Shedding after in situ rAAV delivery Review of the literature and 8 - - PowerPoint PPT Presentation

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Shedding after in situ rAAV delivery Review of the literature and 8 years of experience in Nantes in large animal models Caroline LE GUINER, PhD PhD EMEA/ICH Workshop on viral/vector vector shedding shedding Caroline LE GUINER, EMEA/ICH

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SLIDE 1

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, PhD PhD INSERM U649 (Philippe MOULLIER) INSERM U649 (Philippe MOULLIER) – – Gene Gene Therapy Therapy Laboratory Laboratory Nantes Nantes -

  • FRANCE

FRANCE EMEA/ICH Workshop on viral/ EMEA/ICH Workshop on viral/vector vector shedding shedding 10/30/07 10/30/07 Rotterdam, NL Rotterdam, NL

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SLIDE 2

Mode of delivery Mode of delivery Intracerebral Intracerebral Intrathecal Intrathecal Subretinal Subretinal Intravitreal Intravitreal Isolated limb infusion Isolated limb infusion Intramuscular Intramuscular Intrathymic Intrathymic Animal Models in Nantes Animal Models in Nantes Spinal Muscular Atrophy Cat Spinal Muscular Atrophy Cat Mucopolysaccharidosis Mucopolysaccharidosis Type I Dog Type I Dog Nonhuman primate Nonhuman primate Spinal Muscular Atrophy Cat Spinal Muscular Atrophy Cat RPE65 deficient Dog RPE65 deficient Dog Normal dog Normal dog Nonhuman primate Nonhuman primate Normal dog Normal dog Nonhuman primate Nonhuman primate Nonhuman primate Nonhuman primate Nonhuman primate Nonhuman primate rAAV rAAV serotype serotype 1, 2, 5 1, 2, 5 1 1 2, 4, 5 2, 4, 5 2 2 1, 8 1, 8 1, 2, 8 1, 2, 8 8 8 Maximal time course Maximal time course 8 months 8 months 3 months 3 months 6 years 6 years 4 months 4 months 2 years 2 years 5 years 5 years 1 month 1 month

slide-3
SLIDE 3

nd nd nd nd

  • N/A

N/A

Gonads Gonads

nd nd nd nd

+ +

( (≥ ≥ month month 3) 3) N/A N/A

Liver Liver

nd nd nd nd pending pending N/A N/A

Spleen Spleen

nd nd nd nd

+ +

( (≥ ≥ month month 3) 3) N/A N/A

Lymph Lymph nodes nodes

nd nd nd nd

+ +

(up to (up to month month 2) 2) N/A N/A

PBMC PBMC

nd nd nd nd nd nd nd nd

Urine Urine

nd nd nd nd

+ +

( (≥ ≥ month month 3) 3)

  • Cerebrospinal

Cerebrospinal Fluid Fluid

nd nd nd nd

+ +

(up to (up to day day 10) 10)

+ +

(up to (up to day day 4) 4)

Serum Serum

+ +

( (≥ ≥ month month 8) 8)

+ +

( (≥ ≥ month month 6 6) ) >100 >100 vg vg/ /cell cell ( (≥ ≥ month month 8) 8) N/A N/A

Target Target organ

  • rgan

Transgene Transgene product product Transgene Transgene mRNA mRNA Vector Vector genome genome Infectious Infectious particles particles

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

+ ? + ?

α α-

  • capsid

capsid

+ ? + ?

α α-

  • transgene

transgene Cellular Cellular response response

+ +

α α-

  • capsid

capsid

+ +

α α-

  • transgene

transgene Humoral Humoral response response nd = not done

slide-4
SLIDE 4

nd nd nd nd pending pending N/A N/A

Gonads Gonads

nd nd nd nd pending pending N/A N/A

Liver Liver

nd nd nd nd pending pending N/A N/A

Spleen Spleen

nd nd nd nd pending pending N/A N/A

Lymph Lymph nodes nodes

nd nd nd nd

+ +

(up to (up to month month 2) 2) N/A N/A

PBMC PBMC

nd nd nd nd nd nd nd nd

Urine Urine

nd nd nd nd

+ +

(up to (up to month month 3) 3)

+ +

(up to (up to day day 3) 3)

Cerebrospinal Cerebrospinal Fluid Fluid

nd nd nd nd

+ +

(up to (up to month month 2) 2) nd nd

Serum Serum

+ +

( (≥ ≥ month month 3) 3) nd nd

+ +

( (≥ ≥ month month 3) 3) N/A N/A

Target Target organ

  • rgan

Transgene Transgene product product Transgene Transgene mRNA mRNA Vector Vector genome genome Infectious Infectious particles particles

Mode of delivery:

Intrathecal

Animal Model:

Cat

rAAV serotype:

1

rAAV dose (total):

4,6x1011vg to 1,4x1012vg

Joussemet et al., Manuscript in preparation

nd nd

α α-

  • capsid

capsid

nd nd

α α-

  • transgene

transgene Cellular Cellular response response

nd nd

α α-

  • capsid

capsid

nd nd

α α-

  • transgene

transgene Humoral Humoral response response nd = not done

slide-5
SLIDE 5

nd nd nd nd

  • N/A

N/A

Gonads Gonads

nd nd nd nd

  • N/A

N/A

Liver Liver

nd nd nd nd nd nd N/A N/A

Spleen Spleen

nd nd nd nd

+ +

( (≥ ≥ month month 3) 3) N/A N/A

Lymph Lymph nodes nodes

nd nd nd nd

+ +

( (≥ ≥ month month 3 3) ) N/A N/A

PBMC PBMC

nd nd nd nd nd nd nd nd

Urine Urine

nd nd nd nd

  • nd

nd

Cerebrospinal Cerebrospinal Fluid Fluid

nd nd nd nd

+ +

(up to (up to day day 14) 14) nd nd

Serum Serum

+ +

( (≥ ≥ y year ear 6) 6) nd nd 44 44 vg vg/ /cell cell ( (≥ ≥ y year ear 1,5) 1,5) N/A N/A

Target Target organ

  • rgan

Transgene Transgene product product Transgene Transgene mRNA mRNA Vector Vector genome genome Infectious Infectious particles particles

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

nd nd

α α-

  • capsid

capsid

nd nd

α α-

  • transgene

transgene Cellular Cellular response response

  • α

α-

  • capsid

capsid

+ +

α α-

  • transgene

transgene Humoral Humoral response response nd = not done

slide-6
SLIDE 6

nd nd nd nd + + (up to (up to month month 3) 3) N/A N/A

Gonads Gonads

nd nd + + ( (≥ ≥ year year 2) 2) + + ( (≥ ≥ year year 2) 2) N/A N/A

Liver Liver

+ + ( (≥ ≥ year year 1) 1) pending pending + + ( (≥ ≥ year year 2 2) ) N/A N/A

Spleen Spleen

nd nd pending pending + + ( (≥ ≥ year year 2) 2) N/A N/A

Lymph Lymph nodes nodes

nd nd pending pending + + ( (≥ ≥ month month 1 1) ) N/A N/A

PBMC PBMC

nd nd nd nd nd nd nd nd

Urine Urine

nd nd nd nd nd nd nd nd

Cerebrospinal Cerebrospinal Fluid Fluid

+ +

( (≥ ≥ year year 2) 2) nd nd

+ +

(up to (up to month month 3) 3)

+ +

(up to (up to day day 7) 7)

Serum Serum

+ +

( (≥ ≥ year year 2) 2)

+ +

( (≥ ≥ year year 2) 2) 1 to 20 1 to 20 vg vg/ /cell cell ( (≥ ≥ year year 2) 2) N/A N/A

Target Target organ

  • rgan

Transgene Transgene product product Transgene Transgene mRNA mRNA Vector Vector genome genome Infectious Infectious particles particles

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

pending pending

α α-

  • capsid

capsid

pending pending

α α-

  • transgene

transgene Cellular Cellular response response

+/ +/-

  • α

α-

  • capsid

capsid

pending pending

α α-

  • transgene

transgene Humoral Humoral response response nd = not done

slide-7
SLIDE 7

nd nd nd nd

+ +

(up to (up to month month 3) 3) N/A N/A

Gonads Gonads

nd nd

+ +

( (≥ ≥ year year 3) 3)

+ +

( (≥ ≥ year year 3) 3) N/A N/A

Liver Liver

+ +

( (≥ ≥ year year 2) 2) pending pending

+ +

( (≥ ≥ year year 3) 3) N/A N/A

Spleen Spleen

nd nd

+ +

( (≥ ≥ day day 20) 20)

+ +

( (≥ ≥ year year 3) 3) N/A N/A

Lymph Lymph nodes nodes

nd nd pending pending

+ +

(up to (up to month month 15) 15) N/A N/A

PBMC PBMC

nd nd nd nd

+ +

(up to (up to day day 6) 6) nd nd

Urine Urine

nd nd nd nd nd nd nd nd

Cerebrospinal Cerebrospinal Fluid Fluid

+ +

( (≥ ≥ y year ear 5 5) ) nd nd

+ +

(up to (up to month month 1,5) 1,5)

+ +

(up to (up to day day 7) 7)

Serum Serum

+ +

( (≥ ≥ year year 5) 5)

+ +

( (≥ ≥ year year 3) 3) 5 to 50 5 to 50 vg vg/ /cell cell ( (≥ ≥ year year 3) 3) N/A N/A

Target Target organ

  • rgan

Transgene Transgene product product Transgene Transgene mRNA mRNA Vector Vector genome genome Infectious Infectious particles particles

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

+ +

α α-

  • capsid

capsid

+ +

α α-

  • transgene

transgene Cellular Cellular response response

+ +

α α-

  • capsid

capsid

+ +

α α-

  • transgene

transgene Humoral Humoral response response nd = not done

slide-8
SLIDE 8

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

slide-9
SLIDE 9

nd nd nd nd nd nd N/A N/A

Gonads Gonads

+ +

( (≥ ≥ month month 1) 1) pending pending 61 61 vg vg/ /cell cell ( (≥ ≥ month month 1) 1) N/A N/A

Liver Liver

+ +

( (≥ ≥ month month 1) 1) pending pending 6 6 vg vg/ /cell cell ( (≥ ≥ month month 1) 1) N/A N/A

Spleen Spleen

  • pending

pending 0,1 0,1 vg vg/ /cell cell ( (≥ ≥ month month 1) 1) N/A N/A

Lymph Lymph nodes nodes

  • pending

pending 0,02 0,02 vg vg/ /cell cell ( (≥ ≥ month month 1) 1) N/A N/A

PBMC PBMC

nd nd nd nd nd nd nd nd

Urine Urine

nd nd nd nd nd nd nd nd

Cerebrospinal Cerebrospinal Fluid Fluid

nd nd nd nd pending pending pending pending

Serum Serum

+ +

( (≥ ≥ day day 10) 10) nd nd

+ +

( (≥ ≥ day day 10) 10) N/A N/A

Target Target organ

  • rgan

Transgene Transgene product product Transgene Transgene mRNA mRNA Vector Vector genome genome Infectious Infectious particles particles

Mode of delivery:

Intrathymic

Animal Model:

Primate

rAAV serotype:

8

rAAV dose (total):

2x1013vg

Moreau et al., Manuscript in preparation

pending pending

α α-

  • capsid

capsid

pending pending

α α-

  • transgene

transgene Cellular Cellular response response

pending pending

α α-

  • capsid

capsid

pending pending

α α-

  • transgene

transgene Humoral Humoral response response nd = not done

slide-10
SLIDE 10
slide-11
SLIDE 11

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 cells GFP Merge + Nuclei

slide-12
SLIDE 12

vg vg

+ + (

(≥ ≥ month month 3) 3)

Target Target organ

  • rgan

α α-

  • capsid

capsid α α-

  • transgene

transgene α α-

  • capsid

capsid α α-

  • transgene

transgene

nd nd nd nd 1,5x10 1,5x108

8 to 4,5x10

to 4,5x1012

12 vg

vg

rAAV rAAV dose dose

Subretinal Subretinal

Mode of Mode of delivery delivery

Dog / Dog / Primate Primate

« Animal » model « Animal » model

2 2

rAAV rAAV serotype serotype

nd nd

Cellular Cellular response response

+ +

Humoral Humoral response response

  • Gonads

Gonads

  • Liver

Liver

  • Spleen

Spleen

+ + (

(≥ ≥ month month 3) 3)

Lymph Lymph nodes nodes

nd nd

PBMC PBMC

nd nd

Urine Urine

nd nd

Cerebrospinal Cerebrospinal Fluid Fluid

nd nd

Serum Serum

+ + (

(≥ ≥ month month 3) 3)

Transgene Transgene expression expression

Jacobson Jacobson et al., et al., HGT, 2006 HGT, 2006 + + Mol.

  • Mol. Ther

Ther., 2006 ., 2006

And in other labs?

slide-13
SLIDE 13

vg vg

nd nd 4,1 4,1 vg vg/ /cell cell ( (≥ ≥ year year 3,7) 3,7)

+ + (

(≥ ≥ month month 8 8) )

Target Target organ

  • rgan

+ + ?

?

+ + ?

?

+ +

  • +

+ (

(≥ ≥ month month 8) 8)

+ + (

(≥ ≥ month month 8 8) )

+ + (

(≥ ≥ month month 8 8) ) nd nd nd nd nd nd nd nd

+ + (

(≥ ≥ month month 8 8) ) 1,2x10 1,2x1012

12 to 6x10

to 6x1012

12 vg

vg 2 2 Dog Dog Intramuscular Intramuscular Monahan Monahan et al., et al., Gene Gene Ther Ther., 1998; ., 1998; Chao Chao et al., et al., Gene Gene Ther Ther., 1999 ., 1999

  • +

+

  • (

(semen semen) ) nd nd nd nd nd nd

+ + (up to

(up to day day 14) 14) nd nd nd nd

+ + (up to

(up to day day 14) 14)

+ + (

(up to up to month month 1) 1) 2,1x10 2,1x1012

12 to 6,9x10

to 6,9x1013

13

vg vg 2 2 Human Human Intramuscular Intramuscular Brantly Brantly et al., et al., HGT, 2006 HGT, 2006

α α-

  • capsid

capsid α α-

  • transgene

transgene α α-

  • capsid

capsid α α-

  • transgene

transgene

nd nd nd nd 1,4x10 1,4x1013

13 to 7x10

to 7x1014

14 vg

vg

rAAV rAAV dose dose

Intramuscular Intramuscular

Mode of Mode of delivery delivery

Human Human

« Animal » model « Animal » model

2 2

rAAV rAAV serotype serotype

nd nd

Cellular Cellular response response

+ +

Humoral Humoral response response

  • (

(semen semen) )

Gonads Gonads

nd nd

Liver Liver

nd nd

Spleen Spleen

nd nd

Lymph Lymph nodes nodes

nd nd

PBMC PBMC

+ + (up to

(up to day day 1) 1)

Urine Urine

nd nd

Cerebrospinal Cerebrospinal Fluid Fluid

+ + (up to

(up to month month 3) 3)

Serum Serum

+ + (

(≥ ≥ year year 3,7) 3,7)

Transgene Transgene expression expression

Kay Kay et al., et al., Nat.

  • Nat. Gen

Gen., 2000; ., 2000; Manno Manno et al., et al., Blood Blood, 2003; , 2003; Jiang Jiang et al., et al., Mol.

  • Mol. Ther

Ther., 2006 ., 2006

slide-14
SLIDE 14

vg vg

+ + (≥

(≥ month month 13 13) )

+ + (up to

(up to month month 2 2) )

+ + (≥

(≥ month month 6 6) )

Transgene Transgene expression expression

nd nd nd nd

+ + + + + + (

(≥ ≥ month month 1) 1) = Target = Target Organ Organ

+ + (

(≥ ≥ month month 1) 1) nd nd nd nd nd nd nd nd

+ + (

(up up to to day day 3) 3) 29 to 62 29 to 62 vg vg/ /cell cell ( (≥ ≥ month month 1) 1) 5x10 5x1012

12

8 8 Primate Primate Intravenous Intravenous Nathwani Nathwani et al., et al., Blood Blood, 2007 , 2007

α α-

  • capsid

capsid α α-

  • transgene

transgene α α-

  • capsid

capsid α α-

  • transgene

transgene

  • nd

nd

  • +

+

5,6x10 5,6x1012

12 to 1,4x10

to 1,4x1014

14 vg

vg 2x10 2x1012

12 to 1x10

to 1x1014

14 vg

vg

rAAV rAAV dose dose

Intraportal Intraportal Intraportal Intraportal

Mode of Mode of delivery delivery

Human Human Primate Primate

« Animal » model « Animal » model

2 2 2, 5, 8 2, 5, 8

rAAV rAAV serotype serotype

+ +

nd nd

Cellular Cellular response response

+ + + +

Humoral Humoral response response

+ + (

(semen semen, , up up to to month month 4) 4) 0,02 0,02 vg vg/ /cell cell ( (≥ ≥ month month 6) 6)

Gonads Gonads

= Target = Target Organ Organ = Target = Target Organ Organ

Liver Liver

nd nd 2 2 vg vg/ /cell cell ( (≥ ≥ month month 6) 6)

Spleen Spleen

nd nd nd nd

Lymph Lymph nodes nodes

+ + (

(up up to to month month 4,5) 4,5) nd nd

PBMC PBMC

+ + (

(up up to to month month 1) 1) nd nd

Urine Urine

nd nd nd nd

Cerebrospinal Cerebrospinal Fluid Fluid

+ + (

(up up to to month month 3) 3) nd nd

Serum Serum

nd nd 16 16 vg vg/ /cell cell ( (≥ ≥ month month 6) 6)

Target Target organ

  • rgan

Manno Manno et al., et al., Nat.

  • Nat. Med

Med., 2006 ., 2006 Davidoff Davidoff et et al al., ., Mol.

  • Mol. Ther

Ther., 2005; ., 2005; Nathwani Nathwani et et al al., ., Blood Blood, 2006; , 2006; Jiang Jiang et et al al., ., Blood Blood, 2006 , 2006

slide-15
SLIDE 15

Conclusions

Regardless of the mode of delivery mode of delivery, the serotype serotype and the dose dose, in situ in 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 α α-

  • capsid

capsid and and α α− −transgene transgene host response as well as the potential host response as well as the potential hepatocarcinogenic hepatocarcinogenic effect of effect of rAAV rAAV. .

  • extra target shedding

extra target shedding

  • presence of infectious

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

  • constant detection of vector genome

constant detection of vector genome at distant sites including the at distant sites including the immune system immune system and the and the liver liver

slide-16
SLIDE 16

Acknowledgment

INSERM U649 - Nantes, FRANCE: Alice Alice Toromanoff Toromanoff David Favre David Favre Pierre Pierre Chenuaud Chenuaud Aur Auré élie Moreau lie Moreau Carine Ciron Carine Ciron B Bé éatrice atrice Joussemet Joussemet Nathalie Nathalie Provost Provost Alexandra Alexandra Mendes Mendes-

  • Madeira

Madeira Knut Knut Stieger Stieger Guyl Guylè ène ne le le Meur Meur Caroline Le Caroline Le Guiner Guiner Fabienne Rolling Fabienne Rolling Philippe Philippe Moullier Moullier VETERINARY SCHOOL VETERINARY SCHOOL -

  • Nantes, FRANCE :

Nantes, FRANCE : Laurence Dubreuil Laurence Dubreuil Jack Jack-

  • Yves

Yves Deschamps Deschamps Fran Franç çoise Roux

  • ise Roux

Marie Marie-

  • Anne Colle

Anne Colle Yan Yan Ch Ché érel rel INSERM U643 - Nantes, FRANCE: Ignacio Ignacio Anegon Anegon UNIVERSITY HOSPITAL - Nantes, FRANCE Guillaume Guillaume Podevin Podevin Sylvie Raoul Sylvie Raoul Michel Weber Michel Weber Genethon Genethon -

  • Evry, FRANCE:

Evry, FRANCE: Sandra Sandra Duqu Duqué é Martine Martine Barkats Barkats Naomi Taylor Naomi Taylor lab lab (IGM) (IGM) -

  • Montpellier, FRANCE:

Montpellier, FRANCE: Rita Vicente Rita Vicente Val Valé érie Zimmermann rie Zimmermann Naomi Taylor Naomi Taylor Pasteur Institute - Paris, FRANCE: Jean Michel Heard Jean Michel Heard