Southern blots of liver DNA from FVIII KO mice treated with various AAV serotypes. One mouse per serotype treated at a dose of 1 × 1011 gc/vector/mouse, injected either intraportally or via tail vein, was killed at 4 months after injection. A total of 12 μg gDNA was used for Southern blotting. Untreated FVIII KO mouse liver DNA either unspiked or spiked with 20, 10, or 1 copy of canine FVIII-AAV was used as control. Liver DNA from mice treated with the 2 vectors (heavy and light chains of FVIII) was digested with XhoI to determine the molecular status of the vector genome and XhoI plus NotI to determine the copy numbers. Similarly liver DNA from the single-vector treatment was digested with AscI for molecular status and AscI plus KpnI for copy numbers. Panels A and C (Southern blot: canine FVIII-heavy chain and Southern blot: canine FVIII-light chain, respectively) represent mice treated intraportally with either AAVs 8, 7, 5, or 2, administered in 2 vectors. The blot in panel A was probed with a 700-bp heavy chain probe (B, canine FVIII heavy chain in AAV vector) encoding the A1 domain of the canine FVIII cDNA. A single digest with XhoI in the blot in panel A revealed 2 different molecular configurations, present in equal ratio, 4-kb head-to-tail molecules and 5.9-kb tail-to-tail molecules for all serotypes. The copy numbers were 9, 7, 1, and 5 copies/cell for AAVs 8, 7, 5, and 2. The blot in panel C was probed with 1-kb light-chain probe (D, canine FVIII light chain in AAV vector), encoding the A3 domain. The copy numbers for the blot in panel C were 9, 8, 0.2, and 5 for AAVs 8, 7, 5, and 2. Also, an equal numbers of head-to-tail (3.9 kb) and tail-to-tail (5.6 kb) concatamers were present. The blot in panel E (Southern blot: canine FVIII-single chain) represents mice treated both intraportally and via tail vein with AAV8 and AAV2 single-chain vectors. The blot in panel E was probed with 1.2-kb single-chain probe (F, canine FVIII single chain in AAV vector), which spans the IGBP promoter and the 5′ end of the FVIII transgene. The copy numbers were 8, 5, 6, and 0.5 for AAV8 intraportal, AAV8 tail vein, AAV2 intraportal, and AAV2 tail vein injections, respectively. DNA digested with AscI revealed 5.6-kb head-to-tail concatemers.
