Gene transfer in astrocytes: comparison between different delivering methods and expression of the HIV-1 protein Nef

To identify a good system to introduce foreign genes into normal and tumoral astrocytes, we studied the efficiency of two chemical methods, calcium phosphate precipitation and lipofection, and of a viral-mediated transfer by a vector derived from the highly attenuated modified vaccinia virus Ankara (MVA). Using the beta-galactosidase (beta-gal) gene (lacZ) as reporter, we searched for optimal experimental conditions to obtain an efficient gene transfer into human embryonic and neonatal rat astrocytes and into a human astrocytoma cell line (U373 MG). The beta-gal protein production was evaluated by cytochemical staining and enzymatic activity assay. Among chemical methods, lipofection was the most efficient system to transfect astrocytes in providing up to 60% of beta-gal-positive cells in all the cell types analyzed. MVA infection also proved to be an efficient system to introduce heterologous genes into human embryonic astrocytes that appeared 80-100% positive 48-96 hr after an infection at a multiplicity of 1-10. In contrast, only a limited infection was observed with rat astrocytes, human astrocytoma cells, and human leptomeningeal cells. A recombinant MVA vector expressing the human immunodeficiency virus-1 (HIV-1) regulatory protein Nef was used to transfect human embryonic astrocytes, and the resulting Nef expression was compared with that detected after lipofection in the same cells. By Western blot analysis, Nef expression was observed in human astrocytes 24-96 hr after infection and was similar to that present in stably HIV-1-infected astrocytoma cells. Lipofection resulted in lower Nef expression. In spite of these promising results, the negative effects of MVA infection on cell viability and the possibility that a productive infection occurs in human embryonic astrocytes limit the use of this vector for gene delivery in developmentally immature human glial cells.