162. Neonatal Gene Therapy Induces Tolerance in Dogs but Not Cats
Authors: Lingfei Xu, Patty O'Donnell, Tom O'Malley, Mark E. Haskins, Wan-Ching Chan, Yuli Liu, Katherine P. Ponder
Journal: Molecular Therapy (2004)

Immune responses after gene therapy could include an antibody response that blocks the activity of a blood protein, or a cytotoxic T lymphocyte (CTL) response that destroys transduced cells. Either could reduce the efficacy of gene therapy. We have previously shown that high dose neonatal gene therapy resulted in tolerance to canine Factor IX (cFIX), human FIX (hFIX), and canine β-glucuronidase in mice. However, since large animals may have a more mature immune system at birth, we evaluated immune responses after neonatal gene therapy in dogs and cats. Neonatal normal dogs that were transduced with a medium (3 × 109 transducing units (TU)/kg) or a low (8 × 107 TU/kg) dose of an RV expressing hFIX achieved stable expression of hFIX for over 6 months at 494 +/− 132 ng/ml (10% of normal) and 26 +/− 12 ng/ml (0.5% of normal), respectively. None of the neonatal RV-treated dogs developed anti-hFIX IgG. Further, the low dose group did not develop antibodies after infusion of 10 doses of hFIX (30 IU/kg/dose, given once per week starting at 2 months after birth), and thus were truly tolerant. Similar results were obtained in one hemophilia B dog that was transduced with 3 × 109 TU/kg of RV at birth and achieved 220 ng/ml of hFIX, as he did not develop anti-hFIX antibodies either before or after stimulation. In contrast, normal dogs that did not receive neonatal gene therapy developed high levels of anti-hFIX antibodies in response to an identical regimen of hFIX protein infusion. We conclude that neonatal gene therapy results in tolerance to hFIX in dogs. A similar gene therapy approach was tested in cats with mucopolysaccharidosis I (MPS I). Six MPS I cats received 1 × 109 TU/kg of an RV expressing canine α-L-iduronidase (IDUA) shortly after birth. All achieved detectable IDUA activity in blood within 2 weeks after gene transfer, with average serum levels of 25.2 units (U)/ml. However, serum activity declined to undetectable levels (<0.2 U/ml) by 2 months. This decline was not associated with anti-cIDUA antibodies. None of 5 cats analyzed at 2 months had detectable IDUA activity or RV DNA sequences in the liver, although these were high at 10 days in 1 cat (DNA copy number 0.25 copies/cell). Cats are capable of expressing this RV long-term, as MPS VI cats that received neonatal injection of an otherwise-identical vector expressing the feline N-acetylgalactosamine 4-sulfatase have maintained expression for 4 months. We infer that the cats most likely developed a CTL response to cIDUA after neonatal gene therapy. Interestingly, 3 of 18 MPS I mice that received 1 × 108 TU/kg of the cIDUA-expressing RV at birth probably developed a CTL response, although 0 of 25 MPS I mice that received 1 × 109 TU/kg did so. We conclude that cats develop a potent immune response to cIDUA but dogs do not develop an immune response to hFIX after neonatal gene therapy. It is possible that the intracellular cIDUA is more potent at inducing a CTL response than is the secreted hFIX. Alternatively, cats may have more mature immune systems at birth than do dogs. Further experiments will determine if cats produce immune responses to hFIX after neonatal gene therapy, and will further evaluate the CTL response to cIDUA in cats.