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Darrel W. Stafford - One of the best experts on this subject based on the ideXlab platform.
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human Factor IX corrects the bleeding diathesis of mice with hemophilia b
Blood, 1998Co-Authors: Szu Hao Kung, Darrel W. Stafford, J N Hagstrom, Darrell L Cass, Katherine A HighAbstract:Mice with hemophilia B have been engineered using gene targeting techniques. These animals exhibit severe Factor IX deficiency and a clinical phenotype that mirrors the human disease. We have bred the founder animals onto two different strains of mice, C57B1/6 and CD-1, and have sought to determine whether adenoviral vectors expressing human Factor IX could correct the bleeding diathesis of mice with hemophilia B. Initial experiments showed that purified plasma-derived human Factor IX added to murine Factor IX–deficient plasma resulted in complete correction of the activated partial thromboplastin time (aPTT), and that injection of 10 11 particles of an adenoviral vector expressing human Factor IX resulted in normalization of a modified aPTT in mouse plasma. As an additional method of assessing the function of human Factor IX in the murine coagulation system, bleeding times were performed in normal, hemophilic, and adenoviral-treated hemophilic mice. By two different bleeding-time techniques, the treated hemophilic mice gave values identical to normal littermate controls, whereas the untreated hemophilic mice exhibited heavy blood loss and prolonged bleeding. There was a marked difference in antibody formation in the two strains of mice; 100% of the hemophilic CD-1 mice formed antibodies to human Factor IX, but none of the C57B1/6 mice did. These data suggest that the C57B1/6 hemophilic mice will be more useful for gene transfer studies, while the CD-1 hemophilic mice may be of greater utility in studying the development of inhibitors.
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a coagulation Factor IX deficient mouse model for human hemophilia b
Blood, 1997Co-Authors: Nobuyo Maeda, David L Straight, Oliver Smithies, Darrel W. StaffordAbstract:Coagulation Factor IX deficiency causes hemophilia B in humans. We have used gene targeting to develop a coagulation Factor IX-deficient (Factor IX-knockout) mouse strain. Mouse embryonic stem (ES) cells were targeted by a socket-containing vector that replaces the promoter through exon 3 of the Factor IX gene by neoΔHPRT, which is a functional neo gene plus a partially deleted hypoxanthine phosphoribosyl transferase minigene. Chimeric mice generated using these socket-containing ES cells transmitted the targeted Factor IX gene to their female offspring. Male offspring from these females were characterized and shown to exhibit a phenotype similar to hemophilia B. This Factor IX-deficient mouse strain will be useful for studying gene therapy methods and structure-function relationships of recombinant Factor IX proteins in vivo.
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The endothelial cell binding determinant of human Factor IX resides in the gamma-carboxyglutamic acid domain.
Biochemistry, 1992Co-Authors: John R. Toomey, Harold R. Roberts, Kenneth J. Smith, Darrel W. StaffordAbstract:The blood coagulation Factor IX(a) binds specifically to a site on endothelial cells with a K{sub d} of 2.0-3.0 nM. A number of previous studies have attempted to define the region(s) of Factor IX(a) that mediate this interaction. These studies suggested that there are two regions of Factor IX(a), the {gamma}-carboxyglutamic acid (Gla) domain and the epidermal growth Factor like (EGF-like) domains, that mediate high-affinity binding to endothelial cells. Recently, however, the participation of the EGF1 domain has been excluded from the interaction. This indicated that if there was an EGF component of Factor IX contributing to the binding affinity, then it must be in the second EGF-like domain. In order to further evaluate this relationship, the authors performed competitive binding experiments between {sup 125}I plasma Factor IX and a set of sIX chimeric proteins composed of portions of Factor VII and Factor IX. The data suggest that the high-affinity interaction between Factor IX and the endothelial cell binding site is mediated by the Factor IX Gla domain and that the Factor IX EGF domains are not involved in binding specificity.
Randal J Kaufman - One of the best experts on this subject based on the ideXlab platform.
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pace furin can process the vitamin k dependent pro Factor IX precursor within the secretory pathway
Journal of Biological Chemistry, 1993Co-Authors: Louise C Wasley, Alnawaz Rehemtulla, J A Bristol, Randal J KaufmanAbstract:Abstract Factor IX is synthesized as a precursor polypeptide which requires proteolytic cleavage of the propeptide for functional activity. Expression of Factor IX at high levels in Chinese hamster ovary (CHO) cells results in the secretion of a mIXture of proFactor IX and mature Factor IX. We have studied whether the processing of proFactor IX may be mediated by the recently discovered subtilisin-like serine proteases PACE/furin and/or PACE4. Co-transfection of a PACE expression vector with a proFactor IX expression vector resulted in the secretion of fully processed Factor IX. In contrast, co-transfection of a PACE4 expression vector with a proFactor IX expression vector did not increase processing of proFactor IX to the mature form. A Factor IX Arg-to-Thr mutation at the P1 position (residue 39) destroyed the ability for PACE to process proFactor IX. Amino-terminal sequence analysis demonstrated that processing mediated by PACE occurred at the authentic site within proFactor IX. The specificity of proFactor IX processing by PACE was also evaluated by transfection of a vector encoding the serine protease inhibitor alpha 1-antitrypsin. Expression of wild-type alpha 1-antitrypsin, which does not inhibit PACE, did not influence processing of proFactor IX mediated by co-expression of PACE. In contrast, the alpha 1-antitrypsin Pittsburgh mutant, which inhibits PACE, inhibited proFactor IX processing activity mediated by transfected PACE as well as the endogenous CHO cell propeptide processing enzyme. Pulse-chase labeling indicated that PACE processed proFactor IX late within the secretory pathway, although a secreted soluble mutant PACE was also capable of processing proFactor IX in the conditioned medium. The results implicate PACE as a candidate for the enzyme that processes proFactor IX in vivo.
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pace furin can process the vitamin k dependent pro Factor IX precursor within the secretory pathway
Journal of Biological Chemistry, 1993Co-Authors: Louise C Wasley, Alnawaz Rehemtulla, J A Bristol, Randal J KaufmanAbstract:Factor IX is synthesized as a precursor polypeptide which requires proteolytic cleavage of the propeptide for functional activity. Expression of Factor IX at high levels in Chinese hamster ovary (CHO) cells results in the secretion of a mIXture of proFactor IX and mature Factor IX. We have studied whether the processing of proFactor IX may be mediated by the recently discovered subtilisin-like serine proteases PACE/furin and/or PACE4. Co-transfection of a PACE expression vector with a proFactor IX expression vector resulted in the secretion of fully processed Factor IX. In contrast, co-transfection of a PACE4 expression vector with a proFactor IX expression vector did not increase processing of proFactor IX to the mature form. A Factor IX Arg-to-Thr mutation at the P1 position (residue 39) destroyed the ability for PACE to process proFactor IX. Amino-terminal sequence analysis demonstrated that processing mediated by PACE occurred at the authentic site within proFactor IX. The specificity of proFactor IX processing by PACE was also evaluated by transfection of a vector encoding the serine protease inhibitor alpha 1-antitrypsin. Expression of wild-type alpha 1-antitrypsin, which does not inhibit PACE, did not influence processing of proFactor IX mediated by co-expression of PACE. In contrast, the alpha 1-antitrypsin Pittsburgh mutant, which inhibits PACE, inhibited proFactor IX processing activity mediated by transfected PACE as well as the endogenous CHO cell propeptide processing enzyme. Pulse-chase labeling indicated that PACE processed proFactor IX late within the secretory pathway, although a secreted soluble mutant PACE was also capable of processing proFactor IX in the conditioned medium. The results implicate PACE as a candidate for the enzyme that processes proFactor IX in vivo.
Steven J Freedman - One of the best experts on this subject based on the ideXlab platform.
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Identification of the Phospholipid Binding Site in the Vitamin K-dependent Blood Coagulation Protein Factor IX
The Journal of biological chemistry, 1996Co-Authors: Steven J Freedman, James D. Baleja, Margaret Jacobs, Barbara C Furie, Mark D. BlosteinAbstract:Abstract The blood coagulation and regulatory proteins that contain γ-carboxyglutamic acid are a part of a unique class of membrane binding proteins that require calcium for their interaction with cell membranes. Following protein biosynthesis, glutamic acids on these proteins are converted to γ-carboxyglutamic acid (Gla) in a reaction that requires vitamin K as a coFactor. The vitamin K-dependent proteins undergo a conformational transition upon metal ion binding, but only calcium ions mediate protein-phospholipid interaction. To identify the site on Factor IX that is required for phospholipid binding, we have determined the three-dimensional structure of the Factor IX Gla domain bound to magnesium ions by NMR spectroscopy. By comparison of this structure to that of the Gla domain bound to calcium ions, we localize the membrane binding site to a highly ordered structure including residues 1–11 of the Gla domain. In the presence of Ca2+, Factor IX Gla domain peptides that contain the photoactivatable amino acid p-benzoyl-L-phenylalanine at positions 6 or 9 cross-link to phospholipid following irradiation, while peptides lacking this amino acid analog or with this analog at position 46 did not cross-link. These results indicate that the NH2 terminus of the Gla domain, specifically including leucine 6 and phenylalanine 9 in the hydrophobic patch, is the contact surface on Factor IX that interacts with the phospholipid bilayer.
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Structure of the calcium ion-bound gamma-carboxyglutamic acid-rich domain of Factor IX.
Biochemistry, 1995Co-Authors: Steven J Freedman, James D. BalejaAbstract:: We have determined the Ca(II)-bound structure of Factor IX, residues 1-47, by nuclear magnetic resonance (NMR) spectroscopy. The amino-terminal 47 residues include the gamma-carboxyglutamic acid-rich and aromatic amino acid stack domains, and this region is responsible for Ca(II)-dependent phospholipid binding in Factor IX. Protons in the 1-47 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. A total of 851 distance restraints and 57 torsion angle restraints were used to generate 17 final structures by distance geometry and simulated annealing methods. The backbone RMSD to the geometric average is 0.6 +/- 0.1 A. The Ca(II)-bound structure is substantially more ordered with increased helical content compared to the apo-Factor IX (1-47) structure. The global fold is similar to the crystal structure of the Ca(II)-bound Gla domain of prothrombin fragment I from residues 12 to 47 (RMSD approximately 1.3 A), but the backbone conformation differs in the first 11 residues, particularly between residues 3 and 6. The amino-terminal nine Gla residues are oriented to the interior of the protein and suggest an internal Ca(II) binding pocket. The carboxyl-terminal three Gla residues are exposed to solvent. The majority of hydrophobic residues are required to stabilize a globular core in the carboxyl-terminal three-quarters of the molecule. However, a hydrophobic surface patch in the amino-terminal region may represent a phospholipid binding site in Factor IX.
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Membrane binding properties of the Factor IX gamma-carboxyglutamic acid-rich domain prepared by chemical synthesis.
Journal of Biological Chemistry, 1994Co-Authors: Margaret Jacobs, Steven J FreedmanAbstract:Abstract The fully gamma-carboxylated peptides based upon the complete and truncated Gla/aromatic amino acid stack domains of human Factor IX were prepared by solid phase peptide synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. A 47-residue peptide Factor IX-(1-47) and a 42-residue peptide Factor IX-(1-42), both containing 12 residues of L-gamma-carboxyglutamic acid, were purified by high performance liquid chromatography and oxidized to form the disulfide bond. Quantitative gamma-carboxyglutamic acid analysis of Factor IX-(1-47) and Factor IX-(1-42) indicated the presence of 12.1 and 11.2 gamma-carboxyglutamic acid residues/mol of peptide, respectively; no glutamic acid was detected. As monitored by fluorescence quenching, calcium ions induced the prototypical conformational transition in Factor IX-(1-47), but not in Factor IX-(1-42), that is observed with Factor IX. Half-maximal quenching of the intrinsic fluorescence of Factor IX-(1-47) was observed at Ca(II) concentrations of about 50 microM. Factor IX-(1-47) bound to the conformation-specific antibodies, anti-Factor IX:Mg(II) and anti-Factor IX:Ca(II)-specific in the presence of metal ions. Factor IX-(1-47) bound to phospholipid membranes, as monitored by energy transfer from intrinsic fluorophores to dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-phosphatidylethanolamine incorporated into a lipid bilayer composed of phosphatidylserine:phosphatidylcholine. In contrast, Factor IX-(1-42) bound poorly to these same membranes. Factor IX-(1-47) did not inhibit Factor XIa activation of Factor IX but did inhibit the activation of Factor X by Factor IXa bound to Factor VIII in the presence of calcium ions and phospholipid. These results show that phospholipid membrane binding is a property of the Gla/aromatic amino acid stack domain and that the Factor IX-(1-47) peptide, prepared by chemical synthesis, preserves the membrane binding properties and the metal-induced conformational transitions observed in native Factor IX. These results indicate that Factor IX-(1-47) but not Factor IX-(1-42) is a suitable model for structural studies of Factor IX-membrane interaction.
Takashi Morita - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of Mg2+- and Ca2+-bound Gla domain of Factor IX complexed with binding protein.
The Journal of biological chemistry, 2003Co-Authors: Yasuo Shikamoto, Takashi Morita, Zui Fujimoto, Hiroshi MizunoAbstract:Abstract Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a γ-carboxyglutamic acid (Gla) domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla domain and in doing so augment the biological activity of Factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) Factor IX Gla domain (IXGD1–46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1–46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1–46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1–46 and IX-bp, forced IXGD1–46 to rotate 4° relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of Factor IX Gla domain during blood coagulation.
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Magnesium(II) Is a Crucial Constituent of the Blood Coagulation Cascade POTENTIATION OF COAGULANT ACTIVITIES OF Factor IX BY Mg IONS
Journal of Biological Chemistry, 1996Co-Authors: Fujio Sekiya, Misa Yoshida, Toshiko Yamashita, Takashi MoritaAbstract:Abstract We recently showed that not only Ca ions but also Mg ions play a crucial role in stabilizing the native conformation of coagulation Factor IX. We here report that Mg ions at physiological concentrations greatly augment the biological activities of Factor IX. In clotting assays with dialyzed plasma, addition of Mg ions enhanced the apparent coagulant activity of Factor IXa, while that of Factor Xa was scarcely affected. Activation of Factor X by Factor IXa in the presence of Factor VIIIa, phospholipids, and Ca ions was accelerated by Mg ions. It appeared that the cation increased the affinity between Factor IXa and Factor VIIIa, thereby increasing the apparent catalytic efficacy of the enzyme. We also evaluated the effect of Mg ions in the coagulation pathway initiated by tissue Factor and found that activation of Factor IX by Factor VIIa•tissue Factor was accelerated by the cation. Consequently, clotting of normal plasma induced by Factor VIIa•tissue Factor was shortened by the cation, while no such effect was observed in plasma deficient in Factor IX or VIII. These results indicate that the previously unrecognized plasma component, Mg ions, plays crucial roles in blood coagulation and, moreover, that contributions of Factors IX and VIII in the coagulation cascade have been seriously underestimated in previous investigations.
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blood coagulation Factor IX binding protein from the venom of trimeresurus flavoviridis purification and characterization
Journal of Biochemistry, 1995Co-Authors: Hideko Atoda, Fujio Sekiya, Midori Ishikawa, Eiji Yoshihara, Takashi MoritaAbstract:: The coagulation Factor IX/Factor X-binding protein (IX/X-bp) from the venom of Trimeresurus flavoviridis is a heterogeneous two-chain protein, and the structure of each chain is similar to that of the carbohydrate-recognition domain of C-type lectins, such as asialoglycoprotein receptors, pancreatic stone protein, and the Fc epsilon receptor for immunoglobulin E. Analysis of the binding properties of IX/X-bp revealed that it binds to the gamma-carboxyglutamic acid (Gla)-containing domains of Factors IX and X [Atoda, H. et al. (1994) Eur. J. Biochem. 224, 703-708]. In the present study, we isolated another anticoagulant protein that binds to Factor IX but is not to Factor X. This protein, designated IX-bp, inhibited Factor IXa-induced clotting but not Factor Xa-induced clotting, whereas IX/X-bp inhibits both. The concentration of IX-bp for half-maximal binding to solid-phase bovine Factor IX was 0.4 nM whereas IX-bp did not bind to Factor X even at 40 nM. The binding of IX-bp to solid-phase Factor IX was inhibited by the addition of Gla-domain peptide of Factor IX, indicating that IX-bp binds to the Gla-domain region of Factor IX. IX-bp had two Ca(2+)-binding sites with different affinities for Ca2+ ions. At pH 7.5, the apparent Kd values for these sites were 14 and 130 microM, respectively. IX-bp was a two-chain protein (27.5-kDa band before reduction and 16.8- and 15.7-kDa bands after reduction on SDS-PAGE) and it reacted with immunoglobulin G against IX/X-bp. The complete amino acid sequence of IX-bp was determined. The 16.8-kDa chain (A chain) of IX-bp consisted of 129 residues, of which 19 were different from those in the A chain of IX/X-bp (129 residues). The sequence of the 15.7-kDa chain (B chain) was identical to that of the B chain of IX/X-bp (123 residues). We conclude that IX-bp is a protein that is structurally similar to but functionally different from IX/X-bp. The difference of binding specificity between IX-bp and IX/X-bp presumably arises from the sequence differences in the A chains.
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regulation of the tertiary structure and function of coagulation Factor IX by magnesium ii ions
Journal of Biological Chemistry, 1995Co-Authors: Fujio Sekiya, Toshiko Yamashita, Yutaka Komiyama, Takashi MoritaAbstract:The indispensable role of Ca2+ ions in the maintenance of the functional tertiary structures of vitamin K-dependent coagulation Factors has been definitively established but the participation of Mg2+ ions, another alkaline-earth metal that is present abundantly in blood plasma, in such a process is not yet understood. We show here that the Ca2+-stabilized conformation of coagulation Factor IX undergoes a further conformational change upon binding of Mg2+ ions using three independent structural probes. The probes we used were (i) IX/X-bp, a snake venom anticoagulant that recognizes the Gla domains in coagulation Factors IX and X, (ii) conformation-specific polyclonal antibodies against bovine Factor IX, and (iii) monoclonal antibodies against the Gla domain of human Factor IX. The binding of all these probes had an absolute requirement for Ca2+ ions, and Mg2+ ions alone were ineffective. However, when added together with Ca2+ ions, Mg2+ ions at physiological concentrations greatly augmented the binding of these probes to Factor IX; the required concentration of Ca2+ ions was much reduced, and the affinity of each probe for Factor IX was increased even in the presence of an excess of Ca2+ ions. These results suggest the presence of a Mg2+-specific binding site that does not interact with Ca2+ ions in Factor IX. Furthermore, Mg2+ ions potentiated the susceptibility of Factor IX to activation by Factor XIa, concomitant with their effect on the conformation. Similarly, the required Ca2+ concentration was reduced by Mg2+ ions, and the rate of conversion to Factor IXa was increased by Mg2+ ions in the presence of an excess of Ca2+ ions. At a saturating concentration of Ca2+ ions (5 mM), addition of 1 mM Mg2+ reduced the apparent Km value for Factor IX from 0.31 to 0.18 μM, and in the presence of a physiological concentration of Ca2+ ions (1 mM), the reduction in Km by Mg2+ ions was far more striking (from 0.91 to 0.24 μM). The apparent Vmax values were hardly affected by Mg2+ ions. Our present data reveal a hitherto novel physiological role of the Mg2+ ions in plasma. Not only Ca2+ ions but also Mg2+ ions are important regulators of the stabilization of the native conformation of Factor IX as well as of its efficient activation.
Inder M Verma - One of the best experts on this subject based on the ideXlab platform.
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systemic delivery of Factor IX messenger rna for protein replacement therapy
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Suvasini Ramaswamy, Nina Tonnu, Kiyoshi Tachikawa, Pattraranee Limphong, Jerel Vega, Priya Prakash Karmali, Pad Chivukula, Inder M VermaAbstract:Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4–6 h) that remains stable for up to 4–6 d and is therapeutically effective, like the recombinant human Factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA–LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.
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a Factor IX deficient mouse model for hemophilia b gene therapy
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Lili Wang, Monica Zoppe, Tilman M Hackeng, John H Griffin, Inder M VermaAbstract:We have generated a mouse where the clot- ting Factor IX (FIX) gene has been disrupted by homologous recombination. The FIX nullizygous (2y2) mouse was devoid of Factor IX antigen in plasma. Consistent with the bleeding disorder, the Factor IX coagulant activities for wild-type (1y1), heterozygous (1y2), and homozygous (2y2) mice were 92%, 53%, and <5%, respectively, in activated partial thromboplastin time assays. Plasma Factor IX activity in the deficient mice (2y2) was restored by introducing wild-type murine FIX gene via adenoviral vectors. Thus, these Factor IX-deficient mice provide a useful animal model for gene therapy studies of hemophilia B.
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circulating human or canine Factor IX from retrovirally transduced primary myoblasts and established myoblast cell lines grafted into murine skeletal muscle
Somatic Cell and Molecular Genetics, 1992Co-Authors: Mark Roman, J H Axelrod, Yifan Dai, Robert K Naviaux, Theodore Friedmann, Inder M VermaAbstract:We have used retroviral vectors to introduce human or canine Factor IX cDNAs into cultured primary murine and canine myoblasts and into the established murine myoblast cell line C2C12. In all cases, the stably infected cells produced biologically active Factor IX in culture and secreted detectable amounts into the culture medium both before and after differentiation of the cells into myotubes. Myoblasts and differentiated myotubes are therefore capable of performing all the posttranslational modifications of the coagulation Factor required for biological activity. We have grafted the genetically modified myoblasts into skeletal muscles of nude mice and have detected stable levels of circulating human Factor IX for up to two months after grafting. We propose that grafting genetically modified primary myoblasts or established myoblast cell lines into skeletal muscle may represent a useful approach to gene therapy for a variety of genetic diseases, including intrinsic muscle disease and defects in circulating proteins as in the hemophilias.
