Figure S1dm5migu4zj3pb.cloudfront.net/manuscripts/83000/83185/JCI... · 2016. 10. 20. · R6/2 mice...

5’- -3’23CAGrepeatsT7

PCRamplifica9onofHTT-exon-1,usingaforwardprimerthatincorporatesT7sequence

HTT-e1DNA

5’- -3’ HTT-e1RNA

Transcrip9on T7transcriptase

Incuba9onwithLNA-ASOs

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RTwithsequencespecificprimers

5’- -3’ 5’- -3’RT-primer RT-primer

5’- -3’

Superscript-III

Full-lengthHTT-e1cDNA(387nt)

-3’Par9alHTT-e1cDNA(191nt)

191nt

387nt

PCRamplifica9onwithapairofprimersspanningtheCAGrepeat

5’- -3’ -3’

5’-

5’-

Product(387nt) No-product

b cRetrotranscribedcDNAfromaHTT-e1RNA

PCRproductswithprimersspanningtheCAGrepeat

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Figure S1 relatedtoFigure1andFigure2

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LNA-CTGSuperscriptIII +++-

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Sanger-sequencingofthePCRproductswithprimersspanningtheCAGrepeatd

Upper 6 TTAGAC - A-CGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGG 63 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 7 TTAGACAACCGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGG 66 Upper 64 GGAGGGAGAGGGGCGGATCCCGGGCCCGCGGTACCGTCGACTGCAGAATTCCGGCTGAGG 123 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 67 GGAGGGAGAGGGGCGGATCCCGGGCCCGCGGTACCGTCGAC 126 Upper 124 AAGCTGAGGAGGCGGCGGCGGCGGCGGCGGCGGTGGTGGCTGTTGCTGCTGCTGCTGCTG 183 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 127 186 Upper 184 CTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGGAAGGACTTGAG 243 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 187 246 Upper 244 GGACTCGAAGGCCTTCATGGATCCCGGGCCCGCGGTACCGTCGACTGCAGAATTCGAAGC 303 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Lower 247 GGATCCCGGGCCCGCGGTACCGTCGACTGCATTATTC T AAGC 306 Upper 304 TTGAGCTCGAGATCTGAGTCCGGTAGCGCTACCTATAGTGAGTCGTATTAATTTCA 359 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | || Lower 307 TTGAGCTCAAGATCTGAGTCCGGTACCGCTACCTATAGTGAGTCGTATTAATTTCA 362

UpperLower

HTT-e1


Figure S1. LNA-CTGs interfere with retrotranscrip9on of HTT-e1. (a) Schema9c representa9on of theexperimentalworkflow.ApureHTT-e1DNAwith23CAGrepeatswastranscribedandtheresultantHTT-e1RNAwasincubatedwithLNA-CTGorLNA-SCB.Ineachcondi9on,HTT-e1RNAwasretrotranscribedwithasequence specific primer. Strong binding of LNA-CTG to CAG repeats impeded full-lengthretrotranscrip9on, resul9ng in a truncated product of 191 nt. Incuba9on with LNA-SCB allowedretrotranscrip9on of a full-length product of 387 nt. PCR was subsequently performed using a pair ofprimers that span the CAG repeat. (b) Gel electrophoresis showing the retrotranscribed cDNA, in eachcondi9on, e.g. in the in the absence (–) or presence (+) of LNA-SCB, LNA-CTG or SuperscriptIII. (c) Gelelectrophoresis showingRT-PCRproducts using a pair of primers that span the CAG repeats; a productcorrespondingtofull-lengthHTT-e1ofabout~380nt(d)wasnotamplifiedinsamplesincubatedwithLNA-CTG.Anaddi9onalproductofabout~200ntthatcorrespondstoafragmentlackingtheCAGrepeatandadjacent nucleo9deswas present (d), sugges9ng that secondary structures containing the CAG repeatsmay be compa9ble with PCR amplifica9on with the set of primers spanning the CAG repeat in theseexperimentalcondi9ons.(d)AlignementoftheSangersequencingoftheupper(~380nt)andlower(~200nt)PCRproductsshowninthefirstlanein(c).TheyellowboxindicatesthemiddlefragmentwithinHTT-e1thatcontainstheCAGrepeat(highlightedinred)thatisnotpresentinthelower(~200nt)PCRproducts.SimilarsequencestothelowerproductweredetectedinthePCRproductsoflanes2and3in(c).

a%ofcellswith

foci

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Numberoffoci≤34-6≥7

LNA-SCB

LNA-CTG

LNA-CT

GLN

A-SCB

Ac9n

HTT-e29-30

WT

Mut

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HTT-e1*

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Figure S2 relatedtoFigure1

c d

Figure S2. RNA foci in HD fibroblasts (68_CAG repeats) transfected with 15 nM of LNA-ASOs. (a)Representa9ve FISH image in HD fibroblasts transfected with LNA-SCB or LNA-CTG using a probecomplementary to theCAG repeat (n=3 independent transfec9ons).NucleiwerevisualizedbyDAPIstaining (right images). Scale bar, 5 µm (b) Representa9ve gel electrophoresis showingHTT RT-PCRproducts from HD fibroblasts transfected with LNA-ASOs (n = 3 independent transfec9ons). (c)Percentageoffibroblastspresen9ngnuclearRNAfociand(d)focidistribu9on,uponexposuretoLNA-CTGorLNA-SCB.Thenumberofcellswithfoci(c)andthenumberoffocipercell(d)wasdeterminedin150-300 cells per random field in LNA-SCB and LNA-CTG transfected cells (n = 3 independenttransfec9ons,in(d)*P<0,001;Chi-Squaretest).

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X1.3.foci.cellX1.3.foci.cell.1 V3 X4.6.foci.cellX4.6.foci.cell.1 V6 X.7.foci.cellX.7.foci.cell.1

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23xCAG 80xCAG

GFP

Actin

23xCAG-exon1

LNA-CTG LNA-SCB 10 (nM)

CMVp IRES GFP

(CAG)n

Control: 23xCAG_HTT-e1 WT

Mutant: 80xCAG_HTT-e1 Mut

Protein(PolyQ)HTT-exon1expressionvectorsa

LNA-CTG LNA-SCB

80xCAG-exon1

RNAExpression,24hpost-transfec9onb

HTT-e1


Figure S3, LNA-CTG bind to HTT-e1 CAG repeats in a human neuronal cell context. (a) Schema9crepresenta9on of HTT-e1-GFP expression vectors. HTT-e1 constructs containing 23 CAG repeats(23×CAG)wereusedasthewild-type(unexpanded)HTT-e1,andanHTT-e1constructcontaining80CAGrepeats(80×CAG)wasusedasamodelofmutantHTT.(b)GelelectrophoresisshowingPCRproductsintwo independentexperiments.HTT-e1expressionvectorswereco-transfectedwithLNA-CTGor LNA-SCB (10nM) inneuronal cells.RT-PCRof totalRNAwasperformed,usingprimers that span theCAGrepeatwithinHTT-e1 and primers targe9ngGFP. β-ac9n amplifica9onwas used as an endogenouscontrol.

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80xCAG

_HTTe1/T

ubulin

Tubulin

polyQ

LNA-CTG LNA-SCB

45nM 45nM

HTT-exon1proteinexpressiona

80*CAG_HTT-e1proteinlevels,48hpost-transfec9on

Figure S4 relatedtoFigure1andFigureS3

FigureS4.LNA-CTGsblockthetoxiceffectofmutantHTT-e1withoutaffec9ngHTT-e1proteinlevels.(a)ExpandedHTT-e1(80×CAG_HTTe1)proteinlevels,48haqerco-transfec9onwithLNA-CTGorLNA-SCB(45nM).ValuesobtainedbydensitometricanalysisofWesternblots.An9-polyQdetectstheexpressionof 80×CAG_HTTe1 using an an9body that recognizes glutamines (MAB1574); a representa9veimmunoblot is shown. Data are expressed as a 80×CAG_HTT-e1/α-Tubulin ra9o, and are shown asmean±SEM.ThedatawereanalyzedbyaMann-WhitneyU test (n=5). (b)Lactatedehydrogenase(LDH)celltoxicityassayindifferen9atedSH-SY5Yneuronalcells24hand36haqerco-transfec9onwith80×CAG_HTT-e1expressionvectorandLNA-SCBorLNA-CTG(45nM).Values represent themean foldchangewithrespecttothecontrolnon-transfectedcells±SEM.ThedatawereanalyzedbyaKruskal-WallistestfollowedbyaMann-WhitneyUtestwithBonferronicorrec9onasapost-hoctest(n=5,*P<0.05)

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80XCAGLNA-SCB

80XCAGLNA-CTG

Rela:v

ecelltoxicy(LDH

assay)

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80XCAGLNA-SCB

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ecelltoxicy(LDH

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MutantHTTexon1toxicityb

24hpost-transfec9on 36hpost-transfec9on

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WTLNA-SCBR6/2LNA-SCBR6/2LNA-CTG

Surgery

Bodyweight(g)

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FigureS5.ExperimentaldesignandLNA-CTGeffectsonphenotypicparametersinanR6/2mousemodelofHD.(a)Schemeshowingtheexperimentalprotocolforthebehaviouralanalysisinwild-type(WT)andR6/2miceintrastriatallyinjectedwithLNA-SCBorLNA-CTG.HWT,hangingwiretest.Thebodyweight(b)andlatencytofallinthehangingwiretest(c)areshowngraphicallyfortheWTLNA-SCB,R6/2LNA-SCBandR6/2LNA-CTGmicegroupsatdifferent9mepointsaqersurgery.Resultsareexpressedasmean±SEM(n=7).Bodyweightdatawasanalyzedbytwo-wayANOVAwithBonferronitestasaposthoc.*P<0.05ascomparedwithLNA-SCB–orLNA-CTG–injectedR6/2mice.

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W T L N A -S C B

W T L N A -C T G

Num

bero

ffalls

(60second

s)

Age(weeks)

16rpm 24rpm 32rpm

a

c

FigureS6.Intrastriatalinjec9onofLNA-CTGdoesnotaltermotorbehaviorinWTmicenorthelevelsofseveralstriatalproteins(a)Resultsfromtherotarodtaskperformedat16,24and32rpmfrom8to15weeksofage.Valuesrepresentthenumberoffallswithin60s.(b)Bodyweightatdifferentages,aqerinjec9onofLNA-CTG.In(a)and(b)data,arepresentedasthemean±SEM(WTLNA-SCBn=10;WTLNA-CTGn=9)andanalyzedbytwo-way ANOVA test. (c) Levels of HTT, DARPP-32, ENJ, PHLPP1, CBP, STEP46 and PSD-95 proteins. StriatalhomogenatesofWTmice5weeksaqerinjec9onofLNA-SCBorLNA-CTGwereanalyzedbyWesternblot.Box-plotsrepresentthedensitometricproteinquan9fica9onnormalizedtotubulinandexpressedrela9vetoaLNA-SCB-injectedWTmice.Representa9veimmunoblotsareshown.DatawereanalyzedwithMann-WhitneyUtest(n=7ineachgroups).


WTLNA-SCB

WTLNA-CTG

PHLPP1

Proteinlevels

(%W

T-SCB)

Proteinlevels

(%W

T-SCB)

STEP46CBP

Proteinlevels

(%W

T-SCB)

Proteinlevels

(%W

T-SCB)

PSD-95

DARPP-32

Proteinlevels

(%W

T-SCB)

HTT

Proteinlevels

(%W

T-SCB)

Proteinlevels

(%W

T-SCB)

ENK

HTTTubulin

DARPP-32Tubulin

ENKTubulin

PHLPP1Tubulin

CBPTubulin

STEP46Tubulin

PSD95Tubulin

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Hoechst33258 EM48 Merge

LNA-CT

Ginjected

R6

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R6

/2stria

tum


FigureS7.Intrastriatalinjec9onofLNA-CTGdoesnotmodifymHTTdistribu9oninR6/2micestriatum,5weeksaqeradministra9on.NucleiwerestainedwithHoechst,andmHTTwiththeEM48an9body,48haqerLNA-SCBorLNA-CTGadministra9oninR6/2mice.Scalebar,20µm.

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ckin

g

Htt full length

Exon-1

1 2 3 1 2 3 1 2 3 1 2 3

Tubulin

1 2 3 1 2 3 1 2 3 1 2 3

1: WT LNA-SCB

2: R6/2 LNA-SCB

3: R6/2 LNA-CTG

Sta

ckin

g

Htt full length

Exon-1

Tubulin


a

b

FigureS8.Wild-typeandmutantHTTexpressionlevelsarenotalteredaqerintrastriataladministra9onofLNA-CTG. Representa9ve immunoblots (obtained by incuba9on with MAB5492 an9-HTT an9body (aa1-82)showingwild-typeandexon-1proteinlevelsinthestriatumofR6/2miceat5(a)and(b)11daysaqerLNA-SCBorLNA-CTGadministra9on(n=4percondi9on).

RT-PCRproductsofcor9calsamples,2daysaqeradministra9on

R6/2+LNA-SCB R6/2+LNA-CTG

Ac9n

Gdx

Hu_E1*

Hu_E1


Hu_E1*

Ac9n

Gdx

RT-PCRproductsofcor9calsamples,10daysaqeradministra9on

Hu_E1


RT-PCRproductsofcor9calsamples,5weeksaqeradministra9on

Hu_E1*

Ac9n

Gdx

Hu_E1

a

b

c


Figure S9. LNA-CTGs strongly bind to HTT-e1 CAG repeats in the cortex of R6/2 mice at 2–10 daysfollowing injec9on into the striatum. The gel electrophoresis shows RT-PCR products of the HTT-e1transgene using primers that span the CAG repeat (HTT_E1*) and primers outside the CAG repeat(HTT_E1) at 2 days (a), 10 days (b) and 5 weeks (c) aqer intrastriatal injec9on of LNA-ASOs (n=5).AmplifiedofGdxandβ-ac4nwereusedasinternalcontrols.

b

Mo_ac9n

Hx_e41

HTT_e1*

12345

a

Proteinlevels(%

)

LNA-SCB

LNA-CTG

STHdhQ7/Q7 STHdhQ111/Q111

HTT

Tubulin

LNA-SCB

LNA-CTG


Figure 10. LNA-CTG transfec9on in mouse striatal cells does not alter wild-type and mutant HTTexpressionlevels.(a)HTTandmHTTproteinlevelsinSTHdhQ7/Q7andSTHdhQ111/Q111cells,respec9vely,24h aqer transfec9onwith25nMLNA-SCBor LNA-CTG.Box-plots representHTTexpression levels (fromdensitometricanalysesofWesternblots)thatwerenormalizedtoα-tubulinandreferredtoHTTlevelsincontrolSTHdhQ7/Q7cells transfectedwithLNA-SCB.DatawereanalyzedwithMann-WhitneyUtestwithBonferronicorrec9on(n=3inSTHdhQ7/Q7cellsandn=4inSTHdhQ111/Q111).Arepresenta9veimmunoblotis shown. (b) Gel electrophoresis showingHTT andβ-ac9n RT-PCR products from STHdhQ111/Q111 cellstransfectedwithLNA-CTGorLNA-SCB(n=5).TheprimersetsHTT_e1*(forHx-e1),Hx_e41(formouseHxexon41)andMo_ac9n(formouseβ-ac9n)wereused(seeTableS2fordetails).

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Pif1 Pf4 Pde3b

P2ry12 Med1 Git2

Gpr34 P2ry13 D130043k22Rik

WTR6/2R6/2

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WTR6/2R6/2

LNA-SCBLNA-CTGWTR6/2R6/2

LNA-SCBLNA-CTG

WTR6/2R6/2

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WTR6/2R6/2

LNA-SCBLNA-CTG

WTR6/2R6/2

LNA-SCBLNA-CTG

WTR6/2R6/2

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WTR6/2R6/2

LNA-SCBLNA-CTG

WTR6/2R6/2

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*+

+

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+* *

Figure S11relatedtoFigure3andTableS4

FigureS11.qPCRanalysestovalidatemicroarraytop-deregulatedgenesinthestriatumofR6/2miceinjectedwithLNA-CTGorLNA-SCB.Pif1,Pf4,Pde3b,P2ry12,Med1,Git2,Gpr34,P2ry13andD130043k22RikexpressionlevelswereanalyzedinWTanimalsinjectedwithLNA-SCBandR6/2miceinjectedwitheitherLNA-SCBorLNA-CTG.Rela9vequan9fica9ons(RQ) inthebox-plotsshowexpression levelsreferredtowild-typeanimals.Hprtwas used as a reference gene. Data were analyzed using a linear mixed effects model and Bonferroni’scorrec9onwasappliedformul9plecomparisons(n=5-9pergroup,*P<0.01withrespecttowild-typeLNA-SCBmice;+P<0.01withrespecttoR6/2LNA-SCBmice).

LNA-SCBLNA-CTG

Figure S12relatedtoFigure3

aM

aml2

mR

NA

leve

ls

PrimersspanningtheCAGrepeats,p1 PrimersmappingoutsidetheCAGrepeats,p2

*+

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+*

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2a m

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r3c1

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ls

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vels

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LNA-SCBLNA-CTG

WTR6/2R6/2

LNA-SCBLNA-CTG

WTR6/2R6/2

LNA-SCBLNA-CTG

WTR6/2R6/2

LNA-SCBLNA-CTG

WTR6/2R6/2 WTR6/2R6/2

LNA-SCBLNA-CTG

WTR6/2R6/2

LNA-SCBLNA-CTG

WTR6/2R6/2

LNA-SCBLNA-CTG

CAGrepeat Setofprimersp1:F-p1,R-p1

Setofprimersp2:F-p1,R-p1

F-p1

R-p1

F-p2

R-p2

b

FigureS12.LNA-CTGbindtoseveraltranscriptscontainingCAGrepeats,withoutalteringthecorrespondingproteinlevels. (a) Schema showing the binding site of the primers spanning the CAG repeats (p1) and primersmappingoutsidetheCAGrepeats(p2).(b)RT-qPCRquan9fica9onofofMaml2,Mef2a,Nr3c1andSatb1mRNAinthestriatumofWTanimals injectedwithLNA-SCBandR6/2mice injectedwithLNA-SCBorLNA-CTG,at5dayspost-injec9on.PrimersspanningtheCAGrepeat (leqpanels)andoutsidetheCAGrepeat (rightpanels)wereused.AWTsamplewasusedasareferenceforrela9vequan9fica9on(RQ).Quan9fica9onwasnormalizedtoHprtasareferencegene.PlotsshowmeanRQdistribu9on.DatawereanalyzedusingalinearmixedeffectsmodelandBonferronicorrec9onwasappliedformul9plecomparisons(n=4pergroup,*P<0.01withrespecttoWT-LNA-SCBmice;+P<0.01withrespecttoR6/2-LNA-SCBmice).(c)Protein levels inthestriatumofWT andR6/2miceat2 (upperpanels)and10(lowerpanels)daysaqerLNA-SCBorLNA-CTGintrastriatal injec9on.Box-plotsrepresentthedensitometricproteinquan9fica9onnormalizedtotubulinandexpressedrela9vetoWTmice.DatawereanalyzedbyaKruskal-Wallistest(n=4-5pergroup).Representa9veimmunoblotsareshown.

NR3C1Tubulin

WTLNA-SCB

R6/2LNA-SCB

R6/2LNA-CTG

Proteinlevels(%

WT)

MEF2ATubulin

SATB1Tubulin

MAML2Tubulin

SATB1Tubulin

NR3C1Tubulin

MEF2ATubulin

MAML2Tubulin

c

Figure S12relatedtoFigure3

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NR3C1 SATB1MEF2AMAML2

NR3C1 SATB1MEF2AMAML2

Proteinlevels(%

WT)

CAGrepeat Setofprimersp1:F-p1,R-p1

Setofprimersp2:F-p1,R-p1

F-p1

R-p1

F-p2

R-p2

a

b

LNA-SCB

*

Figure S13relatedtoFigure3andFigureS6

Figure S13. LNA-CTGs bind to several transcripts containing CAG repeats in WT mice, without altering thecorrespondentexpression levels (a)Schemeshowingthebindingsiteof theprimersspanningtheCAGrepeats(p1) and primers mapping outside the CAG (p2). (b) The expression levels are shown in the striatum ofWTanimals injectedwithLNA-SCBorLNA-CTG,at5weekspost-injec9on.Quan9fica9onwasnormalizedtoHprtasanindependentreferencegene.Box-plotsshowrela9vequan9fica9on(RQ)referredtoaLNA-SCBinjectedmiceanddatawereanalyzedusing linearmixedeffectsmodel (n=7pergroup,*P< 0.001with respectLNA-SCBinjectedmice).

LNA-CTG LNA-SCB LNA-CTG

Satb1,p1 Satb1,p2

LNA-SCB LNA-CTG LNA-SCB LNA-CTG

Mef2a,p1 Mef2a,p2


Nr33c1,p1 Nr3c1,p2


Arhgap17,p1 Arhgap,p2


Maml2,p1 Maml2,p2


Crebbp,p1 Crebbp,p2


Mdh2,p1 Mdh2,p2

*

*

*

* *

*

RQ

RQ

RQ

RQ

RQ

RQ

RQ

Figure S14, related with Figure 5

0.4

0.6

0.8

1.0

1.2

1.4

WT.LNA.SCB R6.2.LNA.SCB

●

●

●

●

●

●

●

●

●

●

●

●

●

RQ

a

FigureS14.sCAGexpression in the striatumofWTandR6/2mice injectedwith LNA-SCB. (a) sCAGexpressionlevels are shown in totalRNA isolated from the striatumofWTandR6/2animals injectedwith LNA-SCB, at5weeks post-injec9on. Rela9ve quan9fica9on (RQ) represents the densitometric determina9ons of sCAG PCRproductsnormalizedtoU6andreferredtoaWTsamplewithavalueof1.sCAGquan9fica9onisnotshowninLNA-CTGinjectedR6/2mice,sinceLNA-CTGinterferewithRT-PCR.DatawereanalyzedbyaMann-WithneyUtest(n = 4WTmice andn = 9 R6/2mice). (b) sCAGproducts in total RNA and a frac9on enriched in small RNAs.SamplescorrespondtopooledRNAsisolatedfromthestriatumof13WTand11R6/2animalsinjectedwithLNA-SCB,at5weekspost-injec9on.ThepooledsmallRNAfrac9onswereusedinthetransfec9onexperimentsshownin Figure 5. In the RT-PCR amplifica9on we included (CAG)7x and (CTG)7x pure trinucleo9de repeatoligonucleo9desthatwereusedasposi9veandnega9vecontrols,respec9vely.

LNA-SCB LNA-SCBWT R6/2

b

LNA-SCB LNA-SCBWT R6/2

100nt

200nt

C+ C-

5 days post-injection striatum

+

*

*

*

+

Figure S15

FigureS15.LNA-CTGsincreasethelevelsofRn45Sinthestriatumof R6/2mice,5weeksaqeradministra9onofLNA-ASOs.Theexpressionlevelsareshowninthestriatumof WTanimalsinjectedwithLNA-SCBandR6/2miceinjectedwithLNA-SCBorLNA-CTG,at5days(a)or5weeks(b)post-injec9on.Quan9fica9onwasnormalizedtoHprt and/or Tbp as independent reference genes and a WT sample was used as a reference for rela9vequan9fica9on(RQ).DatawereanalyzedusinglinearmixedeffectsmodelandBonferronicorrec9onwasappliedformul9plecomparisons(n=4,miceat5dayspost-injec9on;n=4-9,miceat5weekspost-injec9on;*P<0.05withrespecttoWT-LNA-SCBmice;+P<0.05withrespecttoR6/2-LNA-SCBmice)

b

a

5 weeks post-injection striatum

Hprt,asareferencegene Tbp,asareferencegene

Hprt,asareferencegene

Gene Name Fold change p value adj. P value

A_55_P1980214 -5.85 0.00 0.00Arhgap17 -5.85 0.00 0.00Pif1 -5.60 0.00 0.00Crebbp -5.09 0.00 0.00A_55_P2077546 -4.22 0.00 0.00Trp63 -4.18 0.00 0.00D130043K22Rik -4.08 0.00 0.00Git2 -4.05 0.00 0.00Pf4 -3.10 0.00 0.10Fabp4 -3.00 0.00 0.12Fam48a -2.88 0.00 0.00Mdh2 -2.81 0.00 0.01A230006K03Rik -2.73 0.00 0.00BC051142 -2.69 0.00 0.00chr17:88890039-88905680_F -2.30 0.00 0.00Las1l -2.15 0.00 0.17Gpr137b -2.05 0.00 0.00Mmp27 -1.87 0.00 0.14chr9:30529709-30589042_R -1.83 0.00 0.01Tob1 -1.82 0.00 0.05chr13:112717680-112733330_R -1.81 0.00 0.13NAP017915-001 -1.73 0.00 0.16Gm5124 -1.72 0.00 0.10Angptl4 -1.68 0.00 0.10Ecm1 -1.64 0.00 0.14Tgm2 -1.62 0.00 0.10Gpr137b-ps -1.56 0.00 0.05Tgfbr2 -1.55 0.00 0.12Gadd45b -1.44 0.00 0.18Foxp1 -1.42 0.00 0.10Secisbp2 -1.42 0.00 0.20D11Wsu99e -1.35 0.00 0.11Vwa1 -1.34 0.00 0.12Lpin2 -1.34 0.00 0.01Tmem150c -1.32 0.00 0.10Wfikkn1 -1.30 0.00 0.05Chl1 -1.27 0.00 0.19chr19:59000015-59016865_R -1.26 0.00 0.101700029G01Rik -1.24 0.00 0.16Ier2 -1.19 0.00 0.20Phlpp1 1.22 0.00 0.16Guf1 1.25 0.00 0.20chr4:128727332-128735227_F 1.30 0.00 0.11Ppfia4 1.31 0.00 0.09Pcdhb12 1.38 0.00 0.13P2ry13 1.39 0.00 0.05chr4:105035450-105059250_F 1.39 0.00 0.16chr19:21653968-21671718_F 1.39 0.00 0.02Pde3b 1.43 0.00 0.12Rpl15 1.44 0.00 0.01Med1 1.46 0.00 0.17chr14:61321789-61325711_F 1.46 0.00 0.12Gm10790 1.51 0.00 0.05LOC100861634 1.63 0.00 0.11P2ry12 1.74 0.00 0.05Gpr34 1.74 0.00 0.02

Table S1. Microarray gene expression analysis in the striatum of R6/2 mice injected with LNA-ASOs. Differently expressed genes in LNA-CTG vs LNA-SCB injected mice are shown (Fold Change greater or less than 1.2; adjusted P < 0.2). The dysegulation pattern and the corresponding fold change are indicated for each gene

Target gene/fragment Primer name, Forward (Fw) or Reverse (Rev) Sequence 5'--->3' Annealing T(ºC) Figure

GCAGCAGCAGCAGCAACAGCCGCCACCGCC

Table S2. Primers for qPCR or retrotranscription of human or mouse endogenous HTT, actin, Gdx or exogenously expressed HTT-exon1. Forward (Fw) and Reverse (Rev) primers are shown. The target gene and or fragment are indicated for each pair of primers. Annealing temperature (T) and efficiency for each pair of primers are also shown

Figure 1A, C

Figure 1A,B,C, Figure S1. Figure S2

Figure 1A,B,C; Figure S2

Figure 1B, Figure S2, Figure S3

Human HTT-exon 1, outside the CAG repeat

Human HTT-exon 1, spanning CAG repeat

HTT_e1_Fw

Human HTT-exon 1 with 23_CAG repeats (HTT-e1 expression vector), spanning the CAG repeat

Figure 2D, Figure S9, Figure S10

Human HTT-exon 29-30HTT_e29_Fw

HTT_e30_Rev

TGGATCTTCAGAACAGCACG

TCGACTAAAGCAGGATTTCAGG

Human actinHu_Actin_Fw

Hu_Actin_Rev

CTGGAACGGTGAAGGTGACA

GGGAGAGGACTGGGCCATT

Human HTT-exon 1 transgene, spanning CAG repeat

HTT_e1*_Fw

HTT_e1*_Rev

CGGCTGAGGCAGCAGCGGCTGT

Mouse GdxMo_Gdx_Fw GGCAGCTGATCTCCAAAGTCCTGG

Mouse actinMo_Actin_Fw ACCTTCTACAATGAGCTGCG

Mouse Htt exon 41Htt_e41_Fw

Htt_e41_Rev

Human HTT-exon 1 transgene, outside the CAG repeat

HTT_e1_Fw

Figure 2D, Figure S9Mo_Gdx_Rev

ATGGCGACCCTGGAAAAGFigure 2D, Figure S9, Figure

S10HTT_e1_Rev CTGCTGCTGGAAGGACTTG

AACGTTCGATGTCATCCAGTGTTA

58

62

60

Figure S10CTTGGCATGAGCGAGATTGC

ACTTGTTCCAGTAGGCCGTG

Figure 2D, Figure S9, Figure S10

Mo_Actin_Rev CTGGATGGCTACGTACATGG60

HTT_e1*_Fw ATGGCGACCCTGGAAAAGCTGAT

HTT_e1*_Rev TGAGGCAGCAGCGGCTG

HTT_e1_Rev

GTTCTGCTTTTACCTGCGGC

ATCAGCTTTTCCAGGGTCGC

60

64

60

60

65

60 Figure S1HTT_e1_T7_Rev CCAAAAGACGGCAATATGGT

Human HTT-exon 1 55 Figure S1HTT_e1_T7_Rev-RT CCAAAAGACGGCAATATGGT (specific primer for retrotranscription of HTT-e1)

Human HTT-exon 1 with 23 CAG repeats containing T7 sequence

HTT_e1_T7_Fw GAAATTAATACGACTCACTATAGGTAGCGCTACCGGACTCAGAT

HTT_e1_23CAG_Fw GTCCCTCAAGTCCTTCCAGC 60 Figure S3

HTT_e1_23CAG_Rev GCTGTTGCTGCTGCTGCT

GFP_Fw TGCAGTGCTTCAGCCGCTAC GFP (HTT-e1 expression vector) 60 Figure S3

GFP_Rev TCGCCCTCGAACTTCACCTC

Gene symbol Gene full name Type of primer Sequence 5'--->3' Annealing T(ºC) Efficiency

Forward GCTCTGGTCCCTGTTCACCReverse ACAGAGCTCTCCTGGCACAForward (spanning the CAG repeat) TGTGGGCACTGAGGATGTAAForward (spanning the CAG repeat) ATCCATCGCAAAGTGAGACCForward CTGCTGGAACTGGCTGTGTReverse TGAACATCATGAACCCAGGAForward (spanning the CAG repeat) GGAGCATTGCACTCTGTTCAForward (spanning the CAG repeat) CAATCCCAACCTCCACATTCForward AGGCGAGGACTTTGTCAAGAReverse TCAGAGCCTGCTTTACTGTCAForward (spanning the CAG repeat) GCTTTGTCTTCTCCCTCGTGForward (spanning the CAG repeat) CAAAGTCCTCGCCTTTCTTGForward TGGTGCCTAAGAAATACCACTTGReverse TGAATGTCTGCTGCCTTCTGForward (spanning the CAG repeat) ATCCTTAGCTCCCCCTGGTAForward (spanning the CAG repeat) CGAAAGTCTGTTTCCCCAGAForward CCTGTCCTGCAGTAAGCACAReverse TTCTCCAGCTCCAGGTCACTForward (spanning the CAG repeat) TTCTCTTTTGCCTTCCCAGAForward (spanning the CAG repeat) AGGGAGCCTTGCTGTTGTTForward AGCGTCAAGGACTGTTTGGTReverse AGTGTGGCGGGTTACAGAAGForward (spanning the CAG repeat) TGCCATCTATGAGCAGGAGAForward (spanning the CAG repeat) TCCAGCTGTGCAGACAGAGTForward CCTCACTGGTGGTGAATGTG Reverse TACCATTTTTCCCCCACAAA Forward (spanning the CAG repeat) CACCTCCCCGTGATAGAATG Forward (spanning the CAG repeat) GCCAAGCACAATTGGAGAATForward TGTGGGCACTGAGGATGTAAReverse ATCCATCGCAAAGTGAGACCForward (spanning the CAG repeat) GCTCTGGTCCCTGTTCACCForward (spanning the CAG repeat) ACAGAGCTCTCCTGGCACAForward GGAGCATTGCACTCTGTTCAReverse CAATCCCAACCTCCACATTCForward (spanning the CAG repeat) CTGCTGGAACTGGCTGTGTForward (spanning the CAG repeat) TGAACATCATGAACCCAGGAForward GCTTTGTCTTCTCCCTCGTGReverse CAAAGTCCTCGCCTTTCTTGForward (spanning the CAG repeat) AGGCGAGGACTTTGTCAAGAForward (spanning the CAG repeat) TCAGAGCCTGCTTTACTGTCAForward CGTGAGACGTACGAGATGTTGReverse TGAAGTAGGTGCTGGTGCTGForward GTAGCACTGCCGTGGAAAATReverse TGTGAGCGCTAGGAACTGAAForward TGCTGTTCTCTTTGGTGGTGReverse ACACGTTGCTGTAACCGAGAForward TTCATGGATTTAACAACAAAACAGAReverse CATGTTCACTGACATTTCAACTCAForward CAAGTTCAGATGCTGGGTCAReverse CCTGTGACGATGAGGGCTATForward TTGCTGCCTCTAGTGTTAAGGAReverse GACAATGGATAAGGCCTGGAForward GAGGTTGGGCAGGTAAGGTTReverse TCCTGGCTTCTCTCTTTCCAForward CAGCCCCACAGACAGAATTTReverse TTTCACAACTGGGGCACATAForward GCGATGGAGATCTTAGCTGTGReverse GGCAAATTTTCCTCCCATTCForward TCTTCGTGTGCTGGACTTTGReverse GCCTCTCCCCAGTTAATTCCForward CGCGGTTCTATTTTGTTGGTReverse TCGTCTTCGAAACTCCGACTForward TGTTGTTGGATATGCCCTTGReverse GGACGCAGCAACTGACATTForward TGCTGTTGGTGATTGTTGGTReverse GTCCTGCAATACTGGGAAGG

Nr3c1

Nr3c1

Glucocorticoid Nuclear Receptor Variant 1

Glucocorticoid Nuclear Receptor Variant 1

60

60

Crebbp CREB Binding Protein 60

Maml2 Mastermind-Like Transcriptional Coactivator 2 56 2043

Mef2A Myocyte Enhancer Factor 2A 56

1617

Crebbp CREB Binding Protein 60 1821

Arhgap17 Rho GTPase Activating Protein 17 60 1683

Arhgap17 Rho GTPase Activating Protein 17 60 1856

1963

Satb1 SATB Homeobox 1 60 2035

Trp63 Transformation related protein 63 64º 1.978

Mdh2 Malate Dehydrogenase 2, NAD (Mitochondrial)

1909

Mef2A Myocyte Enhancer Factor 2A 60 1906

Tbp TATA box binding protein 60º 1.894

RIKEN cDNA D130043K22 gene 60º 1.925

Git2 G protein-coupled receptor kinase-interactor 2 60º 1.886

Med1 Mediator complex subunit 1 60º 1.992

60ºP2ry12 Purinergic receptor P2Y, G-protein coupled 12

Rn45S 45S Pre-Ribosomal RNA 60 1976

1.71560º

Table_S3. Selected genes for qPCR validation, and the pair of primers used for each gene. Annealing temperature (T) and efficiency for each pair of primers are shown.

Arhgap17 Rho GTPase activating protein 17 60º 1.683

Crebbp CREB binding protein 60º 1.617

Rho GTPase activating protein 17Arhgap17 60º 1.856

Crebbp CREB binding protein 60º 1.821

P2ry13 Purinergic receptor P2Y, G-protein coupled 13 60º 1.77

Pde3b Phosphodiesterase 3B, cGMP-inhibited 60º 1.934

D130043K22Rik

G protein-coupled receptor 34Gpr34

Hprt Hypoxanthine guanine phosphoribosyl transferase 60º 1.929

Pf4 Platelet factor 4 60º 1.862

Pif1 PIF1 5'-to-3' DNA helicase homolog (S. cerevisiae) 60º 1.741

1.861

Mdh2 Malate dehydrogenase 2, NAD (mitochondrial) 60º 1.933

1.691

Satb1 SATB Homeobox 1 60 1854

Mdh2 Malate dehydrogenase 2, NAD (mitochondrial) 60º

60 1691

Mdh2 Malate Dehydrogenase 2, NAD (Mitochondrial) 60 1933

2021

1521

Maml2 astermind-Like Transcriptional Coactivator 2 60

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