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Titolo:
Posttranslational modifications of recombinant myotube-synthesized human factor IX
Autore:
Arruda, VR; Hagstrom, JN; Deitch, J; Heiman-Patterson, T; Camire, RM; Chu, K; Fields, PA; Herzog, RW; Couto, LB; Larson, PJ; High, KA;
Indirizzi:
Childrens Hosp Philadelphia, Div Hematol, Abramson Res Ctr 310A, Philadelphia, PA 19104 USA Childrens Hosp Philadelphia Philadelphia PA USA 19104 lphia, PA 19104 USA Univ Penn, Med Ctr, Dept Pediat & Pathol, Philadelphia, PA 19104 USA Univ Penn Philadelphia PA USA 19104 & Pathol, Philadelphia, PA 19104 USA Allegheny Univ Hlth Sci, Dept Neurol, Philadelphia, PA 19102 USA AlleghenyUniv Hlth Sci Philadelphia PA USA 19102 ladelphia, PA 19102 USA Univ Penn, Inst Human Gene Therapy, Philadelphia, PA 19104 USA Univ Penn Philadelphia PA USA 19104 e Therapy, Philadelphia, PA 19104 USA Avigen, Alameda, CA USA Avigen Alameda CA USAAvigen, Alameda, CA USA
Titolo Testata:
BLOOD
fascicolo: 1, volume: 97, anno: 2001,
pagine: 130 - 138
SICI:
0006-4971(20010101)97:1<130:PMORMH>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
COAGULATION FACTOR-IX; ADENOASSOCIATED VIRUS VECTORS; PREVIOUSLY UNTREATED PATIENTS; LONG-TERM CORRECTION; FRESH-FROZEN PLASMA; HEMOPHILIA-B; FACTOR-VIII; GENE-TRANSFER; TYROSYLPROTEIN SULFOTRANSFERASE; BLOOD-COAGULATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Clinical Medicine
Life Sciences
Citazioni:
60
Recensione:
Indirizzi per estratti:
Indirizzo: High, KA Childrens Hosp Philadelphia, Div Hematol, Abramson Res Ctr 310A, 34th St &Civic Ctr Blvd, Philadelphia, PA 19104 USA Childrens Hosp Philadelphia 34th St & Civic Ctr Blvd Philadelphia PA USA 19104
Citazione:
V.R. Arruda et al., "Posttranslational modifications of recombinant myotube-synthesized human factor IX", BLOOD, 97(1), 2001, pp. 130-138

Abstract

Recent data demonstrate that the introduction into skeletal muscle of an adenoassociated 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 withan AAV vector expressing F.IX was used. F.IX was purified from conditionedmedium 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 gamma -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 gamma -carboxylation of the N-terminal glutamic acid residues,are also similar, but carbohydrate analysis and assessment oftyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but nothalf-life of muscle-synthesized F.IX. (C) 2001 by The American Society of Hematology.

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Documento generato il 21/09/20 alle ore 12:32:31