Vector design. (A) Skeletal muscle-directed gene transfer: The expression cassette contains the CMV enhancer/promoter, the simian virus 40 (SV40) polyadenylation signal, and is flanked by the AAV-2 inverted terminal repeats (ITRs). The human transgene contains cDNA of exon 1 from the human F.IX gene as WT or variants (K5A or K5A/V10K) interrupted by a 1.4-kb fragment of F.IX intron I, and exons 2 to 8 of the F.IX-WT or variant (R338A). The substitutions introduced in the mature F.IX sequence are indicated. (B) Liver-directed gene transfer: The expression cassette contains the human α1-antitrypsin promoter coupled to the human apolipoprotein E enhancer, exon 1 from the human F.IX gene, a truncated human F.IX intron 1, exons 2 to 8 of the F.IX gene WT or variant (R338A), and the bovine growth hormone polyadenylation signal sequence. The expression cassette is flanked by ITRs derived from AAV type 2.