Regulated Delivery of Glial Cell Line-Derived Neurotrophic Factor into Rat Striatum, Using a Tetracycline-Dependent Lentiviral Vector

Abstract

In this study, a tetracycline-regulated lentiviral vector system, based on the tetracycline-dependent transactivator rtTA2(S)-M2, was developed for controlled expression of glial cell line-derived neurotrophic factor (GDNF) in the rat brain. Expression of the marker gene green fluorescent protein (GFP) and GDNF was tightly regulated in a dose-dependent manner in neural cell lines in vitro. Injection of high-titer lentiviral vectors into the rat striatum resulted in a 7-fold induction of GDNF tissue levels (1060 pg/mg tissue), when doxycycline (a tetracycline analog) was added to the drinking water. However, low levels of GDNF (150 pg/mg tissue) were also detected in animals that did not receive doxycycline, indicating a significant background leakage from the vector system in vivo. The level of basal expression was markedly reduced when a 10-fold lower dose of the tetracycline-regulated GDNF vector was injected into the striatum (3-11 pg/mg tissue), and doxycycline-induced GDNF tissue levels obtained in these animals were about 190 pg/mg tissue. Doxycycline-induced expression of GDNF resulted in a significant downregulation of the tyrosine hydroxylase (TH) protein in the intact striatum. Removal of doxycycline from the drinking water rapidly (within 3 days) turned off transgenic GDNF mRNA expression and GDNF protein levels in the tissue were completely reduced by 2 weeks, demonstrating the dynamics of the system in vivo. Accordingly, TH protein expression returned to normal by 2-8 weeks after removal of doxycycline, indicating that GDNF-induced downregulation of TH is a reversible event.

Human neural progenitors deliver glial cell line-derived neurotrophic factor to parkinsonian rodents and aged primates

“…This result is similar to other reports where inducible vectors have been used to control the release of GDNF following direct injection of viral vectors to the brain, but only met with limited Human neural progenitors releasing GDNF S Behrstock et al success. 43 There are a number of reasons why regulation cannot be achieved in vivo. Perhaps the most parsimonious of these is that GDNF may have a long-half life when compared to TH or destabilized GFP although there is limited evidence in the literature at present.…”

Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9

“…Lentiviral vectors were produced as previously described 27 . These vectors were titrated using flow cytometry and quantitative PCR analysis as previously described 28 . The titres of the vectors in this study were in the range of: 5 Â 10 8 -1 Â 10 9 TU per ml.…”

Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics

“…With the exception of two reports, 46,48 all the tetracycline-inducible HIV-1 vectors listed in the literature were constructed using the tet-on system. 49,50 The authors showed that the tetracycline approach applied to HIV-1 gene transfer vectors allows inducible expression, that is dose-dependent and rapidly switched on and off.…”