A new nucleic acid–based agent inhibits cytotoxic T lymphocyte–mediated immune disorders Chuang-Wei Wang, MS, Wen-Hung Chung, MD, PhD, Yi-Fang Cheng,

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Presentation transcript:

A new nucleic acid–based agent inhibits cytotoxic T lymphocyte–mediated immune disorders Chuang-Wei Wang, MS, Wen-Hung Chung, MD, PhD, Yi-Fang Cheng, PhD, Nien-Wen Ying, MS, Konan Peck, PhD, Yuan-Tsong Chen, MD, PhD, Shuen-Iu Hung, PhD Journal of Allergy and Clinical Immunology Volume 132, Issue 3, Pages 713-722.e11 (September 2013) DOI: 10.1016/j.jaci.2013.04.036 Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Specificity of the CD8AP17 aptamer. A and B, The binding capacity of selected pools (Fig 1, A) and CD8AP17 (Fig 1, B) for CD8 transfectants and nontransfectants was observed by using flow cytometry. An unselected initial DNA library pool (Random pool) served as a negative control. C, Percentage of DNA bound and Kd of CD8AP17. D, Binding of the CD8AP17 aptamer to Sup-T1 cells, Sup-T1 cells with CD8a siRNA (CD8KD), and C1R cells. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Shortening and identifying the CD8-binding region of the CD8AP17 aptamer by using a microarray. A, The aptamer sequence was permuted by using the 1-base-shift method. B, Fluorescence intensity of the 15 highest C/S ratios of CD8 binding. C and D, Predicted secondary structures of CD8AP17 and CD8AP17.29.15 (as CD8AP17s), respectively. E and F, Binding capacity and Kd of CD8AP17s and its 2 shortened stem-loop subsequences. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 The CD8AP17s aptamer was internalized into live CD8+ cells but not into CD8− cells. The nuclei of Sup-T1 and C1R cells were stained with Hoechst 33258 (blue). Alexa Fluor 647–labeled CD8AP17s aptamer (red; 100 nmol/L) was incubated with cells at 37°C for 20 minutes. Images of Z plane scanning for CD8AP17s on Sup-T1 cells are also shown. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Design of the aptamer-siRNA chimera. A, Best knockdown efficacy of GNLY siRNA. B, Structure of the aptamer (CD8AP17s)-stick-siRNA chimera. C, Aptamer with the sticky sequence and the siRNAs formed stable base pairs (lane 5). D, Binding capacity of the aptamer-siRNA chimera on Sup-T1 and C1R cells. E, Silencing of GNLY mRNA expression in CD8+ cells. Elec., Electroporation transfected. F, Median effective concentration (EC50) of the aptamer-siRNA chimera. Shown are means ± SDs (n = 3). **P < .01, 2-tailed t test. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 The aptamer-siRNA chimera inhibits drug-induced SJS T-cell responses in vitro. A, Release of GNLY was measured in the coculture model of carbamazepine (CBZ)–induced SJS. B, Release of GNLY was measured in the coculture model of allopurinol-induced SJS. C, Cytolytic activity of allopurinol-specific T cells. N.D., Not detectable. Shown are means ± SDs (n = 3). *P < .05 and **P < .01, 2-tailed t test. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 The aptamer-siRNA chimera inhibits alloreactive responses in vitro. A, Human CD8+ T cells were alone or cocultured with allologous B cells (Allo-Ag). GNLY release was measured. B, Best knockdown efficacy of NF-κB p65 siRNA in THP-1XBlue cells. C and D, NF-κB protein expression was measured in Sup-T1 or C1R cells, respectively. Elec., Electroporation transfected. E, Cytolytic activity of CD8+ T cells. Shown are means ± SDs (n = 3). *P < .05, **P < .01, and ***P < .001, 2-tailed t test. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Proposed mechanism of the aptamer-siRNA chimera. The aptamer-siRNA chimera is capable of cell type–specific binding and delivery of functional siRNA into cells. The aptamer-siRNA chimera binds to the CD8 receptor, internalizes through endocytosis, and subsequently is released by dicer enzyme from the endosome to start the RNAi process. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Flowchart of the SELEX process. Starting the SELEX process was a synthetic random ssDNA oligonucleotide library consisting of 1015 different sequences. The ssDNA library pool was bound with intact CD8+ T cells in the first round of selection, followed by heat elution. The selected ssDNA pool was directly incubated with CD8 pure protein, which was immunoprecipitated by protein G beads from cell lysate. The SELEX procedures, such as elution of bound ssDNA and PCR amplification, were similar to those used in the conventional SELEX process. Negative selection was performed in the third and eighth rounds to remove the ssDNA that was bound to protein G beads. The last SELEX round was finished on the amplification step. The enriched ssDNA pool was cloned, and individual aptamers were characterized. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Knockdown of CD8 expression in Sup-T1 cells. For examination of the binding specificity of aptamers, Sup-T1 cells were transfected with CD8 siRNA to yield CD8 knockdown (CD8KD) cells. The flow cytometric assay was used to measure the knockdown efficiency of CD8 expression on the cell surface. The cells treated with nonsilencing siRNA (CON siRNA) were also shown. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 The median effective concentration (EC50) of the aptamer-siRNA chimera. The CD8AP17s-GNLY chimera inhibited the GNLY gene expression of CD8+ cells in a concentration-dependent manner. The figure shows the chimera concentrations of 1, 2.5, 5, 10, and 20 nmol/L. Knockdown efficiency of GNLY mRNA expression was assessed by using quantitative PCR. The EC50 of the CD8AP17s-GNLY chimera was 14.65 nmol/L. Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 The aptamer-siRNA chimera did not silence gene expression in CD8− cells. C1R B cells (CD8−) were used to determine the specific effect of the CD8AP17s-GNLY chimera. GNLY mRNA levels were very low in B cells, and B cells stimulated with IL-2 and IL-15 did not enhance GNLY mRNA expression. Therefore we first transfected the GNLY gene into B cells for 24 hours and then treated it with aptamer or the aptamer-siRNA chimera for 48 hours. GNLY mRNA amounts were assessed by using quantitative PCR. The level of GNLY mRNA expression in B-cell transfectants was set at 100%. All data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Shown are means ± SDs (n = 3). Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 The aptamer-siRNA chimera did not trigger nonspecific immune responses. A and B, Human PBMCs (2 × 106) treated with 500 nmol/L CD8AP17s, GNLY siRNA (by using electroporation transfection), or the CD8AP17s-GNLY chimera were assessed for production of IFN-α (Fig E6, A) and IL-6 (Fig E6, B) by means of ELISA at 48 hours after treatment. C, Cell viability was also determined by using the MTT assay. Cells treated with IL-2 (100 U/mL) were used as a positive control. Shown are means ± SDs (n = 3). Journal of Allergy and Clinical Immunology 2013 132, 713-722.e11DOI: (10.1016/j.jaci.2013.04.036) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions