrangement in which HIV-1 sequences are removed from the 5' end, to enable ribosomal entry from the 5' 7-methylguanylate cap for efficient translation of the vector payload. We have shown that this LV-mediated mRNA delivery platform provides transient transgene expression in vitro and in vivo. This has a potential application in gene and cell therapy scenarios requiring temporary payload expression in cells and tissues that can be targeted with pseudotyped LV vectors.

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications.

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D-such as their cellular tropism, receptor usage, and in vivo biodistribution profile-remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)-a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

Dystrophin involvement in peripheral circadian SRF signalling.

Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-µ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

Gene therapy for inherited metabolic diseases.

Over the last two decades, gene therapy has been successfully translated to many rare diseases. The number of clinical trials is rapidly expanding and some gene therapy products have now received market authorisation in the western world. Inherited metabolic diseases (IMD) are orphan diseases frequently associated with a severe debilitating phenotype with limited therapeutic perspective. Gene therapy is progressively becoming a disease-changing therapeutic option for these patients. In this review, we aim to summarise the development of this emerging field detailing the main gene therapy strategies, routes of administration, viral and non-viral vectors and gene editing tools. We discuss the respective advantages and pitfalls of these gene therapy strategies and review their application in IMD, providing examples of clinical trials with lentiviral or adeno-associated viral gene therapy vectors in rare diseases. The rapid development of the field and implementation of gene therapy as a realistic therapeutic option for various IMD in a short term also require a good knowledge and understanding of these technologies from physicians to counsel the patients at best.

Effects of corticosterone within the hypothalamic arcuate nucleus on food intake and body weight in male rats.

OBJECTIVE: Obesity is a major cause of morbidity and mortality. Few weight-reducing medications are available, and these have limited efficacy. Cushing's Syndrome (caused by elevated glucocorticoid levels) and obesity have similar metabolic features. Though circulating glucocorticoid levels are not elevated in obesity, tissue-specific glucocorticoid levels have been implicated in the development of the metabolic phenotype of obesity. Tissue glucocorticoid levels are regulated by 11ß-hydroxysteroid dehydrogenase type1 (11ßHSD1), which increases the local concentration of active glucocorticoids by the production of corticosterone from 11-dehydrocorticosterone. 11ßHSD1 is expressed in the hypothalamic arcuate nucleus (ARC), a major weight and appetite-regulating centre, and therefore represents a target for novel anti-obesity therapeutic agents. Thus, we sought to investigate the effect of chronic alterations of ARC corticosterone levels (mediated by 11ßHSD1) on food intake and body weight in adult male rats. METHODS: Recombinant adeno-associated virus particles bearing sense 11ßHSD1 (rAAV-S11ßHSD1) and small interfering 11ßHSD1 (rAAV-si11ßHSD1), respectively, were stereotactically injected into the ARC (bilaterally) of adult male Wistar rats. rAAV-GFP was injected into control groups of male Wistar rats. Food intake and body weight were measured three times a week for 70 days. Terminal brain, plasma and intrascapular brown adipose tissue (iBAT) samples were taken for measurement of mRNA expression and hormone levels. RESULTS: Compared to controls, rAAV-S11ßHSD1 injection resulted in higher ARC corticosterone levels, hyperphagia and increased weight gain. Conversely, rAAV-si11ßHSD1 injection (compared to controls) resulted in lower ARC corticosterone levels, higher iBAT uncoupling protein-1 mRNA expression and less weight gain despite similar food intake. CONCLUSIONS: Therefore ARC corticosterone, regulated by 11ßHSD1, may play a role in food intake and body weight regulation. These data have important implications for the development of centrally-acting 11ßHSD1 inhibitors, which are currently being developed for the treatment of obesity, metabolic disorders, and other conditions.