[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jan 2006 · European Journal of Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The studies of neural stem cell fate, as well as the possibility to genetically manipulate them, represent important tools for modern neuroscience research. Furthermore, the potential use of these cells in treatment of neurological disorders makes these methods valuable for the development of new treatment paradigms. Here we report a method to genetically mark and modify neuroblasts and progenitor cells in the subventricular zone of post-natal rats using retroviral vectors. Using GFP as a marker gene we were able to follow the cells as they migrate and differentiate into olfactory interneurons. The cells were found in the olfactory bulb already 1 week after injection of the vector and after 3 weeks all cells had reached this area. There was a higher efficiency of the labeling of cells in neonatal rats compared to adults but injecting directly into the subventricular zone could to some extent counteract this effect. However, the cell types generated by the GFP positive cells were the same in neonatal and adult animals. This method will be a powerful tool to study the genetic interplay involved in neural stem cell differentiation and may be instrumental in finding a way to instruct these cells to participate in brain repair in the adult central nervous system.
No preview · Article · Apr 2005 · Journal of Neuroscience Methods
[Show abstract][Hide abstract] 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.
No preview · Article · Nov 2004 · Human Gene Therapy
[Show abstract][Hide abstract] ABSTRACT: The ability to regulate transgene expression will be crucial for development of gene therapy to the brain. The most commonly used systems are based on a transactivator in combination with a drug, e.g. the tetracycline-regulated system. Here we describe a different method of transgene regulation by the use of the human glial fibrillary acidic protein (GFAP) promoter. We constructed a lentiviral vector that directs transgene expression to astrocytes. Using toxin-induced lesions we investigated to what extent transgene expression could be regulated in accordance with the activation of the endogenous GFAP gene. In animals receiving excitotoxic lesions of the striatum we detected an eightfold increase of green fluorescent protein (GFP)-expressing cells. The vast majority of these cells did not divide, suggesting that the transgene was indeed regulated in a similar fashion as the endogenous GFAP gene. This finding will lead to the development of lentiviral vectors with autoregulatory capacities that may be very useful for gene therapy to the brain.
No preview · Article · Mar 2004 · European Journal of Neuroscience
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Oct 2003 · Journal of Neuroscience Research
[Show abstract][Hide abstract] ABSTRACT: Implantation of cells genetically modified to express therapeutic genes into the brain has been proposed as a potential treatment for neurodegenerative diseases. In the current study embryonic rat-derived astrocytes were cultured and transduced with a lentiviral vector expressing the reporter gene green fluorescent protein (GFP) and subsequently grafted into the adult rat brain. The proportion of GFP expressing cells was stable, albeit small (1%), at all survival times, up to 6 weeks, the longest time point studied. In parallel in vitro studies, the astrocytes were lentivirally transduced to express either one of the two isoforms of glutamate decarboxylase (GAD(65) or GAD(67)) or glial cell line-derived neurotrophic factor (GDNF). When transducing 293T cells with the two GAD vectors, released GABA could be measured using high-performance liquid chromatography. Further studies of rat astrocytes transduced with the same vectors resulted in a level of GAD activity about 10 times higher than the activity of an intact rat striatum. One hundred thousand astrocytes transduced with LV-GDNF released approximately 27 ng of GDNF per hour. Thus, taken together, our observations provide support for the use of rat astrocytes in ex vivo gene transfer of these proteins in animal models of CNS disorders, e.g., Parkinson's disease or epilepsy.
No preview · Article · Feb 2002 · Experimental Neurology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Long-term attached cultures, prepared from mouse embryonic days 15-17 lateral ganglionic eminence, were grown in a medium including epidermal growth factor and serum, and the survival, differentiation, and migration of cells from either early or late passages were analyzed following transplantation. The cultured cells had the morphology of type I astroglial cells, with the vast majority of the cells immunoreactive for glial fibrillary acidic protein (around 90%), the intermediate filament marker nestin, and also the mouse-specific neural markers M2 and M6. The cultures were kept over 25 passages (7 months). During the first 8 passages, the growth rate gradually declined, but it increased again after passage 9 and thereafter stabilized at values similar to those observed during the initial culture period. After passages 4-6 and 18, cell suspensions were implanted cross-species into the intact or lesioned striatum of adult (passages 4-5 only) or intact striatum of neonatal rats (passages 4-6 or 18). Both early and late passage cells formed M2 (and M6)-positive transplants. In the neonatal recipients, widespread migration was seen from the needle tract throughout most of the striatum, along the internal capsule, and into the globus pallidus. In the adult striatum, the cells remained mostly around the injection tract, or within 0.4-0.6 mm from the graft core. These long-term attached cultures are interesting to compare to nonattached neurosphere cultures, and might also offer a means of propagating relatively pure populations of astroglia-like cells for basic transplantation studies or for use in experimental trials with ex vivo gene transfer.
No preview · Article · Aug 2000 · Experimental Neurology