Study of Human Bocavirus Gene Expression for Development of a Parvoviral Vector

人类博卡病毒基因表达的细小病毒载体开发研究

• 批准号: 8968485
• 负责人: Jianming Qiu
• 金额: $ 20.15万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8968485
• 关键词: Acute; Affect; Air; Animals; Antibodies; Apical; Attenuated; Baculoviruses; Capsid; Capsid Proteins; Cells; Child; Chloride Ion; Chlorides; Clinical Research; Clinical Treatment; Clinical Trials; Codon Nucleotides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA Viruses; Dependovirus; Development; Diagnosis; Diagnostic; Disease; Epithelial; Family; Gene Expression; Gene Expression Profile; Gene Transfer; Generations; Genes; Genetic Transcription; Genome; Goals; Human; Hybrids; Infection; Insecta; Length; Liquid substance; Lung; Lung diseases; Messenger RNA; Molecular Profiling; Open Reading Frames; Outcome Study; Parvoviridae; Parvovirus; Play; Polyadenylation; Process; Production; Proteins; RNA Splicing; Recombinant adeno-associated virus (rAAV); Recombinants; Regulator Genes; Research; Respiratory Tract Infections; Role; Safety; Site; Surface; System; Tissues; Translations; Tropism; Vaccines; Viral; Viral Load result; Viral Nonstructural Proteins; Virus; Virus-like particle; Work; airway epithelium; base; clinical application; combat; cystic fibrosis patients; design; expression vector; gene therapy; gene transfer vector; human disease; improved; mRNA Precursor; member; novel; pre-clinical; preclinical study; prevent; promoter; public health relevance; respiratory; reverse genetics; stoichiometry; tool; vector; virus development

 DESCRIPTION (provided by applicant): Human bocavirus 1 (HBoV1) is a small DNA virus of the family Parvoviridae, and its natural host tissue is the lung airway tract. In an effort to harnss the tropism of this virus for the development of gene therapies and vaccines to combat airway diseases (tools that are in high demand), we have cloned a full-length HBoV1 genome, established a reverse genetics system for introducing the virus into cells, and demonstrated that it can productively infect polarized primary human airway epithelium air-liquid interface (HAE-ALI) cultures. The HBoV1 capsid shares key features of the T=1 parvovirus icosahedral capsid of adeno-associated virus (AAV), and we have discovered that this makes it possible to efficiently package the recombinant AAV2 (rAAV2) genome into HBoV1 capsid via cross-genus pseudopackaging. Taking advantage of this finding, we developed a novel parvoviral cross-genus hybrid vector, rAAV2/HBoV1, to facilitate gene transfer to the human airway. This vector combines the high apical tropism of the HBoV1 capsid with the safety and persistence of the rAAV2 genome in the human airway epithelium, and expands the capacity of the rAAV genome by 20%. We have demonstrated two key advantages of this vector. Firstly, it transduces HAE-ALI from the apical surface more efficiently than rAAV vectors do. Secondly, it is capable of delivering a 5.5-kb oversized rAAV2 genome carrying the cystic fibrosis transmembrane conductance regulator (CFTR) open reading frame behind a strong promoter, and of correcting the CFTR-specific deficiency in chloride transport that characterizes HAE derived from cystic fibrosis (CF) patients. Thus, the rAAV2/HBoV1 hybrid vector is an ideal vector for CF gene therapy. Our preliminary analyses of the first-generation HBoV1 packaging helper in 293 cells have revealed a novel gene expression profile for the genes of this virus. Increasing the capsid expression yielded the second- generation HBoV1 helper, with which the rAAV2/HBoV1 vector production has been increased by 8-fold, approaching a yield of ~50-80% that of rAAV2/2. These findings will guide us to further understand the process of parvovirus cross-genus pseudopackaging in order to produce the third-generation helper and production systems that enable significantly improved vector yield. Our long-term goal is to utilize the novel rAAV2/HBoV1 vector for gene transfer to the human airway, in particular, for use in CF gene therapy. The proposed study is designed to build on these findings and expertise by: i) determining the roles of HBoV1 proteins in parvovirus cross-genus pseudopackaging; ii) optimizing an HBoV1 packaging helper construct for rAAV2/HBoV1 vector production in 293 cells (for pre-clinical applications); and iii) establishing an efficient baculovirus/insect cell sstem for high-yield and large-scale rAAV2/HBoV1 vector production in Sf9 cells (for preclinical study with animal modes and clinical applications).

 描述（由适用提供）：人类Bocavirus 1（HBOV1）是parvoviridae家族的小型DNA病毒，其天然宿主组织是肺气道。 In an effort to harns the tropism of this virus for the development of gene therapies and vaccines to combat airway diseases (tools that are in high demand), we have cloned a full-length HBoV1 genome, established a reverse genetics system for introducing the virus into cells, and demonstrated that it can productively infected primary human airway epithelium air-liquid interface (HAE-ALI) cultures. HBOV1 CAPSID具有T = 1粉红色的腺体相关病毒（AAV）的t = 1个小骨的关键特征，并且我们发现这使得有可能有​​效地包装重组AAV2（RAAV2）基因组中的HBOV1基因组中的HBOV1通过交叉果皮capsid capsid capsid capsid capsid capsid capsid capsid capsid。利用这一发现，我们开发了一种新型的小谷横断杂交载体RAAV2/HBOV1，以促进基因转移到人类气道。该矢量将HBOV1衣壳的高顶端归因于人类气道上皮中RAAV2基因组的安全性和持久性，并将RAAV基因组的能力扩大20％。我们已经证明了该向量的两个关键优势。首先，它比RAAV向量更有效地从顶部表面翻译了HAE-ALI。其次，它能够提供5.5 kb超大的RAAV2基因组，该基因组携带囊性纤维化跨膜电导调节剂（CFTR）在强启动子后面的开放式阅读框架，并纠正氯化物运输的CFTR特异性缺乏，从而表征了HAE源自HAE的CATSTIC纤维化（CF）患者（CF）患者。这是RAAV2/HBOV1混合载体是CF基因治疗的理想载体。我们对293个细胞中第一代HBOV1包装辅助器的初步分析揭示了该病毒基因的新基因表达谱。增加的衣壳表达产生了第二代HBOV1助手，而Raav2/Hbov1载体的产生已增加了8倍，接近RAAV2/2的收率约为50-80％。这些发现将指导我们进一步了解细小病毒交叉伪造的过程，以产生第三代助手和生产系统，从而显着提高了矢量产量。我们的长期目标是利用新型的RAAV2/HBOV1载体将基因转移到人类气道中，特别是用于CF基因治疗。拟议的研究旨在以以下方式建立在这些发现和专业知识的基础上： ii）在293个细胞中优化用于RAAV2/HBOV1矢量产生的HBOV1包装辅助构建体（用于临床前应用）；和iii）在SF9细胞中建立有效的杆状病毒/昆虫细胞系统，用于高产和大规模RAAV2/HBOV1载体的产生（用于动物模式和临床应用的临床前研究）。