Safety and efficacy of an engineered hepatotropic AAV gene therapy for ornithine transcarbamylase deficiency in cynomolgus monkeys.

X-linked inherited ornithine transcarbamylase deficiency (OTCD) is the most common disorder affecting the liver-based urea cycle, a pathway enabling detoxification of nitrogen waste and endogenous arginine biosynthesis. Patients develop acute hyperammonemia leading to neurological sequelae or death despite the best-accepted therapy based on ammonia scavengers and protein-restricted diet. Liver transplantation is curative but associated with procedure-related complications and lifelong immunosuppression. Adeno-associated viral (AAV) vectors have demonstrated safety and clinical benefits in a rapidly growing number of clinical trials for inherited metabolic liver diseases. Engineered AAV capsids have shown promising enhanced liver tropism. Here, we conducted a good-laboratory practice-compliant investigational new drug-enabling study to assess the safety of intravenous liver-tropic AAVLK03 gene transfer of a human codon-optimized OTC gene. Juvenile cynomolgus monkeys received vehicle and a low and high dose of vector (2 × 1012 and 2 × 1013 vector genome (vg)/kg, respectively) and were monitored for 26 weeks for in-life safety with sequential liver biopsies at 1 and 13 weeks post-vector administration. Upon completion of monitoring, animals were euthanized to study vector biodistribution, immune responses, and histopathology. The product was well tolerated with no adverse clinical events, predominant hepatic biodistribution, and sustained supra-physiological OTC overexpression. This study supports the clinical deployment of intravenous AAVLK03 for severe OTCD.

Lentiviral Hematopoietic Stem Cell Gene Therapy Rescues Clinical Phenotypes in a Murine Model of Pompe Disease.

Pompe disease is a lysosomal storage disorder caused by malfunctions of the acid alpha-glucosidase (GAA) enzyme with a consequent toxic accumulation of glycogen in cells. Muscle wasting and hypertrophic cardiomyopathy are the most common clinical signs that can lead to cardiac and respiratory failure within the first year of age in the more severe infantile forms. Currently available treatments have significant limitations and are not curative, highlighting a need for the development of alternative therapies. In this study, we investigated the use of a clinically relevant lentiviral vector to deliver systemically GAA through genetic modification of hematopoietic stem and progenitor cells (HSPCs). The overexpression of GAA in human HSPCs did not exert any toxic effect on this cell population, which conserved its stem cell capacity in xenograft experiments. In a murine model of Pompe disease treated at young age, we observed phenotypic correction of heart and muscle function with a significant reduction of glycogen accumulation in tissues after 6 months of treatment. These findings suggest that lentiviral-mediated HSPC gene therapy can be a safe alternative therapy for Pompe disease.

Inhibition of Mitochondrial Complex I Impairs Release of α-Galactosidase by Jurkat Cells.

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.

Vitamin D intake, calcium intake and physical activity among children with wrist and ankle injuries and the association with fracture risk.

BACKGROUND: Several studies have revealed a substantial increase in the incidence of fractures in children in the past few decades. AIM: To assess the strength of the association between suggested risk factors and fracture prevalence in children. METHOD: A cross sectional observational study. Children aged 6-15 years and their guardians presenting to the Emergency Department of a single tertiary paediatric hospital were recruited. Self-reported data on vitamin D intake, calcium intake and physical activity were collected. All participants had a radiograph of their injured limb reported by a consultant radiologist, on the basis of which they were classified into fracture or no fracture groups. Statistical analysis included descriptive statistics and binary logistic regression. RESULTS: Of the 130 patients recruited, 53 (41%) had sustained a fracture. The overwhelming majority of children (98%) did not consume the recommended daily dietary amount of vitamin D (400 IU/day). Low calcium intake and low levels of physical activity were also ascertained. However, there were no significant differences between fracture and no fracture groups for vitamin D intake, calcium intake or physical activity. Both site of injury (wrist) and sex (male) were associated with increased fracture risk ( p = 0.001 and p = 0.05, respectively). Logistic regression showed a statistically significant relationship between calcium intake and fracture risk (every additional unit of calcium consumption (mg/day) decreased the likelihood of fracture by 0.002, 95% confidence interval, 0.001-0.003). CONCLUSIONS: Low dietary intake of calcium and vitamin D and low levels of physical activity were evident. Fracture risk was significantly associated with reduced calcium intake but showed no association with vitamin D intake or physical activity.

Fetal gene therapy for neurodegenerative disease of infants.

For inherited genetic diseases, fetal gene therapy offers the potential of prophylaxis against early, irreversible and lethal pathological change. To explore this, we studied neuronopathic Gaucher disease (nGD), caused by mutations in GBA. In adult patients, the milder form presents with hepatomegaly, splenomegaly and occasional lung and bone disease; this is managed, symptomatically, by enzyme replacement therapy. The acute childhood lethal form of nGD is untreatable since enzyme cannot cross the blood-brain barrier. Patients with nGD exhibit signs consistent with hindbrain neurodegeneration, including neck hyperextension, strabismus and, often, fatal apnea1. We selected a mouse model of nGD carrying a loxP-flanked neomycin disruption of Gba plus Cre recombinase regulated by the keratinocyte-specific K14 promoter. Exclusive skin expression of Gba prevents fatal neonatal dehydration. Instead, mice develop fatal neurodegeneration within 15 days2. Using this model, fetal intracranial injection of adeno-associated virus (AAV) vector reconstituted neuronal glucocerebrosidase expression. Mice lived for up to at least 18 weeks, were fertile and fully mobile. Neurodegeneration was abolished and neuroinflammation ameliorated. Neonatal intervention also rescued mice but less effectively. As the next step to clinical translation, we also demonstrated the feasibility of ultrasound-guided global AAV gene transfer to fetal macaque brains.

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.

Inhibition of neuronal mitochondrial complex I or lysosomal glucocerebrosidase is associated with increased dopamine and serotonin turnover.

Parkinson's disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic and serotoninergic signalling. A number of pathogenic mechanisms have been implicated including loss of mitochondrial function at the level of complex I, and lysosomal metabolism at the level of lysosomal glucocerebrosidase (GBA1). In order to investigate further the potential involvement of complex I and GBA1 in PD, we assessed the impact of loss of respective enzyme activities upon dopamine and serotonin turnover. Using SH-SY5Y cells, complex I deficiency was modelled by using rotenone whilst GBA1 deficiency was modelled by the use of conduritol B epoxide (CBE). Dopamine, its principal metabolites, and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the extracellular medium were quantified by HPLC. Inhibition of complex I significantly increased extracellular concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-HIAA. Comparable results were observed with CBE. These results suggest increased monoamine oxidase activity and provide evidence for involvement of impaired complex I or GBA1 activity in the dopamine/serotonin deficiency seen in PD. Use of extracellular media may also permit relatively rapid assessment of dopamine/serotonin metabolism and permit screening of novel therapeutic agents.

ARNT2 mutation causes hypopituitarism, post-natal microcephaly, visual and renal anomalies.

We describe a previously unreported syndrome characterized by secondary (post-natal) microcephaly with fronto-temporal lobe hypoplasia, multiple pituitary hormone deficiency, seizures, severe visual impairment and abnormalities of the kidneys and urinary tract in a highly consanguineous family with six affected children. Homozygosity mapping and exome sequencing revealed a novel homozygous frameshift mutation in the basic helix-loop-helix transcription factor gene ARNT2 (c.1373_1374dupTC) in affected individuals. This mutation results in absence of detectable levels of ARNT2 transcript and protein from patient fibroblasts compared with controls, consistent with nonsense-mediated decay of the mutant transcript and loss of ARNT2 function. We also show expression of ARNT2 within the central nervous system, including the hypothalamus, as well as the renal tract during human embryonic development. The progressive neurological abnormalities, congenital hypopituitarism and post-retinal visual pathway dysfunction in affected individuals demonstrates for the first time the essential role of ARNT2 in the development of the hypothalamo-pituitary axis, post-natal brain growth, and visual and renal function in humans.

Glucocerebrosidase deficiency in substantia nigra of parkinson disease brains.

OBJECTIVE: Mutations in the glucocerebrosidase gene (GBA) represent a significant risk factor for developing Parkinson disease (PD). We investigated the enzymatic activity of glucocerebrosidase (GCase) in PD brains carrying heterozygote GBA mutations (PD+GBA) and sporadic PD brains. METHODS: GCase activity was measured using a fluorescent assay in cerebellum, frontal cortex, putamen, amygdala, and substantia nigra of PD+GBA (n = 9-14) and sporadic PD brains (n = 12-14). Protein expression of GCase and other lysosomal proteins was determined by western blotting. The relation between GCase, α-synuclein, and mitochondria function was also investigated in vitro. RESULTS: A significant decrease in GCase activity was observed in all PD+GBA brain areas except the frontal cortex. The greatest deficiency was in the substantia nigra (58% decrease; p < 0.01). GCase activity was also significantly decreased in the substantia nigra (33% decrease; p < 0.05) and cerebellum (24% decrease; p < 0.05) of sporadic PD brains. GCase protein expression was lower in PD+GBA and PD brains, whereas increased C/EBP homologous protein and binding immunoglobulin protein levels indicated that the unfolded protein response was activated. Elevated α-synuclein levels or PTEN-induced putative kinase 1 deficiency in cultured cells had a significant effect on GCase protein levels. INTERPRETATION: GCase deficiency in PD brains with GBA mutations is a combination of decreased catalytic activity and reduced protein levels. This is most pronounced in the substantia nigra. Biochemical changes involved in PD pathogenesis affect wild-type GCase protein expression in vitro, and these could be contributing factors to the GCase deficiency observed in sporadic PD brains.