Shedding after in situ rAAV delivery

Review of the literature and 8 years of experience in Nantes in large animal models

Caroline LE GUINER, Caroline LE GUINER, PhDPhDINSERM U649 (Philippe MOULLIER) INSERM U649 (Philippe MOULLIER) –– GeneGene TherapyTherapy LaboratoryLaboratoryNantes Nantes -- FRANCEFRANCE

EMEA/ICH Workshop on viral/EMEA/ICH Workshop on viral/vectorvector sheddingshedding10/30/0710/30/07

Rotterdam, NLRotterdam, NL

Mode of deliveryMode of delivery

IntracerebralIntracerebral

IntrathecalIntrathecal

SubretinalSubretinal

IntravitrealIntravitreal

Isolated limb infusionIsolated limb infusion

IntramuscularIntramuscular

IntrathymicIntrathymic

Animal Models in NantesAnimal Models in Nantes

Spinal Muscular Atrophy CatSpinal Muscular Atrophy Cat

Mucopolysaccharidosis Mucopolysaccharidosis Type I DogType I Dog

Nonhuman primateNonhuman primate

Spinal Muscular Atrophy CatSpinal Muscular Atrophy Cat

RPE65 deficient DogRPE65 deficient Dog

Normal dogNormal dog

Nonhuman primateNonhuman primate

Normal dogNormal dog

Nonhuman primateNonhuman primate

Nonhuman primateNonhuman primate

Nonhuman primateNonhuman primate

rAAV rAAV serotypeserotype

1, 2, 51, 2, 5

11

2, 4, 52, 4, 5

22

1, 81, 8

1, 2, 81, 2, 8

88

Maximal time courseMaximal time course

8 months8 months

3 months3 months

6 years6 years

4 months4 months

2 years2 years

5 years5 years

1 month1 month

ndndndnd--N/AN/AGonadsGonads

ndndndnd++((≥ ≥ monthmonth 3)3)

N/AN/ALiverLiver

ndndndndpendingpendingN/AN/ASpleenSpleen

ndndndnd++((≥ ≥ monthmonth 3)3)

N/AN/ALymphLymph nodesnodes

ndndndnd++(up to (up to monthmonth 2)2)

N/AN/APBMCPBMC

ndndndndndndndndUrineUrine

ndndndnd++((≥ ≥ monthmonth 3)3)

--CerebrospinalCerebrospinalFluidFluid

ndndndnd++(up to (up to dayday 10)10)

++(up to (up to dayday 4)4)

SerumSerum

++((≥ ≥ monthmonth 8)8)

++((≥ ≥ monthmonth 66))

>100 >100 vgvg//cellcell((≥ ≥ monthmonth 8)8)

N/AN/ATarget Target organorgan

TransgeneTransgeneproductproduct

TransgeneTransgenemRNAmRNA

VectorVectorgenomegenome

InfectiousInfectiousparticlesparticles

Mode of delivery:Intracerebral

Animal Model:Cat, Dog, Primate

rAAV serotype:1, 2, 5

rAAV dose (total):1,4x1010vg to 4,6x1011vg

Ciron et al.,Annals of Neurol., 2006

Ciron et al.,Manuscript in preparation

Joussemet et al.,Manuscript in preparation

+ ?+ ?αα--capsidcapsid

+ ?+ ?αα--transgenetransgeneCellular Cellular responseresponse

++αα--capsidcapsid

++αα--transgenetransgeneHumoral Humoral responseresponse

nd = not done

ndndndndpendingpendingN/AN/AGonadsGonads

ndndndndpendingpendingN/AN/ALiverLiver

ndndndndpendingpendingN/AN/ASpleenSpleen

ndndndndpendingpendingN/AN/ALymphLymph nodesnodes

ndndndnd++(up to(up to monthmonth 2)2)

N/AN/APBMCPBMC

ndndndndndndndndUrineUrine

ndndndnd++(up to(up to monthmonth 3)3)

++(up to (up to dayday 3)3)

CerebrospinalCerebrospinalFluidFluid

ndndndnd++(up to(up to monthmonth 2)2)

ndndSerumSerum

++((≥ ≥ monthmonth 3)3)

ndnd++((≥ ≥ monthmonth 3)3)

N/AN/ATarget Target organorgan

TransgeneTransgeneproductproduct

TransgeneTransgenemRNAmRNA

VectorVectorgenomegenome

InfectiousInfectiousparticlesparticles

Mode of delivery:Intrathecal

Animal Model:Cat

rAAV serotype:1

rAAV dose (total):4,6x1011vg to 1,4x1012vg

Joussemet et al.,Manuscript in preparation

ndndαα--capsidcapsid

ndndαα--transgenetransgeneCellular Cellular responseresponse

ndndαα--capsidcapsid

ndndαα--transgenetransgeneHumoral Humoral responseresponse

nd = not done

ndndndnd--N/AN/AGonadsGonads

ndndndnd--N/AN/ALiverLiver

ndndndndndndN/AN/ASpleenSpleen

ndndndnd++((≥ ≥ monthmonth 3)3)

N/AN/ALymphLymph nodesnodes

ndndndnd++((≥ ≥ monthmonth 33))

N/AN/APBMCPBMC

ndndndndndndndndUrineUrine

ndndndnd--ndndCerebrospinalCerebrospinalFluidFluid

ndndndnd++(up to (up to dayday 14)14)

ndndSerumSerum

++((≥ ≥ yyearear 6)6)

ndnd44 44 vgvg//cellcell((≥ ≥ yyearear 1,5)1,5)

N/AN/ATarget Target organorgan

TransgeneTransgeneproductproduct

TransgeneTransgenemRNAmRNA

VectorVectorgenomegenome

InfectiousInfectiousparticlesparticles

Mode of delivery:Subretinal/Intravitreal

Animal Model:Dog, Primate

rAAV serotype:2, 4, 5

rAAV dose (total):1,6x1010vg to 5x1011vg

Provost et al.,Mol. Ther., 2005 + unpub. data

Stieger et al.,Mol. Ther., 2006

Le Meur et al.,Gene Ther., 2006

ndndαα--capsidcapsid

ndndαα--transgenetransgeneCellular Cellular responseresponse

--αα--capsidcapsid

++αα--transgenetransgeneHumoral Humoral responseresponse

nd = not done

ndndndnd++

(up to (up to monthmonth 3)3)N/AN/AGonadsGonads

ndnd++

((≥≥ yearyear 2)2)++

((≥≥ yearyear 2)2)N/AN/ALiverLiver

++((≥≥ yearyear 1)1)

pendingpending++

((≥≥ yearyear 22))N/AN/ASpleenSpleen

ndndpendingpending++

((≥≥ yearyear 2)2)N/AN/ALymphLymph nodesnodes

ndndpendingpending++

((≥≥ monthmonth 11))N/AN/APBMCPBMC

ndndndndndndndndUrineUrine

ndndndndndndndndCerebrospinalCerebrospinalFluidFluid

++((≥≥ yearyear 2)2)

ndnd++(up to (up to monthmonth 3)3)

++(up to (up to dayday 7)7)

SerumSerum

++((≥≥ yearyear 2)2)

++((≥≥ yearyear 2)2)

1 to 20 1 to 20 vgvg//cellcell((≥≥ yearyear 2)2)

N/AN/ATarget Target organorgan

TransgeneTransgeneproductproduct

TransgeneTransgenemRNAmRNA

VectorVectorgenomegenome

InfectiousInfectiousparticlesparticles

Mode of delivery:Isolated limb infusion

Animal Model:Primate

rAAV serotype:1, 8

rAAV dose (total):6,7x1012vg to 1,2x1013vg

Toromanoff et al.,Manuscript in preparation

pendingpendingαα--capsidcapsid

pendingpendingαα--transgenetransgeneCellular Cellular responseresponse

+/+/--αα--capsidcapsid

pendingpendingαα--transgenetransgeneHumoral Humoral responseresponse

nd = not done

ndndndnd++(up to (up to monthmonth 3)3)

N/AN/AGonadsGonads

ndnd++((≥≥ yearyear 3)3)

++((≥≥ yearyear 3)3)

N/AN/ALiverLiver

++((≥≥ yearyear 2)2)

pendingpending++((≥≥ yearyear 3)3)

N/AN/ASpleenSpleen

ndnd++((≥≥ dayday 20)20)

++((≥≥ yearyear 3)3)

N/AN/ALymphLymph nodesnodes

ndndpendingpending++(up to (up to monthmonth 15)15)

N/AN/APBMCPBMC

ndndndnd++(up to (up to dayday 6)6)

ndndUrineUrine

ndndndndndndndndCerebrospinalCerebrospinalFluidFluid

++((≥≥ yyearear 55))

ndnd++(up to (up to monthmonth 1,5)1,5)

++(up to (up to dayday 7)7)

SerumSerum

++((≥≥ yearyear 5)5)

++((≥≥ yearyear 3)3)

5 to 50 5 to 50 vgvg//cellcell((≥≥ yearyear 3)3)

N/AN/ATarget Target organorgan

TransgeneTransgeneproductproduct

TransgeneTransgenemRNAmRNA

VectorVectorgenomegenome

InfectiousInfectiousparticlesparticles

Mode of delivery:Intramuscular

Animal Model:Primate

rAAV serotype:1, 2, 8

rAAV dose (total):4x1011vg to 1,4x1013vg

Favre et al.,Mol. Ther., 2001

Chenuaud et al.,Unpublished data

Toromanoff et al.,Manuscript in preparation

++αα--capsidcapsid

++αα--transgenetransgeneCellular Cellular responseresponse

++αα--capsidcapsid

++αα--transgenetransgeneHumoral Humoral responseresponse

nd = not done

One single IM injection of rAAV1, rAAV2, rAAV8 in primate results in widespread detection of vector genome in lymph nodes for years

Positive lymph nodes

ndndndndndndN/AN/AGonadsGonads

++((≥ ≥ monthmonth 1)1)

pendingpending61 61 vgvg//cellcell

((≥ ≥ monthmonth 1)1)N/AN/ALiverLiver

++((≥ ≥ monthmonth 1)1)

pendingpending6 6 vgvg//cellcell

((≥ ≥ monthmonth 1)1)N/AN/ASpleenSpleen

--pendingpending0,1 0,1 vgvg//cellcell((≥ ≥ monthmonth 1)1)

N/AN/ALymphLymph nodesnodes

--pendingpending0,02 0,02 vgvg//cellcell((≥ ≥ monthmonth 1)1)

N/AN/APBMCPBMC

ndndndndndndndndUrineUrine

ndndndndndndndndCerebrospinalCerebrospinalFluidFluid

ndndndndpendingpendingpendingpendingSerumSerum

++((≥ ≥ dayday 10)10)

ndnd++((≥ ≥ dayday 10)10)

N/AN/ATarget Target organorgan

TransgeneTransgeneproductproduct

TransgeneTransgenemRNAmRNA

VectorVectorgenomegenome

InfectiousInfectiousparticlesparticles

Mode of delivery:Intrathymic

Animal Model:Primate

rAAV serotype:8

rAAV dose (total):2x1013vg

Moreau et al.,Manuscript in preparation

pendingpendingαα--capsidcapsid

pendingpendingαα--transgenetransgeneCellular Cellular responseresponse

pendingpendingαα--capsidcapsid

pendingpendingαα--transgenetransgeneHumoral Humoral responseresponse

nd = not done

GFP Nuclei Merge

Primate Spleen, Day 30 pi: (Intrathymic injection / rAAV8-PGK-GFP)

Moreau et al., Manuscript in preparation

Primate Liver, Day 30 pi: (Intrathymic injection / rAAV8-PGK-GFP)

Endothelial cellsGFP Merge + Nuclei

vgvg

+ + ((≥ ≥ monthmonth 3)3)Target Target organorgan

αα--capsidcapsid

αα--transgenetransgene

αα--capsidcapsid

αα--transgenetransgene

ndnd

ndnd

1,5x101,5x1088 to 4,5x10to 4,5x101212 vgvgrAAVrAAV dosedose

SubretinalSubretinalMode of Mode of deliverydelivery

Dog / Dog / PrimatePrimate« Animal » model« Animal » model

22rAAVrAAV serotypeserotype

ndnd

Cellular Cellular responseresponse

++Humoral Humoral responseresponse

--GonadsGonads

--LiverLiver

--SpleenSpleen

+ + ((≥ ≥ monthmonth 3)3)LymphLymph nodesnodes

ndndPBMCPBMC

ndndUrineUrine

ndndCerebrospinalCerebrospinal FluidFluid

ndndSerumSerum

+ + ((≥ ≥ monthmonth 3)3)TransgeneTransgene expressionexpression

JacobsonJacobson et al., et al., HGT, 2006HGT, 2006

+ + Mol. Mol. TherTher., 2006., 2006

And in other labs?

vgvg

ndnd4,1 4,1 vgvg//cellcell ((≥ ≥ yearyear 3,7)3,7)++ ((≥ ≥ monthmonth 88))Target Target organorgan

++ ??

++ ??

++----

++ ((≥ ≥ monthmonth 8)8)

++ ((≥ ≥ monthmonth 88))

++ ((≥ ≥ monthmonth 88))

ndnd

ndnd

ndnd

ndnd

++ ((≥ ≥ monthmonth 88))

1,2x101,2x101212 to 6x10to 6x101212 vgvg

22

DogDog

IntramuscularIntramuscular

MonahanMonahan et al., et al., GeneGene TherTher., 1998;., 1998;

Chao Chao et al., et al., GeneGene TherTher., 1999., 1999

----++

---- ((semensemen))

ndnd

ndnd

ndnd

++ (up to (up to dayday 14)14)

ndnd

ndnd

++ (up to (up to dayday 14)14)

+ + ((up to up to monthmonth 1)1)

2,1x102,1x101212 to 6,9x10to 6,9x101313

vgvg

22

HumanHuman

IntramuscularIntramuscular

BrantlyBrantly et al.,et al.,

HGT, 2006HGT, 2006

αα--capsidcapsid

αα--transgenetransgene

αα--capsidcapsid

αα--transgenetransgene

ndnd

ndnd

1,4x101,4x101313 to 7x10to 7x101414 vgvgrAAVrAAV dosedose

IntramuscularIntramuscularMode of Mode of deliverydelivery

HumanHuman« Animal » model« Animal » model

22rAAVrAAV serotypeserotype

ndnd

Cellular Cellular responseresponse

++Humoral Humoral responseresponse

-- ((semensemen))GonadsGonads

ndndLiverLiver

ndndSpleenSpleen

ndndLymphLymph nodesnodes

ndndPBMCPBMC

+ + (up to (up to dayday 1)1)UrineUrine

ndndCerebrospinalCerebrospinal FluidFluid

+ + (up to (up to monthmonth 3)3)SerumSerum

+ + ((≥ ≥ yearyear 3,7)3,7)TransgeneTransgene expressionexpression

Kay Kay et al., et al., Nat. Nat. GenGen., 2000;., 2000;

MannoManno et al., et al., BloodBlood, 2003;, 2003;

Jiang Jiang et al., et al., Mol. Mol. TherTher., 2006., 2006

vgvg

++ (≥ (≥ monthmonth 1313))++ (up to (up to monthmonth 22))++ (≥ (≥ monthmonth 66))TransgeneTransgene expressionexpression

ndnd

ndnd

++

++

++ ((≥ ≥ monthmonth 1)1)

= Target = Target OrganOrgan

++ ((≥ ≥ monthmonth 1)1)

ndnd

ndnd

ndnd

ndnd

++ ((upup to to dayday 3)3)

29 to 62 29 to 62 vgvg//cellcell ((≥ ≥ monthmonth 1)1)

5x105x101212

88

PrimatePrimate

IntravenousIntravenous

NathwaniNathwani et al.,et al.,

BloodBlood, 2007, 2007

αα--capsidcapsid

αα--transgenetransgene

αα--capsidcapsid

αα--transgenetransgene

--ndnd

--++

5,6x105,6x101212 to 1,4x10to 1,4x101414 vgvg2x102x101212 to 1x10to 1x101414 vgvgrAAVrAAV dosedose

IntraportalIntraportalIntraportalIntraportalMode of Mode of deliverydelivery

HumanHumanPrimatePrimate« Animal » model« Animal » model

222, 5, 82, 5, 8rAAVrAAV serotypeserotype

++ndnd

Cellular Cellular responseresponse

++++Humoral Humoral responseresponse

++ ((semensemen, , upup to to monthmonth 4)4)0,02 0,02 vgvg//cellcell ((≥ ≥ monthmonth 6)6)GonadsGonads

= Target = Target OrganOrgan= Target = Target OrganOrganLiverLiver

ndnd2 2 vgvg//cellcell ((≥ ≥ monthmonth 6)6)SpleenSpleen

ndndndndLymphLymph nodesnodes

++ ((upup to to monthmonth 4,5)4,5)ndndPBMCPBMC

++ ((upup to to monthmonth 1)1)ndndUrineUrine

ndndndndCerebrospinalCerebrospinal FluidFluid

++ ((upup to to monthmonth 3)3)ndndSerumSerum

ndnd16 16 vgvg//cellcell ((≥ ≥ monthmonth 6)6)Target Target organorgan

MannoManno et al.,et al.,

Nat. Nat. MedMed., 2006., 2006

DavidoffDavidoff et et alal., ., Mol. Mol. TherTher., 2005;., 2005;

NathwaniNathwani et et alal., ., BloodBlood, 2006;, 2006;

Jiang Jiang et et alal., ., BloodBlood, 2006, 2006

Conclusions

Regardless of the mode of deliverymode of delivery, the serotypeserotype and the dosedose,

in situin situ administration of administration of rAAV rAAV in large animal models and patients is associated with :in large animal models and patients is associated with :

These findings should be discussed in the context of the These findings should be discussed in the context of the αα--capsid capsid and and αα−−transgene transgene

host response as well as the potential host response as well as the potential hepatocarcinogenic hepatocarcinogenic effect of effect of rAAVrAAV. .

-- extra target sheddingextra target shedding

-- presence of infectious presence of infectious rAAV rAAV in the circulation with slow clearancein the circulation with slow clearance (days)(days)

-- constant detection of vector genomeconstant detection of vector genome at distant sites including the at distant sites including the

immune systemimmune system and the and the liverliver

Acknowledgment

INSERM U649 - Nantes, FRANCE:Alice Alice ToromanoffToromanoff

David FavreDavid FavrePierre Pierre ChenuaudChenuaudAurAuréélie Moreaulie MoreauCarine CironCarine Ciron

BBééatrice atrice JoussemetJoussemetNathalie Nathalie ProvostProvost

Alexandra Alexandra MendesMendes--MadeiraMadeiraKnut Knut StiegerStieger

GuylGuylèènene le le MeurMeurCaroline Le Caroline Le GuinerGuinerFabienne RollingFabienne RollingPhilippe Philippe MoullierMoullier

VETERINARY SCHOOL VETERINARY SCHOOL -- Nantes, FRANCE :Nantes, FRANCE :Laurence DubreuilLaurence Dubreuil

JackJack--YvesYves DeschampsDeschampsFranFranççoise Rouxoise Roux

MarieMarie--Anne ColleAnne ColleYanYan ChChéérelrel

INSERM U643 - Nantes, FRANCE:Ignacio Ignacio AnegonAnegon

UNIVERSITY HOSPITAL - Nantes, FRANCEGuillaume Guillaume PodevinPodevin

Sylvie RaoulSylvie RaoulMichel WeberMichel Weber

GenethonGenethon -- Evry, FRANCE:Evry, FRANCE:Sandra Sandra DuquDuquéé

Martine Martine BarkatsBarkatsNaomi Taylor Naomi Taylor lablab (IGM) (IGM) -- Montpellier, FRANCE:Montpellier, FRANCE:

Rita VicenteRita VicenteValValéérie Zimmermannrie Zimmermann

Naomi TaylorNaomi TaylorPasteur Institute - Paris, FRANCE:

Jean Michel HeardJean Michel Heard