Optimized adeno-associated virus (AAV)-protein phosphatase-5 helper viruses for efficient liver transduction by single-stranded AAV vectors: Therapeutic expression of factor IX at reduced vector doses

Our studies have shown that coinjection of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors carrying the enhanced green fluorescent protein (EGFP) gene with self-complementary (sc) AAV2-T cell protein tyrosine phosphatase (TC-PTP) and scAAV2-protein phosphatase-5 (PP5) vectors resulted in an ∼16-fold increase in EGFP expression in primary murine hepatocytes in vivo [Jayandharan, G.R., Zhong, L., Li, B., Kachniarz, B., and Srivastava, A. (2008). Gene Ther. 15, 1287-1293]. In the present studies, this strategy was further optimized to achieve transgene expression at reduced vector/helper virus doses. These included the use of scAAV helper viruses containing (1) hepatocyte-specific promoters, (2) tyrosine-mutant AAV2 capsids, and (3) additional AAV serotype vectors known to efficiently transduce hepatocytes. The hepatocyte-specific transthyretin (TTR) promoter was ∼6-to 7-fold more efficient than the Rous sarcoma virus (RSV) promoter; tyrosine-mutant AAV2 capsids were ∼6-to 11-fold more efficient than the wild-type AAV2 capsids; and the AAV8 serotype helper virus was ∼16-fold more efficient than AAV2 serotype helper virus. With these modifications, the vector dose of the helper virus could be further reduced by ∼50-fold. Last, coadministration of scAAV8-PP5 helper virus increased coagulation factor IX expression from an ssAAV2 vector by ∼7-to 10-fold, thereby achieving therapeutic levels at lower vector doses. No adverse effect on hepatocytes was observed under any of these experimental conditions. The strategy presented here should be adaptable to any ssAAV transgene cassette and, specifically, liver-directed applications of ssAAV2 vectors containing larger genes that cannot be encapsidated in scAAV vectors. Although recombinant adeno-associated viral (AAV) vectors containing single-stranded genomes have been used in clinical trials, the single-stranded DNAs are transcriptionally inactive, and relatively large vector doses are needed to achieve expression of therapeutic genes. We have developed optimized recombinant self-complementary AAV vectors carrying the protein phosphatase-5 gene (scAAV-PP5) as helper viruses, coadministration of which leads to efficient transgene expression from conventional single-stranded AAV (ssAAV) vectors at reduced doses. These optimized helper viruses should prove useful in achieving high levels of expression at lower doses of therapeutic genes from ssAAV vectors containing large genes, such as factor VIII, which exceed the packaging capacity of scAAV vectors, for the potential gene therapy of hemophilia A.