Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity.

Self-complementary adeno-associated viral (scAAV) vectors bypass the requirement for viral second-strand DNA synthesis, but the packaging capacity of these vectors (∼2.4 kb) is significantly smaller than that of conventional AAV vectors (∼4.8 kb). We constructed human recombinant green fluorescent protein (hrGFP) expression cassettes ranging from 2.3 to 4.1 kb. Each vector was biologically active, but the transduction efficiency of vectors containing <3.3-kb genomes was significantly higher than those containing 3.5-kb genomes or larger. However, scAAV vectors containing up to ∼3.3-kb genomes also contained single-stranded genomes, and 3.5-kb and larger genomes were packaged only as single-stranded DNA. These data suggest that the maximum packaging capacity of scAAV vectors is ∼3.3 kb. The production of single-stranded genomes was not due to repair of the terminal resolution site (trs) in the inverted terminal repeats in the AAV genome, but rather was partly due to the use of AAV helper plasmid, known to ...