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Roger Kaspar TransDerm rogerkaspar@transderm

Dundee Patient Support Meeting On the road to the clinic: Development of siRNA-based therapeutics for PC. Roger Kaspar TransDerm rogerkaspar@transderm.org. PC is what we call a dominant negative genetic disorder. Translation. Bad keratin. Good keratin. Translation.

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Roger Kaspar TransDerm rogerkaspar@transderm

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  1. Dundee Patient Support MeetingOn the road to the clinic:Development of siRNA-based therapeutics for PC Roger Kaspar TransDerm rogerkaspar@transderm.org

  2. PC is what we call a dominant negative genetic disorder

  3. Translation

  4. Bad keratin Good keratin Translation

  5. 1 gene comes from each parent 50% chance of passing it along to kids

  6. Multiple strategies to developing a PC therapeutic

  7. Multiple strategies to developing a PC therapeutic • Block the bad gene from being made

  8. Multiple strategies to developing a PC therapeutic • Block the bad gene from being made • Block the bad gene’s effects

  9. Multiple strategies to developing a PC therapeutic • Block the bad gene from being made • Block the bad gene’s effects • Block both the bad and good genes

  10. Multiple strategies to developing a PC therapeutic • Block the bad gene from being made • Block the bad gene’s effects • Block both the bad and good genes • Increase the amount of the good gene to try and swamp out the effects of the bad

  11. Multiple strategies to developing a PC therapeutic • Block the bad gene from being made • Block the bad gene’s effects • Block both the bad and good genes • Increase the amount of the good gene to try and swamp out the effects of the bad

  12. Multiple strategies to developing a PC therapeutic • Block the bad gene from being made • Block the bad gene’s effects • Block both the bad and good genes • Increase the amount of the good gene to try and swamp out the effects of the bad

  13. Transfected cells stained with DAPI and visualized by fluorescence microscopy K6a-wt/YFP K6a-N171K mut/YFP

  14. RNAi

  15. shRNA siRNA RNA interference (RNAi) Dicer RISC RISC

  16. Screening for effective N171K siRNA inhibitors K6A WT GTGAACAGATCAAGACCCTCAACAACAAGTTTGCCTCCTTC K6A N171K GTGAACAGATCAAGACCCTCAAAAACAAGTTTGCCTCCTTC Inhibitors: K6a_513.1 ACAGAUCAAGACCCUCAAaUU K6a_513.2 CAGAUCAAGACCCUCAAaAUU K6a_513.3 AGAUCAAGACCCUCAAaAAUU K6a_513.4 GAUCAAGACCCUCAAaAACUU K6a_513.5 AUCAAGACCCUCAAaAACAUU K6a_513.6 UCAAGACCCUCAAaAACAAUU K6a_513.7 CAAGACCCUCAAaAACAAGUU K6a_513.8 AAGACCCUCAAaAACAAGUUU K6a_513.9 AGACCCUCAAaAACAAGUUUU K6a_513.10 GACCCUCAAaAACAAGUUUUU K6a_513.11 ACCCUCAAaAACAAGUUUGUU K6a_513.12 CCCUCAAaAACAAGUUUGCUU K6a_513.13 CCUCAAaAACAAGUUUGCCUU K6a_513.14 CUCAAaAACAAGUUUGCCUUU K6a_513.15 UCAAaAACAAGUUUGCCUCUU K6a_513.16 CAAaAACAAGUUUGCCUCCUU K6a_513.17 AAaAACAAGUUUGCCUCCUUU K6a_513.18 AaAACAAGUUUGCCUCCUUUU K6a_513.19 aAACAAGUUUGCCUCCUUCUU

  17. K6a(N171K) siRNA #4 K6a(N171K) siRNA #12 Filaments Mixture Aggregates WT plasmid N171K plasmid WT + N171K Tissue culture model of dominant negative genetic disorder (K6a wt + K6a N171K expression plasmids + indicated siRNA) Control siRNA

  18. Inhibition of K6a(N171K)/fLuc expression

  19. K6a-wt/Tomato

  20. K6a-wt/Tomato

  21. K6a-wt/Tomato + K6a-mut/YFP

  22. K6a-wt/Tomato + K6a-mut/YFP + NSC4 siRNA

  23. K6a-wt/Tomato + K6a-mut/YFP + NSC4 siRNA

  24. K6a-wt/Tomato + K6a-mut/YFP + NSC4 siRNA

  25. K6a-wt/Tomato + K6a-mut/YFP + N171K.12wt siRNA

  26. K6a-wt/Tomato + K6a-mut/YFP + N171K.12wt siRNA

  27. K6a-wt/Tomato + K6a-mut/YFP + N171K.12wt siRNA

  28. K6a-wt/Tomato + K6a-mut/YFP + N171K.12wt siRNA

  29. K6a-wt/Tomato + K6a-mut/YFP + N171K.12wt siRNA

  30. K6a-wt/Tomato + K6a-mut/YFP + N171K.12 siRNA

  31. K6a-wt/Tomato + K6a-mut/YFP + N171K.12 siRNA

  32. K6a-wt/Tomato + K6a-mut/YFP + N171K.12 siRNA

  33. K6a-wt/Tomato + K6a-mut/YFP + N171K.12 siRNA

  34. K6a-wt/Tomato + K6a-mut/YFP + N171K.12 siRNA

  35. It’s great to have potent inhibitors, but . . .

  36. It’s great to have potent inhibitors, but . . . How do we deliver them to the cells that need them?

  37. Transgenic L2G mouse model Keratinocytes express Green Fluorescent Protein (eGFP) Skin GFP Transgenic Lines Fluorescence (Green Pseudocolor) 6.4x109 2.4x109 0.4x109 Tg Newborn Skin Tg Adult Skin Balb C Control 200x 400x 200x Yuan Cao, Stephan Shultz & Andreas Beilhack

  38. Can we inhibit gene expression in the skin? Day 0 Day 3 Day 6 Day 14 Top paw--Inhibitor of Green Fluorescent Protein (eGFP) Bottom paw--Non-specfic control inhibitor

  39. NSC4 3.9 wt* chol 3.9 wt* N/A PBS

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