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

描述（由申请人提供）：通过端粒酶或端粒替代延长（ALT）途径的激活实现端粒稳定是肿瘤获得无限复制潜力所必需的。大多数人ALT阳性细胞系和肿瘤具有几个共同的独特特征，包括长且高度异质的端粒，称为ALT相关PML核体（APB）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，以及由端粒重复序列组成的染色体外环状DNA分子。然而，染色体外端粒环，APB和端粒延长之间的关系尚未建立，由于缺乏在人类细胞中的遗传学上易于处理的系统来研究ALT途径的激活和调节。这也使靶向这种端粒酶非依赖性途径的治疗剂的开发复杂化并阻碍其发展。作为开发易于处理的遗传测定系统的第一步，我们已经生物工程化了哺乳动物人工染色体，以按需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技术结合，以测试特定途径（基因）在ALT激活中的作用，该领域迄今为止一直难以在人源模型系统中进行受控操作和探索。

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.