Widespread distribution of beta-hexosaminidase activity in the brain of a Sandhoff mouse model after coinjection of adenoviral vector and mannitol.

Sandhoff disease is a severe inherited neurodegenerative disorder resulting from deficiency of the beta-subunit of hexosaminidases A and B, lysosomal hydrolases involved in the degradation of G(M2) ganglioside and related metabolites. Currently, there is no viable treatment for the disease. Here, we show that adenovirus-mediated transfer of the beta-subunit of beta-hexosaminidase restored Hex A and Hex B activity after infection of Sandhoff fibroblasts. Gene transfer following intracerebral injection in a murine model of Sandhoff disease resulted in near-normal level of enzymatic activity in the entire brain at the different doses tested. The addition of hyperosmotic concentrations of mannitol to the adenoviral vector resulted in an enhancement of vector diffusion in the injected hemisphere. Adenoviral-induced lesions were found in brains injected with a high dose of the vector, but were not detected in brains injected with 100-fold lower doses, even in the presence of mannitol. Our data underline the advantage of the adjunction of mannitol to low doses of the adenoviral vector, allowing a high and diffuse transduction efficiency without viral cytotoxicity.

Endothelial nitric oxide synthase gene polymorphisms in Fabry's disease.

The gene encoding endothelial nitric oxide synthase (eNOS) is involved in abnormalities in nitric oxide (NO) synthesis that mediates functional damage of vascular cells, especially of endothelial cells (ECs), a common characteristic in cardiovascular diseases. In Fabry's disease, the characteristic mutation in the alpha-galactosidase A (alpha-gal A) gene induces large deposits of glycosphingolipids, particularly concentrated in ECs, a process associated with endothelial dysfunction. To determine whether in addition to alpha-gal A gene mutations, eNOS genetic variations are implicated in this process, we examined the genotypes of the missense Glu298Asp (G894T) variant in exon 7 and 27-bp tandem repeats in intron 4 (4b/a) in 19 patients with Fabry's disease, and 39 normal volunteers. The results showed that both varials have a significant association with Fabry's disease. The frequencies of mutant Glu/Asp + Asp/Asp genotypes and Asp allele are significantly higher in Fabry's disease (68.4%, p = 0.044, and 47.4%, p = 0.022, respectively) than in controls (46.7% and 25%, respectively). The frequencies of eNOS 4b/a polymorphisms are also significantly different in Fabry's disease when compared to controls. The mutant 4b/a + 4a/a genotype frequencies are 55.5% (p = 0.032) and 4a allele 27.8% (p = 0.05) compared with controls (23.1% and 12.8%, respectively). These results indicate that more than half of the patients with Fabry's disease carry the Glu298Asp variant ( approximately 68%) and/or the 4b/a polymorphism ( approximately 55%). To the best of our knowledge, this is the first report showing an influence of eNOS gene polymorphisms in patients with Fabry's disease.

Muscle as a putative producer of acid alpha-glucosidase for glycogenosis type II gene therapy.

Glycogenosis type II (GSD II) is a lysosomal disorder affecting skeletal and cardiac muscle. In the infantile form of the disease, patients display cardiac impairment, which is fatal before 2 years of life. Patients with juvenile or adult forms can present diaphragm involvement leading to respiratory failure. The enzymatic defect in GSD II results from mutations in the acid alpha-glucosidase (GAA) gene, which encodes a 76 kDa protein involved in intralysosomal glycogen hydrolysis. We previously reported the use of an adenovirus vector expressing GAA (AdGAA) for the transduction of myoblasts and myotubes cultures from GSD II patients. Transduced cells secreted GAA in the medium, and GAA was internalized by receptor-mediated capture, allowing glycogen hydrolysis in untransduced cells. In this study, using a GSD II mouse model, we evaluated the feasibility of GSD II gene therapy using muscle as a secretary organ. Adenovirus vector encoding AdGAA was injected in the gastrocnemius of neonates. We detected a strong expression of GAA in the injected muscle, secretion into plasma, and uptake by peripheral skeletal muscle and the heart. Moreover, glycogen content was decreased in these tissues. Electron microscopy demonstrated the disappearance of destruction foci, normally present in untreated mice. We thus demonstrate for the first time that muscle can be considered as a safe and easily accessible organ for GSD II gene therapy.

From gene transfer to gene therapy in lysosomal storage diseases affecting the central nervous system.

Gene transfer into the central nervous system (CNS) is one of the foremost scientific challenges today. To give a brief survey of possible approaches to gene therapy in diseases affecting the CNS, we have selected the lysosomal storage diseases (LDS), which are an excellent model of both early-onset infantile neurological forms and late-onset adult psychiatric forms. Lysosomal storage diseases represent a group of about 50 monogenic metabolic disorders resulting from a deficiency in intralysosomal enzymes involved in macromolecule catabolism. The clinical severity, including neuropsychiatric symptoms, and the absence of an efficient therapy for the majority of these disorders prompted the various trials of gene therapy now in progress. Most of the genes encoding the normal lysosomal enzymes have been cloned, and the size of the corresponding cDNAs is generally compatible with their transfer by recombinant vectors. New vectors with improved immunogenicity, transduction efficacy, insert capacity, and specificity of targeting are under development. Here we discuss several gene therapy strategies for the correction of LSD-induced anomalies in the CNS. Interesting results have been obtained by animal model brain, which raises hopes that large-scale clinical trials may soon be started.

Gene therapy in lysosomal diseases.

Lysosomal storage diseases are monogenic metabolic disorders resulting from a deficiency in intralysosomal enzymes involved in macromolecule catabolism. Various groups have been delineated according to the affected pathway and the accumulated substrate: mucopolysaccharidoses, lipidoses, glycoproteinoses and glycogenosis type II. Their clinical severity and the absence of efficient therapy for the majority of these disorders justify the development of gene transfer methods. Most of the genes encoding the normal lysosomal enzymes have been cloned and recently numerous animal models have been obtained for nearly all these diseases. Due to the clinical heterogeneity of lysosomal diseases, showing multivisceral involvement or affecting predominantly the reticuloendothelial system, muscle or central nervous system, various gene therapy strategies have to be developed. Vectors, ways of access, results and limits will be reviewed. Interesting results have already been obtained in the gene transfer for lysosomal diseases, but improvements are needed before a future application to humans.

Juvenile and adult-onset acid maltase deficiency in France: genotype-phenotype correlation.

OBJECTIVE: To characterize the phenotypes of patients with juvenile and adult-onset acid maltase deficiency (AMD) in the French population and correlate them with genetic defects. BACKGROUND: AMD is an autosomal recessive disorder caused by the absence of the enzyme acid a-glucosidase (GAA). Patients are generally compound heterozygotes for various mutations in the GAA gene. The most common mutant allele is a -13T to G transversion in intron 1. METHODS: The authors performed a clinical, biochemical, and genetic study on 21 unrelated patients with juvenile and adult-onset AMD. RESULTS: Although onset of progressive muscle weakness occurred during adulthood in all cases but one, presence of mild, nonprogressive muscular symptoms appearing during childhood was detected in 16 patients. Eighteen patients had a similar clinical pattern with pelvic girdle muscle weakness predominating in glutei and thigh adductors. Restrictive respiratory insufficiency with vital capacity less than 60% was noted in eight patients, and respiratory failure was the first manifestation in two cases. All patients but one were compound heterozygotes, and 17 carried the IVS1 (-13T ---> G) transversion (one patient was homozygous for this mutation). The two mutated alleles were identified in 10 cases, with 13 different mutations detected in the GAA gene. There was no clear correlation between the type of mutation and phenotype. CONCLUSIONS: This study shows a high genetic heterogeneity of juvenile and adult AMD in the French population. The absence of genotype-phenotype correlation suggests a complex physiopathology that requires further investigations.

Approach to gene therapy of glycogenosis type II (Pompe disease).

Pompe disease is a generalized lysosomal glycogenosis affecting essentially the skeletal muscles and the heart. It is due to the deficiency of acid alpha-glucosidase, also called acid maltase, involved in glycogen degradation by the cleavage of alpha-1,4 and alpha-1,6 glycosidic linkages. The severe infantile, milder juvenile, and late-onset or adult forms are associated under the generic name of glycogenoses type II. The clinical picture can differ according to these variants, forming a clinical spectrum from cardiorespiratory failure with early death in the infantile variant to late muscular weakness or respiratory problems in the adult variant. Enzymatic pre- and postnatal diagnoses and mutation characterization are available. Different therapeutic attempts have been conceived and some of them have come to clinical trials. Several pilot studies have demonstrated the feasibility of gene therapy and remarkable advances have been realized. Of particular interest, strategies for gene therapy in a generalized disease like Pompe disease must be accompanied by the secretion and uptake of the corrective enzyme by more distant cells or tissues in order to obtain efficient results. Preliminary positive results have been obtained in animal models, and new approaches with improvements in the access to muscle and heart, in the efficacy and innocuity of vectors, and in the clinical evolution are proposed. Gene therapy is a promising strategy for Pompe disease. However, several steps must be explored before this method becomes clinically successful.

Gaucher disease: the origins of the Ashkenazi Jewish N370S and 84GG acid beta-glucosidase mutations.

Type 1 Gaucher disease (GD), a non-neuronopathic lysosomal storage disorder, results from the deficient activity of acid beta-glucosidase (GBA). Type 1 disease is panethnic but is more prevalent in individuals of Ashkenazi Jewish (AJ) descent. Of the causative GBA mutations, N370S is particularly frequent in the AJ population, (q approximately .03), whereas the 84GG insertion (q approximately .003) occurs exclusively in the Ashkenazim. To investigate the genetic history of these mutations in the AJ population, short tandem repeat (STR) markers were used to map a 9.3-cM region containing the GBA locus and to genotype 261 AJ N370S chromosomes, 60 European non-Jewish N370S chromosomes, and 62 AJ 84GG chromosomes. A highly conserved haplotype at four markers flanking GBA (PKLR, D1S1595, D1S2721, and D1S2777) was observed on both the AJ chromosomes and the non-Jewish N370S chromosomes, suggesting the occurrence of a founder common to both populations. Of note, the presence of different divergent haplotypes suggested the occurrence of de novo, recurrent N370S mutations. In contrast, a different conserved haplotype at these markers was identified on the 84GG chromosomes, which was unique to the AJ population. On the basis of the linkage disequilibrium (LD) delta values, the non-Jewish European N370S chromosomes had greater haplotype diversity and less LD at the markers flanking the conserved haplotype than did the AJ N370S chromosomes. This finding is consistent with the presence of the N370S mutation in the non-Jewish European population prior to the founding of the AJ population. Coalescence analyses for the N370S and 84GG mutations estimated similar coalescence times, of 48 and 55.5 generations ago, respectively. The results of these studies are consistent with a significant bottleneck occurring in the AJ population during the first millennium, when the population became established in Europe.

Fabry disease: identification of novel alpha-galactosidase A mutations and molecular carrier detection by use of fluorescent chemical cleavage of mismatches.

Fabry disease (FD) (angiokeratoma corporis diffusum) is an X-linked inborn error of glycosphingolipid metabolism caused by defects in the lysosomal alpha-galactosidase A gene (GLA). The enzymatic defect leads to the systemic accumulation of neutral glycosphingolipids with terminal alpha-galactosyl moieties. Clinically, affected hemizygous males have angiokeratoma, severe acroparesthesia, renal failure, and vasculopathy of the heart and brain. While demonstration of alpha-galactosidase deficiency in leukocytes is diagnostic in affected males, enzymatic detection of female carriers is often inconclusive, due to random X-chromosomal inactivation, underlining the need of molecular investigations for accurate genetic counseling. By use of chemical cleavage of mismatches adapted to fluorescence-based detection systems, we have characterized the mutations underlying alpha-Gal A deficiency in 16 individuals from six unrelated families with FD. The mutational spectrum included five missense mutations (C202W, C223G, N224D, R301Q, and Q327K) and one splice-site mutation [IVS3 G(-1) --> C]. Studies at the mRNA level showed that the latter led to altered pre-mRNA splicing with consequent alteration of the mRNA translational reading frame and generation of a premature termination codon of translation. By use of this strategy, carrier status was accurately assessed in all seven at-risk females tested, whereas enzymatic dosages failed to diagnose or exclude heterozygosity.

Adenoviral gene therapy of the Tay-Sachs disease in hexosaminidase A-deficient knock-out mice.

The severe neurodegenerative disorder, Tays-Sachs disease, is caused by a beta-hexosaminidase alpha-subunit deficiency which prevents the formation of lysosomal heterodimeric alpha-beta enzyme, hexosaminidase A (HexA). No treatment is available for this fatal disease; however, gene therapy could represent a therapeutic approach. We previously have constructed and characterized, in vitro, adenoviral and retroviral vectors coding for alpha- and beta-subunits of the human beta-hexosaminidases. Here, we have determined the in vivo strategy which leads to the highest HexA activity in the maximum number of tissues in hexA -deficient knock-out mice. We demonstrated that intravenous co-administration of adenoviral vectors coding for both alpha- and beta-subunits, resulting in preferential liver transduction, was essential to obtain the most successful results. Only the supply of both subunits allowed for HexA overexpression leading to massive secretion of the enzyme in serum, and full or partial enzymatic activity restoration in all peripheral tissues tested. The enzymatic correction was likely to be due to direct cellular transduction by adenoviral vectors and/or uptake of secreted HexA by different organs. These results confirmed that the liver was the preferential target organ to deliver a large amount of secreted proteins. In addition, the need to overexpress both subunits of heterodimeric proteins in order to obtain a high level of secretion in animals defective in only one subunit is emphasized. The endogenous non-defective subunit is otherwise limiting.

Adenovirus-mediated transfer of the acid alpha-glucosidase gene into fibroblasts, myoblasts and myotubes from patients with glycogen storage disease type II leads to high level expression of enzyme and corrects glycogen accumulation.

Glycogen storage disease type II (GSD II) is an autosomal recessive disorder caused by defects in the lysosomal acid alpha-glucosidase (GAA) gene. We investigated the feasibility of using a recombinant adenovirus containing the human GAA gene under the control of the cytomegalovirus promoter (AdCMV-GAA) to correct the enzyme deficiency in different cultured cells from patients with the infantile form of GSD II. In GAA-deficient fibroblasts infected with AdCMV-GAA, transduction and transcription of the human transgene resulted in de novo synthesis of GAA protein. The GAA enzyme activity was corrected from the deficient level to 12 times the activity of normal cells. The transduced cells overexpressed the 110 kDa precursor form of GAA, which was secreted into the culture medium and was taken up by recipient cells. The recombinant GAA protein was correctly processed and was active on both an artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (4MUG) and glycogen. In GAA-deficient muscle cells, a significant increase in cellular enzyme level, approximately 20-fold higher than in normal cells, was also observed after viral treatment. The transduced muscle cells were also able to efficiently secrete the recombinant GAA. Moreover, transfer of the human transgene resulted in normalization of cellular glycogen content with clearance of glycogen from lysosomes, as assessed by electron microscopy, in differentiated myotubes. These results demonstrate phenotypic correction of cultured skeletal muscle from a patient with infantile-onset GSD II using a recombinant adenovirus. We conclude that adenovirus-mediated gene transfer might be a suitable model system for further in vivo studies on delivering GAA to GSD II muscle, not only by direct cell targeting but also by a combination of secretion and uptake mechanisms.

Exhaustive screening of the acid beta-glucosidase gene, by fluorescence-assisted mismatch analysis using universal primers: mutation profile and genotype/phenotype correlations in Gaucher disease.

Gaucher disease (GD) is one of the most prevalent lysosomal storage disorders and one of the rare genetic diseases now accessible to therapy. Outside the Ashkenazi Jewish community, a high molecular diversity is observed, leaving approximately 30% of alleles undetected. Nevertheless, very few exhaustive methods have been developed for extensive gene screening of a large series of patients. Our approach for a complete search of mutations was the association of fluorescent chemical cleavage of mismatches with a universal strand-specific labeling system. The glucocerebrosidase (GBA) gene was scanned by use of a set of six amplicons, comprising 11 exons, all exon/intron boundaries, and the promoter region. By use of this screening strategy, the difficulties due to the existence of a highly homologous pseudogene were easily overcome, and both GD mutant alleles were identified in all 25 patients studied, thus attesting to a sensitivity that approaches 100%. A total of 18 different mutations and a new glucocerebrosidase haplotype were detected. The mutational spectrum included eight novel acid beta-glucosidase mutations: IVS2 G(+1)-->T, I119T, R170P, N188K, S237P, K303I, L324P, and A446P. These data further indicate the genetic heterogeneity of the lesions causing GD. Established genotype/phenotype correlations generally were confirmed, but notable disparities were disclosed in several cases, thus underlining the limitation in the prognostic value of genotyping. The observed influence of multifactorial control on this monogenic disease is discussed.

Retrovirus-mediated enzymatic correction of Tay-Sachs defect in transduced and non-transduced cells.

Tay-Sachs disease is a severe neurodegenerative disorder due to mutations in the HEXA gene coding for the alpha-chain of the alpha-beta heterodimeric lysosomal enzyme beta-hexosaminidase A (HexA). Because no treatment is available for this disease, we have investigated the possibility of enzymatic correction of HexA-deficient cells by HEXA gene transfer. Human HEXA cDNA was subcloned into a retroviral plasmid generating to G.HEXA vector. The best Psi-CRIP producer clone of G.HEXA retroviral particles was isolated, and murine HexA-deficient fibroblasts derived from hexa -/- mice were transduced with the G.HEXA vector. Transduced cells overexpressed the alpha-chain, resulting in the synthesis of interspecific HexA (human alpha-chain/murine beta-chain) and in a total correction of HexA deficiency. The alpha-chain was secreted in the culture medium and taken up by HexA-deficient cells via mannose-6-phosphate receptor binding, allowing for the restoration of intracellular HexA activity in non-transduced cells.

Glycogen-storage disease type II (acid maltase deficiency): identification of a novel small deletion (delCC482+483) in French patients.

Glycogen-storage disease type II (GSD II, acid maltase deficiency, Pompe's disease) is caused by defects in the lysosomal acid alpha-glucosidase (GAA) gene. Clinically, patients with the severe infantile form of GSD II have muscle weakness and cardiomyopathy eventually leading to death before the age of two years. Patients with the juvenile or the adult form of GSD II present with myopathy with a slow progression over several years or decades. Apart from a common base substitution in intron1, designated IVS1(-13T-->G) and resulting in the aberrant splicing of exon 2, the other mutations recently discovered in the GAA gene are rare and often unique to single patients. In this paper, we identified a two-base frameshift deletion in three unrelated adult-onset GSD II patients. This small deletion lies in the first coding exon (exon 2) and results in a premature stop codon at the very 5' end of the coding sequence of the GAA gene. The three patients were compound heterozygotes and two of them had the common IVS1(-13G-->T) mutation on the second allele. We speculate that this novel deletion may be relatively frequent among French patients, possibly leading to the severe infantile phenotype of GSD II if it occurs in homozygous form.

[Beta mannosidosis: a new case]. / Beta mannosidose: une nouvelle observation.

BACKGROUND: Only 11 cases of beta mannosidase deficiency have been reported until now. We report a new case. CASE HISTORY: J was born at full term to consanguineous parents; her weight was 2,080 g and her height was 44 cm. During the first months of life she was hypotonic and had feeding difficulties. At the age of 7 months, she was admitted to an intensive care unit because of a serious inhalation. Standard blood analysis, chest X-ray, abdominal ultrasonography, electroencephalogram, cerebral nuclear magnetic resonance and electromyography were normal. Blood and urine amino acids and urine organic acids were also normal. The only detected abnormality was a marked deficiency of beta mannosidase in her serum and leukocytes. Later on, she suffered from recurring respiratory infections, and she had abnormalities of esophageal mobility, hypotoria of the lower esophageal sphincter, and at the age of 2 years, achalasia requiring surgery. To date, her motor development is retarded. CONCLUSIONS: The main clinical manifestations of beta mannosidosis are various degrees of mental retardation, speech disorders and hearing loss. Our patient presented with abnormalities of swallowing and esophageal motility resulting in recurring respiratory infections, previously reported in some other cases.

Metachromatic leukodystrophy: identification of the first deletion in exon 1 and of nine novel point mutations in the arylsulfatase A gene.

Metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA), is inherited as an autosomal recessive trait, and its frequency is estimated to be 1 in 40,000 live births. Genomic DNA from 21 MLD patients (14 late-infantile and 7 juvenile cases) was amplified in four overlapping PCR fragments and tested by allele-specific oligonucleotide (ASO) for the two common mutations 459 + 1G-->A and P426L. These mutations were found in only 28.6% of the alleles studied. The remaining alleles were analyzed by chemical mismatch cleavage (CMC) and automatic sequencing. In addition to five previously reported mutations (459 + 1G-->A, A212V, R244C, R390W, P426L), 10 novel mutations were identified: 9 missense mutations (S95N, G119R, D152Y, R244H, S250Y, A314T, R384C, R496H, K367N) and one 8 bp deletion in exon 1, the first mutation reported in this exon. These methods allowed us to identify 76% of the alleles tested. Genotype-phenotype correlations could be established for some of these mutations. These results confirm the heterogeneity of mutations causing MLD and suggest that CMC is a reliable and informative screening method for point mutation detection in the arylsulfatase A gene.

Fluorescence-assisted mismatch analysis (FAMA) for exhaustive screening of the alpha-galactosidase A gene and detection of carriers in Fabry disease.

We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the alpha-galactosidase A gene in patients with Fabry disease in order to identify unknown mutations and help define genotype-phenotype correlations in this X-linked lysosomal storage disorder. Chemical cleavage at mismatches on heteroduplex DNA end-labeled with strand-specific fluorescent dyes, reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. Exhaustive scanning of the alpha-galactosidase gene was accomplished on four polymerase chain reaction-generated amplicons, covering all seven exons, the exon-intron boundaries, and 700 bp of 5'-flanking sequence. Mutations were identified in each of the 15 patients studied from nine unrelated kindreds. Among the seven previously undescribed sequence changes, three are obviously pathogenic because they lead to premature protein termination. The other four, a splicesite mutation and three missense mutations, were the only changes found upon complete scanning of the gene and its promoter. In addition, FAMA also detects female heterozygous carriers more dependably than direct sequencing, and thus provides a valuable diagnostic test. In Fabry disease, this molecular criterion is especially important for genetic counseling since heterozygotes can be asymptomatic and their enzymatic values within the normal range.

Restoration of hexosaminidase A activity in human Tay-Sachs fibroblasts via adenoviral vector-mediated gene transfer.

Tay-Sachs disease (TSD) is a lysosomal storage disease due to hexosaminidase A deficiency caused by mutations in the gene for alpha-chain (Hex alpha). A human Hex alpha cDNA was subcloned into the adenoviral plasmid pAdRSV. Hex alpha. Replication-deficient adenovirus was generated by homologous recombination in 293 cells. Human fibroblasts from a patient suffering from TSD were infected with the recombinant adenovirus. TSD fibroblasts expressing the recombinant alpha-chain had an enzyme activity on the natural substrate ranging from 40 to 84% of the normal. The corrected cells secreted up to 25 times more Hex alpha than control fibroblasts. The Hex alpha encoded by the adenovirus was shown to be correctly transported into the lysosomes and to normalize the impaired degradation of GM2 ganglioside in TSD fibroblasts.