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Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA

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Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA

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Título Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA
Autor Balestrin, Raquel Cristina
Baldo, Guilherme
Vieira, Matheus Barbosa
Sano, Renata
Coelho, Janice Carneiro
Giugliani, Roberto
Matte, Ursula da Silveira
Resumo GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 μL lipofectamine 2000 and 1.5-2.0 μg DNA. Cells (2 x 105/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß- Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients’ skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.
Contido em Brazilian Journal of medical and biological research. Vol. 41, n. 4 (2008), p. 283-288
Assunto Gangliosidose GM1
[en] Gene therapy
[en] GM1 gangliosidosis
[en] Lipofectamine
[en] Lysosomal storage disorder
[en] ß-galactosidase deficiency
Origem Nacional
Tipo Artigo de periódico
URI http://hdl.handle.net/10183/21223
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