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Blood, 1 January 2001, Vol. 97, No. 1, pp. 130-138
GENE THERAPY
Posttranslational modifications of recombinant
myotube-synthesized human factor IX
Valder R. Arruda,
James N. Hagstrom,
Jeffrey Deitch,
Terry Heiman-Patterson,
Rodney M. Camire,
Kirk Chu,
Paul A. Fields,
Roland W. Herzog,
Linda B. Couto,
Peter J. Larson, and
Katherine
A. High
From the Department of Pediatrics and Pathology,
University of Pennsylvania Medical Center; The Children's Hospital of
Philadelphia; the Department of Neurology, Allegheny University of the
Health Sciences; and the Institute for Human Gene Therapy, University
of Pennsylvania, Philadelphia, PA; and Avigen, Alameda, CA.
Recent data demonstrate that the introduction into skeletal muscle
of an adeno-associated viral (AAV) vector expressing blood coagulation
factor IX (F.IX) can result in long-term expression of the transgene
product and amelioration of the bleeding diathesis in animals with
hemophilia B. These data suggest 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 in
the circulation, and the immunogenicity of the protein. Before
initiating a human trial of an AAV-mediated, muscle-directed approach
for treating hemophilia 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 dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal
sequence analysis, chemical  -carboxyglutamyl analysis, carbohydrate
analysis, assays for tyrosine sulfation, and serine phosphorylation,
and for specific activity. Results show that myotube-synthesized F.IX
has specific activity similar to that of liver-synthesized F.IX.
Posttranslational modifications critical for specific activity,
including removal of the signal sequence and propeptide, and
-carboxylation of the N-terminal glutamic acid 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.
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