Joanne Ng

Biallelic Mutations in PDE10A Lead to Loss of Striatal PDE10A and a Hyperkinetic Movement Disorder with Onset in Infancy

Deficits in the basal ganglia pathways modulating cortical motor activity underlie both Parkinson disease (PD) and Huntington disease (HD). Phosphodiesterase 10A (PDE10A) is enriched in the striatum, and animal data suggest that it is a key regulator of this circuitry. Here, we report on germline PDE10A mutations in eight individuals from two families affected by a hyperkinetic movement disorder due to homozygous mutations c.320A>G (p.Tyr107Cys) and c.346G>C (p.Ala116Pro). Both mutations lead to a reduction in PDE10A levels in recombinant cellular systems, and critically, positron-emission-tomography (PET) studies with a specific PDE10A ligand confirmed that the p.Tyr107Cys variant also reduced striatal PDE10A levels in one of the affected individuals. A knock-in mouse model carrying the homologous p.Tyr97Cys variant had decreased striatal PDE10A and also displayed motor abnormalities. Striatal preparations from this animal had an impaired capacity to degrade cyclic adenosine monophosphate (cAMP) and a blunted pharmacological response to PDE10A inhibitors. These observations highlight the critical role of PDE10A in motor control across species.

Perinatal systemic gene delivery using adeno-associated viral vectors

Neurodegenerative monogenic diseases often affect tissues and organs beyond the nervous system. An effective treatment would require a systemic approach. The intravenous administration of novel therapies is ideal but is hampered by the inability of such drugs to cross the blood–brain barrier (BBB) and precludes efficacy in the central nervous system. A number of these early lethal intractable diseases also present devastating irreversible pathology at birth or soon after. Therefore, any therapy would ideally be administered during the perinatal period to prevent, stop, or ameliorate disease progression. The concept of perinatal gene therapy has moved a step further toward being a feasible approach to treating such disorders. This has primarily been driven by the recent discoveries that particular serotypes of adeno-associated virus (AAV) gene delivery vectors have the ability to cross the BBB following intravenous administration. Furthermore, safety has been demonstrated after perinatal administration mice and non-human primates. This review focuses on the progress made in using AAV to achieve systemic transduction and what this means for developing perinatal gene therapy for early lethal neurodegenerative diseases.

Continual conscious bioluminescent imaging in freely moving somatotransgenic mice

Luciferase bioimaging in living animals is increasingly being applied in many fields of biomedical research. Rodent imaging usually involves anaesthetising the animal during data capture, however, the biological consequences of anaesthesia have been largely overlooked. We have evaluated luciferase bioimaging in conscious, unrestrained mice after neonatal intracranial or intravascular administration of lentiviral, luciferase reporter cassettes (biosensors); we present real-time analyses from the first day of life to adulthood. Anaesthetics have been shown to exert both neurotoxic and neuroprotective effects during development and in models of brain injury. Mice subjected to bioimaging after neonatal intracranial or intravascular administration of biosensors, targeting the brain and liver retrospectively showed no significant difference in luciferase expression when conscious or unconscious throughout development. We applied conscious bioimaging to the assessment of NFκB and STAT3 transcription factor activated reporters during the earliest stages of development in living, unrestrained pups. Our data showed unique longitudinal activities for NFκB and STAT3 in the brain of conscious mice. Conscious bioimaging was applied to a neonatal mouse model of cerebral palsy (Hypoxic-Ischaemic Encephalopathy). Imaging of NFκB reporter before and after surgery showed a significant increase in luciferase expression, coinciding with secondary energy failure, in lesioned mice compared to controls.

Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. To further restrict biosensor activity to the CNS, we performed intracerebroventricular injection of each vector. We observed greater restriction of bioluminescence to the head and spine with both vectors. Immunohistochemistry revealed strongest expression in cells of neuronal morphology. LPS administration stimulated a 4-fold increase over baseline bioluminescence. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner using a model of LPS-induced inflammation. This technology employs the 3R’s of biomedical animal research, complements existing germline-transgenic models and may be applicable to other rodent disease models with the use of different response elements.

Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer

Argininosuccinate lyase (ASL) belongs to the hepatic urea cycle detoxifying ammonia, and the citrulline-nitric oxide (NO) cycle producing NO. ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia, multiorgan disease and neurocognitive impairment despite treatment aiming to normalise ammonaemia without considering NO imbalance. Here we show that cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress independent of hyperammonaemia. Intravenous injection of AAV8 vector into adult or neonatal ASL-deficient mice demonstrates long-term correction of the hepatic urea cycle and the cerebral citrulline-NO cycle, respectively. Cerebral disease persists if ammonaemia only is normalised but is dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This correlates with behavioural improvement and reduced cortical cell death. Thus, neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Disease amelioration by simultaneous brain and liver gene transfer with one vector, to treat both metabolic pathways, provides new hope for hepatocerebral metabolic diseases.Patients with mutations in the ASL gene present with argininosuccinic aciduria characterised by hyperammonaemia and cognitive impairment. Here, the authors show that cerebral disease involves neuronal nitrosative/oxidative stress that is not induced by hyperammonaemia, and that it can be reversed using AAV-ASL directed to liver and brain in mice.

Foamy Virus Vectors Transduce Visceral Organs and Hippocampal Structures following In Vivo Delivery to Neonatal Mice

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders.

Ascending Vaginal Infection Using Bioluminescent Bacteria Evokes Intrauterine Inflammation, Preterm Birth, and Neonatal Brain Injury in Pregnant Mice

Preterm birth is a serious global health problem and the leading cause of infant death before 5 years of age. At least 40% of cases are associated with infection. The most common way for pathogens to access the uterine cavity is by ascending from the vagina. Bioluminescent pathogens have revolutionized the understanding of infectious diseases. We hypothesized that bioluminescent Escherichia coli can be used to track and monitor ascending vaginal infections. Two bioluminescent strains were studied: E. coli K12 MG1655-lux, a nonpathogenic laboratory strain, and E. coli K1 A192PP-lux2, a pathogenic strain capable of causing neonatal meningitis and sepsis in neonatal rats. On embryonic day 16, mice received intravaginal E. coli K12, E. coli K1, or phosphate-buffered saline followed by whole-body bioluminescent imaging. In both cases, intravaginal delivery of E. coli K12 or E. coli K1 led to bacterial ascension into the uterine cavity, but only E. coli K1 induced preterm parturition. Intravaginal administration of E. coli K1 significantly reduced the proportion of pups born alive compared with E. coli K12 and phosphate-buffered saline controls. However, in both groups of viable pups born after bacterial inoculation, there was evidence of comparable brain inflammation by postnatal day 6. This study ascribes specific mechanisms by which exposure to intrauterine bacteria leads to premature delivery and neurologic inflammation in neonates.

TBC1D24 Mutations in a Sibship with Multifocal Polymyoclonus

Background Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus. Case Report A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in TBC1D24, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G>A (p.Glu157Lys), and a novel frameshift mutation c.545del (p.Thr182Serfs*6). Discussion We propose that TBC1D24-related diseases should be in the differential diagnosis for children with polymyoclonus.

Novel therapeutic approaches for childhood parkinsonism

Abstract Background Dopamine transporter deficiency syndrome (DTDS) is a primary neurotransmitter disorder caused by loss-of-function mutations in SLC6A3 , which encodes the dopamine transporter (DAT). The syndrome is characterised by progressive infantile-onset dystonia-parkinsonism with raised dopamine metabolite homovanillic acid in the cerebrospinal fluid. There are no disease modifying therapies for this life-limiting disorder. The aims of this study were to evaluate the clinical disease spectrum and develop a novel gene therapy approach for DTDS. Methods Patients aged 1–36 years with childhood-onset dystonia-parkinsonism and suggestive neurotransmitter profile had SLC6A3 sequenced. In-vitro functional studies were undertaken for identified missense mutations. We specifically phenotyped DAT knockout (–/–) mice motor behaviour as a model of DTDS and developed a preclinical viral gene therapy construct. We evaluated effects of intracranial delivery of DAT gene therapy in neonatal DAT–/– mice as a proof of concept study. Findings We identified ten new patients harbouring seven novel missense mutations, and found a novel atypical phenotype of juvenile parkinsonism. In-vitro functional characterisation revealed multifactorial disease mechanisms, including abnormal DAT trafficking, glycosylation, and impaired substrate recognition and uptake function. The DAT–/– mouse clearly recapitulated many features of human disease, including reduced survival, early hyperkinesia with later parkinsonism, raised homovanillic acid concentrations, and neurodegeneration. We have evaluated viral gene therapy approaches to target dopaminergic neurons and subsequently delivered DAT via adeno-associated virus type 9 to neonatal DAT–/– mice, with improved survival rates and motor phenotype. Interpretation We report an expanding disease spectrum in DTDS, in which the clinical presentation mimics both cerebral palsy and juvenile parkinsonism. Genotype–phenotype correlation is evident, with in-vitro functional studies demonstrating greater residual DAT function in later-onset milder forms of disease. The preclinical study of viral gene therapy in neonatal mice is promising and will facilitate the longer term aim towards clinical translation for this untreatable disorder. Funding Medical Research Council Clinical Research Training Fellowship, Great Ormond Street Hospital Childrens Charity.

305. Generation of Light-Producing, Somatic-Transgenic Mice Using Lentivirus and Adeno-Associated Virus Vectors

Germ line light producing transgenic mice, where luciferase expression is controlled by a surrogate promoter or by a minimal promoter downstream of tandem, synthetic, transcription factor binding elements, are used to provide an in vivo readout of disease processes. However, as every cell within the organism contains the luciferase reporter gene, it is therefore not specific to individual organs. We have developed a novel technology for the generation of light emitting somatic transgenic animals using lentiviral vectors. This allows signalling pathways in diseased organs to be monitored specifically, continually and in a non-invasive manner [1]. In this study, we aimed to deliver NFkB driving a luciferase reporter constructs to the nervous system of neonatal mice to generate somatic-transgenic mice using both lentivirus and adeno-associated viral (AAV) vectors. Lentivirus vector pseudotyped with VSV-G viral envelope glycoproteins or AAV8 serotyped vector carrying an NFkB response element was injected intracranially or intravenously to outbred CD1 neonatal (P1) mice and luciferase expression was monitored continually by whole body bioluminescence imaging of conscious mice. The ability to image conscious mice holds advantages when studying neuropathology. After weaning, pathological activation of NFKB was induced by intraperitoneal injection of lipopolysaccharide (LPS). Following intracranial injection, the VSV-G lentivirus NFkB biosensor showed transduction of the brain and spinal cord. Luciferase expression was upregulated 24 hours after administration of LPS. Immunohistochemistry revealed only modest spread throughout the brain. Conversely, intracranial injection of AAV8 NFkB biosensor showed a much wider spread and increased luciferase expression. Finally we administered AAV8 NFkB biosensor intravenously at P1. Whereas previous studies show AAV8 serotype transduces many systemic tissues [2] through this route; we observed luciferase expression predominantly in the brain and spine (see figurefigure). Using a standard Gateway® cloning system we have established a library of more than 25 lentivirus biosensors where some have been tested in vitro and in vivo. We plan on incorporating these response elements into the AAV backbone described above. Therefore, enabling the generation of somatic-transgenic mice which have a wider spread of AAV biosensor. This will complement existing germ line transgenic, light producing technology by maximising the use, and reducing the numbers, of animals used in biomedical research.View Large Image | Download PowerPoint Slide1. Buckley, SMK et al. 2015. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters. Scientific Reports. 2. Inagaki, K et al. 2008. Frequency and spectrum of genomic integration of recombinant AAV serotype 8 vector in neonatal mouse liver. Journal of virology.