Status of rAAV genome in rhesus liver after rAAV-mediated gene transfer.

(A) Southern blot analysis of DNA derived from liver biopsy specimens from monkey 2 (M2), monkey 3 (M3), and monkey 4 (M4) at 11, 9, and 6 months after liver-targeted delivery of rAAV CAGG-FIX. Each lane contains 10 μg uncut (UC) DNA or DNA digested with EcoRI, which cuts once within the transgene. The rAAV transgene was not detectable in DNA from the liver of a naive macaque (control). Four different rAAV species were detected in UC genomic DNA from the livers of monkeys 3 and 4, and these were deduced to represent HMW concatamers as well as double-stranded dimer, monomer, and circular forms (arrows). The EcoRI digest indicated that the rAAV transgene persists mainly as head-to-tail tandems with approximately a third maintained in the head-to-head orientation. (B) Schematic representation of the rAAV CAGG-FIX vector showing the position of the probe and the expected fragment size after EcoRI digestion of the vector.