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

R.C Balestrin,R Sano,R Giugliani,U Matte,M.B Vieira,J.C Coelho,G Baldo

doi:10.1590/s0100-879x2008000400005

Abstract

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase beta-galactosidase (beta-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 beta-Gal gene (Glb1) to fibroblasts in culture using liposomes. Beta-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 microL lipofectamine 2000 and 1.5-2.0 microg DNA. Cells (2 x 10(5)/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-beta-Gal: 621.5 +/- 323.0, pSCTOP-beta-Gal: 714.5 +/- 349.5, pREP9-beta-Gal + pSCTOP-beta-Gal: 1859.0 +/- 182.4, and pREP9-ss-Gal + pTRACER: 979.5 +/- 254.9 nmol x h-1 x mg-1 protein) compared to untreated cells (18.0 +/- 3.1 for cell and 32.2 +/- 22.2 nmol x h-1 x 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 beta-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.

Highlights

GM1 gangliosidosis (OMIM #230500) is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of the acid hydrolase ß-galactosidase (GLB1; EC 3.2.1.23)
Gene transfer to cells inside the central nervous system (CNS) may represent a powerful tool to treat a large number of diseases, including neurological disorders and lysosomal diseases with CNS involvement [4]
Research in this area has been mainly based on the use of viral vectors due to their high gene transfer efficiency

Summary

Introduction

GM1 gangliosidosis (OMIM #230500) is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of the acid hydrolase ß-galactosidase (GLB1; EC 3.2.1.23). The disorder leads to progressive neural and visceral accumulations of GM1 ganglioside, its asialo derivative GA1, and other minor glycolipids and glycopeptides [1]. GM1 gangliosidosis is one of the most common LSD in Southern Brazil, with an estimated incidence of 1:17,000 live births [2]. GM1 gangliosidosis is characterized by generalized www.bjournal.com.br involvement of the central nervous system (CNS), progressive neurodegeneration, and early death [3]. Three clinical phenotypes can be distinguished, classified by age of onset: infantile, late infantile/juvenile, and adult. Infantile GM1 gangliosidosis, the severe form of the disease, is characterized by facial and skeletal abnormalities and neurological deterioration starting at about 6 months of age. Most patients die within the first 2 years of age [3]

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