Successful treatment of the childhood cancer neuroblastoma is difficult, particularly in patients with advanced stage disease. One novel treatment approach is genetic tumor modulation in which a "susceptibility gene" is introduced into tumor cells to increase their sensitivity to treatment. While this approach holds significant promise, it is unlikely that the introduction of a single gene will lead to eradication of all the cancer cells carrying the introduced gene due to occasional mutation or modification of the gene by the host cell. During the past six months, my laboratory has succeeded in transferring a number of yeast artificial chromosomes (YACs) into several human neuroblastoma cell lines by yeast spheroplast x neuroblastoma cell fusion. The main objective of this proposal is to develop a multimodal genetic therapy approach using the YAC vector, which has the unique ability of accommodating a large cloned insert. This will allow multiple copies of a single, or more, "susceptibility gene(s)" to be transferred into human neuroblastoma cells simultaneously such that the tumor cells become susceptible to multiple modalities of therapy. Using this gene transfer technique, I will explore the target-specificity for YAC transfer, and test whether YACs with a circular configuration are more like to be transferred. Furthermore, I will introduce one or more copies of the genes representing the herpes simplex virus thymidine kinase (HS-tk), a mouse histocompatibility antigen, H-2Kb, and the human deoxycytidine kinase (dCK) into human neuroblastoma cells through a YAC and determine whether these genes can be expressed simultaneously. These genes, when expressed, will impart to the neuroblastoma cells sensitivities to the anti-viral agent ganciclovir, anti-H-2Kb antibodies and complement, and the chemotherapeutic agent cytarabine. Hybrids carrying one or more copies of HS-tk gene(s) will be exposed to different concentrations of ganciclovir and the percentage of cell kill will be assessed. Similarly, cell hybrids carrying the H-2Kb antigen gene(s) will be exposed to cytotoxic antibodies against H-2Kb and those carrying the human dCK gene(s) will be exposed to varying concentrations of cytarabine and the percentage of cell kill will be quantitated for each treatment. In addition, hybrids containing multiple different genes, together with parental neuroblastoma control cells, will be exposed to these agent sequentially. The goal is to achieve 90% cell kill using each agent individually which will give an overall cell kill of >>99% per cycle of treatment. This will determine whether near-complete cell kill can be achieved using a combined regimen of sub-lethal level of each of these agents. Results from this study are expected to be directly applicable to designing new gene therapy approaches in cancer.