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

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Dundee Patient Support MeetingOn the road to the clinic:Development of siRNA-based therapeutics for PC

Roger Kaspar

TransDerm

[email protected]


PC is what we call a dominant negative genetic disorder


Translation


Bad keratin

Good keratin

Translation


1 gene comes from each parent

50% chance of passing it along to kids


Multiple strategies to developing a PC therapeutic


Multiple strategies to developing a PC therapeutic

  • Block the bad gene from being made


Multiple strategies to developing a PC therapeutic

  • Block the bad gene from being made

  • Block the bad gene’s effects


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


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


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


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


Transfected cells stained with DAPI

and visualized by fluorescence microscopy

K6a-wt/YFP

K6a-N171K mut/YFP


RNAi


shRNA

siRNA

RNA interference (RNAi)

Dicer

RISC

RISC


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 AGACCCUCAAaAACAAGUUUUK6a_513.10 GACCCUCAAaAACAAGUUUUUK6a_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


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


Inhibition of K6a(N171K)/fLuc expression


K6a-wt/Tomato


K6a-wt/Tomato


K6a-wt/Tomato + K6a-mut/YFP


K6a-wt/Tomato

+ K6a-mut/YFP

+ NSC4 siRNA


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


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


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


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


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


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


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


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


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


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


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


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


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


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

How do we deliver them to the cells that need them?


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


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


NSC4

3.9 wt* chol

3.9 wt*

N/A

PBS


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