Uso de vectores derivados de virus adenoasociados para el tratamiento de la enfermedad de Morquio A / Adenoassociated virus-derived vectors for the treatment of Morquio A disease

En el desarrollo de una estrategia de terapia génica para la mucopolisacaridosis IV A (enfermedad de Morquio A), en el presente trabajo se evaluó la capacidad de un vector adenoasociado (AAV) para expresar el gen de la enzima sulfatasa N-acetilgalactosamina-6-sulfato (GALNS, N-acetylgalactosamine-6-sulfate sulfatase) en células HEK293, fibroblastos humanos con mucopolisacaridosis IV A y condrocitos de ratón con mucopolisacaridosis IV A. En el lisado celular, la actividad de la GALNS se incrementó entre 5 y 24 veces los valores observados en las células sin transfectar. La coexpresión con el gen del factor activador de sulfatasas 1 (SUMF1, sultatase modifiying factor 1) permitió un incremento en la actividad de la GALNS en el lisado celular de hasta 4,5 veces los valores observados en las células cotransfectadas con AAV-GALNS. La actividad de la GALNS en el medio de cultivo sólo se detectó en células cotransfectadas con AAV-SUMF1. Estos resultados constituyen evidencia valiosa sobre la posibilidad de realizar la corrección del defecto genético en la mucopolisacaridosis IV A empleando vectores AAV...

Toward the development of a gene therapy strategy for the mucopolysaccharidosis IV A (MPS IV A, Morquio A), in this work we evaluated the capability of an AAV vector to express the N-acetilgalactosamine-6-sulfate sulfatase (GALNS) gene in HEK293 cells, human mucopolysaccharidosis IV A fibroblasts and murine MPS IV A chondrocites. GALNS activity in lysated cells was increased between 5 and 24 folds compared to nontransfected cells. Also, the effect of coexpression with the sultatase modifiying factor 1 (SUMF1) was evaluated.GALNS activity was increased up to 4.5 folds in lysated cells compared to those values observed in AAV-GALNS transduced cells. Noteworthy, enzyme activity was only detected in culture media when cells were cotransfected with AAV-GALNS and AAV-SUMF1. These results represent a valuable step in the development of a gene therapy strategy for mucopolysaccharidosis IV A using AAV vectors...

Identification of a common mutation in mucopolysaccharidosis IVA: correlation among genotype, phenotype, and keratan sulfate.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS was performed by genomic PCR and direct sequence analyses in 20 MPS IVA patients from Latin America. In this study, 12 different gene mutations including nine unreported ones were identified in 16 severe and four attenuated patients and accounted for 90.0% of the unrelated mutant alleles. The gene alterations were missense mutations except one insertion. Six recurrent mutations, p.A75G, p.G116S, p.G139S, p.N164T, p.R380S, and p.R386C, accounted for 5.0, 10.0, 5.0, 7.5, 5.0, and 32.5% of the unrelated mutant alleles, respectively. The p.R386C mutation was identified in all Latin American populations studied. Eleven mutations correlated with a severe form, while one mutation, p.R380S, was associated with an attenuated form. MPS IVA patients had an elevation of urine and plasma keratan sulfate (KS) concentrations compared with those of the age-matched control. KS concentrations in severe patients were higher than those in attenuated patients. These data provide evidence for extensive allelic heterogeneity and presence of a common mutation in Latin American patients. Accumulation of mutations with clinical description and KS concentration will lead us to predict clinical severity of the patient more precisely.

Mucopolysaccharidosis IVA (Morquio A): identification of novel common mutations in the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene in Italian patients.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS gene was performed by RT-PCR with one amplicon and direct sequence analyses using cDNA samples from 15 Italian MPS IVA patients. Each mutation was confirmed at the genomic level. In this study, 13 different gene mutations with four common mutations (over 10% of mutant alleles) were identified in 12 severe and three milder (attenuated) MPS IVA patients. The gene alterations in 12 out of 13 were found to be point mutations and only one mutation was deletion. Ten of 13 mutations were novel. The c.1070C>T (p.Pro357Leu) mutation coexisted with c.1156C>T (p.Arg386Cys) mutation on the same allele. Together they accounted for 100% of the 30 disease alleles of the patients investigated. Four common mutations accounted for 70% of mutant alleles investigated. Urine keratan sulfate (KS) concentrations were elevated in all patients investigated. These data provide further evidence for extensive allelic heterogeneity and importance of relation among genotype, phenotype, and urine KS excretion as a biomarker in MPS IVA.

Mouse model of N-acetylgalactosamine-6-sulfate sulfatase deficiency (Galns-/-) produced by targeted disruption of the gene defective in Morquio A disease.

Mucopolysaccharidosis IVA is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), a lysosomal enzyme required for the stepwise degradation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). To generate a model for studies of the pathophysiology and of potential therapies, we disrupted exon 2 of Galns, the homologous murine gene. Homozygous Galns-/- mice have no detectable GALNS enzyme activity and show increased urinary glycosaminoglycan (GAGs) levels. These mice accumulate GAGs in multiple tissues including liver, kidney, spleen, heart, brain and bone marrow. At 2 months old, lysosomal storage is present primarily within reticuloendothelial cells such as Kupffer cells and cells of the sinusoidal lining of the spleen. Additionally, by 12 months old, vacuolar change is observed in the visceral epithelial cells of glomeruli and cells at the base of heart valves but it is not present in parenchymal cells such as hepatocytes and renal tubular epithelial cells. In the brain, hippocampal and neocortical neurons and meningeal cells had lysosomal storage. KS and C6S were more abundant in the cytoplasm of corneal epithelial cells of Galns-/- mice compared with wild-type mice by immunohistochemistry. Radiographs revealed no change in the skeletal bones of mice up to 12 months old. Thus, targeted disruption of the murine Galns gene has produced a murine model, which shows visceral storage of GAGs but lacks the skeletal features. The complete absence of GALNS in mutant mice makes them useful for studies of pharmacokinetics and tissue targeting of recombinant GALNS designed for enzyme replacement.

Mucopolysaccharidosis IVA: characterization of a common mutation found in Finnish patients with attenuated phenotype.

Mucopolysaccharidosis IVA (MPS IVA) is caused by the deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase encoded by the GALNS gene on chromosome 16. We describe, in detail, the clinical phenotype of five patients from three unrelated Finnish families and have characterized the disease-causing mutations in GALNS. Genotypes of the patients are D60N/A291T, D60N/W230X, and D60N/1374delT. Mutation 1374delT introduces premature termination of GALNS. Cells over-expressing the novel mutation W230X and A291T had no residual GALNS activity, whereas D60N gave 12.2% residual activity compared with the wild type. Co-transfection of D60N/A291T and D60N/W230X showed 5.5% and 6.7% of wild type activity, respectively. The precursor proteins of D60N and A291T were observed at 55 kDa and 57 kDa, respectively, whereas there was no detectable band in cells over-expressing W230X. At 55 degrees C, the mutant protein showed lower thermostability than the wild type protein at pH 3.8 and 7.0. The tertiary structural model of the GALNS protein revealed that aspartic acid at position 60 is located on the surface of the molecule, away from the active site. This makes it unlikely that the enzymatic function of the protein with D60N is severely impaired. On the other hand, A291 and W230 are localized near the active site. The molecular characteristics of the D60N mutation explain the attenuated clinical phenotype of the patients.