Characterization of AAV vector types 1 to 6 for expression of hfIX. (A) Structure of AAV pseudotyping helper plasmids. All helper constructs contain the AAV-2 rep gene, together with the cap gene of one of the 6 AAV serotypes. Other elements shown are the AAV-2 promoters (p5, p19, and p40), the AAV polyA signal, and the frt sequence upstream of the rep gene. Depicted also is the mutation of the p5 TATA box, from the genuine sequence as present in wild-type AAV-2 plasmid pSM620 (top) to a modified sequence (bottom). As a result, the constructs express lower amounts of large Rep but greater amounts of small Rep proteins than a nonmodified helper plasmid (data not shown). (B) Analysis of vector particle purity. A recombinant AAV-2 genome expressing the hfIX gene was cross-packaged into capsids of AAV serotypes 1 to 6. Then 5 × 10¹⁰ genome-containing particles were subjected to SDS-PAGE silver stain. This revealed 3 protein bands (VP1, VP2, and VP3) in each sample, corresponding to the proteins of the particular AAV capsid (as a reference, capsid proteins of AAV-2 are marked on the left side). Notable is the absence of other protein bands demonstrating the high purity of the preparations. The fact that ratios of capsid proteins to genome copy numbers were constant for all AAV types was also evident. Note that the capsid proteins of the 6 AAVs differ slightly in their gel migration pattern, though the corresponding cap genes have nearly identical lengths. (C) Analysis of hFIX expression in cell culture. HepG2 cells were infected with the 6 different vector preparations, and hFIX protein levels in the media were quantified as detailed in “Materials and methods.” ▪ indicates AAV-1; ▪, AAV-2; ▴, AAV-3; ×, AAV-4; ♦, AAV-5; and •, AAV-6. Values shown are mean ± SD (n = 3).