Ideal vectorIdeal vector forfor gene deliverygene delivery::

High transduction efficiencyHigh transduction efficiency

High and longHigh and long--lasting transgene expressionlasting transgene expression

NoNo cytotoxicitycytotoxicity

NoNo induction of immune responseinduction of immune response

CellCell--type specificitytype specificity

High transgene capacityHigh transgene capacity

EasyEasy forfor construction and multiplicationconstruction and multiplication

ComparisonComparison of of gene transfer vectorsgene transfer vectorsWang Y. & Wang Y. & Huang Huang S. 2000. DDT 5: 10.S. 2000. DDT 5: 10.

PlasmidPlasmid RetrovirusRetrovirus LentivirusLentivirus AAVAAV AdenovirusAdenovirus

Host Host tropismtropism

Construction systemConstruction system

Transduction efficiencyTransduction efficiency

Capacity for transgeneCapacity for transgene

Yield Yield ((titretitre))

DurationDuration of of expressionexpression

Cytotoxic responseCytotoxic response

BroadBroad BroadBroad BroadBroadRestrictedRestricted BroadBroad

SimpleSimple EstablishedEstablished EstablishedEstablished EstablishedEstablishedDifficultDifficult

HighHigh LowLow LowLow HighHighModerateModerate

UnlimitedUnlimited 44--5 5 kbkb 9 9 kbkb 44--5 5 kbkb 77--10 10 kbkb

Very lowVery low LowLow LowLow ModerateModerate HighHigh

LowLow LowLow LowLow LowLow HighHigh

Long Long termterm Long Long termterm Long Long termterm weeksweeks--monthsmonthsDaysDays

plazmidplazmid

PlazmidPlazmidz z genemgenem

bakteriebakterie

(na (na wyrosna tylko wyrosna tylko bakteriebakterie z z plazmidemplazmidem))

Kolonie Kolonie bakteriibakteriiz z plazmidemplazmidem

wysianie koloniiwysianie koloniido do bulionubulionu

Bakterie z Bakterie z plazmidemplazmidem

izolacjaizolacja plazmidowplazmidow

Wprowadzenie plazmidu Wprowadzenie plazmidu z z genemgenem do do bakteriibakterii

gen (z gen (z promotorem promotorem i i sygnalem poliadenylacjisygnalem poliadenylacji))

Wysianie bakteriiWysianie bakterii+

Wyizolowane plazmidy z genem

SchemeScheme of of experimentexperiment

VEGFVEGF plasmidplasmid

rabbitsrabbits, 2, 2 weeksweeks

Cell CultureCell CulturePlasmaPlasma VEGFVEGFLocalLocal VEGFVEGF mRNA andmRNA and proteinproteinBlood flow in muscleBlood flow in muscleNumber of microvessels

Expression ofExpression of VEGFVEGF mRNA andmRNA and proteinproteinStimulation of endothelial cell proliferationStimulation of endothelial cell proliferation

Number of microvessels

The simplest of adenoviral life cycleThe simplest of adenoviral life cycle

Entry into the cellEntry into the cell

Uncoating ofUncoating of DNADNA

replicationreplicationtranscriptiontranscription

translationtranslation

Assembly of progeny Assembly of progeny virus particlesvirus particlesand exit from celland exit from cell

DNADNARNARNA

AdenovirusesAdenoviruses~40~40 serotypes of adenovirusesserotypes of adenoviruses (for(for gene therapy typegene therapy type 22 andand 55 were usedwere used),),

causing usually mild ilness in humans causing usually mild ilness in humans

Genome consistsGenome consists a 36a 36 kb doublekb double--stranded linearstranded linear DNADNA withwith ITRITR sequences at each endsequences at each end,, withwith:: Early genesEarly genes ((responsibleresponsible forfor viral gene transcriptionviral gene transcription, DNA, DNA replicationreplication,, host immune suppression host immune suppression

and host cell apoptosisand host cell apoptosis Late genesLate genes ((coding proteins requiredcoding proteins required forfor virus assemblyvirus assembly))

E1E1 early gene is essentialearly gene is essential forfor the subsequent adenoviral gene expressionthe subsequent adenoviral gene expression

transgen

E1

The simplest of adenoviral vector actionThe simplest of adenoviral vector action

Entry into the cellEntry into the cell

Uncoating ofUncoating of DNADNA

replicationreplicationtranscriptiontranscription

translationtranslationRNARNA

TherapeuticTherapeutic proteinproteinproduced from transgeneproduced from transgene

Schematic representationSchematic representationof of adenoviral vector productionadenoviral vector production

Adenovirus Adenovirus 5 5 vector vector ((egeg. . pJMpJM--17)17)E1E1

Shuttle Shuttle vector vector ((egeg. . pXCJLpXCJL--1)1)

Foreign geneForeign gene293 293 cellscells

∆∆E1E1--recombinant vectorrecombinant vector

Adenoviral vectors of the first generation Adenoviral vectors of the first generation GreatGreat::

Very high transduction efficiencyVery high transduction efficiencyBroad host and cell type rangeBroad host and cell type rangeCanCan bebe prepared in high titersprepared in high titersCan transduce mitotic andCan transduce mitotic and postpost--mitotic cellsmitotic cellsDo notDo not integrate with genomeintegrate with genomeCan harbor Can harbor ~ 7~ 7 kb of transgenekb of transgene

But:But:Strong immune responseStrong immune response toto viral proteins eliminate virally transduced cells withinviral proteins eliminate virally transduced cells within 3030 daysdaysNeutralizing antibody response prevents readministration of adenNeutralizing antibody response prevents readministration of adenovirus vector of theovirus vector of the samesameserotypeserotype

ThusThus:: Adenoviral vectors provide the highAdenoviral vectors provide the high butbut transienttransient (<4(<4 weeksweeks)) transgene transgene

expressionexpression

Adenoviral vectorsAdenoviral vectors E1E1 E3E3

E2E2 E4E4

No No viral protein neosynthesisviral protein neosynthesis, , No No viral replicationviral replication, , HelperHelpervirus contaminants virus contaminants (<0.1%)(<0.1%)

∆∆E1E1 E1 (and E3)E1 (and E3) E1 E1 complementing cellscomplementing cells 7.5 7.5 kbkb Viral protein neosynthesisViral protein neosynthesis, , viral replicationviral replicationdespitedespite lack of E1lack of E1

∆∆E1E4E1E4 E1,E4 (and E3)E1,E4 (and E3) E1 and E4E1 and E4complementing cellscomplementing cells

10 10 kbkb Reduced viral proteinReduced viral proteinneosynthesisneosynthesis, ,

∆∆E1E2E1E2 E1,E2A E1,E2A or or E2BE2B(and E3)(and E3)

E1 and E2E1 and E2complementing cellscomplementing cells

9 9 kbkb Block of Block of viral replicationviral replicationsever inhibitionsever inhibition of of viralviralprotein synthesisprotein synthesis

GutlessGutless All All viralviral genesgenes E1 E1 complementing cellscomplementing cells,,helper virushelper virus

36 36 kbkb

VectorVector DeletionsDeletions ProductionProduction CapacityCapacity FeaturesFeatures

Benihoud K. et al. 1999. Current Opinion in Biotechnology 10: 440.

SchemSchematic comparison atic comparison of of adenovirusadenovirus,, first generation and gutless vectorsfirst generation and gutless vectors

ITRITR E1E1 ITRITR

Wild type virusWild type virus

First generation vectorFirst generation vector

Gutless vectorGutless vector

transgenetransgene ITRITRITRITR

transgenetransgene ITRITRstufferstufferITRITR

capsidcapsid genomegenome

Generation of HDGeneration of HD--Ad viral vectorsAd viral vectors

apoE fragmentsapoE fragments

bluescriptbluescript, 3 , 3 kbkb

++

promoterpromoterpBLDLpBLDL, 3 k, 3 kbb++ polyApolyA

intronintron

pp∆∆28, 28 28, 28 kbkb

HDHD--AdAd

bluescriptbluescript

Sygnal pakowania Sygnal pakowania DNA do DNA do kapsydu

Sygnal pakowania Sygnal pakowania DNA do DNA do kapsydu kapsydukapsydu

WirusWirus pomocniczypomocniczy

loxP loxPloxP loxPmolekularne nozyczkimolekularne nozyczki

LeczniczyLeczniczy gengen

WektorWektor gutlessgutless

KomorkiKomorki

Powielanie DNA w komorkach

Produkuje Produkuje bialka kapsydowbialka kapsydow, ,

NIE NIE pakuje sie pakuje sie do do kapsydowkapsydow

Sygnal pakowania Sygnal pakowania DNA do DNA do kapsydukapsydu

zostal wycietyzostal wyciety PakujePakuje siesie do do gotowych kapsydowgotowych kapsydow

Testing of helper virusTesting of helper virus contaminationcontamination

gutlessgutlessplasmid

506506

10181018

2036203616361636

((bpbp))

SouthernSouthern--blotting with the probes blotting with the probes recognizing ITR sequencesrecognizing ITR sequencesin HDin HD--AdAd and helper virusand helper virus

Viral DNA digested with Viral DNA digested with PvuIPvuI

plasmidhelperhelpervirus

gutlessgutlessvectorvirus vector

Structure of Adenoviral VectorsStructure of Adenoviral Vectors

hGH polyAhGH polyAAI AI IntronIntronHDHD--AdPEPCKAdPEPCKapoEapoE (34.3 kb)(34.3 kb)

⇒⇒

ITR ITR ψψ++ HPRT HPRT IntronIntron (16 kb)(16 kb) C346 (11.1 kb)C346 (11.1 kb) ITRITRPEPCKprPEPCKpr ApoEApoE

⇐⇐

HDHD--AdAdgengenApoEApoE (33.4 kb) (33.4 kb) APOEprAPOEprApoEApoE

((exons and intronsexons and introns))

ApoE polyAApoE polyA

⇐⇐ ⇒⇒

ITRITRITR ITR ψψ++ HPRT HPRT IntronIntron (16 kb(16 kb)) C346 (4.6 kb)C346 (4.6 kb)

LCRLCR

ApolipoproteinApolipoprotein E (E (ApoEApoE)) genegene

44 66 193 869

Scheme of ApoE geneScheme of ApoE gene

ApoCI ApoCI’ ApoCIIApoE

LSE

Scheme of ApoEScheme of ApoE//ApoC gene clusterApoC gene cluster ((chromosomechromosome 19)19)

RoleRole of ApoEof ApoE proteinprotein

ProducedProduced byby hepatocyteshepatocytes (>70%)(>70%) andand byby astrocytesastrocytes,, macrophagesmacrophages,, ovarian ovarian granulosa cellsgranulosa cells,, smooth muscle cells and epithelial cellssmooth muscle cells and epithelial cells

MajorMajor structural component of various mammalian lipoproteinsstructural component of various mammalian lipoproteins:: chylomicron chylomicron remnantsremnants, VLDL, IDL, VLDL, IDL andand HDLHDL

Mediates the cellular uptake of chylomicronsMediates the cellular uptake of chylomicrons, VLDL, IDL, VLDL, IDL andand HDL byHDL byinteraction withinteraction with VLDLVLDL--R, LDLR, LDL--R,R, andand LRPLRP

Participates in the remyelination during peripheral nerve regeneParticipates in the remyelination during peripheral nerve regeneration and in the ration and in the accumulation ofaccumulation of cholesterol for steroidcholesterol for steroid hormone synthesishormone synthesis

Decreased apoE binding activity is associated with Decreased apoE binding activity is associated with typetype IIIIII hypercholesterolemia hypercholesterolemia andand accelerated atherosclerosisaccelerated atherosclerosis

ApoEApoE --//-- micemice

Very highVery high cholesterolcholesterol levellevel (500(500--600 mg/dl600 mg/dl versusversus 5050--100 mg/dl100 mg/dl in wild in wild typetype mice)mice)

Abnormal lipoproteinAbnormal lipoprotein profile:profile:

very high concentration ofvery high concentration of VLDLVLDL and chylomicronsand chylomicrons,, very low leve ofvery low leve of HDLHDL

Spontaneous atherosclerosis evenSpontaneous atherosclerosis even onon the regular chowthe regular chow dietdiet

Morphology of atherosclerotic plaques is relatively similarMorphology of atherosclerotic plaques is relatively similar toto human human lessionslessions..

0

10

20

30

40

Therapeutic effect of ApoETherapeutic effect of ApoE

Treatment of Treatment of ApoEApoE--//-- micemice with ApoEwith ApoE proteinprotein or with ApoE or with ApoE adenoviral vectors leadsadenoviral vectors leads toto normalization of cholestrerol level and normalization of cholestrerol level and lipoprotein profiles and decreases the development of atherosclelipoprotein profiles and decreases the development of atherosclerosisrosis

Overexpression of ApoEOverexpression of ApoE ((or treatment with ApoEor treatment with ApoE protein)protein) inin LDLRLDLR--deficient Watanabe rabbitsdeficient Watanabe rabbits (model for(model for familial hypercholesterolemiafamilial hypercholesterolemia))reduces thereduces the cholesterolcholesterol level and decreases the progression of level and decreases the progression of atherosclerosisatherosclerosis..

SchemeScheme of of experimentexperiment

Gutless Gutless aadenovirusdenovirus

Plasma apoEPlasma apoEPlasma CholesterolPlasma CholesterolLipoprotein ProfileLipoprotein ProfileALTALT andand ASTAST levelslevelsAtherosclerosisAtherosclerosis developmentdevelopment

ApoEApoE--//-- mousemouseFed a Fed a regularregular dietdiet

Cell CultureCell Culture

Expression of mRNA andExpression of mRNA and proteinprotein

Reversal of Reversal of HypercholesterolemiaHypercholesterolemia in in ApoEApoE--//-- MiceMiceby Adenovirusby Adenovirus--Mediated Mediated mApoEmApoE Gene TransferGene Transfer

Weeks after TreatmentWeeks after Treatment

Cho

lest

erol

(mg/

dl)

Cho

lest

erol

(mg/

dl)

0

200

400

600

800

1000

0 1 2 3 4

AdmgApoEAdmgApoEΑΑdCMVmApoEdCMVmApoEAd (Control)Ad (Control)PBS (Control)PBS (Control)

AdPEPCKmApoEAdPEPCKmApoE

** ** ** **

Plasma aspartate aminotransferasePlasma aspartate aminotransferase (AST)(AST)activity after adenovirus injectionactivity after adenovirus injection

Weeks after TreatmentWeeks after Treatment0 1 2 3 4 5 6 7

AST

(IU

/L)

AST

(IU

/L)

0

200

400

600

800

1000

2010

AdAd mVRmVRHDHD--AdAd mVRmVRControlControl

*

*

Plasma alanine aminotransferasePlasma alanine aminotransferase (ALT)(ALT)activity after adenovirus injectionactivity after adenovirus injection

Weeks after TreatmentWeeks after Treatment0 1 2 3 4 5 6 7

AL

T (I

U/L

)A

LT

(IU

/L)

0

100

200

300

400

500

600

AdAd mVRmVRHDHD--AdAd mVRmVRControlControl**

*

Aortas ofAortas of micemice stained with oilstained with oil redred817817 days after injection with apoE gutless vectordays after injection with apoE gutless vector

Aortas andAortas and micemice stained with oilstained with oil redred817817 days after injection with apoE gutless vectordays after injection with apoE gutless vector

controlcontrol treatedtreated

HelperHelper--dependentdependent adenoviral vectorsadenoviral vectors

Very high Very high transductiontransduction efficiencyefficiency

Broad host species and cell type rangeBroad host species and cell type range

Can Can transducetransduce mitotic and postmitotic and post--mitotic cellsmitotic cells

Can harbor ~ 35 kb (!) of Can harbor ~ 35 kb (!) of transgenetransgene

Do not integrate with genomeDo not integrate with genome

Do not produce any viral proteinsDo not produce any viral proteins

Show significantly reduced Show significantly reduced immunogenicityimmunogenicity in vivoin vivo

Drawback:Drawback:Difficult for producing in high titersDifficult for producing in high titers