X. Ye1, D.W. Stafford2, A.R. Thompson2, C.H. Miao1
1 Puget Sound Blood Center, Dept. of Medicine, University of Washington; 2 Dept. of Biology, University of North Carolina, Chapel Hill, USA

Abstract

Hemophilia B results from a genetic deficiency of blood clotting factor IX (hFIX).
Successful gene therapy would provide a better treatment or even a cure for the patients with hemophilia B. By rapid infusion of a high-expressing hFIX plasmid in large fluid volume into the tail vein of hemophilia B mice, therapeutic-level gene expression (1-5 µg/ml) of hFIX was achieved.
This high-expressing hFIX plasmid contained a hepatic locus control region (HCR) from ApoE gene locus, a human a1-antitrypsin promoter, hFIX cDNA and its truncated first intron, and a bovine growth hormone polyadenylation signal (bpA) (Miao et al. (2000) Mol. Ther. 1, 522-532).
Recently we have also demonstrated that hFIX gene expression was mainly contributed by the episomal plasmid DNA retained in the liver cells after direct plasmid transfer.
Phenotypic correction of treated hemophilia B mice persisted for at least 12 weeks (duration of the experiments).
APTT was shortened to the same value as those of the normal mice, indicating complete correction of the phenotype.
Transient, acute liver toxicity was induced by the rapid infusion technique as shown by elevation of liver enzyme markers, e.g. ALT and AST levels.
However, these high levels dropped back to normal within 3-10 days, suggesting rapid restoration of normal liver function.
No bleeding episode occurred by the treatment procedure in hemophilia B mice. Repeated infusion of hFIX plasmids boosted the hFIX expression to higher levels.
These results provided the evidence that hemophilia B phenotype can be treated by gene transfer of naked hFIX plasmids.
Even if the expression of the episomal plasmids will drop to sub-therapeutic levels, repeated infusions of plasmids can be applied as non-viral gene medicine in the very slowly dividing liver cells to achieve life-long correction of genetic deficiency.