Minimal ureagenesis is necessary for survival in the murine model of hyperargininemia treated by AAV-based gene therapy.

Hyperammonemia is less severe in arginase 1 deficiency compared with other urea cycle defects. Affected patients manifest hyperargininemia and infrequent episodes of hyperammonemia. Patients typically suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, seizures and intellectual disability; death is less common than with other urea cycle disorders. In a mouse model of arginase I deficiency, the onset of symptoms begins with weight loss and gait instability, which progresses toward development of tail tremor with seizure-like activity; death typically occurs at about 2 weeks of life. Adeno-associated viral vector gene replacement strategies result in long-term survival of mice with this disorder. With neonatal administration of vector, the viral copy number in the liver greatly declines with hepatocyte proliferation in the first 5 weeks of life. Although the animals do survive, it is not known from a functional standpoint how well the urea cycle is functioning in the adult animals that receive adeno-associated virus. In these studies, we administered [1-13C] acetate to both littermate controls and adeno-associated virus-treated arginase 1 knockout animals and examined flux through the urea cycle. Circulating ammonia levels were mildly elevated in treated animals. Arginine and glutamine also had perturbations. Assessment 30 min after acetate administration demonstrated that ureagenesis was present in the treated knockout liver at levels as low at 3.3% of control animals. These studies demonstrate that only minimal levels of hepatic arginase activity are necessary for survival and ureagenesis in arginase-deficient mice and that this level of activity results in control of circulating ammonia. These results may have implications for potential therapy in humans with arginase deficiency

Switching on the Lights for Gene Therapy

Strategies for non-invasive and quantitative imaging of gene expression in vivo have been developed over the past decade. Non-invasive assessment of the dynamics of gene regulation is of interest for the detection of endogenous disease-specific biological alterations (e.g., signal transduction) and for monitoring the induction and regulation of therapeutic genes (e.g., gene therapy). To demonstrate that non-invasive imaging of regulated expression of any type of gene after in vivo transduction by versatile vectors is feasible, we generated regulatable herpes simplex virus type 1 (HSV-1) amplicon vectors carrying hormone (mifepristone) or antibiotic (tetracycline) regulated promoters driving the proportional co-expression of two marker genes. Regulated gene expression was monitored by fluorescence microscopy in culture and by positron emission tomography (PET) or bioluminescence (BLI) in vivo. The induction levels evaluated in glioma models varied depending on the dose of inductor. With fluorescence microscopy and BLI being the tools for assessing gene expression in culture and animal models, and with PET being the technology for possible application in humans, the generated vectors may serve to non-invasively monitor the dynamics of any gene of interest which is proportionally co-expressed with the respective imaging marker gene in research applications aiming towards translation into clinical application

Differentially Expressed RNA from Public Microarray Data Identifies Serum Protein Biomarkers for Cross-Organ Transplant Rejection and Other Conditions

Serum proteins are routinely used to diagnose diseases, but are hard to find due to low sensitivity in screening the serum proteome. Public repositories of microarray data, such as the Gene Expression Omnibus (GEO), contain RNA expression profiles for more than 16,000 biological conditions, covering more than 30% of United States mortality. We hypothesized that genes coding for serum- and urine-detectable proteins, and showing differential expression of RNA in disease-damaged tissues would make ideal diagnostic protein biomarkers for those diseases. We showed that predicted protein biomarkers are significantly enriched for known diagnostic protein biomarkers in 22 diseases, with enrichment significantly higher in diseases for which at least three datasets are available. We then used this strategy to search for new biomarkers indicating acute rejection (AR) across different types of transplanted solid organs. We integrated three biopsy-based microarray studies of AR from pediatric renal, adult renal and adult cardiac transplantation and identified 45 genes upregulated in all three. From this set, we chose 10 proteins for serum ELISA assays in 39 renal transplant patients, and discovered three that were significantly higher in AR. Interestingly, all three proteins were also significantly higher during AR in the 63 cardiac transplant recipients studied. Our best marker, serum PECAM1, identified renal AR with 89% sensitivity and 75% specificity, and also showed increased expression in AR by immunohistochemistry in renal, hepatic and cardiac transplant biopsies. Our results demonstrate that integrating gene expression microarray measurements from disease samples and even publicly-available data sets can be a powerful, fast, and cost-effective strategy for the discovery of new diagnostic serum protein biomarkers

Can we improve outcome of congenital diaphragmatic hernia?

This review gives an overview of the disease spectrum of congenital diaphragmatic hernia (CDH). Etiological factors, prenatal predictors of survival, new treatment strategies and long-term morbidity are described. Early recognition of problems and improvement of treatment strategies in CDH patients may increase survival and prevent secondary morbidity. Multidisciplinary healthcare is necessary to improve healthcare for CDH patients. Absence of international therapy guidelines, lack of evidence of many therapeutic modalities and the relative low number of CDH patients calls for cooperation between centers with an expertise in the treatment of CDH patients. The international CDH Euro-Consortium is an example of such a collaborative network, which enhances exchange of knowledge, future research and development of treatment protocols

Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

Over the last decade, pioneering liver-directed gene therapy trials for haemophilia B have achieved sustained clinical improvement after a single systemic injection of adeno-associated virus (AAV) derived vectors encoding the human factor IX cDNA. These trials demonstrate the potential of AAV technology to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Indeed, with more than ten ongoing or planned clinical trials for haemophilia A and B and dozens of trials planned for other inherited genetic/metabolic liver diseases, clinical translation is expanding rapidly. Gene therapy is likely to become an option for routine care of a subset of severe inherited genetic/metabolic liver diseases in the relatively near term. In this review, we aim to summarise the milestones in the development of gene therapy, present the different vector tools and their clinical applications for liver-directed gene therapy. AAV-derived vectors are emerging as the leading candidates for clinical translation of gene delivery to the liver. Therefore, we focus on clinical applications of AAV vectors in providing the most recent update on clinical outcomes of completed and ongoing gene therapy trials and comment on the current challenges that the field is facing for large-scale clinical translation. There is clearly an urgent need for more efficient therapies in many severe monogenic liver disorders, which will require careful risk-benefit analysis for each indication, especially in paediatrics

Congenital Diaphragmatic hernia – a review

Congenital Diaphragmatic hernia (CDH) is a condition characterized by a defect in the diaphragm leading to protrusion of abdominal contents into the thoracic cavity interfering with normal development of the lungs. The defect may range from a small aperture in the posterior muscle rim to complete absence of diaphragm. The pathophysiology of CDH is a combination of lung hypoplasia and immaturity associated with persistent pulmonary hypertension of newborn (PPHN) and cardiac dysfunction. Prenatal assessment of lung to head ratio (LHR) and position of the liver by ultrasound are used to diagnose and predict outcomes. Delivery of infants with CDH is recommended close to term gestation. Immediate management at birth includes bowel decompression, avoidance of mask ventilation and endotracheal tube placement if required. The main focus of management includes gentle ventilation, hemodynamic monitoring and treatment of pulmonary hypertension followed by surgery. Although inhaled nitric oxide is not approved by FDA for the treatment of PPHN induced by CDH, it is commonly used. Extracorporeal membrane oxygenation (ECMO) is typically considered after failure of conventional medical management for infants ≥ 34 weeks’ gestation or with weight >2 kg with CDH and no associated major lethal anomalies. Multiple factors such as prematurity, associated abnormalities, severity of PPHN, type of repair and need for ECMO can affect the survival of an infant with CDH. With advances in the management of CDH, the overall survival has improved and has been reported to be 70-90% in non-ECMO infants and up to 50% in infants who undergo ECMO

Development of operational immunologic tolerance with neonatal gene transfer in nonhuman primates: preliminary studies

Achieving persistent expression is a prerequisite for effective genetic therapies for inherited disorders. These proof-of-concept studies focused on adeno-associated virus (AAV) administration to newborn monkeys. Serotype rh10 AAV expressing ovalbumin and green fluorescent protein (GFP) was administered intravenously at birth and compared with vehicle controls. At 4 months postnatal age, a second injection was administered intramuscularly, followed by vaccination at 1 year of age with ovalbumin and GFP. Ovalbumin was highest 2 weeks post administration in the treated monkey, which declined but remained detectable thereafter; controls demonstrated no expression. Long-term AAV genome copies were present in myocytes. At 4 weeks, neutralizing antibodies to rh10 were present in the experimental animal only. With AAV9 administration at 4 months, controls showed transient ovalbumin expression that disappeared with the development of strong anti-ovalbumin and anti-GFP antibodies. In contrast, increased and maintained ovalbumin expression was noted in the monkey administered AAV at birth, without antibody development. After vaccination, the experimental monkey maintained levels of ovalbumin without antibodies, whereas controls demonstrated high levels of antibodies. These preliminary studies suggest that newborn AAV administration expressing secreted and intracellular xenogenic proteins may result in persistent expression in muscle, and subsequent vector administration can result in augmented expression without humoral immune responses

Minimal ureagenesis is necessary for survival in the murine model of hyperargininemia treated by AAV-based gene therapy.

Hyperammonemia is less severe in arginase 1 deficiency compared with other urea cycle defects. Affected patients manifest hyperargininemia and infrequent episodes of hyperammonemia. Patients typically suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, seizures and intellectual disability; death is less common than with other urea cycle disorders. In a mouse model of arginase I deficiency, the onset of symptoms begins with weight loss and gait instability, which progresses toward development of tail tremor with seizure-like activity; death typically occurs at about 2 weeks of life. Adeno-associated viral vector gene replacement strategies result in long-term survival of mice with this disorder. With neonatal administration of vector, the viral copy number in the liver greatly declines with hepatocyte proliferation in the first 5 weeks of life. Although the animals do survive, it is not known from a functional standpoint how well the urea cycle is functioning in the adult animals that receive adeno-associated virus. In these studies, we administered [1-13C] acetate to both littermate controls and adeno-associated virus-treated arginase 1 knockout animals and examined flux through the urea cycle. Circulating ammonia levels were mildly elevated in treated animals. Arginine and glutamine also had perturbations. Assessment 30 min after acetate administration demonstrated that ureagenesis was present in the treated knockout liver at levels as low at 3.3% of control animals. These studies demonstrate that only minimal levels of hepatic arginase activity are necessary for survival and ureagenesis in arginase-deficient mice and that this level of activity results in control of circulating ammonia. These results may have implications for potential therapy in humans with arginase deficiency