Liver-restricted expression of the canine factor VIII gene facilitates prevention of inhibitor formation in factor VIII-deficient mice.
BACKGROUND
Gene therapy for hemophilia A with adeno-associated virus (AAV) vectors involves difficulties in the efficient expression of factor VIII (FVIII) and in antibody formation against transgene-derived FVIII.


METHODS
AAV8 vectors carrying the canine B domain deleted FVIII (cFVIII) gene under the control of the ubiquitous beta-actin promoter, the liver-specific human alpha1 anti-trypsin promoter (HAAT) and the liver-specific hepatic control region (HCR) enhancer/human alpha1 anti-trypsin promoter complex (HCRHAAT) were used for the expression of cFVIII in FVIII deficient (fviii(-/-)) mice.


RESULTS
Addition of the hepatic control region enhancer element to the HAAT promoter successfully augmented HAAT promoter activity without loss of liver-specificity in vivo. Using this enhancer/promoter complex, a high cFVIII transgene expression was achieved, resulting in increased blood cFVIII activities to more than 100% of the normal canine FVIII levels in fviii(-/-) mice at a 1 : 10 lower dose of the AAV8 vector carrying the cFVIII gene driven by the HAAT promoter. Under short-term immunosuppression, neutralizing antibodies against cFVIII developed in only one out of six mice when the HAAT promoter was used for cFVIII expression, whereas all the mice developed neutralizing antibodies against cFVIII when the beta-actin promoter was used for cFVIII expression. No neutralizing antibodies against cFVIII developed in fviii(-/-) mice that received the AAV8 vector carrying the cFVIII gene driven by the HCRHAAT enhancer/promoter complex without immunosuppression.


CONCLUSIONS
These data suggest that AAV8 vector-mediated liver-restricted cFVIII gene expression is sufficient for immune hypo-responsiveness to transgene-derived cFVIII in fviii(-/-) mice.
Ishiwata
Akira
A
Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University, School of Medicine, Yakushiji, Shimotsuke, Japan.
Mimuro
Jun
J
Mizukami
Hiroaki
H
Kashiwakura
Yuji
Y
Takano
Katsuhiro
K
Ohmori
Tsukasa
T
Madoiwa
Seiji
S
Ozawa
Keiya
K
Sakata
Yoichi
Y
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000229:Dependovirus / Q000235:genetics; D004285:Dogs; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011401:Promoter Regions, Genetic; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism
D000199:Actins; D000515:alpha 1-Antitrypsin; D005169:Factor VIII