The ependymal route for insulin-like growth factor-1 gene therapy in the brain.

I.c.v. administration of the peptide insulin-like growth factor-1 (IGF-1) has been shown to be an effective neuroprotective strategy in the brain of different animal models, a major advantage being the achievement of high concentrations of IGF-1 in the brain without altering serum levels of the peptide. In order to exploit this therapeutic approach further, we used high performance recombinant adenoviral (RAd) vectors expressing their transgene under the control of the potent mouse cytomegalovirus immediate early (mCMV) promoter, to transduce brain ependymal cells with high efficiency and to achieve effective release of transgenic IGF-1 into the cerebrospinal fluid (CSF). We constructed RAd vectors expressing either a chimeric green fluorescent protein fused to HSV-1 thymidine kinase (TK/GFP)(fus), or the cDNA encoding rat IGF-1, both driven by the mCMV promoter. The vectors were injected into the lateral ventricles of young rats and chimeric GFP expression in brain sections was assessed by fluorescence microscopy. The ependymal cell marker vimentin was detected by immunofluorescence and nuclei were labeled with the DNA dye 4',6-diamidino-2-phenylindole. Blood and CSF samples were drawn at different times post-vector injection. In all cerebral ventricles, vimentin immunoreactive cells of the ependyma were predominantly transduced by RAd-(TK/GFP)(fus), showing nuclear and cytoplasmic expression of the transgene. For tanycytes (TK/GFP)(fus) expression was evident in their cytoplasmic processes as they penetrated deep into the hypothalamic parenchyma. I.c.v. injection of RAd-IGF-1 induced high levels of IGF-1 in the CSF but not in serum. We conclude that the ependymal route constitutes an effective approach for implementing experimental IGF-1 gene therapy in the brain.

Restorative effect of insulin-like growth factor-I gene therapy in the hypothalamus of senile rats with dopaminergic dysfunction.

Insulin-like growth factor-I (IGF-I) is emerging as a powerful neuroprotective molecule that is strongly induced in the central nervous system after different insults. We constructed a recombinant adenoviral vector (RAd-IGFI) harboring the gene for rat IGF-I and used it to implement IGF-I gene therapy in the hypothalamus of senile female rats, which display hypothalamic dopaminergic (DA) neurodegeneration and as a consequence, chronic hyperprolactinemia. Restorative IGF-I gene therapy was implemented in young (5 months) and senile (28 months) female rats, which received a single intrahypothalamic injection of 3 x 10(9) plaque-forming units of RAd-betagal (a control adenoviral vector expressing beta-galactosidase) or RAd-IGFI and were killed 17 days post-injection. In the young animals, neither vector modified serum prolactin levels, but in the RAd-IGFI-injected senile rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase-positive cells in the hypothalamus of experimental as compared with control senile animals (5874+/-486 and 3390+/-498, respectively). Our results indicate that IGF-I gene therapy in senile female rats is highly effective for restoring their hypothalamic DA dysfunction and thus reversing their chronic hyperprolactinemia.

Gene therapy in the neuroendocrine system.

The implementation of experimental gene therapy in animal models of neuroendocrine diseases is an area of growing interest. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, and in Koletsky (fa(k)/fa(k)) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively, were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotropic cell proliferation. Stereotaxic injection of an adenoviral vector expressing insulin-like growth factor I corrected their chronic hyperprolactinemia and restored TIDA neuron numbers. Spontaneous intermediate lobe pituitary tumors in a retinoblastoma (Rb) gene mutant mouse were corrected by injection of an adenoviral vector expressing the human Rb cDNA and experimental prolactinomas in rats were partially reduced by intrapituitary injection of an adenoviral vector expressing the HSV1-thymidine kinase suicide gene. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.