Manipulations of Transgene Expression in Neural Progenitor Cell Lines in Vitro.

Manipulations of Transgene Expression in Neural Progenitor Cell Lines in Vitro.

Field: Disorders of the nervous system

Authors: Rosenqvist, Nina
Hård af Segerstad, Caroline
Lundberg, Cecilia

Address of presenting
author: Wallenberg Neuroscience Center
Division of Neurobiology
Sölveg.17
223 62 LUND

E-mail: nina.rosenqvist@mphy.lu.se

Phone: 46-46 2220528

Fax: 2220561

Text of abstract: Gene therapy in the CNS holds great promise to ameliorate symptoms of e.g neurodegenerative disorders. However, achieving stable, long-term transgene expression in grafted cells has proven problematic. DNA metylation and histone deacetylations have been suggested to be mechanisms for silencing of transgenic DNA. We have established an in vitro model of transgene downregulation in the CNS using the immortalized neural progenitor cell lines HiB5 and RN33B. The cell lines were transduced at 33°C (the permissive temperature) with different GFP constructs, both viral and non-viral, containing either viral or non-viral promoters. GFP expression was then followed by FACS analysis. Cell differentiation in vitro was obtained by culturing the cells at non-permissive temperatures in serum free defined media, which halts cell division. Already at 3 days of culture at non-permissive temperatures the transgene expression decreased markedly and after 7 days GFP expression was greatly reduced (up to 95%) in most cell lines. The effect of sodium butyrate, an inhibitior of histone deacetylation, and 5’azacytidine, a DNA methyltransferase inhibitor, were analyzed in the in vitro model. The addition of sodium butyrate induced GFP expression both at permissive and non-permissive temperatures. 5-azacytidine, on the other hand, was not as effective suggesting less impact of DNA methylation on transgene expression in this model. Furthermore, the clones were grafted to the adult rat striatum and GFP expression evaluated after 2 weeks. At this time-point only the RN33B clones show any transgene expression. Knowledge about mechanisms of transgene silencing appears to be crucial in the development of ex vivo gene therapy to the CNS.

Keywords: Gene therapy, Transplantation, Silencing

	Manipulations of Transgene Expression in Neural Progenitor Cell Lines in Vitro.
Field:	Disorders of the nervous system
Authors:	Rosenqvist, Nina Hård af Segerstad, Caroline Lundberg, Cecilia
Address of presenting author:	Wallenberg Neuroscience Center Division of Neurobiology Sölveg.17 223 62 LUND
E-mail:	nina.rosenqvist@mphy.lu.se
Phone:	46-46 2220528
Fax:	2220561
Text of abstract:	Gene therapy in the CNS holds great promise to ameliorate symptoms of e.g neurodegenerative disorders. However, achieving stable, long-term transgene expression in grafted cells has proven problematic. DNA metylation and histone deacetylations have been suggested to be mechanisms for silencing of transgenic DNA. We have established an in vitro model of transgene downregulation in the CNS using the immortalized neural progenitor cell lines HiB5 and RN33B. The cell lines were transduced at 33°C (the permissive temperature) with different GFP constructs, both viral and non-viral, containing either viral or non-viral promoters. GFP expression was then followed by FACS analysis. Cell differentiation in vitro was obtained by culturing the cells at non-permissive temperatures in serum free defined media, which halts cell division. Already at 3 days of culture at non-permissive temperatures the transgene expression decreased markedly and after 7 days GFP expression was greatly reduced (up to 95%) in most cell lines. The effect of sodium butyrate, an inhibitior of histone deacetylation, and 5’azacytidine, a DNA methyltransferase inhibitor, were analyzed in the in vitro model. The addition of sodium butyrate induced GFP expression both at permissive and non-permissive temperatures. 5-azacytidine, on the other hand, was not as effective suggesting less impact of DNA methylation on transgene expression in this model. Furthermore, the clones were grafted to the adult rat striatum and GFP expression evaluated after 2 weeks. At this time-point only the RN33B clones show any transgene expression. Knowledge about mechanisms of transgene silencing appears to be crucial in the development of ex vivo gene therapy to the CNS.

Keywords:	Gene therapy, Transplantation, Silencing

Index

Created 2000-03-14

Department of Physiological Sciences, Lund University

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