Use of a defective herpes simplex virus for transfer of neurotrophin genes into cells of the nervous system

Abstract

There is a need in neurobiology for an efficient method of transferring genes into cells of the nervous system in order to study the function of neuronal proteins, to create animal models of human disease, and to administer gene therapy. The neurotrophins are a family of growth factors, of which nerve growth factor (NGF) is the prototype, that play a key role in the development and maintenance of the nervous system. We have used a defective herpes simplex type 1 viral vector, called an amplicon, to transfer genes for two of the neurotrophins into cells in vitro, in situ, and in vivo with the goal of determining whether such methods would be efficacious in modifying neuronal function for the aforementioned reasons.;In these studies, the herpes amplicon vector system was used to deliver genes for two of the neurotrophins (NGF and brain-derived neurotrophic factor (BDNF)) into cell lines, primary neurons and explanted nervous tissue, as well as in vivo. Three different vectors, each employing the strong herpes simplex virus immediate early 4/5 (HSV IE 4/5) promoter, were used in these studies. In one vector, HSVngf, the promoter drove a rat/mouse hybrid minigene for NGF and in another, HSVbdnf, the cDNA for human BDNF. The third vector, HSVbdnflac, was bicistronic and also used the IE 4/5 promoter to drive a BDNF cDNA; however, this was followed by an internal ribosome entry site (IRES; from parvoviruses) and the gene for E. coli {dollar}\beta{dollar}-galactosidase allowing for both genes to be translated from a bicistronic mRNA. The use of a bicistronic vector, coding for a functional protein and a marker protein, facilitates identification of transduced cells.;The results presented demonstrate that the HSV amplicons can efficiently transfer neurotrophin genes into cells of the nervous system, resulting in the expression and processing of RNA, the production of biologically-active growth factors, and the modification of neuronal physiology both in vitro and in vivo. This amplicon vector system is not only well-suited as a tool for studying neuronal physiology, but is also a potential mode of gene therapy.