Posttranslational modifications of recombinant myotube-synthesized human factor IX

Recent data demonstrate that the introduction into skeletal muscle of an ad enoassociated viral (AAV) vector expressing blood coagulation factor IX (F. IX) can result in long-term expression of the transgene product and amelior ation of the bleeding diathesis in animals with hemophilia B. These data su ggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. in addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life i n the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating he mophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out, As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium d odecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal se quence analysis, chemical gamma -carboxyglutamyl analysis, carbohydrate ana lysis, assays for tyrosine sulfation, and serine phosphorylation, and for s pecific activity. Results show that myotube-synthesized F.IX has specific a ctivity similar to that of liver-synthesized F.IX. Posttranslational modifi cations critical for specific activity, including removal of the signal seq uence and propeptide, and gamma -carboxylation of the N-terminal glutamic a cid residues,are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX. (C) 2001 by The American Society of H ematology.