Flotte T R, Barraza-Ortiz X, Solow R, Afione S A, Carter B J, Guggino W B
Eudowood Division of Pediatric Respiratory Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21287-2533.
Gene Ther. 1995 Jan;2(1):29-37.
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.
腺相关病毒（AAV）载体对于多种人类疾病的基因治疗具有潜在的应用价值。然而，这些载体的使用受到缺乏稳定的载体包装细胞系的限制。开发包装细胞系的困难在于AAV-p5启动子的rep基因表达水平较低，以及Rep蛋白抑制永生化细胞系持续生长的倾向。我们在此描述了两种能够规避这些问题的新技术。首先，我们证明了从人类免疫缺陷病毒（HIV）长末端重复序列（LTR）启动子表达rep可使包装效率提高10倍。其次，我们通过生成含有可拯救的AAV重组基因组的细胞群体，克服了载体质粒转染效率低下的问题。这些改进使AAVp5neo和AAVp5lacZ重组载体的包装效率净提高了50倍。用这种方法包装的AAVp5lacZ载体经全身给药至刚断奶的C57BL小鼠，介导了β-半乳糖苷酶报告基因在气道上皮细胞和脾细胞中的高效表达。这表明了这些载体储备的体内活性及其在基因治疗中的潜在应用价值。
