90 Ann. N.Y. Acad. Sci. 1028: 90–103 (2004). © 2004 New York Academy of Sciences. doi: 10.1196/annals.1322.010 Targeted Delivery of Oncogene-Selective Antisense Oligonucleotides in Neuroectodermal Tumors: Therapeutic Implications FABIO PASTORINO, a CHIARA BRIGNOLE, a DANILO MARIMPIETRI, DANIELA DI PAOLO, MARTA ZANCOLLI, GABRIELLA PAGNAN, AND MIRCO PONZONI Differentiation Therapy Unit, Laboratory of Oncology, G. Gaslini Children’s Hospital, Genoa, Italy ABSTRACT: Neuroectodermal tumors are highly malignant and increasingly common tumors. Because the cure rate of these neoplasias by conventional treatment is very low, new therapeutic approaches are needed. Entrapping high concentrations of cytotoxic drugs and/or oligonucleotides within stabi- lized liposomal formulations represents an emerging modality of antitumor treatment. Here, we tested the in vitro and in vivo antitumor effects of a novel antisense oligodeoxynucleotide (asODN) liposomal formulation, the coated cat- ionic liposomes (CCL), by targeting the c-myc and the c-myb oncogenes on mel- anoma and neuroblastoma, respectively, through the use of a monoclonal antibody against the disialoganglioside GD 2 , selectively expressed by neuroec- toderma-derived tumors. Our methods produced GD 2 -targeted liposomes that stably entrapped 90 percent of added asODNs. These liposomes showed selec- tive binding for GD 2 -positive tumor cells in vitro. Neuroblastoma cells treated with free myb-as or nontargeted CCL-myb-as showed the same level of c-myb protein expression as control cells. In contrast, c-myb protein expression of cells treated with aGD 2 -CCL-myb-as was inhibited by approximately 70 per- cent. Melanoma and neuroblastoma cell proliferation was inhibited to a great- er extent by GD 2 -targeted liposomes containing c-myc or c-myb asODNs than by nontargeted liposomes or free asODNs. Mice bearing established subcuta- neous human melanoma xenografts treated with aGD 2 -CCL-myc-as exhibited significantly reduced tumor growth and increased survival. The mechanism for the antitumor effects appears to be downregulation of the expression of the c- myc protein, induction of p53, and inhibition of Bcl-2 proteins, leading to ex- tensive tumor cell apoptosis. In contrast, the increased life span obtained in a neuroblastoma pseudometastatic mouse model with the liposomal c-myb asODNs seems to be due to a synergistic mechanism: specific targeting to neu- roblastoma cancer cells, downmodulation of c-myb protein expression, and stimulation of the innate immune system. These results suggest that inhibition of c-myc or c-myb proto-oncogenes by GD 2 -targeted antisense therapy could Address for correspondence: Mirco Ponzoni, Differentiation Therapy Unit, Laboratory of Oncology, G. Gaslini Children’s Hospital, Largo G. Gaslini 5, 16148, Genoa, Italy. Voice: +39- 010-5636342; fax: +39-010-3779820. mircoponzoni@ospedale-gaslini.ge.it a These authors contributed equally to this study.