GENERATION OF RECOMBINANT ADENO-ASSOCIATED VIRUS TYPE 3 BASED VECTORS

腺相关病毒是属于猪病毒科的一种小的、无包膜的单链DNA病毒。AAVs已经从各种不同的物种中分离出来，在这些物种中，它们似乎是非致病的。到目前为止，已经描述了五种灵长类AAVs，它们的抗原性不同的衣壳蛋白在血清学上是不同的。AAV-2、AAV-3和AAV-5的分离株已直接从人类临床标本中获得，人类似乎是自然宿主。直到最近，只有AAV-2在基因组水平上被表征。目前对AAV的大部分兴趣源于它们作为基因治疗载体的潜在用途，几乎所有的研究都使用AAV-2。虽然AAV-2具有广泛的组织宿主范围，并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鉴定了AAV-3的全长基因组，并产生了一个具有感染性的克隆。在本研究中，我们将绿色荧光蛋白(GFP)或β-半乳糖苷酶基因分别插入到AAV-2和AAV-3载体中，获得了重组病毒。用重组病毒转导造血细胞，比较转导效率。与rAAV-2相反，重组AAV-3病毒成功地转导了红系和巨核母细胞。相反，rAAV-2在转导淋巴细胞方面表现出更好的效果。这些结果表明，AAV-2和AAV-3不仅存在不同的细胞受体，而且rAAV-3载体可能更适合于某些类型的造血细胞的转导。我们还可以证明AAV-3载体在一定的分化过程中转导了NT2细胞，表明AAV-3载体可以用于调节神经元的分化。注射AAV-TH载体后7个月，AAV载体介导的基因在大鼠纹状体内持续表达。

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.