Jayandharan G R, Zhong L, Li B, Kachniarz B, Srivastava A
Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida College of Medicine, Gainesville, FL 32610-3633, USA.
Gene Ther. 2008 Sep;15(18):1287-93. doi: 10.1038/gt.2008.89. Epub 2008 May 22.
Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of murine hepatocytes. Viral second-strand DNA synthesis continues to be a rate-limiting step for efficient transduction by the single-stranded AAV (ssAAV) vectors. This is due, in part, to the presence of a cellular chaperone (FK506-binding) protein, FKBP52, phosphorylated forms of which interact with the D-sequence in the inverted terminal repeats of AAV2 genome and inhibit the viral second-strand DNA synthesis. Our previous studies have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), and at serine/threonine residues by protein phosphatase 5 (PP5) enhances viral second-strand DNA synthesis and consequently, the transgene expression. We have also reported that coinfection with a self-complementary AAV (scAAV)-TC-PTP vector results in up to sixfold increase in the transduction efficiency of conventional ssAAV2 vectors in primary murine hepatocytes in vivo. We reasoned that coinfection with scAAV-TC-PTP and scAAV-PP5 vectors may lead to a further increase in the transduction efficiency of ssAAV2 vectors. We demonstrate here that this strategy does indeed lead to approximately 16-fold increase in the transduction efficiency of conventional ssAAV vectors in primary murine hepatocytes in vivo following tail-vein injections. Neither scAAV2-TC-PTP nor scAAV2-PP5 vectors alone or together had any adverse effect on the hepatocytes. Thus, this coinfection strategy may be useful for achieving expression from recombinant ssAAV2 vectors containing larger genes, such as coagulation factor VIII, which exceed the packaging capacity of scAAV vectors, for the potential gene therapy of hemophilia A.
基于2型腺相关病毒（AAV）的重组载体可有效靶向肝脏，但转基因表达仅限于约5%的小鼠肝细胞。病毒第二链DNA合成仍然是单链AAV（ssAAV）载体高效转导的限速步骤。部分原因是存在一种细胞伴侣（FK506结合）蛋白FKBP52，其磷酸化形式与AAV2基因组反向末端重复序列中的D序列相互作用并抑制病毒第二链DNA合成。我们之前的研究表明，细胞T细胞蛋白酪氨酸磷酸酶（TC-PTP）使FKBP52酪氨酸残基去磷酸化，蛋白磷酸酶5（PP5）使FKBP52丝氨酸/苏氨酸残基去磷酸化，可增强病毒第二链DNA合成，从而提高转基因表达。我们还报道，与自我互补AAV（scAAV）-TC-PTP载体共感染可使体内原代小鼠肝细胞中传统ssAAV2载体的转导效率提高多达六倍。我们推测，与scAAV-TC-PTP和scAAV-PP5载体共感染可能会进一步提高ssAAV2载体的转导效率。我们在此证明，在尾静脉注射后，该策略确实可使体内原代小鼠肝细胞中传统ssAAV载体的转导效率提高约16倍。单独或共同使用scAAV2-TC-PTP和scAAV2-PP5载体对肝细胞均无任何不良影响。因此，这种共感染策略可能有助于实现含有较大基因（如凝血因子VIII）的重组ssAAV2载体的表达，凝血因子VIII超过了scAAV载体的包装容量，可用于A型血友病的潜在基因治疗。
