title: Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo.
abstract: Tumor antigen-encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.Molecular Therapy-Nucleic Acids (2013) 2, e134; doi:10.1038/mtna.2013.54; published online 19 November 2013.
authors.affiliation: Laboratory of Molecular & Cellular Therapy, Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Daphné
authors.initials: D
authors.lastname: Benteyn
authors.forename: Sébastien
authors.initials: S
authors.lastname: Anguille
authors.forename: Sandra
authors.initials: S
authors.lastname: Van Lint
authors.forename: Carlo
authors.initials: C
authors.lastname: Heirman
authors.forename: An Mt
authors.initials: AM
authors.lastname: Van Nuffel
authors.forename: Jurgen
authors.initials: J
authors.lastname: Corthals
authors.forename: Sebastian
authors.initials: S
authors.lastname: Ochsenreither
authors.forename: Wim
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authors.lastname: Waelput
authors.forename: Katrien
authors.initials: K
authors.lastname: Van Beneden
authors.forename: Karine
authors.initials: K
authors.lastname: Breckpot
authors.forename: Viggo
authors.initials: V
authors.lastname: Van Tendeloo
authors.forename: Kris
authors.initials: K
authors.lastname: Thielemans
authors.forename: Aude
authors.initials: A
authors.lastname: Bonehill