Gene Therapy for Hemophilia

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Gene Therapy for Hemophilia
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Hemophilia

• Affects 15,000 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

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Gene Therapy Approaches for Hemophilia

• Liver-directed
• Muscle-directed
• Bone marrow-directed
• Transplantation of genetically-modified
  fibroblasts

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Ex Vivo vs. In Vivo Transfer into Liver

• Ex Vivo
• Remove hepatocytes
• Modify in culture
• Reinject
• In Vivo
• Inject vector parenterally

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Blood Vessels Have Fenestrations of 100 nm in
Diameter
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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

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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

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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 992670, 2002
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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

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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

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Oncoretroviral vector Transfer into Adult Mice
Inject 1x1010 IU/kg of RV
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Oncoretroviral Vector Transfer of Canine FIX into
Neonatal Mice
Inject 1x1010 TU/kg of Retroviral Vector IV
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Injection into Neonatal Dogs
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Oncoretroviral Vector Transfer into Newborn
Hemophilia B Dogs
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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

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

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IM injection of AAV Vector
Kay, High in Nature Genetics 24257, 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
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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

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Transplantation of genetically-modified
Fibroblasts
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Implantation of Genetically-modified Fibroblasts
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Implantation of Genetically-modified Fibroblasts
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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

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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

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Remaining Concerns

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

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Acknowledgements

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