HIV-1-derived vector ͉ integrase deficient ͉ stable transgene expression

Glial cell line-derived neurotrophic factor (GDNF) was assayed for its neurotrophic effects against the neuronal atrophy that causes cognitive deficits in old age. Aged Fisher 344 rats with impairment in the Morris water maze received intrahippocampal injections at the dorsal CA1 area of either a lentiviral vector encoding human GDNF or the same vector encoding human green fluorescent protein as a control. Recombinant lentiviral vectors constructed with human cytomegalovirus promotor and pseudotyped with lyssavirus Mokola glycoprotein specifically transduced the astrocytes in vivo. Astrocyte-secreted GDNF enhanced neuron function as shown by local increases in synthesis of the neurotransmitters acetylcholine, dopamine and serotonin. This neurotrophic effect led to cognitive improvement of the rats as early as 2 weeks after gene transduction. Spatial learning and memory testing showed a significant gain in cognitive abilities due to GDNF exposure, whereas control-transduced rats kept their performance at the chance level. These results confirm the broad spectrum of the neurotrophic action of GDNF and open new gene therapy possibilities for reducing age-related neurodegeneration.

Gene transfer may become a powerful clinical tool for the delivery of secreted therapeutic polypeptides, provided that the in situ production of these peptides can be tightly regulated by the administration of a small inducer molecule. Particularly efficient control may be achieved by simultaneously using two regulation systems that interfere with the biosynthesis of the therapeutic factor at two different levels. Therefore, we have developed a set of two lentiviral vectors containing two regulation systems. These systems are induced by nonimmunosuppressive derivatives of rapamycin ("rapalogs") and allow simultaneous control of expression and of exocytosis of secreted therapeutic polypeptides. The set of vectors was used to produce green fluorescent protein (GFP) and glial cell line-derived neurotrophic factor (GDNF); GFP served as a model factor to demonstrate expression and entry into the exocytotic pathway in transduced cells. The constructs allowed robust in vitro expression and secretion of the polypeptides in the presence of rapalog AP21967. Withdrawal of the inducer resulted in efficient downregulation. In vivo, tightly regulated production of GFP and GDNF was observed after injection of the constructs into the striata of mice. The vectors thus fulfill key requirements for application in gene therapy.