860. Sustained Long_Term In Vivo Expression of Human Factor IX from Keratinocytes Transduced with HIV_Based Vectors

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

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Adenovirus-mediated regulatable Expression of human Factor IX in vitro and in vivo

32nd Hemophilia Symposium Hamburg 2001 ◽

10.1007/978-3-642-18150-4_9 ◽

2003 ◽

pp. 72-80

Author(s):

M. A. Srour ◽

H. Fechner ◽

X. Wang ◽

U. Siemetzki ◽

T. Albert ◽

...

Keyword(s):

Human Factor ◽

Factor Ix ◽

Human Factor Ix

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Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽

10.1182/blood.v87.12.5095.bloodjournal87125095 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5095-5103 ◽

Cited By ~ 114

Author(s):

G Hortelano ◽

A Al-Hendy ◽

FA Ofosu ◽

PL Chang

Keyword(s):

Gene Therapy ◽

Human Factor ◽

Ex Vivo ◽

Cost Effective ◽

Factor Ix ◽

Hemophilia B ◽

Therapy Strategy ◽

Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

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Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽

10.1182/blood.v66.6.1302.1302 ◽

1985 ◽

Vol 66 (6) ◽

pp. 1302-1308 ◽

Cited By ~ 13

Author(s):

W Kisiel ◽

KJ Smith ◽

BA McMullen

Keyword(s):

Human Factor ◽

Coagulation Factor ◽

Factor Ix ◽

Light Chains ◽

Factor X ◽

Amino Terminal ◽

Coagulation Factor Ix ◽

Human Factor Ix ◽

Coagulant Activity

Abstract Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

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Sustained Release of Transgenic Human Factor IX: Preparation, Characterization, and in Vivo Efficacy

Molecular Pharmaceutics ◽

10.1021/mp200133s ◽

2011 ◽

Vol 8 (5) ◽

pp. 1767-1774 ◽

Cited By ~ 5

Author(s):

Li-Chien Chang ◽

Chi-Yu Yang ◽

Anna Chian New Chua ◽

Yi-Juain Lin ◽

Sun-Mou Lai

Keyword(s):

Sustained Release ◽

Human Factor ◽

Factor Ix ◽

In Vivo Efficacy ◽

Human Factor Ix

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Mesenchymal Stromal Cells Gene-Enhanced To Express Human Factor IX and Their In Vivo Use for Correction of Hemophilia B Phenotype in R333Q Mice.

Blood ◽

10.1182/blood.v110.11.5136.5136 ◽

2007 ◽

Vol 110 (11) ◽

pp. 5136-5136

Author(s):

Daniel L. Coutu ◽

Jessica Cuerquis ◽

May Griffith ◽

Mark D. Blostein ◽

Jacques Galipeau

Keyword(s):

Gene Therapy ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Monogenic Disease ◽

Human Factor Ix ◽

B Mice

Abstract Hemophilia B is considered an appropriate disease target for gene therapy because it is a well characterized monogenic disease with a large therapeutic index. Despite promising preclinical and clinical trials in the last decade, safety and efficacy concerns associated with the in vivo administration of viral vectors still need to be addressed before gene therapy becomes part of the standard arsenal for clinicians. Our laboratory has developed a cell therapy approach using gene-enhanced autologous Mesenchymal Stromal Cells (MSCs) to deliver a therapeutic plasmatic protein which addresses these safety concerns. In this study, we tested whether MSCs engineered to express human Factor IX (hFIX) can be used to reverse the bleeding phenotype of R333Q hemophilia B mice developed by Stafford et al. We retrovirally engineered MSCs harvested from normal C57Bl/6 to express hFIX. A gene enhanced polyclonal population of MSCs was capable of producing carboxylated and fully active hFIX by in vitro clotting assays. By ELISA, the cells were shown to produce approximately 250ng of hFIX per million cells per 24h. Ten million of these cells were embedded in a collagen I gel matrix and implanted subcutaneously in R333Q hemophilia B mice (n=10). hFIX activity in mouse plasma (test and control groups) were followed weekly by aPTT assays. hFIX activity reached levels as high as 20% normal activity in some animals with an average +/− SEM of 11.2 +/− 2.1 (FIX activity in controls is <1%). The hFIX activity returned to baseline within 4 weeks. In conclusion, we demonstrate that gene-enhanced autologous MSCs can serve as an effective delivery of functional FIX for temporary correction of the hemophilia B phenotype. We hypothesize the presence of GFP co-expression by the implanted MSCs caused their immune rejection and we are currently testing this hypothesis.

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Pharmacokinetics of Human Factor IX in a dog with Severe Hemophilia B.

10.1055/s-0039-1682760 ◽

1977 ◽

Author(s):

P.A. Gentry ◽

A.R. Thompson ◽

A.W. Forrey

Keyword(s):

Human Factor ◽

Numerical Procedure ◽

Factor Ix ◽

Hemophilia B ◽

High Yield ◽

Severe Hemophilia ◽

Activity Data ◽

Human Factor Ix

In preparing a factor IX concentrate with a high yield and low hepatitis and thromboembolic risks, we have tested this material for survival in an in vivo system, the hemophiliac dog. By following the disappearance of radiolabeled, isolated factor IX in addition to the classic clotting assays, data on protein survival and more accurate kinetic parameters were obtained.Crude factor IX concentrate was prepared by batchwise adsorption-elution with DEAE-Sephadex using cryoprecipitate-poor human plasma. Isolated human factor IX was radiolabeled with 125I by chloramine-T without in vitro loss of clotting activity (Thompson, J Clin Invest, in press, 1977). A preparation containing both crude and isolated factor IX was then subjected to filtration (0.22 μm) and lyophilization; clotting and radioactivity were not altered by these steps.Following infusion of the combined preparation into a dog with severe hemophilia B (0% baseline factor IX) 10 post infusion samples were taken over 96 h for determination of radioactivity and factor IX clotting activity. These data were then analyzed by fitting to a two exponential expression using a Marquart non-linear least squares numerical procedure for a two compartment open model. The central volume was 14.5% of the animal’s body weight; the total volume of distribution was 28% with a t 1/2 distribution of 114 min. The t 1/2 elimination was 20 h; the slower phase of elimination (β, or that affected by redistribution) had a t 1/2 of 40 h. Factor IX clotting activity from the crude concentrate closely paralleled radioactivity from the isolated factor IX throughout the 96 h; t 1/2 β was slightly longer from the clotting activity data.

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