FIG. 1.

Evaluation of homologous recombination efficiency of dystrophin R7, R8, and R9 in overlapping dual AAV vectors. (A) Schematic outline of the constructs and the overlapping strategy. A 7 kb GFP-fused mini-dystrophin gene is split into two AAV vectors. The upstream vector contains the CMV promoter, the GFP coding sequence, and the 5′-half of the mini-dystrophin gene. The downstream vector contains the 3′-half of the mini-dystrophin gene and the polyadenylation signal. The region shared by both constructs is marked as Rn, which can be R7, R8, R9, or R20. Homologous recombination between the Rn regions of the two vectors reconstitutes the expression of the GFP-fused mini-dystrophin gene. (B) In vitro evaluation of the reconstitution efficiency in MO59K cells. Representative photomicrographs of the bright-field and GFP images are shown in the top and bottom panels, respectively. The dual vectors used in transduction are marked by the homologous region they shared (R7, R8, R9, or R20). Neg ctrl, uninfected negative control. Arrow marks the same cell in the bright-field image and the corresponding GFP fluorescence image. (C) In vivo evaluation of the reconstitution efficiency in mdx4cv mice. Representative photomicrographs of the GFP images from muscles infected by the indicated set of the dual vectors are shown. Neg ctrl, un-infected negative control. (D) Quantitative evaluation of the reconstitution efficiency in mdx4cv mice. N=3–5 mice/group. R7H, R8H, R9H, and R20H refer to the muscles that were injected only with the indicated head vector instead of the paired dual vectors. Neg, the muscle from uninfected mice. *Significantly higher than all other groups. AAV, adeno-associated virus; CMV, cytomegalovirus.