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

1. 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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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)

10. 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

11. 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

12. 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

13. 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 ?

14. 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