FIG. 2.

Expression of full-length human dystrophin with two different sets of tri-AAV vectors. (A) Schematic outline of the hybrid-overlapping (HO) tri-AAV vectors. The first (WL30) and the middle (WL34) one-third of the dystrophin coding sequences are split at the junction of exons 26 and 27. A highly recombinogenic alkaline phosphatase gene sequence (AP1) is engineered at the end of the first vector (WL30) and the beginning of the second vector (WL34). Splicing donor (circle) and acceptor (triangle) signals are also engineered in WL30 and WL34, respectively, at the indicated position. Reconstitution of these two vectors can be achieved by either homologous recombination of the AP1 sequence (vertical gray lines) or end-to-end joining of the inverted terminal repeat (ITR). In either case, the open reading frame can be re-established in mRNA after splicing (dotted lines). The recombination between the middle one-third of dystrophin (WL34) and the last one-third of dystrophin (WL37) is mediated by homologous recombination of dystrophin spectrin-like repeat 20 (R20) (vertical gray lines). (B) Schematic outline of the hybrid–hybrid (HH) tri-AAV vectors. The full-length dystrophin expression cassette is carried in three AAV vectors (WL30, WL33, and WL34). The recombination between the first (WL30) and middle (WL33) one-third of dystrophin is identical to that of the HO tri-AAV vectors. A similar approach is used to reconstitute the middle (WL33) and the last (WL35) one-third of dystrophin. The dystrophin coding sequence is split at the junction of exons 48 and 49. A different but also highly recombinogenic region of the AP gene (AP2) and a different set of splicing signals (gray circle and gray triangle) are engineered in WL33 and WL35 to mediate recombination. (C) Representative immunofluorescence staining photomicrographs of serial muscle sections that are stained by two different dystrophin antibodies. The Pan-Dys antibody recognizes dystrophin from any species. The Hum-Dys antibody reacts only with human dystrophin. The two panels at the left show human dystrophin expression in a myofiber (positive for both Pan-Dys and Hum-Dys antibodies). The two panels at the right show expression of mouse dystrophin in a revertant myofiber (positive with the Pan-Dys antibody but negative with the Hum-Dys antibody). *The same myofiber in serial sections. (D) Quantitative evaluation of dystrophin expression in muscles infected by the HO or HH tri-AAV vectors. N=4 for each group.