An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction.
An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction.
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发表时间：
1995
期刊：
影响因子：
5.1
通讯作者：
Terence R. Flotte;Ximena Barraza-Ortiz;R. Solow;S. Afione;B. Carter;W. Guggino
中科院分区：
文献类型：
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作者：
Terence R. Flotte;Ximena Barraza-Ortiz;R. Solow;S. Afione;B. Carter;W. Guggino
腺相关病毒（AAV）载体对于许多人类疾病的基因治疗是潜在有用的。然而，由于缺乏稳定的载体包装细胞系，这些载体的使用受到限制。开发包装细胞系的困难涉及来自AAV-p5启动子的rep基因表达的低水平，以及Rep蛋白抑制永生化细胞系的持续生长的倾向。我们在这里描述两个新的技术，使这些问题得到规避。首先，我们已经证明，从人类免疫缺陷病毒（HIV）长末端重复序列（LTR）启动子的REP的表达导致包装效率提高了10倍。第二，我们通过产生含有可挽救的AAV重组基因组的细胞群体克服了载体质粒转染的低效性。这些改进使AAVp 5 neo和AAVp 5lacZ重组载体的包装效率净增加50倍。将用该方法包装的AAVp 5lacZ载体全身施用给最近断奶的C57 BL小鼠，并介导β-半乳糖苷酶报告基因在气道上皮细胞和脾细胞中的有效表达。这表明这些载体储备物的体内活性，以及它们用于基因治疗的潜在效用。
Adeno-associated virus (AAV) vectors are potentially useful for gene therapy of a number of human diseases. However, the use of these vectors has been limited by the lack of stable vector-packaging cell lines. The difficulties in developing packaging cell lines relate to low levels of rep gene expression from the AAV-p5 promoter, and to the propensity of Rep proteins to suppress continued growth of immortalized cell lines. We describe here two new techniques which allow these problems to be circumvented. First, we have demonstrated that expression of rep from the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter results in a 10-fold improvement of packaging efficiency. Second, we have overcome the inefficiency of vector plasmid transfection by generating cell populations containing rescuable AAV recombinant genomes. These improvements yielded a net increase of 50-fold in the packaging efficiency of the AAVp5neo and AAVp5lacZ recombinant vectors. The AAVp5lacZ vector packaged with this method was administered systemically to recently weaned C57BL mice, and mediated efficient expression of the beta-galactosidase reporter gene in cells of the airway epithelium and spleen. This indicates the in vivo activity of these vector stocks, and their potential utility for gene therapy.