RESUMO
Pompe's disease or glycogen storage disease type II (GSDII) belongs to the family of inherited lysosomal storage diseases. The underlying deficiency of acid alpha-glucosidase leads in different degrees of severity to glycogen storage in heart, skeletal and smooth muscle. There is currently no treatment for this fatal disease, but the applicability of enzyme replacement therapy is under investigation. For this purpose, recombinant human acid alpha-glucosidase has been produced on an industrial scale in the milk of transgenic rabbits. In this paper we demonstrate the therapeutic effect of this enzyme in our knockout mouse model of GSDII. Full correction of acid alpha-glucosidase deficiency was obtained in all tissues except brain after a single dose of i.v. enzyme administration. Weekly enzyme infusions over a period of 6 months resulted in degradation of lysosomal glycogen in heart, skeletal and smooth muscle. The tissue morphology improved substantially despite the advanced state of disease at the start of treatment. The results have led to the start of a Phase II clinical trial of enzyme replacement therapy in patients.
Assuntos
Glucana 1,4-alfa-Glucosidase/metabolismo , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Leite/enzimologia , Animais , Animais Geneticamente Modificados , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/uso terapêutico , Doença de Depósito de Glicogênio Tipo II/enzimologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Miocárdio/ultraestrutura , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , alfa-GlucosidasesRESUMO
Glycogen storage disease type II (GSDII; Pompe disease), caused by inherited deficiency of acid alpha-glucosidase, is a lysosomal disorder affecting heart and skeletal muscles. A mouse model of this disease was obtained by targeted disruption of the murine acid alpha-glucosidase gene (Gaa) in embryonic stem cells. Homozygous knockout mice (Gaa -/-) lack Gaa mRNA and have a virtually complete acid alpha-glucosidase deficiency. Glycogen-containing lysosomes are detected soon after birth in liver, heart and skeletal muscle cells. By 13 weeks of age, large focal deposits of glycogen have formed. Vacuolar spaces stain positive for acid phosphatase as a sign of lysosomal pathology. Both male and female knockout mice are fertile and can be intercrossed to produce progeny. The first born knockout mice are at present 9 months old. Overt clinical symptoms are still absent, but the heart is typically enlarged and the electrocardiogram is abnormal. The mouse model will help greatly to understand the pathogenic mechanism of GSDII and is a valuable instrument to explore the efficacy of different therapeutic interventions.
Assuntos
Cardiomegalia/metabolismo , Doença de Depósito de Glicogênio Tipo II/metabolismo , Doença de Depósito de Glicogênio Tipo II/fisiopatologia , Glicogênio/metabolismo , alfa-Glucosidases/deficiência , Animais , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , Feminino , Doença de Depósito de Glicogênio Tipo II/genética , Masculino , Camundongos , Camundongos Knockout , alfa-Glucosidases/genéticaRESUMO
A method was developed to perform PCR directly on mycelial pellets or colonies treated with NOVOzym 234. The method allows rapid screening of large numbers of transformants of both sporulating and non-sporulating fungi for the presence of (co)transforming plasmid copies or for specific genetic modifications such as gene disruption and site specific integration. PCR fragments of at least 3.2 kb can be obtained. Using this method the identification of specific disruption mutants from Aspergillus niger and Beauveria bassiana was carried out.