Ulrike Blömer

In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.

A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.

Ex Vivo and In Vivo Gene Delivery to the Brain

This unit describes methods for grafting genetically modified cells for ex vivo delivery of specific genes into the rat brain and direct delivery of transgenes to brain cells in vivo using recombinant viral vectors. These methods assess the function of a gene in the brain. The ex vivo approach of gene transfer to the nervous system depends on genetic manipulation of cells in vitro prior to grafting of the cells into the brain to enable production of a transgene at physiologically significant levels for a long period of time. This unit also includes procedures for transcardial perfusion to fix the tissue prior to analysis of expression and for sectioning of brains by use of a freezing sledge microtome. Thionine staining of tissue sections is also described.

UNIT 13.3 Ex Vivo and In Vivo Gene Delivery to the Brain

This unit describes methods for grafting genetically modified cells for ex vivo delivery of specific genes into the rat brain and direct delivery of transgenes to brain cells in vivo using recombinant viral vectors. These methods assess the function of a gene in the brain. The ex vivo approach of gene transfer to the nervous system depends on genetic manipulation of cells in vitro prior to grafting of the cells into the brain to enable production of a transgene at physiologically significant levels for a long period of time. This unit also includes procedures for transcardial perfusion to fix the tissue prior to analysis of expression and for sectioning of brains by use of a freezing sledge microtome. Thionine staining of tissue sections is also described.

Lentiviral Vectors for Gene Delivery in the Nervous System

Gene therapy is an attractive approach for the treatment of a number of inherited and acquired disorders of the nervous system. For instance, genetic manipulations could be used to provide locally the neurotransmitters that are defective in conditions such as Parkinson’s disease, or to produce healing levels of growth factors to protect neurons against the irreversible consequences of traumatic or degenerative lesions.