Functional factor VIII made with von Willebrand factor at high levels in transgenic milk

S. W. Pipe, H. Miao, S. P. Butler, J. Calcaterra, W. H. Velander

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background:Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives:To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods:Transgenic mice were made with a mammary-specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS-PAGE, western blot, and one-stage and two-stage clotting assays. The hemostatic activity of immunoaffinity-enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions:With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single-chain molecule that retained high specific activity, similar to therapeutic-grade FVIII. 226/N6 had >450-fold higher IUmL-1 than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma-derived VWF as therapeutic-grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof-of-principle for the study of expression of 226/N6 and perhaps other single-chain bioengineered rFVIIIs in the milk of transgenic livestock.

Original languageEnglish (US)
Pages (from-to)2235-2242
Number of pages8
JournalJournal of Thrombosis and Haemostasis
Volume9
Issue number11
DOIs
StatePublished - Nov 1 2011

Fingerprint

Factor VIII
von Willebrand Factor
Milk
Hemophilia A
Furin
Transgenic Mice
Cell Culture Techniques
Bioengineering
Livestock
Hemostatics
Human Mammary Glands
Intravenous Infusions
Knockout Mice
Polyacrylamide Gel Electrophoresis
Sheep
Breast
Swine
Therapeutics
Western Blotting
Enzyme-Linked Immunosorbent Assay

Keywords

  • Factor VIII
  • Hemophilia
  • Mammary gland
  • Modified B domain
  • Recombinant protein
  • Transgenic animal
  • Von Willebrand factor

ASJC Scopus subject areas

  • Hematology

Cite this

Functional factor VIII made with von Willebrand factor at high levels in transgenic milk. / Pipe, S. W.; Miao, H.; Butler, S. P.; Calcaterra, J.; Velander, W. H.

In: Journal of Thrombosis and Haemostasis, Vol. 9, No. 11, 01.11.2011, p. 2235-2242.

Research output: Contribution to journalArticle

Pipe, S. W. ; Miao, H. ; Butler, S. P. ; Calcaterra, J. ; Velander, W. H. / Functional factor VIII made with von Willebrand factor at high levels in transgenic milk. In: Journal of Thrombosis and Haemostasis. 2011 ; Vol. 9, No. 11. pp. 2235-2242.
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T1 - Functional factor VIII made with von Willebrand factor at high levels in transgenic milk

AU - Pipe, S. W.

AU - Miao, H.

AU - Butler, S. P.

AU - Calcaterra, J.

AU - Velander, W. H.

PY - 2011/11/1

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N2 - Background:Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives:To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods:Transgenic mice were made with a mammary-specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS-PAGE, western blot, and one-stage and two-stage clotting assays. The hemostatic activity of immunoaffinity-enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions:With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single-chain molecule that retained high specific activity, similar to therapeutic-grade FVIII. 226/N6 had >450-fold higher IUmL-1 than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma-derived VWF as therapeutic-grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof-of-principle for the study of expression of 226/N6 and perhaps other single-chain bioengineered rFVIIIs in the milk of transgenic livestock.

AB - Background:Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives:To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods:Transgenic mice were made with a mammary-specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS-PAGE, western blot, and one-stage and two-stage clotting assays. The hemostatic activity of immunoaffinity-enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions:With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single-chain molecule that retained high specific activity, similar to therapeutic-grade FVIII. 226/N6 had >450-fold higher IUmL-1 than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma-derived VWF as therapeutic-grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof-of-principle for the study of expression of 226/N6 and perhaps other single-chain bioengineered rFVIIIs in the milk of transgenic livestock.

KW - Factor VIII

KW - Hemophilia

KW - Mammary gland

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KW - Recombinant protein

KW - Transgenic animal

KW - Von Willebrand factor

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