Rev. Bras. Hematol. Hemoter. vol.38 número3

rev bras hematol hemoter. 2016;38(3):184–185

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

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Scientific

Comment

Bioengineering

coagulation

factors

for

improved

hemophilia

treatments

Comment

on:

the

mutation

F309S

increases

FVIII

secretion

in

human

cell

line

夽

Bidossessi

Wilfried

Hounkpe,

Erich

Vinicius

de

Paula

UniversidadeEstadualdeCampinas(Unicamp),Campinas,SP,Brazil

Theimplementationofprophylaxiswithregular factor VIII

(FVIII) replacement for patients with hemophilia radically

changedtheclinicalpresentationofthiscondition,froma

dis-easecharacterizedbyprogressivedisablingmusculoskeletal

complications,toonecompatiblewithanactiveandvirtually

normallifestyle.InBrazil,althoughmostadultswithsevere

hemophiliastill sufferthe impactofmusculoskeletal

com-plicationsintheirqualityoflife,1_{therecentimplementation}

ofuniversalprophylaxiswillcertainlychangethissituation

forthe next generation ofpatients. Thisscenario posits a

challengetogovernmentandhealthcareproviders,forwhom

guaranteeingareliableandsafesourceofFVIIIconcentrate

hasbecomemorethanever,astrategicissue.Thisisthe

con-textthatunderscorestheimportanceofthestudybyFantacini

etal.inthisissueoftheRevistaBrasileiradeHematologiae

Hemoterapia,inwhichtheauthorsusedacombinedstrategy

toimproverecombinantFVIIIproduction,basedon‘rational

mutagenesis’,chemicalchaperones,andhumancelllines.2

RationalmutagenesisconsistsintheintroductionofDNA

variations(andhence,aminoacidchanges)aimedat

improv-ingthefunctionofarecombinantprotein,basedonprevious

knowledgeaboutproteinstructureandfunction.Thepowerof

rationalmutagenesiswaswell-illustratedbytheidentification

in1988ofaresidueincoagulationfactorIX(FIX)whosechange

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

夽

SeepaperbyFantacinietal.onpages135–40.

∗ _{Correspondingauthorat:UniversidadedeCampinas(Unicamp),CentrodeHematologiaeHemoterapia,RuaCarlosChagas,480,}

13104-184Campinas,SP,Brazil.

E-mailaddress:erich@unicamp.br(E.V.dePaula).

increased FIX function.3 _{This occurred 11 years before the}

reportofananalogouschangeinthisveryresidueinafamily

withextremelyhighlevelsofFIXactivity.4_{Inthisreport,the}

authorsusedaFVIIIvariantwithapointmutation(FVIIIF309S)

inaregionthatdeterminesthemagnitudeofATP-dependence

ofFVIIIsecretion.Thismutationwasrationallydesignedin

1997bySwaroopetal.,based onapreviousdemonstration

that this specific region ofFVIII could bind a protein that

limiteditssecretion.5_{Bychangingasingleaminoacidinthis}

region,basedonthestructureoffactorV, theauthors were

abletodecreasetheATP-dependenceofthesecretionprocess,

therebyenhancingproductionby2.3-fold,withnoapparent

impactonproteinfunction.Fantacinietal.wentfurther,by

introducing this mutationina humancell line(HEK), and

couplingthisstrategywiththeuse ofchemicalchaperones

to further improve FVIII secretion. Although the use of

chaperonesonlyimprovedwildtypeFVIIIsecretion(possibly

becauselittleornoFVIIIF309Swasleft tobesecreted),the

authorswereabletoreproducetheresultsofSwaroopetal.in

ahumancellline,andtodemonstratethatrecombinantFVIII

F309Smaintainednormalfunctionasmeasuredbothinvitro

andinvivo,inatailclipassayinhemophiliaAmice.2

One of the major issues of site-specific mutagenesis

in protein engineering is its still widely unpredictable

http://dx.doi.org/10.1016/j.bjhh.2016.05.001

1516-8484/©2016Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.PublishedbyElsevierEditoraLtda.Thisisan

rev bras hematol hemoter. 2016;38(3):184–185

185

immunologicalconsequences.Infact,theextremesensitivity

oftheimmunesystemtoevenminorchangeswas

demon-stratedbythedevelopmentofinhibitoryantibodiesinpatients

exposedtoafactorVIIavariant(vatreptacogalpha),

character-izedbya3-aminoacidchangethatincreaseditsactivity,but

turnedit intoaimmunogenicmolecule.6_{Thefactthatthis}

outcomewasnotanticipatedinanimalmodelshighlightsthe

importanceofverycarefulpre-clinicalandclinical

develop-mentprogramsforbioengineeredproteins.

AnotherimportantaspectofthereportofFantacinietal.

isthat,asopposedtothevastmajorityofcurrentlyavailable

recombinantFVIIIproducts(expressedinrodentcelllines),

theauthorsuseahumancelllineintheirexpressionsystem.

Besideslimitingtheexposuretoanimalproteins,theuseof

humancelllinescanreducedifferencesinpost-translational

modifications in the expressed protein.This could be

par-ticularlyinterestingin this field,sincethe development of

inhibitoryantibodiestoFVIIIiscurrentlythemainchallenge

ofhemophiliatreatment.Althoughtheclinicalrelevanceof

thisstrategytoinhibitordevelopmentisyettobeproved,the

recentintroductionofthefirstcommercialrecombinantFVIII

productexpressedinahumancellline7_{willallowthis}

hypoth-esistobetested.

Inconclusion,thestudyofFantacinietal.representsan

additional step of a Brazilian group toward mastering the

technology of recombinant coagulation factor concentrate

production,8_{whichaspreviouslystated,shouldberegarded}

asastrategicareaofresearch.

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest.

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