Southern blot analysis of gDNA isolated from injected human skeletal muscle tissue.
(A) Diagram of AAV vector containing the CMV IE enhancer/promoter, P (CMV), exon 1 and a 1.4-kb portion of intron 1 the human F.IX (F9) gene (intron I), exons 2-8 of the human F.IX cDNA (F.IX cDNA) including 0.2-kb of the 3′-untranslated region, and the SV40 polyadenylation signal (SV40). The expression cassette is flanked by AAV-2 ITRs. (B-D) Total gDNA was isolated from biopsied muscle tissue and restricted with BglII to release a vector-specific 0.7-kb fragment (CMV IE enhancer/promoter), or restricted with EcoRI, which cuts once in the middle of the vector resulting in a 4.5-kb fragment (unit length of the vector) for vector sequences present as concatemers or monomeric circles. Alternatively, gDNA was restricted with ClaI (which does not cut in the vector genome) or with BamHI, which cuts once within the vector and thus allowing a distinction between head-to-tail (4.5-kb) and head-to-head/tail-to-tail (8-kb and 1-kb, respectively) concatemeric arrangement. Plasmid standards (10-1000 pg/lane) encoding the AAV vector genome were cut with BglII for estimation of gene copy number. gDNA (15 μg, restricted or undigested) and pDNA were separated on 1% agarose gels, Southern blotted onto a nylon membrane, and probed with a 32P-random prime-labeled 0.7-kb BglII fragment representing the CMV enhancer/promoter. Sizes of bands were estimated by comparison with a size marker (1-kb ladder; Gibco BRL). Indicated are high-molecular-weight (HMW), putative head-to-head (h:h) and head-to-tail (h:t) fragments (note that a tail-to-tail fragment is not recognized by the probe) and circular monomeric forms (°). Southern blot analyses are shown for muscle biopsy of subjects C and D (panel B, 2 months after vector administration), subjects D and F (panel C, 10 and 2 months after vector administration, respectively), and subjects F, G, and H (panel D, 6, 2, and 2 months after vector administration, respectively).
