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

描述(由申请人提供)：BRCA1基因有一个遗传突变的女性患乳腺癌和卵巢癌的易感性大大增加。然而，由于缺乏合适的人类细胞系模型，对特定BRCA1突变以及BRCA1基因杂合性对乳腺上皮细胞生物学和乳腺癌发生的影响的重点研究一直受到阻碍。为了解决这一缺点，我们建议使用基于三螺旋的基因编辑技术来设计新的人类乳房来源的非恶性细胞系，以在BRCA1基因座引入精确定义的基因类型。这些基因类型将包括BRCA1基因的野生型、杂合型或纯合型突变，以及人类家族性癌症中发现的两个常见有害等位基因(185delAG和5382insC)的突变。这些独特的BRCA1野生型、杂合型和纯合子突变细胞系的配对组合将使BRCA1基因相同、乳房来源的细胞与已定义的BRCA1基因类型进行生理学上的比较。我们将描述这些细胞BRCA1功能的关键方面，包括转录调控、DNA损伤反应和DNA修复。 公共卫生相关性：BRCA1基因的遗传突变是乳腺癌发生的主要因素，但由于缺乏合适的人类细胞系，对特定BRCA1突变的影响的重点研究一直受到阻碍。为了解决这一缺点，我们建议使用一种新的基因编辑技术来设计新的人类乳房来源的非恶性细胞系，以在人类乳房来源的细胞中引入精确定义的BRCA1基因突变。这些独特的配对细胞组将使BRCA1基因类型确定的乳腺来源细胞之间进行重要的和生理上相关的比较，并将随时提供给科学界，以促进广泛的癌症发生和癌症生物学研究。

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. PUBLIC HEALTH RELEVANCE: Inherited mutations in the BRCA1 gene are a major factor in breast carcinogenesis, but focused study of the impact of specific BRCA1 mutations has been hampered by the lack of suitable human cell lines. To address this shortcoming, we propose to engineer novel human breast-derived, non-malignant cell lines using a novel gene editing technology to introduce precisely defined mutations in the BRCA1 gene in human breast- derived cells. These unique matched sets of cells will enable important and physiologically relevant comparisons among breast-derived cells with defined genotypes for BRCA1, and they will be made readily available to the scientific community to facilitate a wide range of carcinogenesis and cancer biology studies.