FIG. 2.
Production of hFIX from human muscle cells transduced with rAAV/Me4βAhFIXm1 or rAAV/CMVhFIXm1 vector. (A, C, E, G, I, K, and M) hFIX production after direct transduction of myotubes (solid triangles); (B, D, F, H, J, L, and N) hFIX production from myotubes generated from transduced myoblasts (open squares). Cells were transduced with rAAV/Me4βAhFIXm1 at a MOI of 1 × 104 (A and B) or rAAV/CMVhFIXm1 at a MOI of 1 × 104 (C to F), 1 × 105 (G to L), and 5 × 105 (M and N). Cells were maintained in differentiation medium (A to D, G and H), F10-based medium (E, F, I, J, M, and N), or SKGM-based medium (K and L). hFIX secreted per day was assayed by ELISA and is shown as mean and standard error of the mean (n = 2 for panels A, B, E, and F; n = 3 for panels C, D, K, L, M, and N). For panels G, H, I, and J, the experiment was started with nine 60-mm dishes, and three dishes were used at each time point as described in the text. For panels F, J, L, and N, myoblasts were differentiated by being transferred to differentiation medium for 3 days p.i., and then maintaining the cells in F10- or SKGM-based rich medium as described in the text. No high or transient expression of hFIX was seen in panels K and L, presumably because a different rAAV batch was used for them, which is different from those used for other experiments.