Download AAV User Manual - Vigene Biosciences

RecombinantAdeno-AssociatedVirus(rAAV)
CloningandVirusPackagingServiceManual
Revision201603.02
©VigeneBiosciences2016
RESEARCHUSEONLY.
Notforuseindiagnosticprocedures
Thisproductshallbeusedbythepurchaserforinternalresearchpurposeonlyand
redistributionisstrictlyprohibitedwithoutwrittenpermissionfromViGene
BiosciencesInc.
TableofContent
CONTENTSANDSTORAGE..............................................................................................................3
Introduction....................................................................................................................................4
Whichviralvectortouse-viralvectorselectionguide...............................................................5
AAVserotypesandnativetropism-AAVselectionguide...........................................................6
AAVRelatedServiceDetails............................................................................................................6
AAVvectorcloningservices........................................................................................................6
SelectionsofrAAVvectorsfromViGeneBiosciencesInc.......................................................6
pAV-FHforgeneexpression...................................................................................................7
rAAVShRNAvectors...............................................................................................................7
AAVCustomcloningserviceswithpre-selectedpromoters...................................................8
FLEX-ONofCredependentinducibleexpression.................................................................10
AAVVirusPackagingServices...................................................................................................10
FinalProductsComponentsandQCStandards........................................................................10
ThepurityoftheAAVvirus.......................................................................................................10
Thevirustiter............................................................................................................................11
Recommendedprotocolforinvitrocelltransductionandinvivoanimalinjection.................11
Invitrocelltransduction...........................................................................................................11
Invivoanimaluse.........................................................................................................................13
FAQ...............................................................................................................................................13
BiosafetyConsiderations:.............................................................................................................16
LIMITEDPRODUCTWARRANTY....................................................................................................16
ORDERINGINFORMATIONANDTECHNICALSUPPORT.................................................................16
Ordering....................................................................................................................................16
TechnicalSupport.....................................................................................................................16
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CONTENTSANDSTORAGE
AAVstocksaresuppliedinliquidformatindicatedtiter.Thestoragesolutionis
PBSwith0.001%F68.Storeat-80°C.Ifdesired,aliquotviralstockuponarrival,
andstorethosealiquotsat-80°Cfreezerimmediately.
Dependentondifferentservicetypes,theproductsmaycontainthefollowing
components.
1. SmallscalecrudeAAV.500ulofrAAVat10^12-13GC/ml.*
2. LargescalepurifiedAAV.500ulofrAAVat10^13-14GC/ml.*
3. CustomerLargescalepurifiedAAV.Customamount(upto10^16GC)of
rAAVat10^13-14GC/ml.*
•
GC/mlstandsforAAVgenomecopies/mlmeasuredbyreal-timeqPCRin
comparisonwithastandardreferenceplasmidwithknowngenomecopy
number.ForallthevirusproductsfromViGeneBiosciencesIncthetitervirus
ismeasuredasgenomecopies/ml.InthisManual,weuseGC
interchangeablywithvg(viralgenomes)andvp(viralparticles)asaunitfor
viraltiters.
AAVclonesaresuppliedin5ugDNAinTEbufferofspecifiedamountoutlinedin
theCertificateofAnalysis(CoA).
DONOTFREEZEANDTHAWREPEATEDLY.
Introduction
InViGeneBiosciences,RecombinantAdeno-associatedVirus(rAAV)ExpressionSystems
areutilizedindeliveringandexpressingshRNA,humanORF,CRISPRinvitroandinvivo.
Adeno-associatedvirus(AAV)isasmallsinglestrandDNAviruswhichinfectshumans
andsomeotherprimatespecies.AAVisnotcurrentlyknowntocausediseaseandhas
verymildimmuneresponse.Itcaninfectdividingandnon-dividingcells.Further
removaloftherepandcapfromthevectorhaseliminatedtheAAVintegrativecapacity.
ThosefeaturesmakerAAVidealviralvectorforgenetherapy.Todate,rAAVvectors
havebeenusedinmanyclinicaltrialsingenetherapy,promisingresultshavebeen
achievedfromPhase1andPhase2trialsincluding,CFTR,HemophiliaB,Arthritis,and
Parkinson’sdiseases.Figure1showsthecellentryandtraffickingofrAAV.
AfterrAAVgetsintothecells,itstaysstableasepisomalDNA.Expressionofgeneusually
peaksin5to10daysandcanlastseveralweeksorevenmonthsinvivo.
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Whichviralvectortouse-viralvectorselectionguide
Whencomparingthreemostpopularviralvectorsingenedelivery,youshouldtake
followingconsiderationbeforeyouchooseadeno-associatedviralvector.
1. Doyouneedtransientorstablegeneexpression?
2. Doyouneedtotransducedividingornon-dividingcells?
3. Howimportantispotentialimmuneresponsefromyourtargetcell?
4. Howlargeisthegeneofinterest?
AAVscaninfectdividingandnon-dividingcells.Itislowoncytotoxicityand
immunogenicity,suitableforlongtermgeneexpressioninnon-dividingcellsandshort
termindividingcellswithrelativehighgenedeliveryefficiency.ButAAVvectorhas
limitedcloningcapacity,thespacebetweentwoITRsisonly4.9kb,soyourgeneshould
be3.5kborless.Pleaserefertothistableforchoosingyourviralvectorsystem.
Adenovirus
Genome
Coat
Genomesize
Infection/tropism
dsDNA
Naked
38-39kb
Dividingandnondividingcells
Non-integrating
HostGenome
Interaction
Transgene
expression
PackagingCapacity
ImmuneResponse
RelativeViralTiter
Relative
Transduction
Efficiency
RelativeForeign
GeneExpression
Adeno-associated
Virus(AAV)
ssDNA
Naked
5kb
Dividingandnondividingcells
Non-integrating
Lentivirus
Longlasting
7.5kb
High
10^11GC/ml
without
purification
100%
Potentiallong
lasting
4.5kb
VeryLow
10^7GC/ml
without
concentration
70%
High
Medium
Medium
Transient
ssRNA(+)
Enveloped
9kb
Dividingand
non-dividingcells
Integrating
6kb
Low
10^7GC/ml
without
concentration
70%
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AAVserotypesandnativetropism-AAVselectionguide
Sofarthereare11AAVserotypesdescribed,theyallhavedifferenttropismandcan
infectcellsfrommultiplediversetissuetypes.Tissuespecificityisdeterminedbythe
capsidserotype.Toselecttherightserotypesiscriticalindeliveryofgeneintodifferent
cellsortissues.ThefollowingtablelistsmostpopularrAAVserotypeandtheirtropism.
TissueTropism(Xindicatesrecommendedapplication)
AAV
Serotype
Muscle
AAV1
X
Liver
Lung
AAV6
X
Lung
alveolar
cells
AAV7
X
AAV8
X
X
AAV9
X
X
X
AAV2
AAV5
Brain
Neurons
andglial
cells
Retina
Pancreas
X
X
X
Neurons
andglial
cells
X
Neurons
X
Neurons
X
X
Neurons
X
X
X
X
Kidney
IfyoucannotfindenoughinformationtodecidewhichserotypeofrAAVorwhichvirus
vectorworksbestforyoursystem,youcantryourGFP-VirusTestingKit,Cat#CT10001.
AAVRelatedServiceDetails
WeoffertwoservicesforAAVdevelopmentandproduction.ThefirstisAACvectorcloning
service;theotherisAAVpackagingservices.
AAVvectorcloningservices
SelectionsofrAAVvectorsfromViGeneBiosciencesInc.
CurrentViGeneBiosciencesInc.offerspAV-FHvectorforgeneexpressionandfour
vectorsforshRNAexpression.
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pAV-FHforgeneexpression
Inmostofcase,ORFinsertsareclonedbetweenAsisIandMluIsites.Inother
rarecasethecombinationofAsisI-RsrII,AsisI-NotIorAscI-MluIareusedinthe
cloning.PleasecheckourwebsiteortheCOAforspecificclones.InthepAV-FH
vector,ORFisfusedwithaFlag/Histagatitscarboxylterminus.Thevector
containsanampicillinmarkerforbacterialselection.ViGene’spAV-FHvectorisa
mammalianORFexpressionvector,dualtagsofFlagandHiscouldbeusedto
detectandpurifyproteinsexpressedinmammaliancells.
4559
bp
rAAVShRNAvectors
1. pAV-U6-GFP
2. pAV-U6-RFP
3. pAV-H1-GFP
4. pAV-H1-RFP
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ToexpressshRNAwithrAAV,ViGeneprovidesthechoicesofeitherU6orH1
promotertodrivetheshRNAexpressionandGFPorRFPasmammalianexpression
marker,shownbytheexamplemapofpAV-H1-RFP.
5020bp
AAVCustomcloningserviceswithpre-selectedpromoters
CMVisaverystrongandthemostcommonlyusedpromoterindrivinggene
expressioninvitroandinvivo.CMVpromoterdrivesubiquitousgeneexpression
inmosttissueandcelltypes.Duetothemethylation/silencing,expressionby
CMVpromoterdecreasesinvivoafter10to20weeks.Forbettertissueorcell
typespecificgeneexpressionandforlongtermstrongandstableexpression,
manydifferentpromotersareofferedbyViGeneBiosciences.
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Cat.#
Promoter
Size
Description
PM10001
ALB
2.4kb
Liver specific 10 timer stronger than CMV after 10 weeks
PM10002
GFAP104
845bp
Hybrid of EF1a and GFAP
PM10003
CAG
944bp
Strong promoter, ubiquitous expression in vivo
PM10004
CamKIIa
1.2kb
Specific expression in excitatory neurons in the neocortex and
hippocampus
PM10005
EF1A
1.2kb
Ubiquitous, weaker than CMV but better for in vivo
PM10006
CK1.3
1.1kb
PM10007
CK0.4
217bp
Calcium/Calmodulin-dependent kinase II alpha
PM10008
GFAP
2.0kb
Specific in astrocyte
PM10009
MBP
1.3kb
Myelin basic protein promoter, efficient transduction of
oligodendrocytes by adeno-associated virus type 8 vectors
PM10010
EFFS
253bp
A short version EF1A
PM10011
TBG
460bp
Homo sapiens serpin peptidase inhibitor, clade A
PM10012
aMHC
0.4kb
Mouse myosin heavy chain alpha promoter
PM10013
cTNT
702bp
Specifically transduce cardiomyocytes
PM10014
Synapsin
471bp
Specific in neuron
PM10015
Mecp2
230bp
Truncated Mcep2 neuron specific
PM10016
c-fos
1.7kb
Activity-dependent promoter
PM10017
MCK
1.35kb
Muscle creatine kinase promoter/enhancer
PM10018
UBC
1.1kb
Ubiquitous, weaker than CMV but better for in vivo
PM10019
PGK
400bp
Ubiquitous, weaker than CMV but better for in vivo
PM10020
Somatostat
1.2kb
Restricting expression to GABAergic neuron
PM10021
Rpe65
700bp
Retinal Pigment epithelium-specific expression in vivo and in vitro
PM10022
Insulin1
1.0kb
Specific in beta- cells of the pancreas
PM10023
3Xenhanc
er McK
728bp
Much stronger than CMV in muscle, inactive in nonmuscle cell lines
and mouse liver
PM10025
NSE
1.3kb
Neuron-specific enolase promoter
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FLEX-ONofCredependentinducibleexpression
CombinetissuespecificpromotersandCredependentFLEX-Oninducibledesign
cangetmorerestrictedtissuespecificandtemporalexpression.InFLEX-ON
system,geneisinreverseorientationdownstreamofpromoter,andthegeneis
flankedbytwooppositelyorientatedloxPs.IntheabsenceofCre,genecannot
beexpressedandinthepresenceofCre,geneexpressioncanbeturnonor
induced.
AAVVirusPackagingServices
FinalProductsComponentsandQCStandards
Unlessspecifiedotherwise,AAVvirusesareproducedfrom10^9Hek293cellsandare
purifiedbyIodixanolgradientultracentrifugation.Resultedvirusareconcentratedto
400ulwithvirustiternolessthan10^13GC/ml.Thepurifiedvirusisgoodforinvivo
animalresearch.
VigeneBiosciencesInc.providestherAAVviruspackagingservicesinafewformats.
1. SmallsacletestingAAVpackagingservice.Inthisservice,rAAVispackagedusing
10^07HEK293Tcells.Thevirusisincrudecelllysate,withoutanypurificationor
concentration.Thetiterisaround10^9-11GC/ml.
2. LargescalepurifiedrAAVpackagingservice.Inthisservice,rAAVispackagedusing
2.5X10^8HEK293cells.VirusesarepurifiedbyIodixanolgradient
ultracentrifugation.Resultedvirusareconcentratedto400ulwithvirustiternoless
than10^13GC/ml.Thetiterisaround10^12-15GC/mldependentonvirus
serotypeandthesizeofinsert.
3. CustomlargescalepurifiedrAAVservice.Thepurityandvirusaredeliveredperthe
specification.ViGeneBiosciencescandeliverupto10^16GCwithvirustiternoless
than10^13GC/ml.
ThepurityoftheAAVvirus
TheAAVproteincomponentsareVR182kDa,VR272kDaandVR362kDa.Soa
goodAAVpurificationshouldonlyshowthreemajorproteinbandswhenthe
virusisanalyzebySDA-PAGE.Followingimageshowedtheproteincomponents
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inourpurifiedAAVvirus.WeguaranteethepurityoftheAAVvirusbasedonthe
specificationthatwegivefordifferentservices.
Thevirustiter
Thevirustiterisdeterminedbytheviralgenomecopynumberin1mlsampleby
Q-PCRandcomparedtocopynumberstandardsamples.Followingexampleis
datafortitteringAAV-GFPvirus.
Recommendedprotocolforinvitrocelltransductionandinvivoanimal
injection
Invitrocelltransduction
DeterminetheMOI(multiplicityofinfections)
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•
MOImeansMultiplicityofInfection.MOIequalsnumberofviralparticles
(vp)percell.Inotherwords,andMOIof1meansinfectingwith1viral
genome(vg)percell.InViGeneBiosciencesourpackagingefficiencyis
~100%.GC/mlstandsforAAVgenomecopies/mlmeasuredbyreal-time
qPCRincomparisonwithastandardreferenceplasmidwithknowngenome
copynumber.ForallthevirusproductsfromViGeneBiosciencesIncthetiter
virusismeasuredasgenomecopies/ml.InthisManual,weuseGC,vgand
vpinterchangeablyasaunitforviraltiters.
ForallthevirusproductsfromViGeneBiosciencesIncthetitervirusismeasured
asvirusparticlesorgenomecopies/ml.Althoughmeasurementofvirustiterin
GC/mlisreproducibleineverylab,therealinfectionunitscouldbeverydifferent
whenit’sestimatedindifferentexperiments.Thusbeforeyoudoyour
experiments,youhavetoestimatetheInfectionUnitsofvirusinyour
experiments.InViGeneBiosciencesInc,weusuallydoaten-foldserialdilutionof
virusstartingwith1ulviralstockandendingin10^8GC/ml.Addthedilutedthe
virustoyourcells,2-3dayslater,basedonhowmanycellsbeeninfectedto
calculateyourinfectionunits.
Thegoalistoget100%ofinfectionwithoutcausinganyundesiredeffects.To
determinethisoptimalconcentrationofvirusforyourstudy,wesuggestyouto
conductpilottestinginyourcelllinebyusingreporterAAVlikeAAV-GFP(ViGene
Biosciences,catalognumber#CV10003throughCV10009).
AfteryouknowtheinfectionunitsoftheviralstockanddecidetheMOIyouare
goingtouseinyourexperiments,dilutetheviralstockwithrightMOIfirst.
Removetheoriginalcellculturemedia,andaddtheaboveAAV-containingmedia
tocellculture.Belowisageneralguidelinefortheamountofmediaused:
24-wellplate:0.2-0.3ml
12-wellplate:0.5-0.8ml
6-wellplate:2ml/well
60mm-plate:3-4ml/plate
10cm-plate:8-12ml/plate
Incubatecellswiththevirus-containingmediaforatleast6-12hours.Youdon’t
havetoexchangethevirus-containingmediaforfreshmedia,butyoucandoit
after6-12hours.Itmaytake3-7daysaftertheAAVinfectiontodetectthegene
over-expression.
Recommendedprotocolforinvitrocelltransduction
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1. ThawtheAAVvirusonice,andkeepitonicethroughoutthedurationofthe
experiment.
2. AAVinfectioniscelltypedependent.Somecelltypesexhibitlowtransduction
efficiency,whileotherstransduceveryreadily.
WhendesigningAAVtransductionexperiments,itisrecommendedtouse
differentserotypesofareportervectorsuchanAAVexpressingGFP((ViGene
Biosciences,catalognumber#CV10003throughCV10009)todetermine
optimalserotypefortransductionofyourtissueorcellculture.
3. StartcelltransductionatMOIof10^4and10^6GC/cellwhencellsarereadily
transducible.WithsomecelllinesahigherMOImightbeneeded.Lookforthe
highesttransductionwithminimalcelldeath.Withsomecelllines,high
transductionlevelscannotbeachieved.
4. UsetheminimumconcentrationofFBSthatthecellscanwithstandwhen
performingthetransduction.Forexample,HT1080cellsaremaintainedusing
mediacontaining10%FBS.Transductionsareperformedusingmediacontaining
2%FBS.
5. Usetheminimumamountofmedianecessarytocoverthesurfaceoftheplate.
Forexample,transductionsareperformedin6-wellplates,1mlofmediaper
wellisused.
6. Lookforexpressionat24h,48h,72hand96h,posttransduction.
Invivoanimaluse
•
Therecommendedtiterforinvivoanimalinjectionis10^11GCpergram(bodyweight).
•
DilutetheviruswithPBStoachievetheappropriateGCnumber.
•
Proceedtotheintravenousinjectionortailveininjectionorlocalizedtissueinjectionas
demonstratedbylabs(forreferencespleasevisit
http://www.vigenebio.com/delivery/AAV-Systems/).
FAQ
WhenshouldIuserAAVinmyexperiments?
rAAVcandelivergeneintodividingandnondividingcellsfortransientgeneexpression.
Ithasbeenusedinvivoandinvitro.Refertoourtablewhenyouarenotsurewhich
virualvectorshouldyouchooseforyourexperiments.YoualsocanpurchaseViGene’s
“GFPvirustestingkit”.Cat#CT10001totestinvitroorinvivo.
What'sthebiosafetyrequirementforusingAAV?
RecombinantAAVconstructs,inwhichthetransgenedoesnotencodeeithera
potentiallytumorigenicgeneproductoratoxinmoleculeandareproducedinthe
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absenceofahelperviruscanbehandledinBiosafetyLevel1(BSL-1)facility.Otherwiseit
shouldbehandledasbiohazardousmaterialunderBiosafetyLevel2(BSL-2)
containment.PleasecheckwithyourInstitutionalBiosafetyCommitteeorrelatedNIH
websitefordetailedinformation,ifyouneedmoreinformation.
IsrecombinantAAVreplicationdeficient?
ForwildtypeAAV,replicationisatextremelylowefficiency,withoutthepresenceof
helpervirus,suchasadenovirus.Forrecombinantadeno-associatedvirusproduced
thesedays,thereplicationandcapsidgenesareprovidedintrans(inpRep/Cap
plasmid),andonlythe2ITRsofAAVgenomeisleftandpackagedintovirion,whilethe
adenovirusgenesrequiredareprovidedeitherprovidedbyadenovirusoranother
plasmid,thelikelihoodforarecombinantAAVtoreplicateistheoreticallyimpossible.
Thisisinsimilarschemetolentiviralvectorsproducedthesedays.
What’sthecloningcapacityforrecombinantAAVs?
AAVhasapackagingcapacityof~4.7Kb.SincethetwoITRsofAAVisabout0.2-0.3Kbin
total,theforeignDNAthatcouldbeintroducedbetweenthese2ITRsshouldbe<4.4Kb,
whichismuchsmallerthanthatofrecombinantadenovirus(7.5Kb).Inaddition,when
thelengthofinsertedDNAbetweenthe2ITRsisclosethemaximalallowed,i.e.,44.4Kb,thepackagingefficiencydecreasessignificantly.Forinstance,forgeneoverexpressionfromcDNA,sincetheCMV-poly(A)elementisabout1Kb,sothemaximal
allowablecDNAlengthisabout3Kb.Inaddition,ifyouareinterestedinGFPcoexpression(fromaseparateexpressioncassette),giventheadditionalCMV-EGFPpoly(A)isabout2Kb,sothemaximalcloningcapacityforGFPco-expressingsystemis
about1.0-1.2Kb.
Fordouble-strandedAAV(dsAAV),thecapacityisonlyhalfofthesingle-strandedAAV
(ssAAV).
HowmanyAAVSerotypesdoesViGeneBiosciencesOffer?
Thereare11differentAAVserotypeshavebeenreportedsofar.ViGeneBiosciences
providethepackagingservicesofAAVserotype1,2,5,6,7,8,9.Total7differentAAV
serotypes.
HowstableareAAVvectors?Howshouldtheybestored?
StabilitystudiescarriedoutinhouseandbysomecolleaguesshowthatpurifiedAAV
vectorsarehighlystableattemperaturesof4Corless.Werecommendaliquotingupon
receiptandstoringat-80oC.Onceanaliquotisthaweditcanbestoredat4oCforshorttermstorage,e.g.,2-3weeks,withoutsignificantlossofbiologicalactivity.
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What'sthedifferencebetweenphysicalandgenomicparticles?
AAVgenomeparticlesrelatetotheviralparticlesthathavebeensuccessfullypackaged
withthegenometobedelivered.DuringtheAAVpackagingprocessmanyparticlesare
formedlackingthegenomicDNA,whichlacktheabilitytotransducethecellstheycome
intocontactwithandarethereforenon-functional.Asaresearcheryouprobablywant
theconcentrationoffunctionalAAVparticles.
Whatdoesacustomerneedtoprovide?
IfcustomerisgoingtoordertheclonefromVigeneBioscience,onlythegeneaccession
numberfromNCBIorcatalognumberfromVigeneBioscienceisrequired.Ifcustomer
providesAAVvectorandusingpackagingservicesfromViGeneBiosciences,1mgDNA
frommaxi-prepattheconcentrationof1mg/mlshouldbeprovidedbycustomer.
HowlongdoestherAAVcloning,smallscaleandlargescaleAAVproductiontake?
Ifthe1mgtransfectiongradequalityplasmidscanbesupplied,smallscaleandlarge
scaleAAVproductionwilltake1-2weeks.IfthecustomersrequiresVigenetoconduct
subcloningandplasmidprepfortheAAVplasmid,theentireprocesscantake4-5
weeks.
HowmuchAAVdoIneed?
•
Celltransduction
o StartcelltransductionatMOIof10^4and10^6GC/cellwhencellsarereadily
transducible.WithsomecelllinesahigherMOImightbeneeded.Lookforthe
highesttransductionwithminimalcelldeath.Withsomecelllines,high
transductionlevelscannotbeachieved.
•
Animalinjection
o
Therecommendedtiterforinvivoanimalinjectionis10^11GCpermouseor
2X10^9GC/g(bodyweight).Forintravenousinjectionslargerquantityof
virusesmaybeneededincomparisontolocalinjections.
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BiosafetyConsiderations:
FollowtherecommendedNIHguidelinesforallmaterialscontainingBSL-1organisms.
LIMITEDPRODUCTWARRANTY
Thiswarrantylimitsourliabilitytoreplacementofthisproduct.Nootherwarrantiesof
anykind,expressorimplied,includingwithoutlimitation,impliedwarrantiesof
merchantabilityorfitnessforaparticularpurpose,areprovidedbyViGeneBiosciences.
ViGeneBiosciencesshallhavenoliabilityforanydirect,indirect,consequential,or
incidentaldamagesarisingoutoftheuse,theresultsofuse,ortheinabilitytousethis
product.
ORDERINGINFORMATIONANDTECHNICALSUPPORT
Ordering
•
•
•
•
Email:[email protected]
TollFree(USA):1-800-485-5808
Telephone:301-251-6638
Fax:301-251-6110
TechnicalSupport
•
•
•
•
Email:[email protected]
TollFree(USA):1-800-485-5808
Telephone:301-251-6638
Fax:301-251-6110
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