Time course of vector persistence in exon 17–disrupted hemophiliac mice. Av1H8101 (4 × 10¹⁰ particles/mouse) was administered via tail vein injection to a group of 8 exon 17–disrupted hemophiliac mice. FVIII biological activity was measured before and at 2 and 16 weeks after vector administration. Groups of four mice each were killed at 2 or 16 weeks, and DNA and RNA were isolated from each mouse liver. (A) Southern analysis. Each DNA sample (10 μg) was digested with BamHI. The arrow designates a 3.4-kb fragment containing the vector-derived FVIII sequence. The standards (lanes 1 and 2) were generated by digesting purified Av1H8101 viral DNA in amounts equivalent to 10 and 1 vector copies per cell. Lanes 3 through 6 and 7 through 10 represent liver DNA from mice treated with Av1H8101 or Av3H8101, respectively. No vector was detected in uninjected control mouse liver DNA (data not shown). (B) FVIII protein expression and RNAse protection analysis. Plasma levels of biologically active FVIII protein measured in each mouse are displayed above the lanes. FVIII levels at 2 weeks and 16 weeks are displayed above lanes 4 through 7, and 8 through 11, respectively. For the RNAse protection analysis, 50 μgs of total cellular RNA isolated from the mouse livers were used in each reaction. The arrow labeled FVIII designates the 212-nt human FVIII-specific protected probe fragment. Lane 2 contains undigested full-length probe. Lane 3 contains liver RNA isolated from an uninjected control exon 17–disrupted hemophiliac mouse. Lanes 4 through 7 and 8 through 11 represent RNA from mice at 2 or 16 weeks after vector treatment, respectively. Lanes 1 and 12 contain³²P-labeled DNA molecular-weight markers. The lower panel displays a separate RNAse protection assay using 20 μg of total cellular mouse liver RNA and an antisense RNA probe encoding a portion of the mouse glyceraldehyde 3-phosphodehydrogenase (GAPDH) cDNA. The arrow labeled GAPDH designates the 134-nt mouse GAPDH-specific protected probe fragment.