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Silencing of Foxp3 enhances the antitumor efficacy of GM-CSF genetically modified tumor cell vaccine against B16 melanoma

Authors Miguel A, Sendra L, Noé V, Ciudad CJ, Dasi F, Hervas D, Herrero MJ, Aliño SF

Received 16 January 2016

Accepted for publication 20 May 2016

Published 23 January 2017 Volume 2017:10 Pages 503—514

DOI https://doi.org/10.2147/OTT.S104393

Checked for plagiarism Yes

Review by Single-blind

Peer reviewers approved by Dr Narasimha Reddy Parine

Peer reviewer comments 4

Editor who approved publication: Dr Faris Farassati

Antonio Miguel,1 Luis Sendra,1 Verónica Noé,2 Carles J Ciudad,2 Francisco Dasí,3,4 David Hervas,5 María José Herrero,1,6 Salvador F Aliño17

1Department of Pharmacology, Faculty of Medicine, University of Valencia, 2Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Barcelona, 3Research University Hospital of Valencia, INCLIVA Health Research Institute, 4Department of Physiology, Faculty of Medicine, University of Valencia Foundation, 5Biostatistics Unit, 6Pharmacogenetics Unit, Instituto de Investigación Sanitaria La Fe (IIS La Fe), 7Clinical Pharmacology Unit, ACM Hospital Universitario y Politécnico La Fe, Valencia, Spain

Abstract: The antitumor response after therapeutic vaccination has a limited effect and seems to be related to the presence of T regulatory cells (Treg), which express the immunoregulatory molecules CTLA4 and Foxp3. The blockage of CTLA4 using antibodies has shown an effective antitumor response conducing to the approval of the human anti-CTLA4 antibody ipilimumab by the US Food and Drug Administration. On the other hand, Foxp3 is crucial for Treg development. For this reason, it is an attractive target for cancer treatment. This study aims to evaluate whether combining therapeutic vaccination with CTLA4 or Foxp3 gene silencing enhances the antitumor response. First, the “in vitro” cell entrance and gene silencing efficacy of two tools, 2'-O-methyl phosphorotioate-modified oligonucleotides (2'-OMe-PS-ASOs) and polypurine reverse Hoogsteen hairpins (PPRHs), were evaluated in EL4 cells and cultured primary lymphocytes. Following B16 tumor transplant, C57BL6 mice were vaccinated with irradiated B16 tumor cells engineered to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) and were intraperitoneally treated with CTLA4 and Foxp3 2'-OMe-PS-ASO before and after vaccination. Tumor growth, mice survival, and CTLA4 and Foxp3 expression in blood cells were measured. The following results were obtained: 1) only 2'-OMe-PS-ASO reached gene silencing efficacy “in vitro”; 2) an improved survival effect was achieved combining both therapeutic vaccine and Foxp3 antisense or CTLA4 antisense oligonucleotides (50% and 20%, respectively); 3) The blood CD4+CD25+Foxp3+ (Treg) and CD4+CTLA4+ cell counts were higher in mice that developed tumor on the day of sacrifice. Our data showed that tumor cell vaccine combined with Foxp3 or CTLA4 gene silencing can increase the efficacy of therapeutic antitumor vaccination.

Keywords: gene silencing, antitumor vaccine, Treg, antisense oligonucleotide, cancer immunotherapy

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