EWS Fli-1 Antisense Nanocapsules Inhibits Ewing Sarcoma-Related Tumor in Mice Gregory Lambert,* Jean Re ´mi Bertrand,† Elias Fattal,* Fre ´de ´ric Subra,† Huguette Pinto-Alphandary,* Claude Malvy,† Christian Auclair,† and Patrick Couvreur* ,1 *Laboratoire de physico-chimie, pharmacotechnie et biopharmacie, UMR CNRS 8612, Faculte ´ de Pharmacie, Cha ˆ tenay-Malabry, France; and †Laboratoire de Physicochimie et Pharmacologie des macromole ´cules biologiques, UMR CNRS 8532, Institut Gustave Roussy, 94800 Villejuif, France Received October 25, 2000 EWS Fli-1, a fusion gene resulting from a t(11;22) translocation is found in 90% of both Ewing’s sarcoma and primitive neuroectodermal tumor (PNET). In the present study, we show that recently developed poly- isobutylcyanoacrylate nanocapsules with an aqueous core were able to encapsulate efficiently high amounts of phosphorothioate oligonucleotides (ODN) directed against EWS Fli-1 chimeric RNA. Release of these ODN in serum medium was shown to be biphasic which was explained by the presence of two types of nanocap- sules able to release ODN with different kinetics. In addition, nanocapsules were found to provide protec- tion of these oligonucleotides from the degradation in serum. These ODN nanocapsules permitted to obtain inhibition of Ewing sarcoma-related tumor in mice after intratumoral injection of a cumulative dose as low as 14.4 nanomoles. This new type of non viral vector shows great potential for in vivo administration of oligonucleotides. © 2000 Academic Press Key Words: nanocapsules; polyisobutylcyanoacry- late; oligonucleotides; antisense; ewing; PNET; EWS Fli-1. Ewing’s sarcomas and primitive neuroectodermal tu- mors (PNETs) are malignant tumors histologically characterized by uniform, densely packed, small round cells with round nucleoli-free nuclei and indistinct cy- toplasm. Ewing’s sarcoma and PNETs represents a biological spectrum of the same tumor (1). The Ewing’s karyotype, t(11;22)(q24;q12), occurring in approxi- mately 90 percent of the Ewing’s and PNETs that can be evaluated, is the most diagnostic signature of any solid tumor (2). Cytogenetic studies of Ewing’s sarcoma and PNETs have identified a consistent alteration of the EWS locus on chromosome 22 band q12. Charac- teristically, the amino terminus of the EWS gene is juxtaposed with the carboxy terminus provided by the Fli-1 member of the Ets family of transcription factors gene located on chromosome 11 band q24 (3). The translocation results in the fusion of the EWS gene with the transcription factor gene Fli-1, leading to a hybrid transcript and an oncogenic chimeric protein that may be potent activator of inappropriate DNA transcription (4). Recent studies have shown that sta- ble expression of antisense EWS Fli-1 in Ewing sar- coma cells by transfecting the antisense EWS Fli-1 expression plasmid resulted in partial loss of endoge- nous EWS Fli-1 proteins and suppression of the cell growth (5). This suggested a possible role of the fusion products in tumorigenesis. The remarkable prevalence of the hybrid transcripts reported by Delattre et al. (6) may allow specific gene therapy for Ewing’s-PNET tu- mors. The abnormal fusion transcript could be targeted and scrambled by specific antisense oligonucleotides. Antisense oligonucleotides (ODN) are synthetic frag- ments of DNA with base sequences complementary to a mRNA. As a result of their ability to hybridize to mRNA targets, ODN can selectively modulate the ex- pression of individual genes (7). Thus, they have a great potential for the treatment of several diseases such as cancer (8, 9) and viral infections (10). In addi- tion, they may be useful tools to understand the phys- iological role of number of proteins involved in cellular message transduction (5). However, the intensive use of ODN in therapeutics has been hampered by their poor stability in biological fluids and their low intra- cellular penetration (11). The use of chemically modi- fied ODN such as phosphorothioates or methylphos- phonates (12) has been proposed as a solution to these problems. As an alternative strategy, colloidal carriers such as polymeric nanospheres have been proposed to improve the administration of ODN (13, 14). In most of cases, phosphodiester ODN have been associated with the surface of nanospheres through electrostatic inter- actions; Chavany et al. (15) took advantage of the for- 1 To whom correspondence should be addressed. Biochemical and Biophysical Research Communications 279, 401– 406 (2000) doi:10.1006/bbrc.2000.3963, available online at http://www.idealibrary.com on 401 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.