Site-specific genomic integration produces therapeutic Factor IX levels in mice.
We used the integrase from phage phiC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to approximately 4 microg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.
Olivares
Eric C
EC
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
Hollis
Roger P
RP
Chalberg
Thomas W
TW
Meuse
Leonard
L
Kay
Mark A
MA
Calos
Michele P
MP
D000818:Animals; D005164:Factor IX / Q000096:biosynthesis* / Q000235:genetics*; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods; D016678:Genome; D007275:Injections, Intravenous; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D005164:Factor IX; D019426:Integrases