Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver

Hiroyuki Nakai, Roland W. Herzog, J. Nathan Hagstrom, Johannes Walter, Szu Hao Kung, Edmund Y. Yang, Shing Jen Tai, Yuichi Iwaki, Gary J. Kurtzman, Krishna J. Fisher, Peter Colosi, Linda B. Couto, Katherine A. High

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Abstract

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 x 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 x 1011 particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.

Original languageEnglish (US)
Pages (from-to)4600-4607
Number of pages8
JournalBlood
Volume91
Issue number12
StatePublished - 1998
Externally publishedYes

Fingerprint

Gene transfer
Factor IX
Dependovirus
Liver
Viruses
Genes
Hepatocytes
Plasmas
Portal Vein
Fluorescent Antibody Technique
Human engineering
Staining and Labeling
Cytomegalovirus
Transgenes
Inbred C57BL Mouse
Polymerase chain reaction
Polymerase Chain Reaction
Injections
DNA

ASJC Scopus subject areas

  • Hematology

Cite this

Nakai, H., Herzog, R. W., Hagstrom, J. N., Walter, J., Kung, S. H., Yang, E. Y., ... High, K. A. (1998). Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver. Blood, 91(12), 4600-4607.

Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver. / Nakai, Hiroyuki; Herzog, Roland W.; Hagstrom, J. Nathan; Walter, Johannes; Kung, Szu Hao; Yang, Edmund Y.; Tai, Shing Jen; Iwaki, Yuichi; Kurtzman, Gary J.; Fisher, Krishna J.; Colosi, Peter; Couto, Linda B.; High, Katherine A.

In: Blood, Vol. 91, No. 12, 1998, p. 4600-4607.

Research output: Contribution to journalArticle

Nakai, H, Herzog, RW, Hagstrom, JN, Walter, J, Kung, SH, Yang, EY, Tai, SJ, Iwaki, Y, Kurtzman, GJ, Fisher, KJ, Colosi, P, Couto, LB & High, KA 1998, 'Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver', Blood, vol. 91, no. 12, pp. 4600-4607.
Nakai H, Herzog RW, Hagstrom JN, Walter J, Kung SH, Yang EY et al. Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver. Blood. 1998;91(12):4600-4607.
Nakai, Hiroyuki ; Herzog, Roland W. ; Hagstrom, J. Nathan ; Walter, Johannes ; Kung, Szu Hao ; Yang, Edmund Y. ; Tai, Shing Jen ; Iwaki, Yuichi ; Kurtzman, Gary J. ; Fisher, Krishna J. ; Colosi, Peter ; Couto, Linda B. ; High, Katherine A. / Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver. In: Blood. 1998 ; Vol. 91, No. 12. pp. 4600-4607.
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abstract = "Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25{\%} of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 x 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 x 1011 particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.",
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AU - Couto, Linda B.

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