Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.

The GM2 gangliosidoses are fatal lysosomal storage diseases principally affecting the brain. Absence of ß-hexosaminidase A and B activities in the Sandhoff mouse causes neurological dysfunction and recapitulates the acute Tay-Sachs (TSD) and Sandhoff diseases (SD) in infants. Intracranial coinjection of recombinant adeno-associated viral vectors (rAAV), serotype 2/1, expressing human ß-hexosaminidase α (HEXA) and ß (HEXB) subunits into 1-month-old Sandhoff mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity-as opposed to tremor-ataxia-were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of ß-hexosaminidase isozymes and their global distribution via axonal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue-long-term protein expression by transduced brain parenchyma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective enzyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by intracranial vector delivery. We contend that infusion of rAAV into CSF space and intraparenchymal administration by convection-enhanced delivery at a few strategic sites will optimally treat neurodegeneration in many diseases affecting the nervous system.

Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis.

OBJECTIVE: Novel recombinant human lysosomal ß-hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency. METHODS: A recombinant human HexA (Om4HexA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb⁻/⁻ mice) at different doses (0.5-2.5 mg/kg), and then the replacement and therapeutic effects were examined. RESULTS: The Om4HexA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (< 63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. INTERPRETATION: This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases.

Neurodegenerative lysosomal storage disease in European Burmese cats with hexosaminidase beta-subunit deficiency.

GM2 gangliosidosis is a fatal, progressive neuronopathic lysosomal storage disease resulting from a deficiency of beta-N-acetylhexosaminidase (EC 3.2.1.52) activity. GM2 gangliosidosis occurs with varying degrees of severity in humans and in a variety of animals, including cats. In the current research, European Burmese cats presented with clinical neurological signs and histopathological features typical of a lysosomal storage disease. Thin layer chromatography revealed substantial storage of GM2 ganglioside in brain tissue of affected cats, and assays with a synthetic fluorogenic substrate confirmed the absence of hexosaminidase activity. When the hexosaminidase beta-subunit cDNA was sequenced from affected cats, a 91 base pair deletion constituting the entirety of exon 12 was documented. Subsequent sequencing of introns 11 and 12 revealed a 15 base pair deletion at the 3' end of intron 11 that included the preferred splice acceptor site, generating two minor transcripts from cryptic splice acceptor sites in affected Burmese cats. In the cerebral cortex of affected cats, hexosaminidase beta-subunit mRNA levels were approximately 1.5 times higher than normal (P<0.001), while beta-subunit protein levels were substantially reduced on Western blots.

Lending a helping hand, screening chemical libraries for compounds that enhance beta-hexosaminidase A activity in GM2 gangliosidosis cells.

Enzyme enhancement therapy is an emerging therapeutic approach that has the potential to treat many genetic diseases. Candidate diseases are those associated with a mutant protein that has difficulty folding and/or assembling into active oligomers in the endoplasmic reticulum. Many lysosomal storage diseases are candidates for enzyme enhancement therapy and have the additional advantage of requiring only 5-10% of normal enzyme levels to reduce and/or prevent substrate accumulation. Our long experience in working with the beta-hexosaminidase (EC 3.2.1.52) isozymes system and its associated deficiencies (Tay-Sachs and Sandhoff disease) lead us to search for possible enzyme enhancement therapy-agents that could treat the chronic forms of these diseases which express 2-5% residual activity. Pharmacological chaperones are enzyme enhancement therapy-agents that are competitive inhibitors of the target enzyme. Each of the known beta-hexosaminidase inhibitors (low microm IC50) increased mutant enzyme levels to >or= 10% in chronic Tay-Sachs fibroblasts and also attenuated the thermo-denaturation of beta-hexosaminidase. To expand the repertoire of pharmacological chaperones to more 'drug-like' compounds, we screened the Maybridge library of 50,000 compounds using a real-time assay for noncarbohydrate-based beta-hexosaminidase inhibitors and identified several that functioned as pharmacological chaperones in patient cells. Two of these inhibitors had derivatives that had been tested in humans for other purposes. These observations lead us to screen the NINDS library of 1040 Food and Drug Administration approved compounds for pharmacological chaperones. Pyrimethamine, an antimalarial drug with well documented pharmacokinetics, was confirmed as a beta-hexosaminidase pharmacological chaperone and compared favorably with our best carbohydrate-based pharmacological chaperone in patient cells with various mutant genotypes.

Biochemical characterization of the GM2 gangliosidosis B1 variant.

The deficiency of the A isoenzyme of beta-hexosaminidase (Hex) produced by different mutations of the gene that codes for the alpha subunit (Tay-Sachs disease) has two variants with enzymological differences: the B variant consists of the absence of Hex A isoenzyme and the B1 variant produces an inactive Hex A isoenzyme for the hydrolysis of the GM2 ganglioside and synthetic substrates with negative charge. In contrast to the early childhood form of the B variant, the B1 variant appears at a later clinical stage (3 to 7 years of age) with neurodegenerative symptoms leading to the death of the patient in the second decade of life. The most frequent mutation responsible for the GM2 gangliosidosis B1 variant is R178H, which has a widespread geographic and ethnic distribution. The highest incidence has been described in Portugal, which has been suggested as the point of origin of this mutation. Biochemical characterization of this lysosomal disease is carried out using negatively charged synthetic alpha subunit-specific sulfated substrates, since Hex A isoenzyme heat-inactivation assays are not applicable. However, the determination of the apparent activation energy of Hex using the neutral substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide, may offer a valid alternative. The presence of an alpha subunit in the alphabeta heterodimer Hex A means that its activation energy (41.8 kJ/mol) is significantly lower than that of the betabeta homodimer Hex B (75.1 kJ/mol); however, as mutation inactivates the alpha subunit, the Hex A of the B1 variant presents an activation energy that is similar to that of the Hex B isoenzyme.

Serum hexosaminidase and beta-glucuronidase activities in infants: effects of age and sex.

We investigated the effect of age and sex on the serum activity of hexosaminidase (HEX) and -glucuronidase (BGLU) in 275 normal term infants aged 12 h to 12 months. Up to six weeks of life, HEX was significantly higher in boys (P<=0.023). During the age period of 1-26 weeks, BGLU was also higher in boys, but differences were significant only at 2-6 and 7-15 weeks (P<=0.016). The developmental pattern of HEX and BGLU was sex dependent. HEX activity increased in both sexes from 4-7 days of life, reaching a maximum of 1.4-fold the birth value at 2-6 weeks of age in boys (P<0.001) and a maximum of 1.6-fold at 7-15 weeks in girls (P<0.001). HEX activity gradually decreased thereafter, reaching significantly lower levels at 27-53 weeks than during the first three days of life in boys (P = 0.002) and the same level of this age interval in girls. BGLU increased in both sexes from 4-7 days of age, showing a maximum increase at 7-15 weeks (3.3-fold in boys and 2.9-fold in girls, both P<0.001). Then BGLU decreased in boys to a value similar to that observed at 4-7 days of age. In girls, BGLU remained elevated until the end of the first year of life. These results indicate a variation of HEX and BGLU activities during the first year of life and a sex influence on their developmental pattern. This observation should be considered in the diagnosis of GM2 gangliosidosis and mucopolysaccharidosis type VII.

Laboratory diagnosis of canine GM2-gangliosidosis using blood and cerebrospinal fluid.

In the present study, laboratory techniques were used to diagnose canine GM2-gangliosidosis using blood and cerebrospinal fluid (CSF) that can be collected noninvasively from living individuals. Lysosomal acid beta-hexosaminidase (Hex) was measured spectrofluorometrically using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl 7-(6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside) as substrates. Main isoenzymes A and B of Hex in leukocytes were also analyzed using cellulose acetate membrane electrophoresis. GM2-ganglioside in CSF was detected and determined quantitatively by using thin-layer chromatography/enzyme-immunostaining method with anti-GM2-ganglioside antibody. In normal dogs, Hex activities could be determined in leukocytes, serum, and CSF and the total activities were markedly reduced in all the enzyme sources in a dog with Sandhoff disease. Electrophoresis of a leukocyte lysate from a normal dog showed that the Hex A and Hex B were not separated distinctively with formation of a broad band, whereas there were no bands in electrophoresis of a lysate from a dog with Sandhoff disease, showing a deficiency in the total enzyme activity. GM2-ganglioside could be detected and determined quantitatively in as little as 100 microl of canine CSE GM2-ganglioside in CSF in a dog with Sandhoff disease increased to 46 times the normal level. In conclusion, the methods in the present study are useful for diagnosis of canine GM2-gangliosidosis. These techniques enable definitive and early diagnosis of canine GM2-gangliosidosis even if tissues and organs cannot be obtained.

[Studies on the molecular mechanism of GM(2) gangliosidosis].

OBJECTIVE: To study the molecular mechanism of GM(2) gangliosidosis. METHODS: The skin fibroblasts from 4 patients with GM(2) gangliosidosis were subjected to culture. Enzyme activities assay, Western blot and immunocytochemical analysis were performed using the cultured fibroblasts. RESULTS: The hexosaminidase (Hex) activities of 4 patients with GM(2) gangliosidosis were significantly decreased. The activities were 12% 3% 15% and 6% of control values, respectively. Western blot analysis indicated that the amount of Hex mature alpha- and beta- subunits (alpha m, beta m) was decreased in cells from patients 2 and 3, but only decreased alpha m was found in patient 1 and both alpha m and beta m were normal in cells from patient 4. Immunocytochemical analysis revealed the accumulated GM(2) ganglioside in cells from patients 1-4. CONCLUSION: The pathogenesis of GM(2) gangliosidosis was associated with deficiency of Hex alpha m and beta m and GM(2) activator caused by HEXA, HEXB and GM(2)A gene mutations.

Structural basis of the GM2 gangliosidosis B variant.

To study the structural basis of the GM2 gangliosidosis B variant, we constructed the three-dimensional structures of the human beta-hexosaminidase alpha-subunit and the heterodimer of the alpha- and beta-subunits, Hex A, by homology modeling. The alpha-subunit is composed of two domains, domains I and II. Nine mutant models due to specific missense mutations were constructed as well and compared with the wild type to determine structural defects. These nine mutations were divided into five groups according to structural defects. R178H is deduced to affect the active site directly, because R178 is important for binding to the substrate. C458Y and W420C are predicted to cause drastic structural changes in the barrel structure carrying the active site pocket. R504C/H is deduced to introduce a disruption of an essential binding with D494 in the beta-subunit for dimerization. R499C/H, located in an extra-helix, is deduced to disrupt hydrogen bonds with domain I and the barrel. R170W and L484P are deduced to affect the interface between domains I and II, causing destabilization. The structural defects reflect the biochemical abnormalities of the disease.

Distribution of enzyme-bearing cells in GM2 gangliosidosis mice: regionally specific pattern of cellular infiltration following bone marrow transplantation.

Tissue distribution of beta-hexosaminidase was investigated using 5-bromo-4-chloro-3-indolyl N-acetyl beta-D-glucosaminide (X-Hex) as substrate in wild-type mice, four GM2 gangliosidosis model mice (Hexa-/-, Hexb-/-, Gm2a-/- and Hexa-/-Hexb-/-) and Hexb-/- mice that received bone marrow transplantation (BMT). In wild-type mice histochemical localization of beta-hexosaminidase was detected in the perikarya of the majority of neurons, small process-bearing microglial cells, perivascular macrophages, and macrophages in the choroid plexus and leptomeninges. X-Hex positivity was also noted in the renal tubular epithelium and macrophages in the liver and spleen. The staining pattern in the Gm2a-/- and Hexa-/- mice was generally similar to those of wild type, but in these mice, X-Hex stain was also noted in some storage neurons with swollen perikarya. No X-Hex-positive cells were detected in Hexb-/- or Hexa-/-Hexb-/- (DKO) mice. In Hexb-/- mice that received wild-type BMT (Hexb-/- +WBMT), many X-Hex-positive cells were detected in the spleen, and to a far lesser extent, in liver and kidney. In the CNS of these mice, X-Hex-positive cells were largely detected in the leptomeninges and choroid plexus. Some positive cells were also detected, mostly in the perivascular regions of the cerebrum, in particular in the regions of the posterior thalamus, brain stem and spinal cord. Some of X-Hex-positive cells were immunoreactive with Mac-1 and F4/80 antibodies and, thus, were cells of microglia/macrophage lineage. X-Hex-positive staining was not detected in neurons in these mice despite clinical improvement following BMT. This is the first time, as far as we know, that the regional distribution of the donor cells in the CNS has been investigated in a model of neuronal storage disease. Our study indicated that donor-derived cells of microglia/macrophage lineage infiltrated the CNS in a regionally specific manner following the BMT.