slide1 l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Fabienne Rajas-Gilles Mithieux Inserm u.855/Université Lyon 1 PowerPoint Presentation
Download Presentation
Fabienne Rajas-Gilles Mithieux Inserm u.855/Université Lyon 1

Loading in 2 Seconds...

play fullscreen
1 / 14

Fabienne Rajas-Gilles Mithieux Inserm u.855/Université Lyon 1 - PowerPoint PPT Presentation


  • 458 Views
  • Uploaded on

MRAR. Gene therapy correction in the liver-specific GSD1a mouse model. Fabienne Rajas-Gilles Mithieux Inserm u.855/Université Lyon 1. October 2 nd , 2010, Milan. Introduction . Unique features of liver for gene therapy. Ø : 100 nm.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Fabienne Rajas-Gilles Mithieux Inserm u.855/Université Lyon 1' - Gabriel


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

MRAR

Gene therapy correction

in the liver-specific GSD1a mouse model

Fabienne Rajas-Gilles Mithieux

Inserm u.855/Université Lyon 1

October 2nd, 2010, Milan

slide2

Introduction

Unique features of liver for gene therapy

Ø : 100 nm

  • Accessible to both ex vivo and in vivo gene therapy
  • Easily accessible
  • Dual blood supply (the Systemic and Portal)
  • The adult liver is quiescent (<0.01% of hepatocyte mitosis);

Non-integrating vector can give long-term expression

  • The liver regenerates from differentiated cells, the hepatocytes
  • Fenestrated sinusoidal endothelium, no basal lamina

facilitated access of vectors to hepatocytes

slide3

The liver as a target for gene therapy

Introduction

  • Site of many inherited metabolic diseases:
      • Normal liver histology

Hemophilias (A, B)

Crigler-Najjar type 1

Galactosemia

OTC deficiency

      • Abnormal liver histology

Type1 tyrosinemia,

Alpha-1-antitrypsin deficiency

Wilson disease,

GSD1a

slide4

Introduction

Gene therapy of inherited metabolic liver diseases

using lentiviral and AAV vectors

Crigler-Najjar type 1

  • High levels of unconjugated bilirubin in the plasma
  • Absence of bilirubin-uridine 5’-diphosphate-glucuronosyltransferase activity UGT1A1
  • No effective medication, Phototherapy to await Orthotopic Liver Transplantation
  • Animal model available: Gunn rat
  • Partial therapeutic correction (10%): clinical benefits
  • Correction easily assessed (serum bilirubin level)

criglernajjar.com

Inserm U948, Nantes

“Liver Biotherapy”

In collaboration with T. Nguyen &N. Ferry

Lyon

slide5

Introduction

Gene therapy of inherited metabolic liver diseases

using lentiviral and AAV vectors

No delay of expression due to the production of a double strand DNA (hairpin)

High efficiency of hepatocyte transduction (10-fold of AAV)

No delay of expression

Stable expression of the transgene

Self complementary AAV (scAAV)

  • DNA virus
  • Insert : 2.3 kb
  • Episomal
  • Ø25 nm
  • Transduce quiescent cells

Lentivirus

  • RNA virus
  • Insert : 9 kb
  • Ø 100 nm (20-30% efficiency of transduction)
  • Integrative
  • Transduce dividing (and non-dividing) cells
slide6

Introduction

Treatment of adult Gunn rat using designed miR-142-regulated lentiviral vector

mTTR

hUGT1A1

Gunn Rat

cPPT R U5

4x miR142-3p (23bp) target sequence

 Intraportal injection

WPRE

6-7 weeks-old Gunn rat (150g)

1.5 x 1010 helaTU/kg (MOI 2)

U3 R

 High transduction efficacy

45-55% of transduced hepatocytes

mTTR-GFP LV

Schmitt et al., Gastroenterology, 2010

slide7

Introduction

Complete and long-term correction of adult Gunn rat using designed miR-142-regulated lentiviral vector

250

225

200

175

Immune response against the foreign UGT1A1 protein

150

mTTR.hBUGT1

125

100

75

uninjected rat controls

Serum bilirubin (µM)

50

25

0

Immunologic tolerance induced by miR142

therapeutic threshold level

*

mTTR.hBUGT1.miR142T

*

*

*

*

*

*

0

20

40

60

80

100

120

140

160

180

Days post-injection

Schmitt et al., Gastroenterology, 2010

slide8

Introduction

Objectives of gene therapy in GSD1a

Targets are the liver but also the kidney

  • To obtain a control of blood glucose during fasting
  • To decrease glycogen stores and steatosis in the liver (to avoid HCA)
  • To decrease glycogen stores in the kidney

☼ High transduction efficiency

…To obtain more than 25% of G6Pase activity in the liver

☼ Specific expression of G6pc gene in the liver (and in the kidney)

… Define the choice of the promoter

slide9

Experiments

Liver GSD1a and scAAV8-hG6pc

Induction of G6pc deficiency in the liver at 15-20 days of live

Retro-orbital injection of scAAV8-TTR-hG6pc, at 7 weeks

Follow of blood glucose and metabolic parameters after 6h of fasting

mTTR

hG6PC

ITR

ITR

**

C57Bl6/J

**

**

**

200

L.g6pc-/-

150

Blood glucose (mg/dl)

100

50

0

0

6

12

18

24

30

36

42

48

Time of fasting (h)

slide10

Results

Good control of blood glucose in LG6pc-/- treated

with scAAV8-hG6pc

250

6h of fasting

LG6pc+/+

200

150

Blood glucose (mg/dL)

scAAV-G6pc 6.1011Vg/kg

LG6pc-/-

scAAV-G6pc 1,5.1011Vg/kg

100

scAAV-GFP 6.1011Vg/kg

50

0

Loss of the transgene expression

1

2

3

4

5

6

Time (month)

After 6h of fasting :

  • Blood glucose was maintained > 130 mg/dL for 6 months after gene therapy
slide11

Results

Partial control of metabolic blood parameters in LG6pc-/-

treated with scAAV8-hG6pc

2

*

1.5

*

*

1.5

1

Plasma triglyceride (g/L)

1

Plasma cholesterol (g/L)

0.5

0.5

0

0

+/+

-/-

-/- hG6PC

+/+

-/-

-/- hG6PC

Triglycerides

Cholesterol

  • Normalization of plasma TG, uric acid, lactic acid for 6 months after gene therapy, except for plasma cholesterol levels
slide12

Results

Partial control of liver parameters in LG6pc-/- treated

with scAAV8-hG6pc

Weight of the liver

-/- hG6pc

**

4

3

Liver weight (g)

2

d

1

100

60% of steatosis !!!

0

+/+

-/-

-/- hG6PC

80

60

G6Pase activity

(U/g protein)

40

15%

**

20

**

0

+/+

-/-

-/- hG6PC

After 6 months of gene therapy

slide13

Conclusions & Perspectives

  • Is gene therapy efficient against hepatic adenomas development?

GDS1 liver

Blood glucose after 6h of fasting

  • No hypoglycaemia after 6h of fasting
  • Partial normalization of plasmatic parameters
  • But steatosis was not significantly prevented
  • Long-term effect of lentiviral treatment ?
slide14

For the virus production

Tuan Huy Nguyen

Dominique Aubert

Nicolas Ferry

Nantes

Fabienne Rajas

Armelle Penhoat

Valérie Large

Amandine Stein

Elodie Mutel

Aya Abdul-Wahed

Sylvie Casteras

Anne Stefanutti

Isabelle Houberdon

Gilles Mithieux

UMR Inserm u.855/UCBL, Lyon

THANK YOU FOR YOUR ATTENTION