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Neurotrophic factors (NF) are potent molecules with great promise for treatment of devastating neurodegenerative disorders like Alzheimer´s and Parkinson´s disease. Targeted delivery of NF like NGF or GDNF by means of viral vectors to distinct populations of brain cells may signifi cantly enhance bio-availability and safety of such treatment approaches. However, gene therapy in its current understanding means to introduce e.g. a cDNA coding GDNF into neurons or astrocytes and expressing the factor from that time on continuously, without an option to tune expression levels according to individual patient’s demands or to shut down expression in case of unforeseen side effects. In order to increase effi cacy and safety of gene therapy vectors for treatments of human patients, we here describe the development of a tightly regulatable vector system based on the approved human drug, mifepristone, for applications in the CNS. A two-vector based layout allows modulating levels of the transactivator in order to achieve a very tight off-state while maintaining suffi cient potency for activation of transgene expression. We describe characterization of the vector system in cultured primary brain cells and in the rat striatum and cortex in terms of dependence of vector titres, inducing drug levels, and repeated responsiveness. We believe that such vector system will signifi cantly promote gene therapeutic applications in patients but will also have great impact on basic research applications.
Brain-derived neurotrophic factor (BDNF) and its proBDNF precursor are released both in constitutive and activity-dependent manner. Prodomain itself proved to be necessary for BDNF targeting to regulated secretory pathway [Egan et al. (2003) Cell, Chen et al. (2005) J Neurosci] but its role in constitutive secretion is elusive. As mature BDNF (mBDNF) conveys trophic and prosurvival signals whereas proBDNF may convey growth inhibiting and death signals, an important issue arises: can we control the type of signal being triggered by BDNF? To verify this we cut off the prodomain and generated plasmid coding only for rat mBDNF. To test the constitutive mBDNF construct secretion we have chosen HEK 293 cell line. Two other plasmids coding either for proBDNF (template for both BDNF forms) or proBDNF protected from prodomain cleavage (source of proBDNF only), served as controls. BDNF secretion was evaluated with WB technique using antibodies detecting (1) both BDNF forms, (2) HA tag (mBDNF construct) and (3) MYC tag (proBDNF constructs). We found all three constructs being stably expressed in HEK cells. However, in contrast to both proBDNF constructs, which were secreted and detected in media fraction, mBDNF construct was revealed only in the cell lysate fraction, not being released to the media. This is the fi rst observation showing that mBDNF can be constitutively released only when accompanied by prodomain. Support: ASTF 211-00-2007 for EZ, Polish-German grant to MS and SK.
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