GENERATION OF RECOMBINANT ADENO-ASSOCIATED VIRUS TYPE 3 BASED VECTORS

基于重组腺相关病毒 3 型的载体的产生

• 批准号: 10670598
• 负责人: MURAMATSU Shinichi
• 金额: $ 1.92万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670598/
• 关键词: ASENO-ASSOCIATED VIRUS; GENE THERAPY; VIRAL VECTOR; HEMATOPOIETIC CELLS; PARKINSON'S DISEASE

Adeno-associated viruses (AAVs) are small, non-enveloped, single-stranded DNA viruses belonging to the Porvoviridae family. AAVs have been isolated from a variety of different species, where they appear to be non-pathogenic. To date, five primate AAVs have been described, distinguished serologically by their antigenically distinct capsid proteins. Isolates of AAV-2, AAV-3 and AAV-5 have been obtained directly from human clinical specimens, and man appears to be the natural host. Until recently only AAV-2 had been characterized at the genomic level. Much of the current interest in AAVs stems from their potential use as a vector for gene therapy, with virtually all studies using AAV-2. Although AAV-2 has a broad tissue host range and can transduce a wide variety of tissue types, some cells, specifically erythroid cells have been shown to be non permissive. We have previously cloned and characterized the full-length genome of AAV-3 and produced an infectious clone. In this study, we inserted either the genes for green fluorescence protein (GFP) or beta-galactosidase into AAV-2 and AAV-3 based plasmids, and produced recombinant virus. Recombinant virus was then used to transduce hematopoietic cells, and the transduction efficiencies compared. Recombinant AAV-3 virus successfully transduced erythroid and megakaryoblastoid cells, in contrast to rAAV-2. In contrast rAAV-2 appeared better at transducing lymphocytes. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be better for transduction of some hematopoietic cell types. We could also show that AAV-3 vector transduced NT2 cells in some process of differentiation, indicating that AAV-3 vectors can be used to modulate neuronal differentiation. AAV vector-mediated gene expression was persisted in the rat striatum at 7 months after injection of AAV-TH vectors.

腺相关病毒 (AAV) 是小型、无包膜、单链 DNA 病毒，属于波尔沃病毒科。 AAV 已从多种不同物种中分离出来，它们似乎不具有致病性。迄今为止，已描述了五种灵长类 AAV，通过其抗原性不同的衣壳蛋白在血清学上进行区分。 AAV-2、AAV-3 和 AAV-5 的分离株是直接从人类临床标本中获得的，而人类似乎是其天然宿主。直到最近，只有 AAV-2 在基因组水平上得到了表征。目前对 AAV 的兴趣很大程度上源于它们作为基因治疗载体的潜在用途，几乎所有研究都使用 AAV-2。尽管 AAV-2 具有广泛的组织宿主范围并且可以转导多种组织类型，但一些细胞，特别是红系细胞已被证明是不允许的。我们之前已经克隆并表征了 AAV-3 的全长基因组，并产生了感染性克隆。在这项研究中，我们将绿色荧光蛋白（GFP）或β-半乳糖苷酶的基因插入到基于AAV-2和AAV-3的质粒中，并产生重组病毒。然后使用重组病毒转导造血细胞，并比较转导效率。与 rAAV-2 相比，重组 AAV-3 病毒成功转导红细胞和巨核母细胞。相比之下，rAAV-2 似乎更擅长转导淋巴细胞。这些结果不仅表明 AAV-2 和 AAV-3 存在不同的细胞受体，而且 rAAV-3 载体可能更适合某些造血细胞类型的转导。我们还可以证明AAV-3载体在某些分化过程中转导NT2细胞，表明AAV-3载体可用于调节神经元分化。注射 AAV-TH 载体后 7 个月，AAV 载体介导的基因表达在大鼠纹状体中持续存在。