Missense mutations in general are very rarely associated with inhibitor formation in humans receiving protein therapy. was at best ~1% of normal. The hepatic gene transfer protocol showed higher efficacy in animal models and resulted in a phenotypic change from severe to moderate disease in hemophilia B dogs, which has been sustained for 8 years.17 In a clinical trial based on administration of AAV-2 vector to the hepatic artery of patients with severe hemophilia B, a subject with low pre-existing neutralizing antibodies to AAV-2 gained therapeutic levels of F.IX expression Sulfo-NHS-LC-Biotin (11% of normal) Sulfo-NHS-LC-Biotin after treatment with 2 1012 vg/kg.7 Expression was transient and declined to pregene transfer levels by 2 months. Subsequent studies strongly suggested that CD8+ T cells against viral capsid caused transaminitis and elimination of transduced hepatocytes.18,19 No evidence for an immune response against the F.IX transgene product was found Sulfo-NHS-LC-Biotin even in subjects with nonsense mutations.7 Hepatic gene transfer in mice with a gene deletion exhibited induction of immune tolerance to the F.IX transgene product in several strains.20 Hepatic expression induces transgene product-specific regulatory CD4+CD25+FoxP3+ T cells, which suppress humoral and cellular immune responses against the transgene product.21,22,23 The importance of this regulatory T-cell population in maintaining tolerance to the F.IX transgene product has also been demonstrated in nonhuman primates.24 Tolerance to F.IX, established by hepatic gene transfer, is maintained after subsequent supplementary gene transfer to other organs.25 Tolerance induction with this method was highly effective in several, but not all, strains of mice with targeted gene deletion, suggesting that additional genetic factors influence the immune response.20,26,27 Hemophilia B patients display a large variety of F9 mutations. Those subjects who develop inhibitors during traditional protein alternative therapy typically have a gene deletion, early stop codon, or other mutation that results in extensive loss of coding information.28 Past assessments of the effects of the underlying F9 mutation and the route of vector administration on immune responses in gene therapy have relied on comparisons between different strains of mice and dogs, or have addressed only B-cell responses and a single target tissue.29 The high number of variables between experiments, including genetic effects, limited conclusions. This new study for the first time provides a comprehensive assessment of B- and T-cell responses upon liver- or muscle-directed gene transfer in animals with identical genetic background but distinct F9 mutations. Results The objective of this investigation was to compare human F.IX (hF.IX)Cspecific immune responses upon muscle- and liver-directed AAV-2-mediated gene transfer as a function of the underlying genetic F9 defect. C3H/HeJ mice were chosen as a deliberatively provocative model, because mice on this genetic background, unlike C57BL/6 or BALB/c mice, develop antibodies to hF.IX upon hepatic AAV-2-mediated gene transfer.20,30 Mice transgenic for a liver-specific human mini gene were backcrossed from a C57BL/6 CXADR onto a C3H/HeJ background and finally crossed with hemophilia B C3H/HeJ mice that carry a targeted deletion of the endogenous murine gene. We obtained four lines of hemophilia B C3H/HeJ mice with 1% systemic F.IX activity. These included gene deletion mice without additional transgene (Null mutation), mice expressing hF.IX with a late stop codon at amino acid residue 338 (LS; crm? mutation, = 3 male mice per line, data not shown). Open in a separate window Physique 1 Lines of hemophilia B mice. (a) Primary amino acid sequence of hF.IX and locations of F9 mutations expressed in transgenic lines of hemophilia B mice: late stop codon at amino acid residue Sulfo-NHS-LC-Biotin 338 (LS, crm?, gene deletion (Null mutation, = 6). (b,f,j) Mice expressing F9 with late stop codon at amino acid Sulfo-NHS-LC-Biotin residue 338 (LS mice, = 5). (c,g,k) Mice expressing F9 with crm? missense mutation G381E as found in the hemophilia.