Lentiviral-mediated gene correction of mucopolysaccharidosis type IIIA

BACKGROUND: Mucopolysaccharidosis type IIIA (MPS IIIA) is the most common of the mucopolysaccharidoses. The disease is caused by a deficiency of the lysosomal enzyme sulphamidase and results in the storage of the glycosaminoglycan (GAG), heparan sulphate. MPS IIIA is characterised by widespread storage and urinary excretion of heparan sulphate, and a progressive and eventually profound neurological course. Gene therapy is one of the few avenues of treatment that hold promise of a sustainable treatment for this disorder. METHODS: The murine sulphamidase gene cDNA was cloned into a lentiviral vector and high-titre virus produced. Human MPS IIIA fibroblast cultures were transduced with the sulphamidase vector and analysed using molecular, enzymatic and metabolic assays. High-titre virus was intravenously injected into six 5-week old MPS IIIA mice. Three of these mice were pre-treated with hyperosmotic mannitol. The weight of animals was monitored and GAG content in urine samples was analysed by polyacrylamide gel electrophoresis. RESULTS: Transduction of cultured MPS IIIA fibroblasts with the sulphamidase gene corrected both the enzymatic and metabolic defects. Sulphamidase secreted by gene-corrected cells was able to cross correct untransduced MPS IIIA cells. Urinary GAG was found to be greatly reduced in samples from mice receiving the vector compared to untreated MPS IIIA controls. In addition, the weight of treated mice became progressively normalised over the 6-months post-treatment. CONCLUSION: Lentiviral vectors appear promising vehicles for the development of gene therapy for MPS IIIA

Lentiviral-mediated gene therapy for mucopolysaccharidosis type IIIA

Gene therapy is promising for the treatment of monogenetic disorders because it aims to restore overall homeostasis, not by treating disease symptoms, but by targeting the fundamental cause of disease. Viral vectors are valuable tools for mediating the transfer of therapeutic genes to target cells, and lentiviral vectors in particular are well suited to this role as they chromosomally integrate, can enter dividing and non-dividing cells, and evoke little or no immune response. The aim of the current thesis was to evaluate the potential of using lentiviral-mediated gene therapy for the treatment of mucopolysaccharidosis type IIIA (MPS IIIA), a heritable lysosomal storage disorder affecting the central nervous system (CNS). The chronic and progressive course of MPS IIIA results from lysosomal accumulation of heparan sulphate (a highly sulphated glycosaminoglycan), secondary to deficiency of the lysosomal hydrolase sulphamidase. Accumulation of heparan sulphate within the cells of the reticulo-endothelial system, the monocyte-macrophage system, and neurons, leads to hepatosplenomegaly and severe, progressive neuropathology in affected children, and ultimately to death at around 15 years of age. There are currently no effective treatments for MPS IIIA patients. The somatic and CNS aspects of pathology in a mouse model of MPS IIIA were addressed in this study using two separate methods of therapeutic gene delivery; intravenous gene delivery, which directs gene transfer to the liver, and gene delivery to the brain via the cerebral lateral ventricles which utilises the cerebrospinal fluid to achieve gene distribution throughout the brain. After intravenous administration of a self-inactivating lentiviral vector expressing murine sulphamidase to young adult MPS IIIA mice, the livers of treated animals were effectively modified to express high levels of therapeutic sulphamidase. The resultant widespread delivery of enzyme secreted from transduced cells to somatic tissues via the peripheral circulation corrected most somatic pathology, furthermore, markers of MPS IIIA pathology within the brains of treated mice were significantly reduced. When liver directed gene therapy was repeated in a second cohort of mice, however, similar benefits to the brain were not observed, presumably because the resulting levels of peripherally circulating enzyme were comparatively low. Although, in common with the first study, somatic pathologies still were corrected. Alternatively, lentivirus delivered to the brains of MPS IIIA mice via the cerebral lateral ventricles achieved extensive sulphamidase gene distribution and reduced lysosomal storage throughout the brain. Improvements in behaviour were observed for these animals, as was the complete prevention of pathological urine retention. The blood-brain-barrier (BBB) limits the transfer of therapeutic enzymes from the blood to the brain, therefore gene therapy approaches to treat CNS pathology in the MPS are to either challenge the BBB with high levels of circulating enzyme, or bypass it altogether, by the direct delivery of therapeutic genes into the brain. While both approaches have advantages for the treatment of MPS IIIA, the results presented for the current thesis suggest that gene delivery to the brain via the cerebral lateral ventricles may, at this stage, be the more practical and efficacious approach to the treatment of CNS pathology in MPS IIIA patients.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2015

Neurocognitive testing in a murine model of mucopolysaccharidosis type IIIA

Mucopolysaccharidosis type IIIA (MPS IIIA) is an inherited metabolic disorder caused by a lysosomal enzyme deficiency resulting in heparan sulphate (HS) accumulation and manifests with a progressive neurodegenerative phenotype. A naturally occurring MPS IIIA mouse model is invaluable for preclinical evaluation of potential treatments but the ability to effectively assess neurological function has proved challenging. Here, the aim was to evaluate a set of behaviour tests for their reliability in assessing disease progression in the MPS IIIA mouse model. Compared to wild-type (WT) mice, MPS IIIA mice displayed memory and learning deficits in the water crossmaze from mid-stage disease and locomotor impairment in the hind-limb gait assessment at late-stage disease, supporting previous findings. Declined wellbeing was also observed in the MPS IIIA mice via burrowing and nest building evaluation at late-stage disease compared to WT mice, mirroring the progressive nature of neurological disease. Excessive HS accumulation observed in the MPS IIIA mouse brain from 1 month of age did not appear to manifest as abnormal behaviours until at least 6 months of age suggesting there may be a threshold of HS accumulation before measurable neurocognitive decline. Results obtained from the open field and three-chamber sociability test are inconsistent with previous studies and do not reflect MPS IIIA patient disease progression, suggesting these assessments are not reliable. In conclusion, water cross-maze, hind-limb gait, nest building and burrowing, are promising assessments in the MPS IIIA mouse model, which produce consistent results that mimic the human disease

Large-scale production of lentiviral vectors using multilayer cell factories

Lentiviral-mediated gene therapy has been proposed for the treatment of a range of diseases, and due to its genome integration properties, it offers the potential for long-lasting benefit from a once-off treatment. Production methods for pre-clinical studies in animal models, and ultimately for human clinical trials, must be capable of producing large quantities of high-quality lentiviral vector in an efficient and cost-effective manner. We report here a medium-scale method (from 1.5 L to 6 L of vector supernatant) for lentiviral vector production in adherent cell cultures using the NUNCâ„¢ EasyFillâ„¢ Cell Factoryâ„¢ from Thermo Fisher Scientific. Downstream purification uses a Mustang Q XT5 anion exchange capsule from Pall, and an ultracentrifugation step to concentrate the vector. This method is capable of producing lentiviral vector with concentrated titres of 108–109 TU/ml, with reduced manual handling compared to single monolayer flask methods

Large-scale production of lentiviral vectors using multilayer cell factories

Lentiviral-mediated gene therapy has been proposed for the treatment of a range of diseases, and due to its genome integration properties, it offers the potential for long-lasting benefit from a once-off treatment. Production methods for pre-clinical studies in animal models, and ultimately for human clinical trials, must be capable of producing large quantities of high-quality lentiviral vector in an efficient and cost-effective manner. We report here a medium-scale method (from 1.5 L to 6 L of vector supernatant) for lentiviral vector production in adherent cell cultures using the NUNCâ„¢ EasyFillâ„¢ Cell Factoryâ„¢ from Thermo Fisher Scientific. Downstream purification uses a Mustang Q XT5 anion exchange capsule from Pall, and an ultracentrifugation step to concentrate the vector. This method is capable of producing lentiviral vector with concentrated titres of 108–109 TU/ml, with reduced manual handling compared to single monolayer flask methods

Systematic Review and Methodological Considerations for the Use of Single Prolonged Stress and Fear Extinction Retention in Rodents

Posttraumatic stress disorder (PTSD) is a mental health condition triggered by experiencing or witnessing a terrifying event that can lead to lifelong burden that increases mortality and adverse health outcomes. Yet, no new treatments have reached the market in two decades. Thus, screening potential interventions for PTSD is of high priority. Animal models often serve as a critical translational tool to bring new therapeutics from bench to bedside. However, the lack of concordance of some human clinical trial outcomes with preclinical animal efficacy findings has led to a questioning of the methods of how animal studies are conducted and translational validity established. Thus, we conducted a systematic review to determine methodological variability in studies that applied a prominent animal model of trauma-like stress, single prolonged stress (SPS). The SPS model has been utilized to evaluate a myriad of PTSD-relevant outcomes including extinction retention. Rodents exposed to SPS express an extinction retention deficit, a phenotype identified in humans with PTSD, in which fear memory is aberrantly retained after fear memory extinction. The current systematic review examines methodological variation across all phases of the SPS paradigm, as well as strategies for behavioral coding, data processing, statistical approach, and the depiction of data. Solutions for key challenges and sources of variation within these domains are discussed. In response to methodological variation in SPS studies, an expert panel was convened to generate methodological considerations to guide researchers in the application of SPS and the evaluation of extinction retention as a test for a PTSD-like phenotype. Many of these guidelines are applicable to all rodent paradigms developed to model trauma effects or learned fear processes relevant to PTSD, and not limited to SPS. Efforts toward optimizing preclinical model application are essential for enhancing the reproducibility and translational validity of preclinical findings, and should be conducted for all preclinical psychiatric research models