Human Parvovirus B19 Vectors: Mechanism of Transduction

描述（由申请人提供）：细小病毒是一种独特的小单链DNA病毒，可感染所有脊椎动物。腺相关病毒2 （AAV）是一种非致病性人细小病毒，作为人类基因治疗中更常用的逆转录病毒和腺病毒载体的有用替代品而受到关注。与具有广泛宿主范围的AAV相比，致病性人细小病毒B19已被证明对红系祖细胞具有非常狭窄的组织趋向性，这可能是因为该病毒使用血型P抗原作为细胞表面受体。我们开发了重组人细小病毒B19载体，其中重组AAV基因组被封装在细小病毒B19衣壳中。虽然获得的病毒滴度很低，但使用这些杂交载体，我们已经证明单独的P抗原不足以赋予细小病毒B19红细胞特异性。我们提出了细小病毒B19感染的“三水平红细胞特异性”模型，其中P抗原被用作病毒结合的主要受体，激活的alpha5beta1整合素作为病毒进入的细胞表面共受体，以及一个迄今未知的细胞转录因子，用于从图单元6的病毒启动子（B19p6）特异性表达红细胞。利用多种分子技术和重组细小病毒载体，我们将检验以下假设：适当的转录控制元件和细小病毒B19“包装信号”的加入将提高重组细小病毒B19载体的滴度；2. 2 .成熟红细胞被用于细小病毒B19的全身传播和有效进入红系人造血细胞；一种推测的转录因子，仅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：制备高滴度重组细小病毒B19载体的策略研究。2. 微小病毒B19感染人红细胞原祖细胞的潜在机制的阐明。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性基因表达的阐明，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阐明细小病毒B19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，以用于人类基因治疗。

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.