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

 描述(申请人提供)：人类博卡病毒1型(HBoV1)是细小病毒科的一种小DNA病毒，其自然宿主组织是肺呼吸道。为了利用这种病毒的趋向性来开发抗击呼吸道疾病的基因疗法和疫苗(需求很大的工具)，我们克隆了HBoV1的全长基因组，建立了将病毒引入细胞的反向遗传学系统，并证明了它可以有效地感染极化的原代人呼吸道上皮气液界面(HAE-ALI)培养物。HBoV1衣壳具有腺相关病毒(AAV)T=1细小病毒二十面体衣壳的主要特征，我们发现这使得通过跨属伪袋化将重组AAV2(RAAV2)基因组有效地包装成HBoV1衣壳成为可能。利用这一发现，我们开发了一种新型的细小病毒跨属杂交载体rAAV2/HBoV1，以促进基因转移到人的呼吸道。该载体结合了HBoV1衣壳的高度顶向性和rAAV2基因组在人呼吸道上皮细胞中的安全性和持久性，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。首先，它比rAAV载体更有效地从根尖表面转导HAE-ALI。其次，它能够传递5.5kb的超大rAAV2基因组，在强大的启动子后面携带囊性纤维化跨膜电导调节因子(CFTR)开放阅读框架，并纠正囊性纤维化(CF)患者的HAE特有的CFTR氯离子转运缺陷。因此，rAAV2/HBoV1杂合载体是一种理想的CF基因治疗载体。我们对293细胞中第一代HBoV1包装助手的初步分析揭示了该病毒基因的新基因表达谱。增加衣壳蛋白的表达产生了第二代HBoV1辅助体，使rAAV2/HBoV1载体的产量提高了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细小病毒跨属伪袋化的过程，以产生第三代辅助体和生产系统，使载体产量显著提高。我们的长期目标是利用新型rAAV2/HBoV1载体将基因转移到人的呼吸道，特别是用于CF基因治疗。这项拟议的研究旨在以这些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HBoV1包装辅助结构，以便在293细胞中生产rAAV2/HBoV1载体(用于临床前应用)；以及iii)建立高效的杆状病毒/昆虫细胞系统，用于在Sf9细胞中高产和大规模生产rAAV2/HBoV1载体(用于动物模式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).