Novel triplex-engineered, BRCA1-mutated cell lines for research

描述（由申请人提供）：BRCA1基因中有一个遗传突变的女性患乳腺癌和卵巢癌的易感性显著增加。然而，特定的BRCA1突变的影响，重要的是，在BRCA1基因座的杂合性，对乳腺上皮细胞的生物学和乳腺癌发生的集中研究受到阻碍，缺乏合适的人类细胞系模型。 为了解决这一缺点，我们建议使用基于三链螺旋的基因编辑技术来设计新型人类乳腺来源的非恶性细胞系，以在BRCA1基因座处引入精确定义的基因型。这些基因型将由BRCA1基因上的野生型、杂合或纯合突变组成，这两种突变是在人类家族性癌症中发现的两种常见有害等位基因（185delAG和5382insC）。这些独特的BRCA1野生型、杂合型和纯合型突变细胞系的匹配集将使具有BRCA1定义基因型的其他同基因乳腺衍生细胞之间的生理学相关比较成为可能。我们将描述这些细胞的BRCA1功能的关键方面，包括转录和DNA损伤反应和DNA修复的调节。

DESCRIPTION (provided by applicant): Women with one inherited mutation in the BRCA1 gene have a substantially increased predisposition to breast and ovarian cancer. However, focused study of the impact of specific BRCA1 mutations and, importantly, of heterozygosity at the BRCA1 locus, on the biology of breast epithelial cells and on breast carcinogenesis has been hampered by the lack of suitable human cell line models. To address this shortcoming, we propose to engineer novel human breast-derived, non-malignant cell lines using triplex-helix-based gene editing technology to introduce precisely defined genotypes at the BRCA1 locus. These genotypes will consist of wild-type, heterozygous, or homozygous mutations at the BRCA1 gene for two of the common deleterious alleles found in human familial cancers (185delAG and 5382insC). These unique matched sets of BRCA1 wild-type, heterozygous, and homozygous mutant cell lines will enable physiologically relevant comparisons between otherwise isogenic, breast-derived cells with defined genotypes for BRCA1. We will characterize these cells for key aspects of BRCA1 function, including regulation of transcription and of DNA damage responses and DNA repair.