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.

Figure 1

Schematic of the different WT1-encoding vectors and quality control of the in vitro transcribed mRNA. (a) The T7 promoter, 5′-UTR, 3′-UTR, human β-globin UTR, poly(A)-tail (A64 or A120), the NLS, sig, and the lysosomal targeting sequence (DC-LAMP) are shown. All pGEM constructs have an overhang of nucleotides derived from the vector backbone, which remains as 3′ attachment to the poly(A)-tail after linearization with SpeI. The pST1 vector has a free-ending poly(A)-tail after linearization with SapI. The original WT1 sequence is depicted in white and the optimized WT1 sequence in gray. (b) Electropherogram of the wild-type WT1 mRNA, representative of all mRNA samples, and (c) gel-like image of mRNA derived from the different WT1 constructs. NLS, nuclear localization sequence; sig, signal peptide; UTR, untranslated regions; WT1, Wilms' tumor 1.

Figure 2

Expression and presentation of WT1 protein after electroporation with mRNA derived from the different WT1-encoding vectors. (a) Intracellular staining of WT1 in K562 cells. K562 cells were electroporated with the indicated WT1 mRNA constructs and analyzed for WT1 expression by intracellular staining 4-, 24-, and 48-hour postelectroporation (open histograms). As a control for nonspecific WT1 immunoreactivity, parallel electroporations were performed with a control mRNA (pGEM/NEF-DC-LAMP), which yielded similar expression levels as the isotype controls (gray-filled histograms). This figure is representative of three independent experiments. (b) Comparison of WT1 ΔMFI (MFI of positive population substracted by the basal WT1 epxression) values of the total K562 population and the total immature DC population after electroporation with WT1-sh-DC-L-OPT mRNA encoded by the pGEM vector. Data are presented as mean ± SEM of three independent experiments (**P = 0.0081). (c) Immunocytochemical detection of WT1 in TriMix-matured, WT1 mRNA-electroporated DCs. Immunocytochemistry for WT1 was performed on DCs fixated 24 hours after coelectroporation with WT1-encoding mRNA or control mRNA. Immunocytochemical staining patterns are shown from one representative experiment out of five (upper panel). Expression data were scored according to the staining intensity (middle panel) and according to the percentage of WT1-positive cells (lower panel; data are expressed as mean ± SEM of five independent experiments). (d) TriMix-DCs were coelectroporated with the indicated WT1 mRNA constructs or with the control mRNA. Twenty-four hours later, electroporated DCs were cocultured with the WT1-specific T-cell clone for 20 hours. IFN-γ release during this coculture was measured. Results are shown as mean ± SEM of three independent experiments (*P < 0.05). (e) Human HLA-A2⁺ TriMix-DCs were loaded with the WT1 antigen either by electroporation with the pST1-derived WT1-sh-DC-L-OPT mRNA or by pulsing with the HLA-A2-restricted WT1_126–134 peptide. Negative controls included TriMix-DCs electroporated with the control mRNA specified above or pulsed with the HLA-A2-restricted Melan-A_26-35 peptide. Four, 24, or 48 hours after antigen loading, cells were used to stimulate a human HLA-A2-restricted WT1_126-134-specific CD8⁺ T-cell clone. IFN-γ release was measured by ELISA after a 20-hour coculture. DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ MFI, mean fluorescence intensity; SEM, standard error of the mean; WT1, Wilms' tumor 1.

Figure 3

Functionality of the WT1 protein encoded by the optimized vector in an in vivo mouse model. (a) In vivo CTL assay after immunization with wild-type WT1 mRNA or mRNA encoded by the optimized vector. In vivo cytolysis of the target cells was assessed ex vivo by flow cytometry analysis of CFSE^lo or CFSE^hi cells in the spleen, injected and noninjected lymph nodes, and the peripheral blood. Data shown are from the blood and are representative for the other compartments. This figure is representative of three independent experiments. Data are presented as the percentage of specific lysis. (b) C57BL/6 mice (n = 7) were inoculated with 5 × 10⁵ C1498-WT1 cells s.c. 7 days later, and mice were immunized intranodally with control, wild-type WT1+TriMix, or optimized WT1+TriMix mRNA. Tumor volume was assessed (**P < 0.01; ***P < 0.001). WT1, Wilms' tumor 1.

Figure 4

Autotoxicity-related side effects after WT1 immunization. (a) WT1 expression in the kidney under physiological conditions. (b) No proteinuria was observed in mice immunized with the optimized WT1 mRNA, indicating no damage to the kidney (n = 8 mice/group). (c) No pathological changes, such as lymphocyte infiltration or tissue destruction and repair, were observed in the kidney, lung, or liver (negative control) of WT1 mRNA-immunized mice. WT1, Wilms' tumor 1.