RESUMO
BACKGROUND: Targeting specific tissues remains a major challenge to the promise of gene therapy. For example, several strategies have failed to target adeno-associated virus 2 (AAV2) vectors, to bone. We have evaluated in vitro and in vivo the affinity of an AAV2 vector to bone matrix, hydroxyapatite (HA) to treat Mucopolysacccharidosis IVA. METHODS: To increase vector affinity to HA, an aspartic acid octapeptide (D8) was inserted immediately after the N-terminal region of the VP2 capsid protein. The modified vector had physical titers and transduction efficiencies comparable to the unmodified vector. RESULTS: The bone-targeting vector had significantly higher HA affinity and vector genome copies in bone than the unmodified vector. The modified vector was also released from HA, and its enzyme activity in bone, 3 months post infusion, was 4.7-fold higher than the unmodified vector. CONCLUSION: Inserting a bone-targeting peptide into the vector capsid increases gene delivery and expression in the bone without decreasing enzyme expression. This approach could be a novel strategy to treat systemic bone diseases.
Assuntos
Osso e Ossos/metabolismo , Proteínas do Capsídeo/química , Durapatita/química , Vetores Genéticos , Mucopolissacaridose IV/terapia , Animais , Ácido Aspártico/química , Medula Óssea/metabolismo , Encéfalo/metabolismo , Capsídeo , Dependovirus , Perfilação da Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Células HEK293 , Humanos , Hidroxiapatitas/química , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Parvovirinae , Domínios Proteicos , TransgenesRESUMO
There is limited information regarding the long-term outcomes of hematopoietic stem cell transplantation (HSCT) for mucopolysaccharidosis II (MPS II). In this study, clinical, biochemical, and radiologic findings were assessed in patients who underwent HSCT and/or enzyme replacement therapy (ERT). Demographic data for 146 HSCT patients were collected from 27 new cases and 119 published cases and were compared with 51 ERT and 15 untreated cases. Glycosaminoglycan (GAG) levels were analyzed by liquid chromatography tandem mass spectrometry in blood samples from HSCT, ERT, and untreated patients as well as age-matched controls. Long-term magnetic resonance imaging (MRI) findings were investigated in 13 treated patients (6 ERT and 7 HSCT). Mean age at HSCT was 5.5 years (range, 2 to 21.4 years) in new patients and 5.5 years (range, 10 months to 19.8 years) in published cases. None of the 27 new patients died as a direct result of the HSCT procedure. Graft-versus-host disease occurred in 8 (9%) out of 85 published cases, and 9 (8%) patients died from transplantation-associated complications. Most HSCT patients showed greater improvement in somatic features, joint movements, and activity of daily living than the ERT patients. GAG levels in blood were significantly reduced by ERT and levels were even lower after HSCT. HSCT patients showed either improvement or no progression of abnormal findings in brain MRI while abnormal findings became more extensive after ERT. HSCT seems to be more effective than ERT for MPS II in a wide range of disease manifestations and could be considered as a treatment option for this condition.
Assuntos
Terapia de Reposição de Enzimas/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Mucopolissacaridose II/terapia , Adolescente , Criança , Pré-Escolar , Glicosaminoglicanos/sangue , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Imageamento por Ressonância Magnética , Adulto JovemRESUMO
Mucopolysaccharidosis (MPS) IVA is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate sulfatase (GALNS), which leads to the accumulation of keratan sulfate and chondroitin 6-sulfate, mainly in bone. To explore the possibility of gene therapy for Morquio A disease, we transduced the GALNS gene into HEK293 cells, human MPS IVA fibroblasts and murine MPS IVA chondrocytes by using adeno-associated virus (AAV)-based vectors, which carry human GALNS cDNA. The effects of the promoter and the cotransduction with the sulfatase-modifying factor 1 gene (SUMF1) on GALNS activity levels was evaluated. Downregulation of the cytomegalovirus (CMV) immediate early enhancer/promoter was not observed for 10 days post-transduction. The eukaryotic promoters induced equal or higher levels of GALNS activity than those induced by the CMV promoter in HEK293 cells. Transduction of human MPS IVA fibroblasts induced GALNS activity levels that were 15-54% of those of normal human fibroblasts, whereas in transduced murine MPS IVA chondrocytes, the enzyme activities increased up to 70% of normal levels. Cotransduction with SUMF1 vector yielded an additional four-fold increase in enzyme activity, although the level of elevation depended on the transduced cell type. These findings suggest the potential application of AAV vectors for the treatment of Morquio A disease, depending on the combined choice of transduced cell type, selection of promoter, and cotransduction of SUMF1.
Assuntos
Condroitina Sulfatases/genética , Condroitina Sulfatases/metabolismo , Dependovirus/genética , Mucopolissacaridose IV/genética , Regiões Promotoras Genéticas/genética , Sulfatases/genética , Sulfatases/metabolismo , Animais , Linhagem Celular , Vetores Genéticos/genética , Humanos , Camundongos , Mucopolissacaridose IV/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , TransfecçãoRESUMO
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...
Assuntos
Dependovirus , Mucopolissacaridose IV , Terapia GenéticaRESUMO
Morquio A is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to the lysosomal accumulation of keratan-sulfate and chondroitin-6-sulfate. We evaluated in HEK293 cells the effect of the cytomegalovirus immediate early enhancer/promoter (CMV) or the elongation factor 1alpha (EF1alpha) promoters, and the coexpression with the sulfatase modifying factor 1 (SUMF1) on GALNS activity. Four days postransfection GALNS activity in transfected cells with CMV-pIRES-GALNS reached a plateau, whereas in cells transfected with EF1alpha-pIRES-GALNS continued to increase until day 8. Co-transfection with pCXN-SUMF1 showed an increment up to 2.6-fold in GALNS activity. Finally, computational analysis of transcription factor binding-sites and CpG islands showed that EF1alpha promoter has long CpG islands and high-density binding-sites for Sp1 compared to CMV. These results show the advantage of the SUMF1 coexpression on GALNS activity and indicate a considerable effect on the expression stability using EF1alpha promoter compared to CMV.
Assuntos
Condroitina Sulfatases/metabolismo , Expressão Gênica , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas/genética , Sulfatases/metabolismo , Sítios de Ligação , Linhagem Celular , Condroitina Sulfatases/genética , Biologia Computacional , Ilhas de CpG/genética , Citomegalovirus/genética , Humanos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Plasmídeos/genética , Fator de Transcrição Sp1/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Condroitina Sulfatases/genética , Mutação de Sentido Incorreto/genética , Fenótipo , Adolescente , Adulto , Criança , Pré-Escolar , Condroitina Sulfatases/metabolismo , Primers do DNA , Feminino , Frequência do Gene , Testes Genéticos , Genótipo , Humanos , Sulfato de Queratano/sangue , Sulfato de Queratano/urina , Masculino , Análise de Sequência de DNA , América do SulRESUMO
A neurofibromatose é doença de herança autossômica dominante, caracterizada pela presença de máculas hiperpigmentares, neurofibromas e tumores dos sistemas nervoso central e periférico. Segundo dados recentes da literatura, existem várias formas de neurobibromatose, porém as mais conhecidas säo a neurofibromatose tipo 1 (NF-1) e a neurofibromatose tipo 2 (NF-2). A NF-1 se caracteriza pela presença de múltiplas manchas "café com leite", nódulos de Lisch (hamartomas de íris), gliomas do nervo óptico e neurofibromas dérmicos. O gene responsável está localizado no braço longo do cromossomo 17. A NF-2 tem como principais características a presença de neuromas acústicos bilaterais e opacidade subcapsular posterior do cristalino. O gene responsável por esta patologia encontra-se no centro do braço longo do cromossomo 22. Embora muitos avanços tenham sido feitos até esta data quanto ao estudo dessa doença, devemos reconsiderar todos os dados disponíveis, as conclusöes e, principalmente, as novas pesquisas, visando obter melhor direcionamento do diagnóstico, prognóstico e tratamento da doença