Work package 1: Selection and synthesis of murine siRNA sequences Christa Schmidt

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Work package 1 Selection and synthesis of
murine siRNA sequencesChrista Schmidt
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Tasks

• selection and synthesis of murine synthetic siRNA
  sequences achieved
• construction of murine siRNA sequences for a
  plasmid vector achieved
• analysis of the chosen sequences by real-time PCR
  and Western blot achieved
• establishment of a physiological screening assay
  achieved

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Strategy
selection of three murine siRNA sequences against
each ENaC subunit alpha, beta, gamma
shRNA sequences for cloning in a plasmid vector
synthetic siRNA
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Strategy
shRNA sequences for cloning in a plasmid vector
synthetic siRNA
cloning by Geneart

• against alpha inclusion of three additional
  sequences
• alpha 4
• alpha 5
• alpha 6

(Li and Folkesson 2005 against rat ENaC modified
to fit the murine sequence)
inclusion of the Roman vectors pEPI-A2 against
alpha ENaC (FA)
AON sequences (Münster) modified to fulfill siRNA
criteria
synthesis by Qiagen
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Synthetic siRNA-sequences
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shRNA sequences for cloning
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Methods
cell lines M-1 M-1-GFP
murine epithelial cell line derived from the
kidney collecting duct, well known model for ENaC
studies created at partner 6 by lentiviral
transduction, gt98 GFP-positive routine culture
in serum free medium (PC-1)
stimulation of ENaC expression
stimulation of ENaC expression by 500nM
dexamethasone
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Methods
transfection
siRNA transfection of M-1 cells with HiPerFect
(Qiagen), 50nM siRNA plasmid transfection with
Lipofectamine 2000 (Invitrogen)
real-time PCR
mRNA level
methods to detect ENaC knockdown
Western blot
protein level
functional
membrane potential assay
93 kDa, immature gamma ENaC
42 kDa, unspecific
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Transfection procedure siRNA
transfection of siRNA

• 5x105 cells in PC-1 serum free medium, 6-well
  format
• stimulation of cells with 500nM dexamethasone
• HiPerFect (Qiagen), 50nM siRNA
• transfections in duplicates
• negative control and control for transfection
  efficiency Alexa Fluor 488 labeled unspecific
  siRNA (Qiagen) knockdown analysis after 72h
• half of the cells is used for RNA exctraction,
  real-time PCR
• half of the cells is used for Western blot
• determination of transfection efficiency FACS
  analysis

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Efficiency of siRNA delivery
6h, FACS 79,09
24h, FACS 90,31
48h, FACS 78,81
transfected with 50nM Alexa Fluor 488 negative
control siRNA and HiPerFect
untransfected
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Inhibition of alpha ENaC with siRNA
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Inhibition of alpha ENaC with chol-siRNA
(Alnylam)

• no effect of modification in vitro with
  HiPerFect
• without carrier knockdown of alpha ENaC to 55

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Inhibition of beta ENaC with siRNA
siRNA 2 (and 1) shows good knockdown of beta
ENaC down to 20
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Inhibition of gamma ENaC with siRNA
siRNAs show good knockdown of gamma ENaC
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Transfection of shRNA plasmids
transfection of plasmids

• 6x105 cells in PC-1 serum free medium,
  6-well-format
• stimulation of ENaC expression with 500nM
  dexamethasone
• Lipofectamine 2000 (Invitrogen)
• transfections in duplicates
• negative and transfection control pEGFPLucAttB
• knockdown analysis after 72h
• half of the cells is used for RNA exctraction,
  real-time PCR
• half of the cells is used for Western blot
• determination of transfection efficiency FACS
  analysis
• establishment of stable cell lines under zeozin
  selection

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Inhibition of alpha ENaC with shRNA
plasmid 2 shows a knockdown of alpha ENaC
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alpha shRNA as synthetic siRNA
also functioning as siRNA
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Inhibition of alpha ENaC with pEPI-A2
pEPI-A2-human shows a knockdown of alpha ENaC
despite 2 base mitmatch to murine sequence
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Inhibition of alpha ENaC with pEPI-A2
pEPI-A2-mouse shows efficient knockdown of alpha
ENaC
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Inhibition of beta ENaC with shRNA
plasmid 2 shows a knockdown of beta ENaC
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beta shRNA as synthetic siRNA
also functioning as siRNA
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Inhibition of gamma ENaC with shRNA
plasmid 2 shows a good knockdown of gamma ENaC
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gamma shRNA as synthetic siRNA
also functioning as siRNA
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Stable transfection with shRNA
performance of a kill curve with zeocin to
determine the minimal inhibitory concentration

• transfection of pCpGsiRNA constructs with
  Lipofectamine 2000
• expansion of cells after 24h
• cell selection with 75 and 40µg/ml zeocin after
  48h
• no growth of cell colonies

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Functional assays
Membrane potential assay (Danahay and Gosling,
Novartis)

• fluorescent assay to detect changes in the cells
  membrane potential
• detect ENaC inhibition by RNAi

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Other channel blocker
NHE sodium-proton exchanger NCX
sodium-calcium-exchanger CNG cyclic nucleotide
gated channels DMA dimethylamiloride CDPC
6-chloro-3,5-diamino-pyrazine-2-carboxamide
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ENaC blocker
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amiloride, benzamil and phenamil show an effect
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Weak ENaC blocker
CDPC and triamterene show little effect
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NHE/CNG blocker
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10
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DMA, EIPA and pimozide show an effect but are NHE
blocker
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Membrane assay

• assay is established
• only a part of the signal depends on ENaC
• may be of use when we have
• a really efficient transfection method (viral
  transfection)
• powerful downregulating sequences

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membrane assay after siRNA transfection
no detectable effect of ENaC siRNA on membrane
potential
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membrane assay for pEPI constructs
stably transfected H441 cells
no detectable effect of pEPI-A2 on membrane
potential
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Next steps

• electrophysiological Ussing chamber experiments
  (Genua)
• in vivo experiments
• CF mouse (Rotterdam, Milano)
• ENaC mouse (London)
• experiments with the siRNA 4 (Li and Folkesson)
  synthesized cholesterol-modified from CureVac
• creation and testing of more efficient siRNAs
  (Patzel)