IN VIVO GENE TRANSFER INTO RAT PITUITARY PROLACTINOMAS USING VIRAL VECTORS

RODOLFO G. GOYA,; CLAUDIA B. HEREÑÚ,; OMAR J. RIMOLDI,; NESTOR G. CARRI,; OSCAR A. BROWN

Fundación Instituto Leloir- Capital Federal- Argentina

Simposio; Gene Therapy in Latin America: from the bench to the clinic- 2002-; 2002

United Nations University ( Biotecnology for Latin America and the Caribbean Programme)

Abstract: We are interested in the implementation of gene therapy strategies for the treatment of age-related pathologies in the neuroendocrine system. In the aging rat, the more prevalent neuroendocrine pathology is the development of pituitary prolactinomas. In previous studies we demonstrated that herpetic and adenoviral vectors constitute suitable tools for gene transfer into both normal and neoplastic anterior pituitary (AP) cells in vitro.  In order to extend these studies to in vivo systems, primary pituitary prolactinomas were induced by estrogen treatment in ovariectomized young female Sprague Dawley rats. The development of the prolactinomas was monitored by measuring serum prolactin (PRL) levels in tail vein samples taken at 5-day intervals. When PRL levels went beyond 500 ng/ml (basal serum PRL was 5-10 ng/ml) the animals were stereotaxically injected into each AP lobe, with 1 ml of a suspension containing  either 5 x 105 pfu tsK/b-gal (a temperature-sensitive HSV-1 derived mutant (constructed by Dr. C. Preston) harboring the E. coli b-galactosidase transgene) or 5 x 108 pfu Ad.RSV.nls/b-gal (a replication-defective adenoviral vector (donated by Dr. M. Perricaudet) harboring the above reporter transgene targeted to the nucleus) or vehicle alone (controls). Two days later the animals were sacrificed, their pituitaries removed and b-galactosidase transgene expression assessed by both b-gal immunohistochemistry or enzymohistochemistry (X-gal). Viral suspensions tended to accumulate at the base of the pituitaries, with tsK/b-gal showing a much higher transgene expression levels than Ad.RSV.nls/b-gal. In vivo cytopathic levels were higher in the tsK/b-gal-injected pituitaries, as assessed by measuring the percentage of apoptotic nuclei in the vicinity of the injection areas. When prolactinoma-bearing rats were intra-pituitarily injected with an adenoviral vector (RadTK), harboring the suicide gene for herpes simplex virus type 1 thymidine kinase (HSV1-TK) under the control of the human cytomegalovirus promoter, and surgery was followed by two daily i.p. injections of 25 mg ganciclovir/Kg, during 7 days, a moderate but significant reduction in both serum PRL and pituitary tumor size was observed. In order to improve the delivery of this suicide gene and the detectability of its product, we have recently constructed a new adenoviral vector, (RAd(eGFP-TK)fus), carrying a DNA sequence coding for the enhanced green fluorescent protein (eGFP) fused to the gene for HSV1-TK. This vector was constructed using the two-plasmid method, which is based on an enzyme-directed recombination between a small shuttle plasmid and a human adenovirus type 5 (Ad5) genomic plasmid. The hybrid gene coding for (eGFP-TK)fus  was excised from pJGH2 (a kind gift from Dr. J. Galipeau) and inserted into the multiple cloning site (MCS) of shuttle pDC515. This MCS was flanked upstream by the strong mouse cytomegalovirus promoter and downstream by the simian virus 40 polyadenylation signal. The genomic plasmid used, pBHGrftDelE1,3FLP, encoded the genome of Ad5 minus the E1a and E3 regions, the former having been deleted to render the adenoviral vector replication-deficient. Plasmid pBHGrftDelE1,3FLP also contained an expression cassette for the yeast FLP recombinase, which recognizes frt elements as recombination sites.  Both plasmids were cotransfected into 293 human embryonic kidney cells where FLP-directed recombination at appropriately placed frt sites occurred between the two plasmids, thus giving rise to the desired RAd genome. Agarose gel DNA analysis of lysates from cotransfected 293 cells confirmed the presence of both the viral genome and the (eGFP-TK)fus insert. Incubation of tumor cell lines with RAd(eGFP-TK)fus  during two days showed a high proportion of green fluorescing cells, thus demonstrating successful transgene transfer and expression. As the eGFP-TK fusion protein is known to retain HSV1 kinase activity, we expect that RAd(eGFP-TK)fus  may constitute a convenient and more efficient adenoviral vector to transfer the HSV1-TK suicide gene in vivo in estrogen-induced pituitary tumors as well as in vitro, in pituitary tumor cells. In the latter, the fluorescence of the eGFP-TK fusion protein  should allow us to readily monitor suicide transgene long-term expression by fluorescence microscopy in living cells under different experimental circumstances. From the work so far done by us and others we conclude that, although further technological improvement is necessary, adenoviral vector-mediated suicide gene therapy of pituitary tumors constitutes an achievable medical goal.