Molecular configuration of scAAV genome in murine liver. (A) Southern analysis of modified Hirt DNA extracted from the liver of a subset of mice at day 1 or 42. Approximately 15 μg DNA from each time point was electrophoresed uncut (uncut), or following digestion with single cutters (PstI) or plasmid safe DNase (PS-D). Shown are bands representing monomeric supercoiled circular (MSC) or dimeric supercoiled circular (DSC), ds-linear (L-ds), or high-molecular-weight concatamers (HMWCs) in the head-tail (HT) and head-to-head (HH) formation. For comparison, 5 × 10⁶ and 2 × 10⁷ purified vector particles were loaded directly on the gel after denaturation (last 2 lanes). Bottom panel is a schematic representation of dimeric scAAV-LP1-hFIXco in the HT (top) and HH (bottom) configuration together with the sites of cleavage by PstI. (B) Southern blot analysis of liver DNA isolated before and after partial hepatectomy at 16 and 20 weeks, respectively, after tail-vein administration of 1 × 10¹⁰ scAAV2/8-LP1-hFIXco vector particles into 129/sv HB mice (HBM1 and 2). Each lane contains 10 μg DNA digested with EcoRI and PstI, which releases a 1.1-kb fragment. Proviral copy number (shown at the bottom) was deduced from standards, which consisted of serial dilutions of vector DNA (0.13 to 13 copies) in 10 μg negative genomic DNA.