Mechanistic analysis of the role of extrachromosomal telomeric circles in the ALT

描述（由申请人提供）：端粒稳定，通过激活端粒酶或端粒选择性延长（ALT）途径，是肿瘤获得无限复制潜力的必要条件。大多数人类alt阳性细胞系和肿瘤具有几个共同的显著特征，包括长且高度异质性的端粒，称为alt相关PML核体（APBs）的多蛋白复合物，其中端粒组分（DNA和蛋白质）与PML核体共定位，染色体外环状DNA分子由端粒重复序列组成。然而，染色体外端粒环、apb和端粒延伸之间的关系尚未建立，因为在人类细胞中缺乏一个遗传可处理的系统来研究alt通路的激活和调控。这也使得针对端粒酶独立通路的治疗方法的发展复杂化和受阻。作为开发易于操作的遗传分析系统的第一步，我们已经对哺乳动物人工染色体进行了生物工程改造，以根据需要在多种细胞类型中制造染色体外端粒环。染色体外端粒环的调节产生将允许系统地测试这些环对ALT途径特征的影响。该技术可用于多种细胞类型和/或与RNAi技术相结合，以测试特定途径（基因）在ALT激活中的作用，这是迄今为止难以在人类衍生模型系统中进行控制操作和探索的领域。

DESCRIPTION (provided by applicant): Telomere stabilization, via activation of either telomerase or the Alternative Lengthening of Telomeres (ALT) pathway, is necessary for tumors to acquire an unlimited replicative potential. Most human ALT-positive cell lines and tumors have several distinctive characteristics in common including long and highly heterogeneous telomeres, multiprotein complexes called ALT-associated PML nuclear bodies (APBs) in which telomeric components (DNA and protein) co-localize with the PML nuclear body, and extrachromosomal circular DNA molecules that are composed of telomeric repeats. However, the relationship between extrachromosomal telomeric circles, APBs and telomere elongation has yet to be established due to the lack of a genetically tractable system in human cells to study the activation and regulation of the ALT-pathway. This also complicates and hampers the development of therapeutics targeting this telomerase- independent pathway. As an initial step towards developing a tractable genetic assay system, we have bioengineered a mammalian artificial chromosome to manufacture, on demand, extra- chromosomal telomeric circles in multiple cell types. The regulated production of extra- chromosomal telomeric circles will allow for the systematic testing of the impact of these circles on characteristics of the ALT pathway. This technology can be used in multiple cell types and/or combined with RNAi technology to test the role of specific pathways (genes) in ALT activation, a field that thus far has been refractory to controlled manipulation and exploration in human-derived model systems.