Development of Therapeutic siRNAs for Pachyonychia Congenita

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Presentation transcript:

Development of Therapeutic siRNAs for Pachyonychia Congenita Frances J.D. Smith, Robyn P. Hickerson, Jane M. Sayers, Robert E. Reeves, Christopher H. Contag, Devin Leake, Roger L. Kaspar, W.H. Irwin McLean Journal of Investigative Dermatology Volume 128, Issue 1, Pages 50-58 (January 2008) DOI: 10.1038/sj.jid.5701040 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Sequence specificity and efficacy of K6a 3′-UTR siRNAs. (a) Alignments of portions of the human K6a and K6b 3′-UTR cDNA sequences, showing that the target sequences of the four siRNAs (bold type) are located where several insertion/deletion and single-base substitutions occur between K6a and K6b. (b) Normalized K6a-EYFP expression measured by FACS of 293FT cells transiently transfected with K6a-EYFP and the lead K6a siRNAs (K6a-S1, -S2, -S3, and -S4). A potent EGFP siRNA (which also targets EYFP equally well) was used as a positive control. A non-specific control siRNA (NSC4) did not significantly affect expression, even at high concentrations, whereas all four K6a inhibitors worked as well or better than the EGFP control inhibitor at low sub-nanomolar concentrations. Bars on the graph represent the mean±SE. (c) Visual confirmation of K6a siRNA efficacy by wide-field imaging of 293FT cells transiently transfected with K6a-EYFP and siRNAs over a range of inhibitor concentrations. Journal of Investigative Dermatology 2008 128, 50-58DOI: (10.1038/sj.jid.5701040) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Inhibition of endogenous K6a in HaCaT cells. (a) Coomassie blue staining of cytoskeletal extracts 96hours after transfection with 5nM K6a siRNAs showing near-complete loss of K6a expression with all four K6a 3′-UTR siRNAs; all other keratins were unaffected. (b) Immunoblotting of replicate gels as shown in (a), with a range of anti-keratin antibodies, confirming the loss of K6a expression and normal expression of other keratins present in HaCaT cells. Journal of Investigative Dermatology 2008 128, 50-58DOI: (10.1038/sj.jid.5701040) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 K6a 3′-UTR siRNAs do not significantly affect K6b expression. (a) Immunoblot of total protein extracts from AD293 cells transfected with dual expression vector expressing FLAG epitope-tagged K6a and myc epitope-tagged K6b in combination with test siRNAs. Blot stained with Ponceau S to demonstrate equal protein loading in each lane. (b) Replica immunoblot shown in (a), stained with anti-FLAG antibody to show K6a-FLAG expression. All four K6a siRNAs completely knock out K6a expression, whereas cells transfected with K6a/K6b plasmid alone or an irrelevant siRNA show high levels of K6a-FLAG expression. (c) Replica immunoblot shown in (a), stained with anti-myc antibody to show K6b-myc expression. Only a slight reduction in K6b-myc expression is seen with the four K6a-specific siRNAs. Journal of Investigative Dermatology 2008 128, 50-58DOI: (10.1038/sj.jid.5701040) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 K6a-S1 is effective in a mouse footpad in vivo assay. (a) Mouse footpads were injected with a bicistronic K6a-luciferase reporter gene plasmid in both footpads. The left footpad in each mouse was co-injected with an inert pUC18 plasmid in place of siRNA as a positive control; the right footpad was co-injected with K6a-S1 siRNA. The amount of nucleic acid injected in each footpad was standardized using pUC18 DNA. (b) Owing to the inherent variability in these experiments (for example, the positive control footpad in mouse 4 did not express the reporter gene at all), several replicates were carried out and the bioluminescence signals captured with the Xenogen IVIS system were averaged and plotted. Five mice from the day 2 time point are shown. (b) Quantitation of K6a-luciferase gene expression shows that on average, K6a/luciferase expression is greatly reduced by siRNA K6a-S1. The wide error bars in the control footpads reflect the inherent variability of these experiments; however, the normalized siRNA data show consistent and highly potent knockdown of the K6a reporter gene. Journal of Investigative Dermatology 2008 128, 50-58DOI: (10.1038/sj.jid.5701040) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions