Human Parvovirus B19 Vectors: Mechanism of Transduction

人类细小病毒 B19 载体：转导机制

• 批准号: 6927575
• 负责人: Arun Srivastava
• 金额: $ 36.38万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6927575
• 关键词: Parvoviridae; adeno associated virus group; binding sites; cell surface receptors; clinical research; erythrocytes; erythroid stem cell; flow cytometry; gene delivery system; gene expression; gene therapy; human subject; integrins; proteoglycan; recombinant virus; transcription factor; transfection /expression vector

DESCRIPTION (provided by applicant): Parvoviruses constitute a unique group of small, single-stranded DNA viruses that infect all vertebrates. One of the non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV) has gained attention as a useful alternative to the more commonly used retrovirus- and adenovirus-based vectors in human gene therapy. In contrast to AAV, which possesses a broad host-range, the pathogenic human parvovirus B19 has been shown to have a remarkably narrow tissue-tropism for erythroid progenitor cells presumably because the virus uses the blood group P antigen as a cell surface receptor. We have developed recombinant human parvovirus B19 vectors in which recombinant AAV genomes are encapsidated in parvovirus B19 capsids. Although the viral titers obtained are low, using these hybrid vectors, we have demonstrated that P antigen alone is not sufficient to impart erythroid cell-specificity to parvovirus B19. We have proposed a "triple level of erythroid-specificity" model for parvovirus B19 infection in which P antigen is utilized as a primary receptor for virus binding, activated alpha5beta1 integrin as a cell surface co-receptor for viral entry, and a hitherto unknown cellular transcription factor for erythroid cell-specific expression from the viral promoter at map unit 6 (B19p6). Using a variety of molecular techniques and recombinant parvovirus vectors, we will test the following hypotheses: 1. The inclusion of appropriate transcriptional control elements and parvovirus B19 "packaging signal" wilt lead to improved titers of recombinant parvovirus B19 vectors; 2. Mature RBCs are utilized for systemic dissemination and efficient entry of parvovirus B19 into primary human hematopoietic cells in the erythroid lineage, and 3. A putative transcription factor, which is present only in primary human erythroid progenitor cells, mediates lineage-restricted gene expression from the parvovirus B19p6 promoter. The following three Specific Aims will be pursued: 1. Development of strategies to generate high-titer recombinant parvovirus B19 vectors. 2. Elucidation of underlying mechanisms of parvovirus B19 infection of primary human erythroid progenitor ceils. 3. Elucidation of recombinant parvovirus vector-mediated transduction of primary human hematopoietic stem cells and erythroid lineage-restricted gene expression, and characterization of the putative cellular transcription factor specific for the B19p6 promoter. The knowledge gained from these studies will shed light on parvovirus Bl9-host cell interactions, and will lead to further improvements in recombinant parvovirus B19 vectors for their potential use in human gene therapy.

描述(申请人提供)：细小病毒构成一组独特的小的单链DNA病毒，感染所有脊椎动物。腺相关病毒2(AAV)是一种非致病性的人类细小病毒，作为人类基因治疗中常用的逆转录病毒和腺病毒载体的有效替代品而受到关注。与具有广泛宿主范围的AAV不同，致病性人类细小病毒B19对红系祖细胞的组织嗜性非常有限，可能是因为该病毒使用P血型抗原作为细胞表面受体。我们研制了重组人细小病毒B19载体，其中重组AAV基因组被包裹在细小病毒B19衣壳中。虽然获得的病毒滴度很低，但使用这些杂交载体，我们已经证明P抗原本身不足以赋予细小病毒B19红系细胞特异性。我们提出了细小病毒B19感染的“三水平红系特异性”模型，其中P抗原作为病毒结合的主要受体，激活的α5β1整合素作为病毒进入的细胞表面辅助受体，以及迄今未知的细胞转录因子，用于从MAP单元6(B19p6)的病毒启动子(B19p6)表达红系细胞特异性。使用各种分子技术和重组细小病毒载体，我们将检验以下假设：1.包含适当的转录调控元件和细小病毒B19的“包装信号”将导致重组细小病毒B19载体滴度的提高；2.成熟的红细胞被用于系统传播和有效地进入红系中的原代人造血细胞，以及3.一种假定的转录因子，它只存在于原代人红系祖细胞中，介导细小病毒B19p6启动子的谱系限制性基因表达。将追求以下三个具体目标：1.建立高效价重组细小病毒B19载体的策略。2.探讨细小病毒B19感染原代人红系祖细胞的可能机制。3.重组细小病毒载体介导的原代人造血干细胞转导和红系限制基因的表达，以及B19p6启动子特异性细胞转录因子的鉴定。从这些研究中获得的知识将有助于揭示细小病毒BL9与宿主细胞的相互作用，并将导致重组细小病毒B19载体的进一步改进，为其在人类基因治疗中的潜在应用奠定基础。