Mechanism of Hepatocyte Transduction by AAV Vectors

腺相关病毒2（AAV）载体作为更常用的基于逆转录病毒和腺病毒的载体的替代物已经获得关注。重组AAV载体已被证明有效靶向肝脏，但转基因表达仅限于约5%的肝细胞。因为病毒基因组是单链DNA， 并且两种极性的单链以相等的频率发生双链化，已经提出，不能进行DNA链退火是缺乏有效的转基因表达的原因。另一方面，其他人和我们提出，未能进行病毒第二链DNA合成有助于观察到的转基因表达效率低。我们以前已经证明，一种称为FKBP 52的细胞蛋白，当以磷酸化形式存在时，抑制AAV第二链DNA合成，因此限制了非肝细胞中的转基因表达，而非磷酸化形式的FKBP 52没有影响。为了进一步评估磷酸化的FKBP 52是否也参与调节AAV介导的转基因在鼠肝细胞中的表达，我们产生了过表达细胞T细胞蛋白酪氨酸磷酸酶（TC-PTP）蛋白的转基因小鼠，已知该蛋白催化FKBP 52的去磷酸化，以及FKBP 52缺陷的小鼠。我们已经证明，TC-PTP转基因（TC-PTP-TG）小鼠中FKBP 52的去磷酸化和FKBP 52敲除（FKBP 52-KO）小鼠中FKBP 52的去除导致尾静脉注射重组AAV载体后鼠肝细胞的有效转导。我们还记录了TC-PTP-TG和FKBP 52-KO小鼠肝细胞中有效的病毒第二链DNA合成。因此，我们的数据有力地支持了这样的论点，即病毒 第二链DNA合成而不是DNA链退火是肝细胞有效转导中的限速步骤。本研究将检验以下假设：1. FKBP 52在丝氨酸/苏氨酸残基处被DNA-PKcs磷酸化，并且被PP 5去磷酸化; 2.原代肝细胞的最佳转导受到FKBP 52的丝氨酸/苏氨酸磷酸化形式的限制，但可以通过PP 5的故意过表达来克服; 3.过表达TC-PTP的DNA-PKcs缺陷小鼠允许有效转导鼠肝细胞;和4. AAV前病毒基因组的有效整合发生在体内DNA-PKcs缺陷/ TC-PTP转基因小鼠的原代肝细胞中。从这些研究中获得的知识不仅将揭示AAV-肝细胞相互作用，而且还将适用于进一步改进重组AAV载体，以用于其在人类肝脏疾病的基因治疗中的潜在用途，特别是α 1-抗胰蛋白酶缺乏症（AATD）和糖原累积病（GSD），这是PPG应用的主要焦点。

The adeno-associated virus 2 (AAV) vectors have gained attention as an alternative to the more commonly used retrovirus- and adenovirus-based vectors. Recombinant AAV vectors have been shown to target the liver efficiently, but the transgene expression is restricted to approximately 5% of the hepatocytes. Because the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. Others and we, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis contributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, inhibits AAV second-strand DNA synthesis, when present in phosphorylated forms, and consequently, limits transgene expression in non-hepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice over-expressing the cellular T cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We have demonstrated that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice, results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We have also documented efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes. This proposal will test the following hypotheses: 1. FKBP52 is phosphorylated at serine/threonine residues by DNA-PKcs, and dephosphorylated by PP5; 2. Optimal transduction of primary hepatocytes is limited by serine/threonine phosphorylated forms of FKBP52, but can be overcome by deliberate overexpression of PP5; 3. DNA-PKcs-deficient mice overexpressing TC-PTP allow efficient transduction of murine hepatocytes; and 4. Efficient integration of the AAV proviral genome occurs in primary hepatocytes in DNA-PKcs-deficient/ TC-PTP-transgenic mice in vivo. The knowledge gained from these studies will not only shed light on the AAV-hepatocyte interactions, but will also be applicable in further improvements in recombinant AAV vectors for their potential use in gene therapy of human liver diseases in general, and al-antitrypsin deficiency (AATD) and glycogen storage disease (GSD) in particular, the main focus of this PPG application.