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

 描述（由申请人提供）：人博卡病毒1型（HBoV 1）是细小病毒科的一种小DNA病毒，其天然宿主组织是肺气道。为了利用这种病毒的嗜性来开发基因疗法和疫苗以对抗气道疾病（高需求的工具），我们克隆了全长HBoV 1基因组，建立了用于将病毒引入细胞的反向遗传学系统，并证明它可以有效地感染极化的原代人气道上皮气液界面（HAE-ALI）培养物。HBoVl衣壳共享腺相关病毒（AAV）的T=1细小病毒二十面体衣壳的关键特征，并且我们已经发现，这使得可以通过跨属假包装将重组AAV 2（rAAV 2）基因组有效地包装到HBoVl衣壳中。利用这一发现，我们开发了一种新的细小病毒跨属杂交载体，rAAV 2/HBoV 1，以促进基因转移到人气道。该载体将皿oVl衣壳的高顶端向性与rAAV 2基因组在人气道上皮中的安全性和持久性相结合，并将rAAV基因组的容量扩大20%。我们已经证明了这种载体的两个关键优势。首先，它比rAAV载体更有效地从顶端表面转导HAE-ALI。其次，它能够递送在强启动子后面携带囊性纤维化跨膜传导调节因子（CFTR）开放阅读框的5.5-kb超大rAAV 2基因组，并且能够校正表征源自囊性纤维化（CF）患者的HAE的氯离子转运中的CFTR特异性缺陷。因此，rAAV 2/HBoV 1杂合载体是CF基因治疗的理想载体。我们对293细胞中第一代HBoV 1包装辅助物的初步分析揭示了该病毒基因的新基因表达谱。增加衣壳表达产生了第二代皿oVl辅助物，利用该辅助物，rAAV 2/皿oVl载体生产增加了8倍，接近rAAV 2/2的约50-80%的产率。这些发现将指导我们进一步了解细小病毒跨属假包装的过程，以生产第三代辅助病毒和生产系统，从而显著提高载体产量。我们的长期目标是利用新型rAAV 2/HBoV 1载体将基因转移到人气道，特别是用于CF基因治疗。所提出的研究旨在通过以下方式建立这些发现和专业知识：i）确定HBoVl蛋白在细小病毒跨属假包装中的作用; ii）优化HBoVl包装辅助构建体，用于在293细胞中产生rAAV 2/HBoVl载体。（用于临床前应用）;和iii）建立有效的杆状病毒/昆虫细胞系统，用于在Sf 9细胞中高产量和大规模的rAAV 2/HBoV 1载体生产（用于动物模型和临床应用的临床前研究）。

 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).