High-fidelity correction of mutations at multiple chromosomal positions by adeno-associated virus vectors
High-fidelity correction of mutations at multiple chromosomal positions by adeno-associated virus vectors
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DOI：
10.1128/jvi.73.9.7376-7380.1999
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发表时间：
1999-09-01
影响因子：
5.4
通讯作者：
Russell, DW
中科院分区：
文献类型：
--
作者：
Inoue, N;Hirata, RK;Russell, DW
用来修饰小鼠胚胎干细胞染色体的基因打靶技术在许多其他类型的细胞上取得了有限的成功，特别是在生物体外生长能力有限的正常原代细胞。这在很大程度上是由于传统DNA转移方法的技术问题和/或效率低下，以及在未经选择的细胞群体中获得的同源重组率较低。我们最近描述了另一种方法，使用腺相关病毒(AAV)载体来修饰同源染色体序列，在正常人细胞(D，W，Russell和R，K，Hirata，NAT，Genet，18：325-330,1998)中，在单拷贝次黄嘌呤磷酸核糖基转移酶(HPRT)基因座上观察到接近1%的靶向率。在这里，我们报道了我们使用逆转录病毒穿梭载体系统在人类染色体上引入和表征靶基因座的实验，证明AAV载体可以高保真地纠正几种类型的突变，与染色体位置无关，基因靶向率因所纠正的突变类型而异，由于AAV载体可以有效地将DNA运送到体内和体外的多种细胞类型，我们的结果表明AAV介导的基因打靶将具有广泛的应用前景，包括治疗性基因纠正。
The gene targeting techniques used to modify chromosomes in mouse embryonic stem cells have had limited success with many other cell types, especially normal primary cells with restricted growth capacity outside the organism. This is due in large part to the technical problems and/or inefficiency of conventional DNA transfer methods, as well as the low rates of homologous recombination obtained in unselected cell populations. We recently described an alternative approach in which adeno-associated virus (AAV) vectors were used to modify homologous chromosomal sequences, and targeting rates close to 1% were observed at the single copy hypoxanthine phosphoribosyl transferase (HPRT) locus in normal human cells (D, W, Russell and R, K, Hirata, Nat, Genet, 18:325-330, 1998), Here we report experiments in which we used a retroviral shuttle vector system to introduce and characterize target loci in human chromosomes, and demonstrate that AAV vectors can correct several types of mutations with high fidelity, independent of chromosomal position, The gene targeting rates varied depending on the type of mutation being corrected, implicating cellular mismatch recognition functions in the reaction, Since AAV vectors can efficiently deliver DNA to many cell types both in vivo and ex vivo, our results suggest that AAV-mediated gene targeting will have wide applicability, including therapeutic gene correction.