Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Abstract Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

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Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽

10.1182/blood.v73.2.438.bloodjournal732438 ◽

1989 ◽

Vol 73 (2) ◽

pp. 438-445

Author(s):

TD Palmer ◽

AR Thompson ◽

AD Miller

Keyword(s):

Human Factor ◽

Normal Skin ◽

Human Fibroblasts ◽

Retroviral Vectors ◽

Factor Ix ◽

Skin Fibroblasts ◽

Hemophilia B ◽

Clotting Factor ◽

Human Factor Ix

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

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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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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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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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Development of Monospecific Antibodies to Human Factor IX

10.1055/s-0039-1680660 ◽

1977 ◽

Author(s):

Cheryl Y. Tiarks ◽

Chin-Hai Chang ◽

Liberto Pechet

Keyword(s):

Neutralizing Antibodies ◽

Human Factor ◽

Human Albumin ◽

Factor Ix ◽

Hemophilia B ◽

Red Cross ◽

Factor X ◽

Normal Plasma ◽

Human Factor Ix ◽

Monospecific Antibodies

The purpose of this research was to develop neutralizing and precipitating antibodies to factor IX. Human factor IX, purified by the method of Rosenberg et.al. (J. Biol. Chem. 250:8883, 1975), was electrophoresed on acrylamide gel. Two major bands migrating adjacently were eluted. They contained factor IX activity only. The eluates and their homogenized gel segments 7 and 8 were injected separately into two rabbits, Rl and R2, respectively. On immunodiffusion the antiserum Rl showed one precipitating line with normal plasma. It neutralized human factor IX (20 Bethesda units) and also slightly neutralized factor X. It had no effect on factors II and VII. Following absorption of this antiserum with purified factor X it neutralized factor IX only. With continuous immunization, however, this antiserum revealed two new precipitating contaminants. The antiserum R2 neutralized only factor IX; it reached 220 Bethesda inhibitory units. On immunodiffusion it showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original eluates 7 and 8, and a Hemophilia-B antigen-positive plasma. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative Hemophilia-B plasma. We conclude that the antisera Rl and R2 contain factor IX neutralizing antibodies and that albumin-absorbed R2 has monospecific precipitating antibodies to human non-activated factor IX.

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210. Neonatal Gene Therapy with a Human Factor IX-Expressing Retroviral Vector Prevents Antibody Responses in Dogs and Hemophilia B Mice

Molecular Therapy ◽

10.1016/s1525-0016(16)40652-0 ◽

2003 ◽

Vol 7 (5) ◽

pp. S83

Keyword(s):

Gene Therapy ◽

Human Factor ◽

Retroviral Vector ◽

Factor Ix ◽

Antibody Responses ◽

Hemophilia B ◽

Human Factor Ix ◽

B Mice

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Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX

Gene Therapy ◽

10.1038/sj.gt.3302638 ◽

2005 ◽

Vol 13 (2) ◽

pp. 117-126 ◽

Cited By ~ 38

Author(s):

B W Bigger ◽

E K Siapati ◽

A Mistry ◽

S N Waddington ◽

M S Nivsarkar ◽

...

Keyword(s):

Stem Cell ◽

Gene Delivery ◽

Mouse Model ◽

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Human Factor Ix ◽

Cell Gene ◽

Stem Cell Gene

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Use of a BamHI polymorphism in the factor IX gene for the determination of hemophilia B carrier status

Blood ◽

10.1182/blood.v67.5.1508.1508 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1508-1511 ◽

Cited By ~ 36

Author(s):

CW Hay ◽

KA Robertson ◽

SL Yong ◽

AR Thompson ◽

GH Growe ◽

...

Keyword(s):

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Carrier Status ◽

X Chromosomes ◽

Human Factor Ix ◽

History Of

Abstract A BamHI polymorphism has been identified in the human factor IX gene. This polymorphism, which occurs in approximately 6% of X chromosomes, has been used to determine the carrier status of a female in a family with a history of hemophilia B. This family was uninformative for the previously reported TaqI and Xmnl polymorphisms in the factor IX gene.

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