12. Self-Complementary AAV2 Vectors Transduce Liver with the Same Efficiency as AAV8: The Critical Role of Second-Strand Synthesis in AAV Biology

Abstract

Top of pageAbstract Previous studies have shown that conventional single-stranded (ss) AAV-2 vectors only transduce a limited number of liver cells, which restricts the use of AAV-mediated liver gene transfer. Recently, we demonstrated that self-complementary (sc) AAV vector significantly increased liver transduction by overcoming the rate-limiting step of second-strand synthesis. Additional studies have shown that cross-packaging of rAAV-2 genome into specific serotype AAV capsids (e.g. AAV6 and AAV8) also increase liver transduction. Although the exact mechanism is not totally clear, the increased transduction by specific serotype AAVs over AAV2 might be related to differences in virus binding, intracellular trafficking, nuclear entry, uncoating or all of the above. In this study, we demonstrate that scAAV genomes packaged into serotypes other than type 2 are also superior to their single-strand counterparts in liver transduction. In addition, we extend these studies to determine if serotype scAAVs show the same hierarchy in transgene expression as that of ssAAV counterparts. 1|[times]|1011 ss vs sc vector genomes carrying liver-specific expression cassette of human factor IX (hFIX) were packaged into serotype 2, 6 and 8 and injected via portal vein into the livers of C57BL/6 mice. Expression of hFIX was monitored over a time course lasting eight weeks. The results showed that among ss vectors, AAV8 gave the strongest expression, followed by AAV6, while AAV2 gave much lower expression at all time points. For each serotype, higher expression of hFIX protein was achieved by sc vector over its ss counterpart. However, the increase of expression by scAAV6 or scAAV8 was not as significant as that of scAAV2 (3 fold vs 50 fold respectively). Interestingly, both ss and scAAV2 exhibited a unique kinetics of hFIX expression, which was distinctly different from AAV6 and 8. hFIX expression by AAV2 increased slowly during eight weeks post injection, while AAV6 and AAV8 reached their peaks rapidly. As a result, at two weeks post injection, scAAV8 and scAAV6 expressed more efficiently than scAAV2, however, scAAV2 exceeded scAAV6 after 4 weeks and was equal to scAAV8 by 8 weeks. Based on these results, the total number of AAV2 virus that enter hepatocyte nuclei is not less than that of AAV8 or AAV6 even though there is a lag phase for expression from AAV2 virions. The unique expression profile of AAV2 might reflect a step post viral binding to cell surface that is inefficient. Our results clearly demonstrate that scAAV-2 will perform equivalent to type 8 in mouse liver. Unlike AAV serotype 8, which has been reported to have the property of wide spread dissemination to other organs, the well-characterized AAV2 vector carrying sc genomes may still be among the most promising candidate vectors for liver-directed gene therapy.