Analysis of biodistribution and transgene expression following tail and portal vein administration of rAAV vector.
Mock transduced (neg) and rAAV CAGG-FIX transduced C57BL/6 SCID mice were killed 22 weeks after vector administration. Genomic DNA and RNA were isolated from the indicated organs. Genomic DNA (1 μg) was used for PCR amplification, using primers unique to hFIX designed to amplify a 681-bp product. Proviral copy number was deduced from standards, which consisted of serial dilutions of vector DNA (6.3 × 10−4 to 6.3 copies) in 1 μg negative genomic DNA. Integrity of DNA was determined by amplifying a 604-bp region of the murine β-actin gene and is shown at the bottom of each panel. (A) PCR amplification of genomic DNA from mice following tail vein administration of 5 × 1010 rAAV CAGG-FIX vector genomes compared with control; (B) PCR amplification of genomic DNA extracted from mice following portal vein injection of either 1 × 1011 or 5 × 1010 rAAV CAGG-FIX vector genomes compared with control (neg); (C) Expression analysis of hFIX mRNA by RT-PCR following portal vein injection of 1 × 1011 rAAV CAGG-FIX particles or mock transduced mice (neg). RNA samples were amplified with (+) and without (−) RT to exclude genomic DNA amplification.
