Biodistribution of HC-Ad vectors, gene transfer efficiency, and FVIII mRNA expression analysis.
FVIIIKO mice were injected with 5 × 109 IU Ad-AAT-hFVIII. Semiquantitative PCR (A-B) or RT-PCR (C) using humanFVIII-specific and β-actin–specific primers (n = 3 mice) at an early time point (5 days after injection, T1) in liver, spleen, lung, heart, and kidney (A) and testis, gut, and brain (B). PCR analysis of liver and spleen at a late time point (1 month after injection, T2) is shown in (B). Mice injected with PBS were used as controls (C). For RT-PCR analysis, controls included samples without reverse transcriptase (no RT) to exclude genomic DNA contamination. Molecular weight (MW) marker was the 100-bp ladder. FVIII PCR generated a 0.6-kb fragment, and β-actin PCR generated a 0.2-kb fragment. Standard corresponds to 2-fold serially diluted DNA with a known number of FVIII copies (ranging from 4 to 0 FVIII gene copies per diploid genomic equivalent).
