Tumor Cell Gene Correction by Bacterial Conjugation

通过细菌接合校正肿瘤细胞基因

• 批准号: 6918692
• 负责人: VIRGINA L WATERS
• 金额: $ 13.86万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6918692
• 关键词: CHO cells; DNA; biotechnology; cancer prevention; cell cell interaction; colorectal neoplasms; complementary DNA; enteric bacteria; fibroblasts; gene delivery system; gene expression; gene targeting; gene therapy; genetic disorder; host organism interaction; human genetic material tag; microorganism conjugation; neoplasm /cancer genetics; neoplastic cell; phenotype; therapy design /development; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Tumor Cell Gene Correction Bacterium Conjugation It is estimated that 13 percent of colorectal cancers in the Western world are associated with defects in DNA mismatch repair pathways. About half of these originate as inherited defects, such as in hereditary non-polyposis colorectal cancer (HNPCC). Studies of HNPCC families have led to the association of tumors with mutations in any of five DNA mismatch repair genes. One long-range goal of the proposed work would be to prevent the onset of colorectal cancers such as HNPCC by early in vivo correction of genetic defects by gene replacement therapy, using normal flora bacteria as gene donors. I have published experiments establishing bacterium-to-mammalian cell conjugation, and propose here to apply this novel method of gene delivery to correct tissue culture cells defective in the gene most commonly associated with HNPCC. Experiments will first determine the targeting efficiencies of conjugative gene delivery, as compared to transfection, using a system developed to correct CHO cells defective in two different indicator genes. Correcting one gene will indicate random insertion; correcting the other will require targeting by homologous recombination. Conjugation, which supplies single-stranded DNA, is expected to out-perform transfection in targeting by homologous recombination. Next, bacterial conjugation will be used to correct mouse embryonic fibroblasts defective in mMIhl, the murine homologue of the human gene (hMLH 1) commonly associated with HNPCC. Lastly, conjugation will be used to deliver hMLH1 to human colon tumor cells (HCT116). Delivery of genomic and cDNA forms of the gene will be compared, and it is expected that the genomic form will be necessary for proper gene expression and restored phenotype. These experiments will provide good preparation for the development of bacterial conjugative transfer designed to correct defective genes in vivo.

描述（申请人提供）：肿瘤细胞基因校正细菌结合物 据估计，西方世界13%的结直肠癌与DNA错配修复途径缺陷有关。其中约一半起源于遗传缺陷，如遗传性非息肉病性结直肠癌（HNPCC）。对HNPCC家族的研究已经导致肿瘤与五种DNA错配修复基因中的任何一种突变相关。这项工作的一个长期目标是利用正常植物群细菌作为基因供体，通过基因替代疗法早期体内纠正遗传缺陷，预防结肠直肠癌如HNPCC的发生。我已经发表了建立细菌与哺乳动物细胞接合的实验，并在此提出应用这种新的基因递送方法来纠正组织培养细胞中最常见的与HNPCC相关的基因缺陷。实验将首先确定接合基因递送的靶向效率，与转染相比，使用开发用于校正两种不同指示基因缺陷的CHO细胞的系统。纠正一个基因将表明随机插入;纠正另一个基因将需要通过同源重组进行靶向。提供单链DNA的缀合预期在通过同源重组的靶向中优于转染。接下来，将使用细菌接合来校正mMIhl缺陷的小鼠胚胎成纤维细胞，mMIhl是通常与HNPCC相关的人基因（hMLH 1）的鼠同源物。最后，缀合将用于将hMLH 1递送至人结肠肿瘤细胞（HCT 116）。将比较基因组和cDNA形式的基因的递送，并且预期基因组形式对于适当的基因表达和恢复的表型是必需的。这些实验为进一步发展细菌接合转移技术在体内修复缺陷基因提供了良好的基础。