Tumor Cell Gene Correction by Bacterial Conjugation

描述(申请人提供)：肿瘤细胞基因矫正细菌接合 据估计，在西方世界，13%的结直肠癌与DNA错配修复途径缺陷有关。其中大约一半起源于遗传性缺陷，例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个DNA错配修复基因中的任何一个的突变有关。这项拟议工作的一个长期目标是通过使用正常菌群细菌作为基因供体，通过基因替代疗法在体内早期纠正遗传缺陷，防止HNPCC等结直肠癌的发生。我已经发表了建立细菌到哺乳动物细胞接合的实验，并在这里建议应用这种新的基因传递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。实验将首先确定与转基因相比，接合基因传递的靶向效率，使用开发的系统来纠正两个不同指示基因缺陷的CHO细胞。纠正一个基因将意味着随机插入；纠正另一个基因将需要通过同源重组来定位。结合提供单链DNA，有望通过同源重组在靶向方面优于转染法。接下来，细菌接合将用于纠正mMIh1缺陷的小鼠胚胎成纤维细胞，mMIh1是人类基因(HMLH1)的小鼠同源基因，通常与HNPCC有关。最后，将利用接合作用将hMLH1输送到人结肠肿瘤细胞(HCT116)。将对基因的基因组形式和cDNA形式进行比较，预计基因组形式将是适当的基因表达和恢复表型所必需的。这些实验将为细菌接合转移的发展提供良好的准备，旨在体内纠正缺陷基因。

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.