Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.
Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.
PLoS One. 2014 Jan 15;9(1):e85565. doi: 10.1371/journal.pone.0085565. eCollection 2014.
Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe's disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.
哺乳动物人工染色体是基于天然染色体的载体，可在大小和数量方面携带大量遗传物质。它们具有相当的稳定性，在有丝分裂和减数分裂中都能很好地分离。早些时候，人们描述了一种平台人工染色体表达系统（ACEs），该系统具有多个用于改良的 lambda-整合酶的加载位点。已经表明，这种 ACEs 适合于高水平的工业蛋白生产和一种毁灭性的人类疾病（Krabbe 病）的小鼠模型的治疗。携带治疗基因的 ACEs 治疗突变小鼠的寿命比未治疗的对照小鼠长四倍以上。这种新的基因治疗方法称为组合哺乳动物人工染色体-干细胞治疗。目前，这种方法受到限制，即对于加载到 ACEs 上的每个治疗基因，都应该存在一个新的选择标记基因。复杂疾病需要几个基因的协同作用来治疗，但只有有限数量的选择标记基因可用，并且这些标记基因在哺乳动物细胞、器官和生物体中的非预期表达也存在严重的副作用风险。我们在这里描述了一种仅使用两个可选择标记基因将多个基因加载到 ACEs 中的新方法。在进行治疗应用之前，这些标记物可以从 ACEs 中去除。这项新技术可能会彻底改变针对复杂疾病和癌症治疗的基因治疗应用。它还可以通过允许研究人员用一组预定的遗传因素来设计染色体，将成人干细胞、胚胎干细胞和诱导多能干细胞（iPS 细胞）分化为具有治疗价值的细胞类型，从而加速细胞治疗。它也是研究基本科学中复杂生化途径的合适工具，通过产生具有几个感兴趣的信号转导途径的基因的 ACEs 来实现。
