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w w w . r b h h . o r g

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Original

article

The

F309S

mutation

increases

factor

VIII

secretion

in

human

cell

line

Daianne

Maciely

Carvalho

Fantacini

a,b,

,

Aparecida

Maria

Fontes

a

,

Mário

Soares

de

Abreu

Neto

b

,

Dimas

Tadeu

Covas

a,b

,

Virgínia

Picanc¸o-Castro

a,b

aUniversidadedeSãoPaulo(USP),RibeirãoPreto,SP,Brazil bHemocentrodeRibeirãoPreto,RibeirãoPreto,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29March2016 Accepted1April2016 Availableonline20April2016

Keywords:

HEK293cells

RecombinantfactorVIII F309Smutation Betaine

Sodium-4-phenylbutyrate

a

b

s

t

r

a

c

t

Objectives: ThecapacityofahumancelllinetosecreterecombinantfactorVIIIwithaF309S pointmutationwasinvestigated,aswastheeffectoftheadditionofchemicalchaperones (betaineandsodium-4-phenylbutyrate)onthesecretionoffactorVIII.

Methods:ThisworkusedavectorwithaF309SmutationintheA1domaintoinvestigateFVIII productionintheHEK293humancellline.FactorVIIIactivitywasmeasuredbychromogenic assay.Furthermore,theeffectsofchemicaldrugsontheculturewereevaluated.

Results:TheadditionoftheF309SmutationtoapreviouslydescribedFVIIIvariantincreased FVIIIsecretionby4.5fold.Moreover,theadditionofbetaineorsodium-4-phenylbutyrate increasedthesecretionrateofFVIIIBproteinsinHEK293cells,butthesameeffectwas notseenforFVIIIB-F309Sindicatingthatalltherecombinantproteinproducedhadbeen efficientlysecreted.

Conclusion: BioengineeringfactorVIIIexpressed inhumancellsmayleadtoanefficient productionofrecombinantfactorVIIIandcontributetowardlow-costcoagulationfactor replacementtherapyforhemophiliaA.FVIII-F309Sproducedinhumancellscanbeeffective

invivo.

©2016Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Hemophilia A is an inherited disorder linked to the X chromosomewhich resultsin deficiencyor abnormality of bloodcoagulationfactorVIII(FVIII).Thecurrenttherapyfor hemophiliaApatientsistheintravenousinfusionof plasma-derivedorrecombinantfactorVIII(rFVIII).Themajorproblem ofreplacement therapy with rFVIII is the low productivity

Correspondingauthor.

E-mailaddress:annydamac@yahoo.com.br(D.M.C.Fantacini).

which increases the cost of therapy. The high costs are mostlyattributabletoseveralbiochemicalcharacteristicsof therecombinantproteinsuchastheretentionofFVIIIwithin theendoplasmicreticulum(ER)duetoitsinteractionwith dif-ferentERchaperones.1

Severalstudieshaveprovidedimportantinformationthat helpedinthedesignanddevelopmentoftechniquesto bio-engineerrFVIII withbettersecretionefficiency.2,3 Swaroop4

developedanrFVIIIwithapointmutationintheA1domain

http://dx.doi.org/10.1016/j.bjhh.2016.04.002

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(Phe309Ser) to increase secretion ofthe protein.4 However

despitemanysignificantadvancesintheheterologous expres-sionofFVIII,alowproductionratepersists.

Recently, it was suggested that recombinant concen-trates might be associated with a higher incidence of inhibitor development.5 This may be associated with the

different glycosylation pattern found in hamster cell lines [available productsareproduced inChinese hamsterovary (CHO) and Baby hamster kidney (BHK) cells] compared to humancells:Gal·1-3Gal␤1-(3)4GlcNAc(␣-Gal)epitopesand N-glycolylneuraminicacid(Neu5Gc)arenotpresentinhumans.6

Asanalternative,humancell lines canbeused to pro-duce recombinant coagulation factors. This heterologous expression system is capable of producing proteins with post-translationalmodificationssimilartotheiroriginal coun-terpartwhichmightreducethepossibilityofimmunogenic reactions.6

ThisstudyinvestigatedtheproductionofFVIIIcontaining theF309SpointmutationintheHEK293humancelllineand theefficacyofthemoleculeinvivoinahemophiliaAmouse model.

Methods

Lentiviralvectors

Ahumanimmunodeficiency virus-1(HIV-1)-basedlentiviral vector containing the human FVIII cDNA with a deletion of a large portion of the B domain (FVIIIB) and a sec-ondvectorwiththe Phe309Sermutation(A1 domain)were usedtoincreasethesecretionoftheprotein.Thetransgene expressionofbothvectorswasdrivenbytheinternal myelo-proliferative sarcoma virus (MSV) promoter with selection usingtheneomycingene.Thevectorswerekindlyprovided byDanielGibsonfromtheCraigVenterInstitute.

Transientexpression

FVIIIBandFVIIIB-F309Sconstructsweretransfected(10␮g ofDNA)intoHEK293cellsusinglipofectamine®2000reagent

(LifeTechnologies)followingthemanufacturer’sinstructions. Conditioned mediumwas harvested at48h and 72h after transfectionandanalyzedusingAsserachrom®VIII:Ag

(Diag-nosticaStago).

Productionoflentiviralparticles

Togeneratelentiviralparticles,theconstructDNAswere tran-sientlyintroduced into293FTcells bytripleco-transfection with the packaging construct pCMVR8.91 encoding gag, pol, and rev and the pseudotyping construct pMD2.VSVG codingforthe vesicularstomatitis virusglycoprotein (VSV-G). Transfection of plasmid DNAs was performed using lipofectamine® (Life Technologies) following the

manufac-turer’sinstructions.Viralparticleswereharvestedat48hand 72h post-transfection and filtered through a 0.22␮m filter (Millex®-GV).Theviralparticleswereconcentratedby

ultra-centrifugation(1.40h at31,000×g). Theconcentrated virus wasstoredat−80◦C.

Lentiviral titers were determined by quantitative poly-merasechainreaction(PCR)ofgenomicDNAfromtransduced 293FTcells.Briefly,1×105293FTcellswereseededinasix-well

plate andtransducedwithserialdilutionsofvector supple-mented with 8␮g/mL polybrene (Sigma-Aldrich). Genomic DNAwasextractedfromtransducedcells72hafter transduc-tionusingtheDNeasyBloodandTissueKit(Qiagen)asperthe manufacturer’s instructions, and quantitativePCRwas car-riedoutinduplicateonsamplestodeterminebothtotalviral DNAandhuman␤-actinlevels(ACTB).Theintegratingcopy number per transducedcell wasnormalized assumingtwo ACTBallelespercellandinfectiousunitspermilliliterwere calculated as total viral DNA/(ACTB/2)×(1×105)×dilution

factor. Primers and probe used have been described previously.7

ExpressionofstableHEK293cells

HEK293cellsweremaintainedinDulbecco’smodifiedeagle medium(DMEM–Life Technology)supplementedwith10% (v/v) fetal bovine serum (ThermoScientific), 1%(v/v) peni-cillin/streptomycinsolution(Gibco)in5%CO2at37◦C.Cells

wereplatedonto24-wellplates(approximately100,000cells) andtransducedatamultiplicityofinfection(MOI)of40for transduction 1–4,MOIof80fortransduction5, andMOIof 160fortransduction6–10withviralparticlesinafinalvolume of500␮LofDMEMsupplementedwith8␮g/mLofpolybrene (Sigma). At 24h post-transduction, virus-containing media was replaced with fresh DMEM and cells were allowed to recover by culturing overnight at 37◦C in 5% CO

2.

Subse-quently, transduced cells were replaced foranother round oftransductionuntilthetenthcycleand analyzedforFVIII activity,copynumberintegration,transcriptexpressionand proteinanalysis.

FactorVIIIquantificationandantigenanalysis

FVIIIactivitywasdeterminedbyatwo-stageassayusingthe COAMATIC FVIII (Chromogenix) according tothe manufac-turer’s instructions. Normal human reference plasma was usedtogeneratethestandardcurve.TheFVIIIantigenwas quantifiedbyanenzyme-linkedimmunosorbentassay(ELISA) methodusingtheAsserachromVIII:Ag(Stago)commercialKit accordingtothemanufacturer’sinstructions.

Reversetranscriptionpolymerasechainreactionanalysis

RNAwasextractedusingtheRNeasyMiniKit(Qiagen) accord-ing tothemanufacturer’sinstructions.Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was per-formedusingtheHighCapacitycDNAreversetranscriptionKit (LifeTechnology).ThiswasfollowedbyPCRwithprimersBiPfor

(5′-CCAACGCCAAGCAACCAAAG-3),BiP

rev(5′-CTTCTCCCC

CTCCCTCTTAT-3′),GAPDH

for(5′-GCCTCAAGATCATCAGCA

ATGC-3′),GAPDHrev(5-CATGGACTGTGGTCATGAGTC

CT-3′).SYBRGreenreal-timePCRwascarriedoutusingABIPrism

(3)

theamountofappliedRNA.Relativeimmunoglobulin-binding protein(BiP)transcriptionlevelsweremeasuredbyapplying the2−(Ct)equation.

Westernblotanalysis

Conditionedmediumwascollectedfromtransducedand non-transduced HEK 293 cells after 72h and total protein was quantifiedbytheBCAProteinAssayKit(Pierce).Thecultured cellswere lysedinice-coldlysis buffercontainingprotease inhibitors(Completemini-proteaseinhibitorcocktail,Roche) and centrifuged at 16,000×g for 15min. Total proteinwas quantifiedbythe BCA ProteinAssay Kit(Pierce).Total pro-tein(35␮gand40␮gextractedfromconditionedmediumand lysedcells,respectively)wasseparatedbySDS-PAGE(4–20% Mini-PROTEAN,BIO-RAD),transferredtonitrocellulose mem-branes(40␮m, Hybond-CExtra,AmershamBioscience)and probed with anti-FVIII light chain mouse antibody (Santa Cruz Biotechnology)or anti-BiP/GRP78 mouse antibody(BD Bioscience).Internalcontrolwasachievedusinganti␤-actin mouseantibody(Sigma-Aldrich).

Chemicalchaperonetreatmentoftransducedcells

Transduced cells were seeded at a cell density of about 3.5×106cellsperplateandwereincubated inthepresence

ofchemicalchaperones:betaine(Sigma-Aldrich)andsodium 4-phenylbutyrate(Sigma-Aldrich)atdifferentconcentrations. After72hofincubationwithchemicalchaperones,levelsof active FVIII were determined in the supernatants. Experi-mentswereperformedinduplicate.

Invivoassay

HemophiliaAmice(B6;129S4-F8tm1kaz/J)were obtainedfrom JacksonLaboratory(BarHarbor,ME, USA).Adultmice,aged 8–12weeks, were used fortail clipping. All protocols were conductedinaccordance withthe EthicalCode forAnimal Experimentationofthe CouncilforInternational Organiza-tionsofMedicalSciences(CIOMS)andtheColégioBrasileirode Experimentac¸ãoAnimal(COBEA).Thisstudywasapprovedby theEthicsCommitteeforAnimalResearchoftheUniversidade deSãoPaulo(USP),RibeirãoPreto(#14.1.784.53.8).

Severetail-bleedingmodel

Thetail-clipchallenge wasdesignedtoanalyze hemophilic A mice after receiving one unit of culture supernatant containing FVIIIB-F309S (n=3 mice), FVIIIB (n=3 mice) or phosphate-buffered saline (PBS) (n=3 mice). Mice were anesthetizedwith4.5%isofluraneandreceivedculture super-natantviaretroorbitalinjection.After10min,bleedingwas inducedbycutting1cmofthetailandanimalsurvivalwas monitoredover50h.

Statisticalanalysis

Resultsareexpressedasmeans±standarderrorofthemean (SEM) or standard deviation (SD) as appropriate. Student’s unpaired t-testand 95% confidenceinterval were used for

comparisonsbetweenthegroups.Two-wayanalysisof vari-ance(ANOVA)wasusedtocomparedifferencesbetweenthe indicatedgroupsandthelog-ranktestwasusedtocompare thesurvivalcurvesafterthetail-clippingchallenge.p-Values <0.05wereconsideredstatisticallysignificant.

Results

Transientassay

InordertostudytheroleoftheF309Smutationonthe secre-tionandfunctionofFVIIIinhumancelllines,amoleculewith thismutationwassynthesizedandclonedintoalentiviral vec-tor.After,itssecretionwasanalyzedbytransienttransfection inHEK293cells.FVIIItwo-stageactivityassaysperformedon conditionedmediaharvested48hand72haftertransfection revealed thattherewasabettersecretionofFVIIIB-F309S overtheFVIIIBlevels(Figure1).

TheFVIIIB-F309S presentedon average, 1.5and 2fold highersecretionthanFVIIIBinHEK293cells,after48hand 72hoftransfection.Theseresultswerereproducibleand con-sistentinthreeseparatetransfectionexperiments.

GenerationoftheHEK293celllinestablyexpressing FVIIIBandFVIIIB-F309S

To compare whether the mutation could also result in increasedsecretionofFVIIIinstableexpression,HEK293cell linesstablyexpressingFVIIIBandFVIIIB-F309Swere gen-erated.Thesecelllinesweregeneratedbylentiviralinfection followedbyselectionusinggeneticin.Thenumberof lentivi-ralvectorsintegratedintothegenomewerequantifiedforthe twogeneratedstrains(Figure2A).ThecelllineFVIIIB-F309S hasonecopy/cellofthelentiviralvectorwhereastheFVIIIB has1.4copies/cellintegratedinitsgenome.

Thecell linecontaining theFVIII mutationexpresses 9-foldmoreFVIIImRNAthanthecellwithFVIIIB(Figure2B). ThelevelofproteinsecretedinthecellwithFVIIIB-F309S isgreaterthanthecell linewithFVIIIB.Thecellline con-tainingtheF309SmutationsecretedanaverageoffiveIU/106

cells/72hofFVIII,thatis,4.5-foldmoreefficientlythanFVIIIB asdeterminedbytwo-stageclottingactivityassay(Figure2C).

48h 72h

0 10 20 30 40

FVIIIΔΒ FVIIIΔΒ-F309S

ng/ml

a

a

Figure1–TransientproductionofFVIIIBand

FVIIIB-F309SinHEK293cells.ThefactorFVIIIproduction wasmeasuredbytheenzyme-linkedimmunosorbent assay(ELISA)technique.aStatisticallysignificant

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A

B

C

REU

UI/10

6 cells/72h

800 000.0

p-value=0.0216

p-value=0.0012

600 000.0

400 000.0

200 000.0

0.0

6

4

2

0

Cells Vector copy/cell

FVIIIΔB-F309S

FVIIIΔB-F309S FVIIIΔB FVIIIΔB-F309S FVIIIΔB 1

FVIIIΔB 1.4

Figure2–(A)Numberoflentiviralvectorsintegratedinto genome;(B)relativeexpressionunits(REU)ofboth recombinantFVIIIexpressionsand(C)FactorVIII

second-stageactivitybyachromogenicassay(COAMATIC). Datapresentedarethemeanofthreeindependent experimentsandtheerrorbarsrepresentthestandard deviation.Asterisksdesignatestaticallysignificant differencesbetweengroupsusingtwo-wayANOVA

CharacterizationofsecretedFVIIIB-F309S

FVIII protein has a 110-amino acid region within the A1-domainthatinhibitsitssecretionandcontainsmultipleshort peptide sequences that have potential to bind to BiP. The low level of FVIII secretion correlates with binding to the BiP,withinthelumenoftheER.Toassesswhetherthehigh expression of FVIII induces the production of BiP, the BIP levelwasevaluatedbyWesternblot.Figure3showsthatFVIII

HEK293

BiP

Advate®

HC+LC (without processing)

HC+B domain deleted HC+B domain β actin

Std

200 kDa

A

B

140 kDa

100 kDa

80 kDa

60 kDa

50 kDa

40 kDa

HEK293

FVIIIΔB

FVIIIΔB

FVIIIΔ B-F309S

FVIIIΔ B-F309S

Figure3–CharacterizationofFVIIIproducingcelllinesby Westernblot.(A)Celllysateswerefractionatedby SDS–PAGE,andimmunoblottedwithBiP(78kDa)and b-actin(42kDa)antibodies.(B)Supernatantsof

non-transducedHEK293cells,293cellstransducedwith FVIIIBandFVIIIB-F309S,andthecontrolAdvate®

immunoblottedwithanti-heavychainFVIII.

expressionleadstoanincreasedamountofBiP.However,there arenosignificantdifferencesinBiPamountsbetweenHEK293 cellsexpressingFVIIIBandthoseexpressingFVIIIB-F309S (Figure3A).

InordertocharacterizethesecretedFVIIIr,Westernblot wasperformedwiththeanti-heavychainmonoclonal anti-body.Asexpected,theHEK293cellsexpressingFVIIIB-F309S secretemoreFVIIIthanHEK293cellsexpressingFVIIIB.It waspossibletodetecta90kDabandreferring totheheavy chain(Figure3B).

However,itwasnotpossibletoidentifyclearlytheband referring to the heavy chain in the FVIIIBsample, prob-ably because this cell produces a low amount of FVIII thereby hindering antibodybinding. Inthe band relatedto theheavychain,anotherbandof∼200kDawasdetectedin commercial FVIII corresponding to theheavy chainwithB domain(Advate®,Baxter).FVIIIB-F309S,whichhasthe

par-tial deletion ofB domain,alsoshows a band of∼200kDa, suggesting that part of the FVIIIr released is composed of heavy and light chains without intracellular cleavage (Figure3B).

The FVIIIB-F309S sample also showed fragments of 53kDaand43kDa,whichareprobablyrelatedtothe degra-dationofFVIII.The53kDabandmaycorrespondtopartofthe A2domainwiththeA1domain(∼55kDa)andthe43kDais relatedtotheA2domain(Figure3B).

Chemicalchaperonesupplementationincreasesthe secretionofFVIIIBbutnotofFVIIIB-F309S

Inaddition,twodifferentcompoundstermedchemical chap-erones (CC)were tested. Thesecompounds are responsible fornon-specificallystabilizingnative proteinconformations and supporting escape from the endoplasmic quality con-trol system. Members ofdifferentcompound classes ofCC (Betaineandsodium-4-phenylbutyrate)weretestedfortheir effecton FVIIIBand FVIIIB-F309SexpressioninHEK293 cells.Betaineandsodium-4-phenylbutyratewereusedat con-centrations of 1mM and 100mM. The drugs were tested individually and in combination in the modified cell lines expressingFVIIIBandFVIIIB-F309S.

The addition of betaine or sodium-4-phenylbutyrate increasedthesecretionrateofFVIII-BproteinsinHEK293 cells(Figure4).Thecombinationofbothdrugsledtoafurther increaseinthesecretionofFVIIIB.However,noincreasein thesecretionofFVIIIB-F309Swasobservedwiththeaddition ofbetaineorsodium-4-phenylbutyrateorwiththe combina-tionofthesetwocompounds(Figure4).

FVIIIBandFVIIIB-F309Sarefunctionallyactivein hemophiliaAmice.

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4

a a a

b b

Control Betaine 100 mM

Sodium-4-phenylbutyrate 1 mM

Sodium-4-phenylbutyrate 1 mM Betaine 100 mM+

3

2

F

old change

1

0

FVIII ΔB

FVIII ΔB-F309S

Figure4–Effectofthebetaineand

sodium-4-phenylbutyrateonFVIIIsecretion.HEK293cells expressingFVIIIBorFVIIIB-F309Swereincubatedwith betaineandsodium-4-phenylbutyratealoneorin

combination.FVIIIactivitywasdeterminedincell supernatantsafter72hbychromogenicassay(n=3).

aStatisticallysignificantdifferencesbetweengroupsusing

two-wayANOVA.p-Value=0.0045.bStatisticallysignificant

differencesbetweengroupsusingtwo-wayANOVA. p-Value=0.0005.

0 20 40 60 80100 0

20 40 60 80 100

100 150200 250 300

Control FVIIIΔB FVIIIΔB-F309S

a a

Time (h)

P

e

rc

e

n

t s

u

rv

iv

a

l, %

Figure5–SurvivalcurveofhemophiliaAmicesubmitted totreatmentwithFVIII-B(n=4),FVIIIB-F309S(n=4)or phosphate-bufferedsaline(n=4)andchallengedbytail clipping.aStatisticallysignificantvalues(p-value=0.0136).

Discussion

RecombinantFVIIIproteinisoneofthemostcomplex pro-teinsforindustrialproductionduetothelowefficiencyofgene transcription,proteininteractions withretentionintheER, inappropriatetransportfromtheERtotheGolgiapparatusand theinstabilityofthesecretedprotein.8–10Overtheyears,other

researchgroupshavestudiedwaystoimprovetheexpression, secretionandtoincreasethehalf-lifeofcoagulationfactors, especiallyFVIII.2,11

Inthisstudy,theHEK293celllinewaschosenduetothe variousadvantagesofferedbythisstrain,forexample,robust patternofgrowth,easymaintenance,andhightransfection efficiency and productionof proteins.12 The transient

pro-ductionofrFVIII using theserum-free HEK293cell line in suspensionhasbeen demonstrated yielding approximately 0.64IU/mLFVIIIr.13Morerecently,thepharmaceutical

indus-try hasdeveloped the first rFVIII producedin humancells (usingtheHEK293Fstrain)whichshowedefficiencyinterms ofsafetyandproduction.14

ThecurrentstudyshowedthattheF309Smutationwasable toincreaseFVIIIsecretionby3-foldcomparedtoFVIIIBin

astableHEK293cellline.Thisresultisinaccordancewith previousworks.2,4Itisknownthattheinefficientsecretionof

FVIIIiscorrelatedwithbindingtotheproteinidentifiedasBiP, alsoknownastheglucose-regulatedprotein78(GRP78)within thelumenoftheER.15Marquetteetal.locateda110amino

acidregionwithintheA1domainthatinhibitsFVIIIsecretion. Thisregionisclusteredwithmultipleshortpeptidesequences thathavepotentialtobind toBiP.16 TheresultsofWestern

BlotinthisstudyrevealedthatbothFVIIIB-F309SandFVIIIB increasedBiPexpression,althoughFVIIIproductionwasnot affected.

AnotherstrategyusedinthisworktoimproveFVIII secre-tionwastoaddchemicalchaperonestoculturecells.Betaine isachemicalchaperonethatcaninhibittheaggregationof FVIIIandrestoretheintracellulartraffickingoflooselycoiled proteins.Inadditiontobetaine,sodiumbutyrate,anorganic compoundthathasvariouseffectsoncellcultures,wasalso tested;oneeffectistheinductionofgeneexpressionby his-tone hyperacetylation promotingthe activation ofgenes.17

This study shows that HEK 293 cells expressing FVIIIB that had been treated with betaine and sodium butyrate increasedFVIIIproduction.However,noeffectwasobservedin HEK293cellsexpressingFVIIIB-F309Streatedwithbetaine. Roth18testedseveralchemicalchaperones,includingbetaine

and reported that this compound increased the secretion of FVIII because ofthe increasedsolubility ofintracellular FVIIIaggregatesandimprovedtransportfromtheERtothe Golgi apparatus.18 By contrast, the data ofthis study

sug-gestthatalltheFVIIIproducedbyHEK293cellsexpressing FVIIIB-F309Swassecretedoutofthecellanddidnotform aggregates.

Ontheotherhand,sodiumbutyrateincreasedFVIII pro-ductioninHEK293cellsexpressingFVIIIBeitheralone or incombinationwithbetaine.Previousstudieshaveshowed that sodiumbutyrateproducesawidevarietyofeffectson cellsinculture:arrestofcellgrowth,reversionofthe trans-formationcharacteristicsofcells,andinductionofproteins, including enzymes, peptide hormones and hemoglobin.17

However,the sameeffectwas notnoticedinHEK293 cells expressing FVIIIB-F309S with the reason for this differ-ence not being clear and thus further investigations are needed.

Conclusion

ThemaincontributionofthisworkistheproductionofaFVIII moleculewithahighsecretionrate(withtheF309Smutation intheA1domain),inordertoincreasetheproductivityand decreasetheproductioncost.Furthermore,thisstudyutilized ahumancellline,HEK293,toproduceanrFVIIImoresimilar to the existing FVIII inhuman plasma, and less immuno-genic than the rFVIII commercially produced in hamster cells.

Conflicts

of

interest

(6)

Acknowledgements

WethankCleideAraújoSilvaandSandraNavarroBresciani whohelpedwiththeanimalexperimentsanddrewthefigures, respectively.ThisworkwassupportedbyCAPES,FAPESPand CNPq.

r

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n

c

e

s

1. DornerAJ,WasleyLC,KaufmanRJ.Increasedsynthesisof

secretedproteinsinducesexpressionofglucose-regulated

proteinsinbutyrate-treatedChinesehamsterovarycells.J

BiolChem.1989;264(34):20602–7.

2. MiaoHZ,SirachainanN,PalmerL,KucabP,CunninghamMA,

KaufmanRJ,etal.BioengineeringofcoagulationfactorVIII

forimprovedsecretion.Blood.2004;103(9):3412–9.

3. DoorissKL,DenningG,GangadharanB,JavazonEH,McCarty

DA,SpencerHT,etal.ComparisonoffactorVIIItransgenes

bioengineeredforimprovedexpressioningenetherapyof

hemophiliaA.HumGeneTher.2009;20(5):465–78.

4. SwaroopM,MoussalliM,PipeSW,KaufmanRJ.Mutagenesis

ofapotentialimmunoglobulin-bindingprotein-bindingsite

enhancessecretionofcoagulationfactorVIII.JBiolChem.

1997;272(39):24121–4.

5. CalvezT,ChambostH,Claeyssens-DonadelS,d’OironR,

GouletV,GuilletB,etal.RecombinantfactorVIIIproducts

andinhibitordevelopmentinpreviouslyuntreatedboyswith

severehemophiliaA.Blood.2014;124(23):3398–408.

6. Picanco-CastroV,BiaggioRT,CovaDT,SwiechK.Production

ofrecombinanttherapeuticproteinsinhumancells:current

achievementsandfutureperspectives.ProteinPeptLett.

2013;20(12):1373–8.

7. MatsuiH,ShibataM,BrownB,LabelleA,HegadornC,

AndrewsC,etal.ExvivogenetherapyforhemophiliaAthat

enhancessafedeliveryandsustainedinvivofactorVIII

expressionfromlentivirallyengineeredendothelial

progenitors.StemCells.2007;25(10):2660–9.

8. SoukharevS,HammondD,AnanyevaNM,AndersonJA,

HauserCA,PipeS,etal.ExpressionoffactorVIIIin

recombinantandtransgenicsystems.BloodCellsMolDis.

2002;28(2):234–48.

9.ChuahMK,VandendriesscheT,MorganRA.Developmentand

analysisofretroviralvectorsexpressinghumanfactorVIIIas

apotentialgenetherapyforhemophiliaA.HumGeneTher.

1995;6(11):1363–77.

10.BeckerS,SimpsonJC,PepperkokR,HeinzS,HerderC,GrezM,

etal.Confocalmicroscopyanalysisofnative,fulllengthand

B-domaindeletedcoagulationfactorVIIItraffickingin

mammaliancells.ThrombHaemost.2004;92(1):23–35.

11.DumontJA,LiuT,LowSC,ZhangX,KamphausG,SakorafasP,

etal.ProlongedactivityofarecombinantfactorVIII-Fcfusion

proteininhemophiliaAmiceanddogs.Blood.

2012;119(13):3024–30.

12.ThomasP,SmartTG.HEK293cellline:avehicleforthe

expressionofrecombinantproteins.JPharmacolToxicol

Methods.2005;51(3):187–200.

13.SwiechK,KamenA,AnsorgeS,DurocherY,Picanc¸o-CastroV,

Russo-CarbolanteEM,etal.Transienttransfectionof

serum-freesuspensionHEK293cellcultureforefficient

productionofhumanrFVIII.BMCBiotechnol.2011;11:

114.

14.ValentinoLA,NegrierC,KohlaG,TiedeA,LiesnerR,HartD,

etal.ThefirstrecombinantFVIIIproducedinhumancells–

anupdateonitsclinicaldevelopmentprogramme.

Haemophilia.2014;20Suppl.1:1–9.

15.MunroS,PelhamHR.AnHsp70-likeproteinintheER:identity

withthe78kDaglucose-regulatedproteinand

immunoglobulinheavychainbindingprotein.Cell.

1986;46(2):291–300.

16.MarquetteKA,PittmanDD,KaufmanRJ.A110-aminoacid

regionwithintheA1-domainofcoagulationfactorVIII

inhibitssecretionfrommammaliancells.JBiolChem.

1995;270:10297–303.

17.KruhJ.Effectsofsodiumbutyrate,anewpharmacological

agent,oncellsinculture.MolCellBiochem.1982;42(2):

65–82.

18.RothSD,SchüttrumpfJ,MilanovP,AbrissD,UngererC,

Quade-LyssyP,etal.Chemicalchaperonesimproveprotein

secretionandrescuemutantfactorVIIIinmicewith

Imagem

Figure 1 – Transient production of FVIIIB and
Figure 3 – Characterization of FVIII producing cell lines by Western blot. (A) Cell lysates were fractionated by SDS–PAGE, and immunoblotted with BiP (78 kDa) and b-actin (42 kDa) antibodies
Figure 5 – Survival curve of hemophilia A mice submitted to treatment with FVIII-B (n = 4), FVIIIB-F309S (n = 4) or phosphate-buffered saline (n = 4) and challenged by tail clipping

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