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: 9089981
• 负责人: Jianming Qiu
• 金额: $ 22.72万
• 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 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9089981
• 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; DNA cassette; Dependovirus; Development; Diagnosis; Diagnostic; Disease; Epithelial; Family; Gene Expression; Gene Expression Profile; Gene Transfer; Generations; Genes; Genetic Transcription; Genome; Goals; Health; 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 Replication; 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; 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（由申请人提供）：人类博卡病毒1型（HBoV1）是细小病毒科的一种小型DNA病毒，其天然宿主组织是肺气道，旨在利用该病毒的趋向性来开发基因疗法。和对抗气道疾病的疫苗（需求量很大的工具），我们克隆了全长 HBoV1 基因组，建立了将病毒引入细胞的反向遗传学系统，以及经证实，HBoV1 衣壳具有腺相关病毒 (AAV) 的 T=1 细小病毒二十面体衣壳的关键特征，可以有效地感染极化的人原代气道上皮气液界面 (HAE-ALI)。这使得通过跨属伪包装将重组 AAV2 (rAAV2) 基因组有效包装到 HBoV1 衣壳中成为可能。基于这一发现，我们开发了一种新型细小病毒跨属杂交载体 rAAV2/HBoV1，以促进基因转移至人类呼吸道。该载体结合了 HBoV1 衣壳的高度向顶端性和 rAAV2 基因组在体内的安全性和持久性。人类气道上皮细胞，并将 rAAV 基因组的容量扩大了 20%。我们已经证明了该载体的两个关键优势。其次，来自顶端表面的 HAE-ALI 比 rAAV 载体更有效，它能够在强启动子后面传递携带囊性纤维化跨膜电导调节因子 (CFTR) 开放阅读框的 5.5 kb 超大 rAAV2 基因组，并能够纠正CFTR 特异性氯转运缺陷是囊性纤维化 (CF) 患者 HAE 的特征，因此，rAAV2/HBoV1 杂合载体是一种。我们对 293 细胞中第一代 HBoV1 包装辅助蛋白的初步分析揭示了该病毒基因的新基因表达谱，增加了衣壳表达，产生了第二代 HBoV1 辅助蛋白。 rAAV2/HBoV1 载体产量增加了 8 倍，产量接近 rAAV2/2 的 50-80%。这些发现将指导我们进一步了解该过程。我们的长期目标是利用新型 rAAV2/HBoV1 载体将基因转移到人类呼吸道，特别是，拟议的研究旨在通过以下发现和专业知识为基础：i) 确定 HBoV1 蛋白在细小病毒跨属伪包装中的作用；ii) 优化用于在 293 细胞中生产 rAAV2/HBoV1 载体的 HBoV1 包装辅助构建体（用于临床前应用）；以及 iii) 建立有效的杆状病毒/昆虫细胞干，用于在 Sf9 细胞中进行高产量和大规模的 rAAV2/HBoV1 载体生产（用于动物模式和临床应用的临床前研究）。