Molecular analysis in rhesus macaques. (A) Southern blot analysis of DNA (10 μg) derived from liver biopsy specimens from monkeys M1-sc and M4-sc 1 month after liver-targeted delivery of 1 × 10¹² vg/kg scAAV2/8-LP1-hFIXco and scAAV2/5-LP1-hFIXco, respectively, or at 1 and 6 months after administration of 4 × 10¹¹ vg/kg scAAV2/8-LP1-hFIXco in M3-sc. After digestion with BsrDI, the released 1486-bp fragment was probed with an LP1-specific probe. (B) Limited biodistribution analysis. Genomic DNA (top panel) isolated at 1 month from the indicated organs following liver-targeted administration of either 1 × 10¹² vg/kg or 4 × 10¹¹ vg/kg scAAV2/8-LP1-hFIXco into monkeys M1-sc or M3-sc, respectively, was subjected to PCR amplification using primers unique to hFIXco designed to amplify a 617-bp product. Integrity of DNA was determined by amplifying a 604-bp region of the rhesus β-actin gene and is shown at the bottom of this panel. (C) RT-PCR analysis of RNA extracted from the indicated organs following mesenteric-vein injection of either 1 × 10¹² vg/kg or 4 × 10¹¹ vg/kg scAAV2/8-LP1-hFIXco into monkeys M1-sc and M3-sc, respectively. RNA samples were amplified with (+) and without (–) RT to exclude genomic DNA amplification. Integrity of the RNA was determined by amplifying a 295-bp region of the rhesus GAPDH gene and is shown at the bottom of the panel. (D) Persistence of the scAAV transgene was assessed by subjecting genomic DNA (1 μg) isolated at 1 or 6 months from the indicated organs following liver-targeted administration of 4 × 10¹¹ vg/kg scAAV2/8-LP1-hFIXco in monkey M3-sc to PCR using primers unique to hFIXco designed to amplify a 617-bp product. Integrity of DNA was determined by amplifying a 604-bp region of the rhesus β-actin gene and is shown at the bottom of the panel.