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Aims—Insulin-like growth factor type I (IGF-I) antisense cellular gene therapy of tumours is based on the following data: rat glioma or hepatoma cells transfected with the vector encoding IGF-I antisense cDNA lose their tumorigenicity and induce a tumour specific immune response involving CD8+ T cells. Recently, using the IGF-I triple helix approach in studies of tumorigenicity, major histocompatibility complex class I (MHC-I) antigens were demonstrated in rat glioma transfected cells. This study used comparative IGF-I antisense and triple helix technologies in human primary glioma cells to determine the triple helix strategy that would be most appropriate for the treatment of glioblastoma. Methods—The cells were transfected using the IGF-I triple helix expression vector, pMT-AG, derived from the pMT-EP vector. pMT-AG contains a cassette comprising a 23 bp DNA fragment transcribing a third RNA strand, which forms a triple helix structure within a target region of the human IGF-I gene. Using pMT-EP, vectors encoding MHC-I or B7 antisense cDNA were also constructed. Results—IGF-I triple helix transfected glioma cells are characterised by immune and apoptotic phenomena that appear to be related. The expression of MHC-I and B7 in transfected cells (analysed by flow cytometry) was accompanied by programmed cell death (detected by dUTP fluorescein terminal transferase labelling of nicked DNA and electron microscopic techniques). Cotransfection of these cells with MHC-I and B7 antisense vectors suppressed the expression of MHC-I and B7, and was associated with a pronounced decrease in apoptosis. Conclusion—When designing an IGF-I triple helix strategy for the treatment of human glioblastoma, the transfected tumour cells should have the following characteristics: the absence of IGF-I, thepresence of both MHC-I and B7 molecules, and signs of apoptosis.

Alterations in the expression of growth factors and their receptors are associated with the growth and development of human tumors. One such growth factor is IGF-I (insulin-like growth factor I ), a 70-amino-acid polypeptide expressed in many tissues, including brain. IGF-I is also expressed at high levels in some nervous system-derived tumors, especially in glioblastoma. When using IGF-I as a diagnostic marker, 17 different tumors are considered as expressing the IGF-I gene. Malignant glioma, the most common human brain cancer, is usually fatal. Average survival is less than one year. Our strategy of gene therapy for the treatment of gliomas and other solid tumors is based on: 1) diagnostic using IGF-I gene expression as a differential marker, and 2) application of "triple-helix anti-IGF-I" therapy. In the latter approach, tumor cells are transfected with a vector, which encodes an oligoribonucleotide--an RNA strand containing oligopurine sequence which might be capable of forming a triple helix with an oligopurine and/or oligopyrimidine sequence of the promotor of IGF-I gene (RNA-IGF-I DNA triple helix). Human tumor cells transfected in vitro become down-regulated in the production of IGF-I and present immunogenic (MHC-I and B7 expression) and apoptotic characteristics. Similar results were obtained when IGF-I antisense strategy was applied. In both strategies the transfected cells reimplanted in vivo lose tumorigenicity and elicit tumor specific immunity which leads to elimination of established tumors.

IGF-I antisense gene therapy has been applied successfully to animal models of glioma, hepatoma and teratocarcinoma. The antisense strategy has shown that tumor cells transfected with vectors encoding IGF-I antisense RNA lose tumorigenicity, become immunogenic and are associated with tumor specific immune response involving CD8+ lymphocytes. An IGF-I triple helix approach to gene therapy for glioma was recently described. The approach we have taken is to establish parameters of change using the IGF-I triple helix strategy. PCC-3 embryonal carcinoma cells derived from murine teratocarcinoma which express IGF-I were used as a model. The cells were transfected with vector which encodes an oligoribonucleotide that forms RNA-IGF-I DNA triple-helix structure. The triple-helix stops the production of IGF-I. Cells transfected in this manner underwent changes in phenotype and an increase in MHC-I and B-7 cell surface molecules. They also showed enhancement in the production of apoptotic cells (60-70%). The "triple helix" transfected cells lost the ability to induce tumor when injected subcutaneously in syngeneic 129 Sv mice. When co-transfected in vitro with expression vectors encoding both MHC-I and B-7 cDNA in antisense orientation, the "triple-helix" transfected cells were down-regulated in expression of MHC-I and B-7 and the number of apoptotic cells was significantly decreased. Injection of the doubly co-transfected cells into 129 Sv mice was associated with induction of teratocarcinoma. Comparison between antisense and triple-helix transfected cells strategies showed similar immunogenic and apoptotic changes. The findings suggest that triple-helix technology may offer a new clinical approach to treatement of tumors expressing IGF-I.

Insulin-like growth factor I (IGF-I), has a role in cellular differentiation and is also expressed in neoplastic transformation of glioma cells. We recently demonstrated inhibition in expression of cellular IGF-I after transfection with vectors that incodes a segment of the human IGF-I RNA in antisense orientation. The transfected cells expressed increased levels of both MHC-I and B7 molecules. In this paper we show that IGF-I antisense transfected cells also become apoptotic. Moreover, the phenomenon of programmed cell death is related to the phenomenon that results in increased expression of MHC-I and B7 molecules. Co-transfection of rat glioma cells with the vector expressing IGF-I antisense RNA and with vectors encoding the expression of MHC-I and B7 antisense cDNA suppressed the expression of both of these molecules and was associated with a decrease in apoptosis.