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Fabienne Rajas-Gilles Mithieux Inserm u.855/Université Lyon 1 October 2 nd, 2010, Milan MRAR Gene therapy correction in the liver-specific GSD1a mouse.

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Presentation on theme: "Fabienne Rajas-Gilles Mithieux Inserm u.855/Université Lyon 1 October 2 nd, 2010, Milan MRAR Gene therapy correction in the liver-specific GSD1a mouse."— Presentation transcript:

1 Fabienne Rajas-Gilles Mithieux Inserm u.855/Université Lyon 1 October 2 nd, 2010, Milan MRAR Gene therapy correction in the liver-specific GSD1a mouse model

2  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 Unique features of liver for gene therapy Introduction Ø : 100 nm

3 Site of many inherited metabolic diseases: 1. Normal liver histology Hemophilias (A, B) Crigler-Najjar type 1 Galactosemia OTC deficiency 2. Abnormal liver histology Type1 tyrosinemia, Alpha-1-antitrypsin deficiency Wilson disease, GSD1a The liver as a target for gene therapy Introduction

4 Inserm U948, Nantes “Liver Biotherapy” In collaboration with T. Nguyen &N. Ferry Gene therapy of inherited metabolic liver diseases using lentiviral and AAV vectors criglernajjar.com 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) Crigler-Najjar type 1 Introduction Lyon

5 Gene therapy of inherited metabolic liver diseases using lentiviral and AAV vectors DNA virus Insert : 2.3 kb Episomal Ø 25 nm Transduce quiescent cells No delay of expression due to the production of a double strand DNA (hairpin) High efficiency of hepatocyte transduction (10-fold of AAV) RNA virus Insert : 9 kb Ø 100 nm (20-30% efficiency of transduction) Integrative Transduce dividing (and non-dividing) cells No delay of expression Stable expression of the transgene Introduction Self complementary AAV (scAAV) Lentivirus

6 Introduction Treatment of adult Gunn rat using designed miR-142-regulated lentiviral vector Schmitt et al., Gastroenterology, 2010 4x miR142-3p (23bp) target sequence  cPPT R U5 WPRE  U3 R mTTR hUGT1A1 6-7 weeks-old Gunn rat (150g) 1.5 x 10 10 hela TU/kg (MOI 2) Gunn Rat  Intraportal injection  High transduction efficacy mTTR-GFP LV 45-55% of transduced hepatocytes

7 Complete and long-term correction of adult Gunn rat using designed miR-142-regulated lentiviral vector Schmitt et al., Gastroenterology, 2010 Introduction

8 Objectives of gene therapy in GSD1a Introduction 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

9 Liver GSD1a and scAAV8-hG6pc Experiments 1.Induction of G6pc deficiency in the liver at 15-20 days of live 2.Retro-orbital injection of scAAV8-TTR-hG6pc, at 7 weeks 3.Follow of blood glucose and metabolic parameters after 6h of fasting ITR mTTR hG6PC ITR 0 0612182430364248 Time of fasting (h) 50 100 150 200 Blood glucose (mg/dl) C57Bl6/J L.g6pc -/- **

10 Blood glucose was maintained > 130 mg/dL for 6 months after gene therapy After 6h of fasting : Time (month) Blood glucose (mg/dL) LG6pc -/- LG6pc +/+ scAAV-G6pc 1,5.10 11 Vg/kg scAAV-G6pc 6.10 11 Vg/kg Loss of the transgene expression 6h of fasting scAAV-GFP 6.10 11 Vg/kg Good control of blood glucose in LG6pc-/- treated with scAAV8-hG6pc Results 0 50 100 150 200 250 123456

11 Partial control of metabolic blood parameters in LG6pc-/- treated with scAAV8-hG6pc Results 0 0.5 1 1.5 +/+-/--/- hG6PC Plasma cholesterol (g/L) * * 0 0.5 1 1.5 2 +/+-/--/- hG6PC Plasma triglyceride (g/L) * Normalization of plasma TG, uric acid, lactic acid for 6 months after gene therapy, except for plasma cholesterol levels TriglyceridesCholesterol

12 Partial control of liver parameters in LG6pc -/- treated with scAAV8-hG6pc 0 20 40 60 80 100 +/+-/--/- hG6PC G6Pase activity (U/g protein) ** 15% -/- hG6pc 60% of steatosis !!! 0 1 2 3 4 +/+-/- -/- hG6PC Liver weight (g) d ** Weight of the liver Results After 6 months of gene therapy

13  Long-term effect of lentiviral treatment ? No hypoglycaemia after 6h of fasting Partial normalization of plasmatic parameters But steatosis was not significantly prevented Blood glucose after 6h of fasting  Is gene therapy efficient against hepatic adenomas development? GDS1 liver Conclusions & Perspectives

14 THANK YOU FOR YOUR ATTENTION 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 Tuan Huy Nguyen Dominique Aubert Nicolas Ferry Nantes For the virus production

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