Distribution of AAV vector DNA in murine tissues. (A) Southern blot analysis for determination of double-stranded vector genome copy numbers per diploid genomic equivalent. Total DNA was extracted from livers of C57Bl/6 mice 6 weeks after portal vein (pv) or tail vein (tv) injection with 2 × 1011 hFIX-expressing AAV vector particles of types 1 to 6. Twenty micrograms DNA was digested with a combination of BamHI and XhoI (leaving a 3.1-kb fragment), separated on a 0.8% agarose gel, and hybridized with a vector sequence-specific probe. Lanes labeled 10.0 to 0.00 are vector genome copy number standards, and all other lanes represent samples from individual mice. (B) Genomic DNA was isolated from 6 different tissues of the same mice described in (A), and AAV genomes were detected and quantified by Q-PCR. Tissues were liver (l), heart (h), kidney (k), spleen (s), brain (b), and lung (lu). Shown are relative genome copy numbers (liver samples from mice injected with AAV-6 through the portal vein served as internal standard and were set to 1; see Table 1) per diploid cell (mean ± SD; n = 3), with liver samples highlighted by dark bars.
