Ex vivo Gene Transfer using rat Astrocytes Transduced with Lentiviral Vectors

Ex vivo Gene Transfer using rat Astrocytes Transduced with Lentiviral Vectors

Field: Disorders of the nervous system

Authors: Ericson, Cecilia
Wictorin, Klas
Lundberg, Cecilia

Address of presenting
author: Wallenberg Neuroscience Center
Lund University
Sölvegatan 17
S-223 62 Lund
Sweden

E-mail: Cecilia.Ericson@mphy.lu.se

Phone: 046-222 05 64

Fax: 046-222 05 61

Text of abstract: Astrocytes have many favourable features for use in ex vivo gene therapy to the CNS. They are normal consituents of the brain and have been shown to survive and differentiate well after transplantation to the CNS. In previous studies with retroviral transduction of astrocytes, downregulation of the transgene has occurred within weeks after transplantation. Lentiviral vectors have been shown to be highly efficient for in vivo gene delivery,with stable long-term expression of the transgene in different target cells, including both neurons and glial cells. Therefore, we wanted to explore the use of lentiviral vectors to obtain genetically modified astrocytes with a capacity for long-term gene expression in the rat brain. Astrocytes derived from rat embryonic day 15 lateral ganglionic eminence were infected with a lentiviral vector expressing the marker green fluorescent protein (GFP) and then transplanted into the adult rat striatum. The animals were perfused and immunostained for GFP at 1, 2 and 6 weeks postgrafting, and surviving, GFP expressing astrocytes were detected at all time-points. The GFP expressing cells were mainly found adjacent to the needle tract. Furthermore, the genes encoding glutamic acid decarboxylase (GAD₆5 and GAD₆7) were lentivirally transduced into human embryonic kidney cells, 293T. GAD expression, synthesis and release of GABA into the media could be readily detected. Thus, in ongoing studies, cultured rat astrocytes have been similarly transduced with the lentiviral vector expressing GAD, with the aim to eventually implant these cells into animal models of neurodegenerative diseases.
Supported by the Crafoord and Segerfalk foundations.

Keywords: astrocytes, GAD, ex vivo, lentiviral vectors, gene transfer

	Ex vivo Gene Transfer using rat Astrocytes Transduced with Lentiviral Vectors
Field:	Disorders of the nervous system
Authors:	Ericson, Cecilia Wictorin, Klas Lundberg, Cecilia
Address of presenting author:	Wallenberg Neuroscience Center Lund University Sölvegatan 17 S-223 62 Lund Sweden
E-mail:	Cecilia.Ericson@mphy.lu.se
Phone:	046-222 05 64
Fax:	046-222 05 61
Text of abstract:	Astrocytes have many favourable features for use in ex vivo gene therapy to the CNS. They are normal consituents of the brain and have been shown to survive and differentiate well after transplantation to the CNS. In previous studies with retroviral transduction of astrocytes, downregulation of the transgene has occurred within weeks after transplantation. Lentiviral vectors have been shown to be highly efficient for in vivo gene delivery,with stable long-term expression of the transgene in different target cells, including both neurons and glial cells. Therefore, we wanted to explore the use of lentiviral vectors to obtain genetically modified astrocytes with a capacity for long-term gene expression in the rat brain. Astrocytes derived from rat embryonic day 15 lateral ganglionic eminence were infected with a lentiviral vector expressing the marker green fluorescent protein (GFP) and then transplanted into the adult rat striatum. The animals were perfused and immunostained for GFP at 1, 2 and 6 weeks postgrafting, and surviving, GFP expressing astrocytes were detected at all time-points. The GFP expressing cells were mainly found adjacent to the needle tract. Furthermore, the genes encoding glutamic acid decarboxylase (GAD₆5 and GAD₆7) were lentivirally transduced into human embryonic kidney cells, 293T. GAD expression, synthesis and release of GABA into the media could be readily detected. Thus, in ongoing studies, cultured rat astrocytes have been similarly transduced with the lentiviral vector expressing GAD, with the aim to eventually implant these cells into animal models of neurodegenerative diseases. Supported by the Crafoord and Segerfalk foundations.

Keywords:	astrocytes, GAD, ex vivo, lentiviral vectors, gene transfer

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Created 2000-03-13

Department of Physiological Sciences, Lund University

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