Direct gene transfer to the adult brain is dependent on vectors that transduce non-dividing cells, such as lentiviral vectors. Another aspect of the development of gene therapy to the brain is the need for cell-specific transgene expression. Expression from vesicular stomatitis virus G-protein (VSV-G) pseudotyped lentiviral vectors has been reported to be mainly neuron specific in the brain. We constructed cell-specific lentiviral vectors using the neuron-specific enolase (rNSE) or the glial fibrillary acidic protein (hGFAP) promoters and compared them to the ubiquitous human cytomegalovirus promoter (hCMV), a hybrid CMV/beta-actin promoter (CAG) and the promoter for human elongation factor 1 alpha (EF1 alpha). Our results showed that the hGFAP promoter was expressed only in glial cells, whereas rNSE was purely neuron specific, showing that VSV-G is pantropic in the rat striatum. We conclude that the VSV-G allows transduction of both glial and neuronal cells and the promoter dictates in what cell type the transgene will be expressed. The expression of transgenes exclusively in astrocytes would allow for local delivery of secreted transgene products, such as glial cell line-derived neurotrophic factor (GDNF), circumventing the anterograde transport that may induce unwanted side effects.

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.

A major obstacle in ex vivo gene transfer has been the loss of transgene expression soon after implantation of the grafted transduced cells. Recently, a lentiviral vector system has been developed which has proven to express high levels of transgenes in vivo after direct injection into the tissue. In this study, we have investigated the use of such a vector for ex vivo gene transfer to the brain. A number of neural cell types were found to be permissive to transduction by the lentiviral vector in vitro and a majority of them expressed the transgene after transplantation to the rat brain. Transgene expression was detected up to 8 weeks post-grafting. These findings suggest that recombinant lentiviral vectors may be used for further development of ex vivo gene therapy protocols to the CNS.

Astrocytes are, as normal constituents of the brain, promising vehicles for ex vivo gene delivery to the central nervous system. In the present study, we have used a lentiviral vector encoding glial cell line-derived neurotrophic factor (GDNF) to transduce rat-derived primary astrocytes, in order to evaluate their potential for long-term transgene expression in vivo and neuroprotection in a rat model of Parkinson's disease. Following transplantation of GDNF-transduced astrocytes to the intact striatum, the level of released GDNF was 2.93 +/- 0.28 ng/mg tissue at 1 week post-grafting, reduced to 0.42 +/- 0.12 ng/mg tissue at 4 weeks, and thereafter was maintained at this level throughout the experiment (12 weeks; 0.53 +/- 0.068 ng/mg tissue). Similarly, grafting to the substantia nigra (SN) resulted in a significant overexpression of GDNF ( approximately 0.20 ng/mg tissue) at 1 week. Intact animals receiving transplants of GDNF-transduced astrocytes displayed an increased contralateral turning (5.39 +/- 1.19 turns/min) in the amphetamine-induced rotation test, which significantly correlated with the GDNF tissue levels measured in the striatum, indicating a stimulatory effect of GDNF on the dopaminergic function. Transplantation of GDNF-transduced astrocytes to the SN 1 week prior to an intrastriatal 6-hydroxydopamine lesion provided a significant protection of nigral tyrosine hydroxylase-positive cells. By contrast, when the cells were transplanted to the striatum, the level of released GDNF was not sufficient to rescue the striatal fibers and, hence, to protect the nigral dopaminergic neurons. Overall, our results suggest that genetically modified astrocytes expressing GDNF can provide neuroprotection in a rat model of Parkinson's disease following transplantation to the SN.