342. Self-Complementary Adeno-Associated Virus 2 (AAV)-T Cell Protein Tyrosine Phosphatase Vector as a Helper-Virus To Improve Transduction Efficiency of Conventional AAV Vectors In Vitro and In Vivo

Abstract

Although adeno-associated virus 2 (AAV) vectors target the liver efficiently, the transgene expression is limited to ~5% of hepatocytes. The lack of efficient transduction is due in part to the presence of a cellular protein, FKBP52, phosphorylated forms of which, inhibit the viral second-strand DNA synthesis, and consequently, transgene expression (J. Virol., 75: 8968–8976, 2001). We have documented that dephosphorylation of FKBP52 by cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV transduction of primary murine hematopoietic cells in TC-PTP-transgenic mice (J. Virol., 77: 2741–2746, 2003). Since TC-PTP cDNA size is within the packaging capacity of self-complementary AAV (scAAV) vectors, which obviate the need for viral-second-strand DNA synthesis, we reasoned that scAAV-TC-PTP vectors, if co-infected with a conventional AAV vector, might serve as a helper-virus for conventional single-stranded AAV vectors. Co-infection of HeLa cells, which are transduced poorly by conventional AAV vectors, at a 1:1 ratio with a conventional AAV-EGFP vector and scAAV vectors containing the wild-type (wt) TC-PTP gene (scAAV-wt-TC-PTP) led to a significant increase in AAV-mediated EGFP transgene expression in vitro. This increase was not observed when scAAV vectors containing a catalytically inactive mutant form of TC-PTP (scAAV-mTC-PTP) were used. We also tested the efficacy of scAAV-TC-PTP vectors in a mouse model in vivo. Approximately 1 × 1011 particles of AAV-EGFP vectors alone, or those ad-mixed with either scAAV-wtTC-PTP, or scAAV-mTC-PTP vectors were injected via the tail-vein into C57BL6 mice. Four weeks-post injection, liver tissues were harvested, sectioned and evaluated for EGFP gene expression. Consistent with previously published reports, little transgene expression occurred in hepatocytes following injection of conventional AAV-EGFP vectors, however, co-injection with scAAV-wt-TC-PTP vectors, but not with scAAV-mTC-PT-PTP vectors, led to a significant increase in EGFP expression in murine hepatocytes in vivo. Low Mr DNA samples were isolated from liver tissues, electrophoresed on 1% alkaline-agarose gels, and analyzed on Southern blots using an EGFP probe. Dimer-length AAV genomes, representing viral second-strand DNA synthesis, were detected in cells from mice co-injected with conventional AAV-EGFP+scAAV-wt-TC-PTP vectors, but not in those injected with AAV-EGFP vector alone, or co-injected with scAAV-mTC-PTP vectors. Cohorts of C57BL6 mice were also injected via the tail-vein either with PBS, or with 1 × 1011 particles of scAAV-wtTC-PTP vectors and sacrificed at the end of 4-weeks. All tissues from all PBS-injected controls, and scAAV-wtTC-PTP vector-injected animals showed no evidence of any toxicity, and no pathological lesions were observed in any organ in any of the animals. Thus, this novel co-infection strategy should be useful in circumventing one of the major obstacles in the optimal use of recombinant AAV vectors in human gene therapy.