近日,一篇发表在《美国国家科学院院刊》(Proceedings of the National Academy of Sciences)上的研究称,通过研制人造人类染色体(HAC)修复人类细胞基因缺陷的实验取得了良好的效果。
与基因治疗中采用的病毒载体相比,HAC可以避免其无法控制基因拷贝数目,以及基因突变和基因沉默等缺点。
研究者通过使用相互结合的2个人造染色体来具有基因缺陷的人类细胞,该细胞从有肿瘤相关基因缺陷患者身上分离而来。首先,他们将该肿瘤相关基因从患者的基因组DNA中分离出来,然后使HAC携带该基因,再将HAC导入该细胞进行修复。他们观察到嵌入的肿瘤相关基因能在该细胞中正常表达蛋白质并修复该细胞的缺陷。
随后,研究者又找出了能使该人造染色体失活的方法。表明这种方法可以用来研究HAC介导的细胞治疗。
HAC稳定性较高,且能可逆性地将完整基因导入人类细胞,有望在基因功能研究和临床基因治疗研究等方面发挥重要作用。(生物谷bioon.com)
doi:10.1073/pnas.1114483108
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Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells
Jung-Hyun Kim, Artem Kononenko, Indri Erliandri, Tae-Aug Kim,Megumi Nakano, Yuichi Iida, J. Carl Barrett, Mitsuo Oshimura,Hiroshi Masumoto, William C. Earnshaw, Vladimir Larionov, andNatalay Kouprina.
Human artificial chromosome (HAC)-based vectors offer a promising system for delivery and expression of full-length human genes of any size. HACs avoid the limited cloning capacity, lack of copy number control, and insertional mutagenesis caused by integration into host chromosomes that plague viral vectors. We previously described a synthetic HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. Here, we demonstrate the utility of this HAC, which has a unique gene acceptor site, for delivery of full-length genes and correction of genetic deficiencies in human cells. A battery of functional tests was performed to demonstrate expression ofNBS1 and VHL genes from the HAC at physiological levels. We also show that phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications.