A new potent hFIX plasmid for hemophilia B gene therapy

The purpose of this work was to construct and characterize a new potent hFIX plasmid, p2SV-hFIX, which has two hFIX expression units containing the SV40 promoter/enhancer for hemophilia B gene therapy.

p1SV-hFIX was constructed by insertion of amplified hFIX cDNA at the ECORI and Xbal sites of pSI expression vector containing simian virus 40 (SV40) promoter/enhancer. To construct p2SV-hFIX, the hFIX expression cassette was isolated from p1SV-hFIX by digestion with restriction enzymes, and the purified expression cassette was inserted at the BglII site of another plSV-hFIX. The gene expression of p1SV-hFIX, p2SV-hFIX, and a plasmid containing a liver-specific apoE enhancer and alpha antitrypsin promoter, pAAV-hAAT-hFIX. were evaluated in various cell lines using polyethylenimine (PEI) as a gene carrier in vitro.

The construction of p1SV-hFIX and p2SV-hFIX were confirmed by restriction enzyme studies. The transfection efficiency of p2SV-hFIX was 3.83-fold and 7.16-fold higher than that of pAAV-hAAT-hFIX in C2C12 and NIH3T3 cells, respectively. p2SV-hFIX also showed higher transfection efficiency than p1SV-hFIX in both cells.

In accordance with these results, p2SV-hFIX is a new potent hFIX plasmid that can be transfected in various cells. Systemic delivery of p2SV-hFIX via intravenous or intramuscular injection is feasible for treatment of hemophilia B.