Structure of rAAV hFIX vector plasmids.

(A) Recombinant AAV plasmid pAV CAGG-FIX consists of CMV-IE enhancer, β-actin promoter, a chicken β-actin/rabbit β globin composite intron (CAGG), 1.6-kb human FIX cDNA (hFIX) and a rabbit β globin polyadenylation signal (hatched box) flanked by the AAV inverted terminal repeats (ITR shown as hairpin loop). (B) pAV MSCV-FIX expression cassette consists of the 5′ murine stem cell leukemia virus (MSCV) long terminal repeat (LTR) together with its splice signals (IVS), hFIX cDNA and a human β globin polyadenylation signal (hatched box) flanked by the AAV ITRs. (C) In pAV CMV-FIX-YFP, the CMV IE enhancer/promoter (CMVe/p) is followed by a synthetic intron that promotes efficient splicing (syn IVS). The promoter controls the expression of a bicistronic transcript encoding the hFIX gene and the enhanced yellow fluorescence protein (EYFP) gene separated by the encephalomyocarditis virus internal ribosomal entry site (ires). Transcription in this cassette is terminated by the bovine growth hormone polyadenylation signal (bGH pA, hatched box). (D) pAV HBPC FIX is identical to rAAV CMV-FIX except that the bicistronic transcript is under the control of the hepatitis B enhancer I and II region (ENI and ENII) and pre-core promoter derived from the hepatitis B viral (subtype ADW) genome. The EcoRI restriction enzyme used to generate the hFIX fragment is shown. The arrow beneath the construct indicates the size of the individual rAAV expression cassettes.