859. Cats Produce a CTL Response That Reduces Expression of Human Factor IX and Human Factor VIII after Neonatal Gene Transfer and Represent an Important Model for Translation into Humans

Gene therapy has corrected bleeding manifestations of hemophilia in small and large animals. However, immune responses can abrogate an effect. We previously demonstrated that neonatal gene therapy could induce tolerance to human Factor IX (hFIX) in mice and dogs. However, this approach may not be effective in humans, which probably have a more mature immune system at birth. Immune responses can involve antibody or cytotoxic T lymphocyte (CTL) responses. Antibody formation has been the major problem in animal models that have been evaluated to date. However, CTL responses could also preclude long-term correction of disease. Indeed, a recent study in humans achieved therapeutic expression of hFIX after liver-directed AAV-mediated gene therapy, but levels fell at 1 month due to a CTL response against AAV capsid proteins. We recently demonstrated that cats with mucopolysaccaridosis I mount a potent CTL response to |[alpha]|-L-iduronidase after neonatal gene therapy. We reasoned that cats might be an excellent model to test immune responses to coagulation factors after neonatal gene transfer. These studies used retroviral vectors (RV) expressing hFIX or human Factor VIII (hFVIII), which have differences at |[sim]|69 and |[sim]|212 amino acids, respectively, from the feline proteins. These results might predict responses in animals with null mutations, which represent |[sim]|1% and 20% of patients with hemophilia B and A, respectively. Newborn cats were injected IV with 4x109 transducing units (TU)/kg of hAAT- hFIX-WPRE, which expresses hFIX from the human |[alpha]|1-antitrypsin promoter. This vector does not shut off in vivo, as it directed stable expression of feline 4-sulfatase for over 1 year in cats. Cats achieved 118+/-29 ng/ml (2% of normal) of hFIX in plasma at 1 month. Although expression was stable in 3 of 4 cats, one lost expression at 4.5 months, which was associated with very low levels of RV RNA in the liver, suggesting that a CTL response had occurred. Anti- hFIX antibody levels were very low. Other cats were injected IV with 9x108 or 7x109 TU/kg of hAAT-hFVIII-WPRE at 2 to 3 days after birth, which expresses a B domain-deleted hFVIII. The low dose resulted in 31+/-9 ng/ml of hFVIII in plasma (16% of normal), but expression fell at 4.5 months. Two of 4 cats developed anti- hFVIII antibodies in an ELISA-based assay. All cats had extremely low RV RNA levels in liver, suggesting that a CTL response eradicated expressing cells. Cats that received the high dose RV achieved 125+/-54 ng/ml (63% normal). Although none developed anti-hFVIII antibodies, 2 of 3 lost most activity at 6 months. These data suggest that 25% of cats that received the hFIX gene, and 86% of cats that received the hFVIII gene as newborns developed a CTL response. Future experiments will attempt to block this CTL response with CTLA4-Ig, which was effective in MPS I cats that received neonatal gene therapy. The cat may be an invaluable preclincal model to test gene therapy approaches for hemophilia.