Mechanism of Hepatocyte Transduction by AAV Vectors

AAV 载体转导肝细胞的机制

基本信息

  • 批准号:
    7017369
  • 负责人:
  • 金额:
    $ 21.1万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2005
  • 资助国家:
    美国
  • 起止时间:
    2005-07-01 至 2010-06-30
  • 项目状态:
    已结题

项目摘要

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.
腺相关病毒2(AAV)载体作为一种更常用的逆转录病毒和腺病毒载体的替代品而受到关注。重组AAV载体已被证明可以有效地靶向肝脏,但转基因表达仅限于大约5%的肝细胞。因为病毒基因组是单链DNA, 而两极的单链都以相同的频率被包裹,有人认为没有进行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合成是导致观察到的转基因表达效率低下的原因。我们以前已经证明,一种名为FKBP52的细胞蛋白,当以磷酸化形式存在时,会抑制AAV第二链DNA的合成,从而限制非肝细胞中的转基因表达,而非磷酸化形式的FKBP52则没有任何作用。为了进一步评估磷酸化的FKBP52是否也参与调节AAV介导的小鼠肝细胞中的转基因表达,我们培育了过表达细胞T细胞蛋白酪氨酸磷酸酶(TC-PTP)蛋白的转基因小鼠,以及FKBP52缺失的小鼠。我们已经证明,在TC-PTP转基因(TC-PTP-TG)小鼠中FKBP52去磷酸化,在FKBP52基因敲除小鼠(FKBP52-KO)中去除FKBP52,导致尾静脉注射重组AAV载体后小鼠肝细胞的有效转导。我们还记录了TC-PTP-TG和FKBP52-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病毒 第二链DNA合成,而不是DNA链退火,是肝细胞有效转导的限速步骤。这一建议将检验下列假设:1.DNA-PKcs使FKBP52在丝氨酸/苏氨酸残基上磷酸化,并被PP5去磷酸化;2.原代肝细胞的最佳转导受到丝氨酸/苏氨酸磷酸化形式的FKBP52的限制,但可以被故意过表达PP5所克服;3.过表达TC-PTP的DNA-PKcs缺陷小鼠允许高效转导小鼠肝细胞;4.在DNA-PKcs缺陷/TC-PTP转基因小鼠中,AAV前病毒基因组在体内发生了有效的整合。从这些研究中获得的知识不仅将有助于阐明AAV与肝细胞的相互作用,而且也将适用于进一步改进重组AAV载体,以期将其用于人类肝病,特别是抗胰蛋白酶缺乏症(AATD)和糖原贮存病(GSD)的基因治疗,这是PPG应用的主要焦点。

项目成果

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Arun Srivastava其他文献

Arun Srivastava的其他文献

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{{ truncateString('Arun Srivastava', 18)}}的其他基金

AAV2 and hepatocellular carcinoma
AAV2 与肝细胞癌
  • 批准号:
    9528459
  • 财政年份:
    2017
  • 资助金额:
    $ 21.1万
  • 项目类别:
Mechanism of Hepatocyte Transduction by AAV Vectors
AAV 载体转导肝细胞的机制
  • 批准号:
    7489003
  • 财政年份:
    2007
  • 资助金额:
    $ 21.1万
  • 项目类别:
Human Parvovirus B19 Vectors: Mechanism of Transduction
人类细小病毒 B19 载体:转导机制
  • 批准号:
    7024569
  • 财政年份:
    2004
  • 资助金额:
    $ 21.1万
  • 项目类别:
Human Parvovirus B19 Vectors: Mechanism of Transduction
人类细小病毒 B19 载体:转导机制
  • 批准号:
    6855770
  • 财政年份:
    2004
  • 资助金额:
    $ 21.1万
  • 项目类别:
Human Parvovirus B19 Vectors: Mechanism of Transduction
人类细小病毒 B19 载体:转导机制
  • 批准号:
    7391091
  • 财政年份:
    2004
  • 资助金额:
    $ 21.1万
  • 项目类别:
Human Parvovirus B19 Vectors: Mechanism of Transduction
人类细小病毒 B19 载体:转导机制
  • 批准号:
    7178444
  • 财政年份:
    2004
  • 资助金额:
    $ 21.1万
  • 项目类别:
Human Parvovirus B19 Vectors: Mechanism of Transduction
人类细小病毒 B19 载体:转导机制
  • 批准号:
    6927575
  • 财政年份:
    2004
  • 资助金额:
    $ 21.1万
  • 项目类别:
Hematepoietic Stem Cell Transduction by AAV2 Vectors
AAV2 载体的造血干细胞转导
  • 批准号:
    6337984
  • 财政年份:
    2001
  • 资助金额:
    $ 21.1万
  • 项目类别:
Hematepoietic Stem Cell Transduction by AAV2 Vectors
AAV2 载体的造血干细胞转导
  • 批准号:
    6746916
  • 财政年份:
    2001
  • 资助金额:
    $ 21.1万
  • 项目类别:
Hematepoietic Stem Cell Transduction by AAV2 Vectors
AAV2 载体的造血干细胞转导
  • 批准号:
    6638693
  • 财政年份:
    2001
  • 资助金额:
    $ 21.1万
  • 项目类别:

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  • 批准号:
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  • 财政年份:
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