Tandem mass spectrometry-based assay for heparan-N-sulphatase in paediatric CSF: A potential pharmacodynamic biomarker for mucopolysaccharidosis type IIIA therapy.

Mucopolysaccharidosis type IIIA is a lysosomal storage disorder caused by mutations in the gene coding for heparan-N-sulphatase, a crucial enzyme in the degradation of heparan sulfate. In mucopolysaccharidosis type IIIA, heparan sulfate accumulates in the lysosomes, predominantly affecting the central nervous system. It is the most common and most severe form of mucopolysaccharidosis type III, with onset typically before the age of ten years. There is an ongoing effort to develop therapies that aim at restoring enzyme function in the brain. This study introduces a novel tandem mass spectrometry method for assessing heparan-N-sulphatase activity in pediatric cerebrospinal fluid from healthy and disease individuals. Analysis of cerebrospinal fluid samples revealed marked differences in enzyme activity, with mucopolysaccharidosis type IIIA individuals exhibiting significantly reduced levels. This new method could serve as a valuable tool for evaluating the efficacy of future therapeutic interventions targeting sulphatase activity restoration in the brain.

Design of a stable human acid-ß-glucosidase: towards improved Gaucher disease therapy and mutation classification.

Acid-ß-glucosidase (GCase, EC3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme® ) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step toward developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild-type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine learning-based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single-nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.

Focal lesions following intracerebral gene therapy for mucopolysaccharidosis IIIA.

OBJECTIVE: Mucopolysaccharidosis type IIIA (MPSIIIA) caused by recessive SGSH variants results in sulfamidase deficiency, leading to neurocognitive decline and death. No disease-modifying therapy is available. The AAVance gene therapy trial investigates AAVrh.10 overexpressing human sulfamidase (LYS-SAF302) delivered by intracerebral injection in children with MPSIIIA. Post-treatment MRI monitoring revealed lesions around injection sites. Investigations were initiated in one patient to determine the cause. METHODS: Clinical and MRI details were reviewed. Stereotactic needle biopsies of a lesion were performed; blood and CSF were sampled. All samples were used for viral studies. Immunohistochemistry, electron microscopy, and transcriptome analysis were performed on brain tissue of the patient and various controls. RESULTS: MRI revealed focal lesions around injection sites with onset from 3 months after therapy, progression until 7 months post therapy with subsequent stabilization and some regression. The patient had transient slight neurological signs and is following near-normal development. No evidence of viral or immunological/inflammatory cause was found. Immunohistochemistry showed immature oligodendrocytes and astrocytes, oligodendrocyte apoptosis, strong intracellular and extracellular sulfamidase expression and hardly detectable intracellular or extracellular heparan sulfate. No activation of the unfolded protein response was found. INTERPRETATION: Results suggest that intracerebral gene therapy with local sulfamidase overexpression leads to dysfunction of transduced cells close to injection sites, with extracellular spilling of lysosomal enzymes. This alters extracellular matrix composition, depletes heparan sulfate, impairs astrocyte and oligodendrocyte function, and causes cystic white matter degeneration at the site of highest gene expression. The AAVance trial results will reveal the potential benefit-risk ratio of this therapy.

AAV-delivered diacylglycerol kinase DGKk achieves long-term rescue of fragile X syndrome mouse model.

Fragile X syndrome (FXS) is the most frequent form of familial intellectual disability. FXS results from the lack of the RNA-binding protein FMRP and is associated with the deregulation of signaling pathways downstream of mGluRI receptors and upstream of mRNA translation. We previously found that diacylglycerol kinase kappa (DGKk), a main mRNA target of FMRP in cortical neurons and a master regulator of lipid signaling, is downregulated in the absence of FMRP in the brain of Fmr1-KO mouse model. Here we show that adeno-associated viral vector delivery of a modified and FMRP-independent form of DGKk corrects abnormal cerebral diacylglycerol/phosphatidic acid homeostasis and FXS-relevant behavioral phenotypes in the Fmr1-KO mouse. Our data suggest that DGKk is an important factor in FXS pathogenesis and provide preclinical proof of concept that its replacement could be a viable therapeutic strategy in FXS.

AAVrh10 vector corrects pathology in animal models of GM1 gangliosidosis and achieves widespread distribution in the CNS of nonhuman primates.

GM1 gangliosidosis is a rare, inherited neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes the lysosomal hydrolase acid ß-galactosidase (ß-gal). ß-gal deficiency leads to toxic accumulation of GM1 ganglioside, predominantly in the central nervous system (CNS), resulting in progressive neurodegeneration. LYS-GM101 is an AAVrh.10-based gene therapy vector carrying the human GLB1 cDNA. The efficacy of intra-cerebrospinal fluid injection of LYS-GM101 analogs was demonstrated in GM1 mouse and cat models with widespread diffusion of ß-gal and correction of GM1 ganglioside accumulation in the CNS without observable adverse effects. Clinical dose selection was performed, based on a good-laboratory-practice study, in nonhuman primates (NHPs) using the clinical LYS-GM101 vector. A broadly distributed increase of ß-gal activity was observed in NHP brain 3 months after intra-cisterna magna injection of LYS-GM101 at 1.0 × 1012 vg/mL CSF and 4.0 × 1012 vg/mL CSF, with 20% and 60% increases compared with vehicle-treated animals, respectively. Histopathologic examination revealed asymptomatic adverse changes in the sensory pathways of the spinal cord and dorsal root ganglia in both sexes and at both doses. Taken as a whole, these pre-clinical data support the initiation of a clinical study with LYS-GM101 for the treatment of GM1 gangliosidosis.

Storage vesicles in neurons are related to Golgi complex alterations in mucopolysaccharidosis IIIB.

The accumulation of intracellular storage vesicles is a hallmark of lysosomal storage diseases. Neither the identity nor origin of these implicated storage vesicles have yet been established. The vesicles are often considered as lysosomes, endosomes, and/or autophagosomes that are engorged with undigested materials. Our studies in the mouse model of mucopolysaccharidosis type IIIB, a lysosomal storage disease that induces neurodegeneration, showed that large storage vesicles in cortical neurons did not receive material from either the endocytic or autophagy pathway, which functioned normally. Storage vesicles expressed GM130, a Golgi matrix protein, which mediates vesicle tethering in both pre- and cis-Golgi compartments. However, other components of the tethering/fusion complex were not associated with GM130 on storage vesicles, likely accounting for both the resistance of the vesicles to brefeldin A and the alteration of Golgi ribbon architecture, which comprised distended cisterna connected to LAMP1-positive storage vesicles. We propose that alteration in the GM130-mediated control of vesicle trafficking in pre-Golgi and Golgi compartments affects Golgi biogenesis and gives rise to a dead-end storage compartment. Vesicle accumulation, Golgi disorganization, and alterations of other GM130 functions may account for neuron dysfunction and death.

GAP43 overexpression and enhanced neurite outgrowth in mucopolysaccharidosis type IIIB cortical neuron cultures.

Behavioral manifestations mark the onset of disease expression in children with mucopolysaccharidosis type III (MPSIII, Sanfilippo syndrome), a genetic disorder resulting from interruption of the lysosomal degradation of heparan sulfate. In the mouse model of MPSIII type B (MPSIIIB), cortical neuron pathology and dysfunction occur several months before neuronal loss and are primarily cell autonomous. The gene coding for GAP43, a neurite growth potentiator, is overexpressed in the MPSIIIB mouse cortex, and neurite dystrophy was reported in other types of lysosomal storage diseases. We therefore examined the development of the neuritic trees in pure populations of MPSIIIB mouse embryo cortical neurons grown for up to 12 days in primary culture. Dynamic observation of living neurons and quantification of neurite growth parameters indicated more frequent neurite elongation and branching and less frequent neurite retraction, resulting in a relative overgrowth of MPSIIIB neuron neuritic trees, involving both dendrites and axons, compared with normal controls. Neurite overgrowth was concomitant with more than twofold increased expression of GAP43 mRNAs and proteins. Correction of the genetic defect leads to expression of the missing lysosomal enzyme, normal GAP43 mRNA expression, and reduced neurite outgrowth. These results indicate that heparan sulfate oligosaccharide storage modifies GAP43 expression in MPSIIIB cortical neurons with potential consequences for neurite development and neuronal functions that may be relevant to clinical manifestations.

Enhanced degradation of synaptophysin by the proteasome in mucopolysaccharidosis type IIIB.

The interruption of the lysosomal degradation of heparan sulfate oligosaccharides has deleterious consequences on the central nervous system in children or in animals with mucopolysaccharidosis type III (Sanfilippo syndrome). Behavioural manifestations are prominent at disease onset, suggesting possible early synaptic defects in cortical neurons. We report that synaptophysin, the most abundant protein of the synaptic vesicle membrane, was detected at low levels in the rostral cortex of MPSIII type B mice as early as 10 days after birth. This defect preceded other disease manifestations, was associated with normal neuron and synapse density and corrected after gene transfer inducing re-expression of the missing lysosomal enzyme. Clearance of heparan sulfate oligosaccharides in cultured embryonic MPSIIIB cortical neurons or treatment with proteasome inhibitors restored normal synaptophysin levels indicating that heparan sulfate oligosaccharides activate the degradation of synaptophysin by the proteasome with consequences on synaptic vesicle components that are relevant to clinical manifestations.

AAVrh10 Vector Corrects Disease Pathology in MPS IIIA Mice and Achieves Widespread Distribution of SGSH in Large Animal Brains.

Patients with mucopolysaccharidosis type IIIA (MPS IIIA) lack the lysosomal enzyme sulfamidase (SGSH), which is responsible for the degradation of heparan sulfate (HS). Build-up of undegraded HS results in severe progressive neurodegeneration for which there is currently no treatment. The ability of the vector adeno-associated virus (AAV)rh.10-CAG-SGSH (LYS-SAF302) to correct disease pathology was evaluated in a mouse model for MPS IIIA. LYS-SAF302 was administered to 5-week-old MPS IIIA mice at three different doses (8.6E+08, 4.1E+10, and 9.0E+10 vector genomes [vg]/animal) injected into the caudate putamen/striatum and thalamus. LYS-SAF302 was able to dose-dependently correct or significantly reduce HS storage, secondary accumulation of GM2 and GM3 gangliosides, ubiquitin-reactive axonal spheroid lesions, lysosomal expansion, and neuroinflammation at 12 weeks and 25 weeks post-dosing. To study SGSH distribution in the brain of large animals, LYS-SAF302 was injected into the subcortical white matter of dogs (1.0E+12 or 2.0E+12 vg/animal) and cynomolgus monkeys (7.2E+11 vg/animal). Increases of SGSH enzyme activity of at least 20% above endogenous levels were detected in 78% (dogs 4 weeks after injection) and 97% (monkeys 6 weeks after injection) of the total brain volume. Taken together, these data validate intraparenchymal AAV administration as a promising method to achieve widespread enzyme distribution and correction of disease pathology in MPS IIIA.

Directed evolution of motor neurons from genetically engineered neural precursors.

Stem cell-based therapies hold therapeutic promise for degenerative motor neuron diseases, such as amyotrophic lateral sclerosis, and for spinal cord injury. Fetal neural progenitors present less risk of tumor formation than embryonic stem cells but inefficiently differentiate into motor neurons, in line with their low expression of motor neuron-specific transcription factors and poor response to soluble external factors. To overcome this limitation, we genetically engineered fetal rat spinal cord neurospheres to express the transcription factors HB9, Nkx6.1, and Neurogenin2. Enforced expression of the three factors rendered neural precursors responsive to Sonic hedgehog and retinoic acid and directed their differentiation into cholinergic motor neurons that projected axons and formed contacts with cocultured myotubes. When transplanted in the injured adult rat spinal cord, a model of acute motor neuron degeneration, the engineered precursors transiently proliferated, colonized the ventral horn, expressed motor neuron-specific differentiation markers, and projected cholinergic axons in the ventral root. We conclude that genetic engineering can drive the differentiation of fetal neural precursors into motor neurons that efficiently engraft in the spinal cord. The strategy thus holds promise for cell replacement in motor neuron and related diseases. Disclosure of potential conflicts of interest is found at the end of this article.

An Improved Adeno-Associated Virus Vector for Neurological Correction of the Mouse Model of Mucopolysaccharidosis IIIA.

Patients with the lysosomal storage disease mucopolysaccharidosis IIIA (MPSIIIA) lack the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), one of the many enzymes involved in degradation of heparan sulfate. Build-up of un-degraded heparan sulfate results in severe progressive neurodegeneration for which there is currently no treatment. Experimental gene therapies based on gene addition are currently being explored. Following preclinical evaluation in MPSIIIA mice, an adeno-associated virus vector of serotype rh10 designed to deliver SGSH and sulfatase modifying factor 1 (SAF301) was trialed in four MPSIIIA patients, showing good tolerance and absence of adverse events with some improvements in neurocognitive measures. This study aimed to improve SAF301 further by removing sulfatase modifying factor 1 (SUMF1) and assessing if expression of this gene is needed to increase the SGSH enzyme activity (SAF301b). Second, the murine phosphoglycerate kinase (PGK) promotor was exchanged with a chicken beta actin/CMV composite (CAG) promotor (SAF302) to see if SGSH expression levels could be boosted further. The three different vectors were administered to MPSIIIA mice via intracranial injection, and SGSH expression levels were compared 4 weeks post treatment. Removal of SUMF1 resulted in marginal reductions in enzyme activity. However, promotor exchange significantly increased the amount of SGSH expressed in the brain, leading to superior therapeutic correction with SAF302. Biodistribution of SAF302 was further assessed using green fluorescent protein (GFP), indicating that vector spread was limited to the area around the injection tract. Further modification of the injection strategy to a single depth with higher injection volume increased vector distribution, leading to more widespread GFP distribution and sustained expression, suggesting this approach should be adopted in future trials.

Adeno-Associated Virus-Based Gene Therapy for CNS Diseases.

Gene therapy is at the cusp of a revolution for treating a large spectrum of CNS disorders by providing a durable therapeutic protein via a single administration. Adeno-associated virus (AAV)-mediated gene transfer is of particular interest as a therapeutic tool because of its safety profile and efficiency in transducing a wide range of cell types. The purpose of this review is to describe the most notable advancements in preclinical and clinical research on AAV-based CNS gene therapy and to discuss prospects for future development based on a new generation of vectors and delivery.

Intracerebral administration of adeno-associated viral vector serotype rh.10 carrying human SGSH and SUMF1 cDNAs in children with mucopolysaccharidosis type IIIA disease: results of a phase I/II trial.

Mucopolysaccharidosis type IIIA is a severe degenerative disease caused by an autosomal recessive defect of a gene encoding a lysosomal heparan-N-sulfamidase, the N-sulfoglycosamine sulfohydrolase (SGSH), the catalytic site of which is activated by a sulfatase-modifying factor (SUMF1). Four children (Patients 1-3, aged between 5.5 and 6 years; Patient 4 aged 2 years 8 months) received intracerebral injections of an adeno-associated viral vector serotype rh.10-SGSH-IRES-SUMF1 vector in a phase I/II clinical trial. All children were able to walk, but their cognitive abilities were abnormal and had declined (Patients 1-3). Patients 1-3 presented with brain atrophy. The therapeutic vector was delivered in a frameless stereotaxic device, at a dose of 7.2×10(11) viral genomes/patient simultaneously via 12 needles as deposits of 60 µl over a period of 2 hr. The vector was delivered bilaterally to the white matter anterior, medial, and posterior to the basal ganglia. Immunosuppressive treatment (mycophenolate mofetil and tacrolimus) was initiated 15 days before surgery and maintained for 8 weeks (mycophenolate mofetil) or throughout follow-up (tacrolimus, with progressive dose reduction) to prevent elimination of transduced cells. Safety data collected from inclusion, during the neurosurgery period and over the year of follow-up, showed good tolerance, absence of adverse events related to the injected product, no increase in the number of infectious events, and no biological sign of toxicity related to immunosuppressive drugs. Efficacy analysis was necessarily preliminary in this phase I/II trial on four children, in the absence of validated surrogate markers. Brain atrophy evaluated by magnetic resonance imaging seemed to be stable in Patients 1 and 3 but tended to increase in Patients 2 and 4. Neuropsychological evaluations suggested a possible although moderate improvement in behavior, attention, and sleep in Patients 1-3. The youngest patient was the most likely to display neurocognitive benefit.

alpha-L-Iduronidase transport in neurites.

Effective therapeutic strategies for mucopolysaccharidosis type I (MPSI) rely on mannose-6-phosphate receptor-mediated uptake of extracellular alpha-l-iduronidase (IDUA), the missing lysosomal enzyme in this disease, by deficient cells. Intravenously infused recombinant human IDUA does not reach the central nervous system, whereas neuropathology and neurological manifestations are prominent in Hurler syndrome, the most severe and most frequent form of MPSI. The creation of a single intracerebral source of IDUA by gene therapy was proved efficient to deliver enzyme throughout the brain of MPSI mice. IDUA spreading far beyond areas where the enzyme was synthesized suggested transport along neuronal processes. To examine the mechanisms of IDUA spreading in the brain, we constructed a chimeric protein in which GFP is fused at the C-terminus of IDUA. The fusion protein was expressed in rat primary neurons using lentivirus vectors. Fluorescent IDUA retained full catalytic activity including on natural substrates, interacted with mannose-6-phosphate receptors and was appropriately addressed to lysosomes. Fluorescent vesicles were broadly distributed over neuronal soma and processes. Time-lapse fluorescent video-microscopy showed that 54% of fluorescent vesicles exhibited either retrograde or anterograde displacements along neurites. Most moving organelles showed complex movements with frequent direction changes and arrests. Motility depended on microtubule integrity. Efficient axono-dendritic transport of IDUA provides a rationale for gene therapy based on the release of therapeutic enzyme at discrete locations within the central nervous system of patients with severe form of MPSI.

Forced expression of the motor neuron determinant HB9 in neural stem cells affects neurogenesis.

In contrast to mouse embryonic stem cells and in spite of overlapping gene expression profiles, neural stem cells (NSCs) isolated from the embryonic spinal cord do not respond to physiological morphogenetic stimuli provided by Sonic hedgehog and retinoic acid and do not generate motor neurons upon differentiation. Transcription factors expressed in motor neuron progenitors during embryogenesis include Pax6, Ngn2, Nkx6.1 and Olig2, whose expression precedes that of factors specifying motor neuron fate, including HB9, Islet1 and LIM3. We showed that all these factors were present in neural progenitors derived from mouse ES cells, whereas NSCs derived from the rat embryonic spinal cord expressed neither HB9 nor Islet1 and contained low levels of Nkx6.1 and LIM3. We constructed a lentivirus vector to express HB9 and GFP in NSCs and examined the consequences of HB9 expression on other transcription factors and cell differentiation. Compared to cell expressing GFP alone, NSCs expressing GFP and HB9 cycled less rapidly, downregulated Pax6 and Ngn2 mRNA levels, produced higher proportions of neurons in vitro and lower numbers of neurons after transplantation in the spinal cord of recipient rats. Oligodendrocytic and astrocytic differentiations were not affected. HB9 expressing NSCs did not express Islet1 or upregulate LIM3. They neither responded to Sonic hedgehog and retinoic acid nor produced cholinergic neurons. We concluded that forced HB9 expression affected neurogenesis but was not sufficient to confer motor neuron fate to NSCs.