ponder110102

Gene Therapy for Hemophilia: 

Gene Therapy for Hemophilia

Hemophilia:: 

Hemophilia: Affects 1:5,000 males 80% hemophilia A due to Factor VIII deficiency 20% hemophilia B due to Factor IX deficiency Results in spontaneous bleeding, which can be fatal Treated with prophylactic or therapeutic infusion of the deficient factor Correction to 1% of normal activity would reduce spontaneous bleeding Correction to 10% of normal activity would eliminate most spontaneous bleeding

Gene Therapy Approaches for Hemophilia:: 

Gene Therapy Approaches for Hemophilia: Liver-directed Muscle-directed Bone marrow-directed Transplantation of genetically-modified fibroblasts

Ex Vivo vs. In Vivo Transfer into Liver : 

Ex Vivo vs. In Vivo Transfer into Liver Ex Vivo: Remove hepatocytes Modify in culture Reinject In Vivo: Inject vector parenterally

Blood Vessels Have Fenestrations of 100 nm in Diameter: 

Blood Vessels Have Fenestrations of 100 nm in Diameter

Summary of Success with Different Vectors in the Liver: 

Summary of Success with Different Vectors in the Liver AAV (Adenovirus associated virus) Vectors Good expression for hemophilia B Safe Retroviral vectors Good expression for hemophilia A and B Safe Adenoviral vectors Great expression for hemophilia A or B Toxic (caused one death in a human patient with OTC) No stable efficacy in large animals

Adeno-associated virus (AAV) Vectors: 

Adeno-associated virus (AAV) Vectors Small single-stranded DNA (4.5 kb) virus of the parvovirus family Does not require replicating cells for gene transfer

Effect of AAV-mediated Hepatic Gene Therapy in Hemophilia B Dogs : 

Effect of AAV-mediated Hepatic Gene Therapy in Hemophilia B Dogs Inject 1x1012 vector particles per kg into the portal vein of Hemophilia B dogs Mount, Nichols, High, and others; Blood 99:2670, 2002

Retroviral Vectors: 

Retroviral Vectors Single stranded RNA virus that gets copied into DNA that integrates into the chromosome Classes of retroviral vectors: Oncoretroviral vectors require replication for gene transfer Lentiviral vectors do not require replication for gene transfer

Ways for Oncoretroviral Vectors to Transduce Hepatocytes:: 

Ways for Oncoretroviral Vectors to Transduce Hepatocytes: Adults: Inject hepatocyte growth factor (HGF) prior to injection of retroviral vectors Newborns: Replication is already sufficient for gene transfer due to the rapid rate of growth

Oncoretroviral vector Transfer into Adult Mice:: 

Oncoretroviral vector Transfer into Adult Mice: Inject 1x1010 IU/kg of RV

Oncoretroviral Vector Transfer of Canine FIX into Neonatal Mice:: 

Oncoretroviral Vector Transfer of Canine FIX into Neonatal Mice: Inject 1x1010 TU/kg of Retroviral Vector IV

Injection into Neonatal Dogs:: 

Injection into Neonatal Dogs:

Oncoretroviral Vector Transfer into Newborn Hemophilia B Dogs:: 

Oncoretroviral Vector Transfer into Newborn Hemophilia B Dogs:

Summary of Results in Hemophilia B Dogs with Neonatal Gene Therapy: 

Summary of Results in Hemophilia B Dogs with Neonatal Gene Therapy Achieved 10% to 35% of normal antigen; about 30% was functional There was a reduction in bleeding in one dog No antibody responses occurred

Neonatal Gene Transfer may Result in Tolerance to Human FIX in Dogs:: 

Neonatal Gene Transfer may Result in Tolerance to Human FIX in Dogs:

Clinical Trials in Patients:: 

Clinical Trials in Patients: IM injection of AAV for hemophilia B IV injection of RV for hemophilia A Implantation of genetically-modified fibroblasts for hemophilia A Hepatic artery injection of AAV for hemophilia A

IM injection of AAV Vector: 

IM injection of AAV Vector Kay, High in Nature Genetics 24:257, 2000 Inject 10 to 12 sites with 0.5 ml Of AAV vector with CMV promoter (low dose; 2x1011 vg/kg; dogs got 1x1013 vg/kg for 2% of normal) Patients that got higher doses had no evidence of expression

IV injection of RV for hemophilia A: 

IV injection of RV for hemophilia A Based on inconsistent results in animals, in which some animals with high antibody levels had high antigen levels, but no coagulation activity Injected RV IV without a stimulus for hepatocyte replication Activity was less that 1% of normal Trial has been stopped No adverse effects were noted

Transplantation of genetically-modified Fibroblasts: 

Transplantation of genetically-modified Fibroblasts

Implantation of Genetically-modified Fibroblasts: 

Implantation of Genetically-modified Fibroblasts

Implantation of Genetically-modified Fibroblasts: 

Implantation of Genetically-modified Fibroblasts

Hepatic Artery Injection of AAV Vector for Hemophilia B:: 

Hepatic Artery Injection of AAV Vector for Hemophilia B: Inject 1/10 of dose in dogs into the hepatic artery No evidence of expression to date AAV was noted in semen for several months due to contaminating WBC Trial recently resumed with a medium dose, but there are concerns about germline transmission

Conclusions:: 

Conclusions: No gene therapy approaches have evidence of long-term efficacy in patients IM injection of AAV is too inefficient Implantation of fibroblasts is very laborious and not very effective Liver delivery of AAV or RV should work

Remaining Concerns:: 

Remaining Concerns: Insertional mutagenesis or other mechanisms causing cancer Immune response to the transduced cells or a secreted transgene Germline transmission

Acknowledgements:: 

Acknowledgements: Ponder Lab Lingfei Xu Robert Mango Jun Zhang Mark Sands, Washington University Mark Haskins, University of Pennsylvania Tim Nichols, University of North Carolina