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