Eradication of Human Ovarian Cancer Cells by Transgenic Expression of Recombinant DNASE1, DNASE1L3, DNASE2, and DFFB Controlled by EGFR Promoter: Novel Strategy for Targeted Therapy of Cancer.
Eradication of Human Ovarian Cancer Cells by Transgenic Expression of Recombinant DNASE1, DNASE1L3, DNASE2, and DFFB Controlled by EGFR Promoter: Novel Strategy for Targeted Therapy of Cancer.
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DOI：
10.4172/2157-7412.1000152
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发表时间：
2013-07-21
期刊：
影响因子：
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通讯作者：
Malecki R
中科院分区：
文献类型：
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作者：
Malecki M;Dahlke J;Haig M;Wohlwend L;Malecki R
卵巢癌在所有妇科癌症中是最致命的。接受晚期卵巢癌全身治疗的患者遭受着可怕的副作用。癌症幸存者及其后代遭受全身治疗的医源性后果：基因突变。我们工作的最终目标是开发出能选择性且完全消灭癌细胞，但不伤害健康细胞的治疗方法。实现这一目标的一个重要考虑因素是卵巢癌细胞过度表达表皮生长因子受体（EGFR）或其突变体，这成为将它们与健康细胞区分开来的因素——一种个性化治疗的潜在促进因素。
该项目有三个具体目标：（1）利用针对表皮生长因子受体变异体III（EGFRvIII）和表皮生长因子受体（EGFR）的合成抗体引导，对携带自杀基因的载体进行生物工程改造；（2）对由表皮生长因子受体启动子控制的人源重组DNA酶1（DNASE1）、DNASE1L3、DNASE2和DFFB的DNA构建体进行基因工程改造；（3）通过对转基因表达的重组DNA酶进行核内靶向，选择性地根除卵巢癌细胞。
从人源文库中筛选出针对表皮生长因子受体（EGFR）和表皮生长因子受体变异体III（EGFRvIII）的合成抗体，并用于对生物标签引导的转基因载体进行生物工程改造。由表皮生长因子受体启动子控制的人源DNASE1、DNASE1L3、DNASE2、DFFB的编码序列从人源cDNA中扩增出来，并通过基因工程技术构建到同时编码与核定位信号（NLS）和绿色荧光蛋白（GFP）融合的质粒构建体中。携带DNA酶转基因的载体在体外被导入来自腹水和培养物的人卵巢癌细胞中。
合成抗体引导的载体有效地将重组DNA酶的转基因导入卵巢癌细胞。这些细胞中DNA酶的转基因表达和核靶向导致其基因组被破坏并致使细胞死亡，这通过分子死亡标签标记得到验证。在不过度表达表皮生长因子受体（EGFR）的健康细胞中，未记录到任何变化。
在体外卵巢癌中，重组DNASE1、DNASE1L3、DNASE2、DFFB的靶向表达导致癌细胞被完全根除，但对健康细胞没有影响。这种新的治疗策略有可能作为卵巢癌和其他癌症的个性化、靶向治疗被简化用于体内试验。
Ovarian cancer is the most deadly among all gynecological cancers. Patients undergoing systemic therapies of advanced ovarian cancers suffer from horrendous side effects. Cancer survivors and their offspring suffer from iatrogenic consequences of systemic therapies: genetic mutations. The ultimate goal of our work is development of therapies, which selectively and completely eliminate cancer cells, but do not harm healthy cells. An important consideration for attaining this goal is the fact that ovarian cancer cells over-express EGFR or its mutants, what becomes the factor discriminating them from healthy cells - a potential facilitator of personalized therapy. The specific aim of this project was threefold: (1) to bioengineer suicide genes’ carrying vectors guided by synthetic antibodies for EGFRvIII and EGFR; (2) to genetically engineer DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, and DFFB controlled by the EGFR promoter; (3) to selectively eradicate ovarian cancer cells by intranuclear targeting of the transgenically expressed recombinant DNases. Synthetic antibodies for EGFR and EGFRvIII were selected from the human library and used to bioengineer biotag-guided transgenes’ vectors. Coding sequences for the human DNASE1, DNASE1L3, DNASE2, DFFB controlled by the EGFR promoter were amplified from the human cDNA and genetically engineered into the plasmid constructs also coding for the fusions with NLS and GFP. The vectors carrying transgenes for the DNases were delivered in vitro into human ovarian cancer cells from ascites and cultures. Synthetic antibody guided vectors delivered the transgenes for the recombinant DNases efficiently into the ovarian cancer cells. Transgenic expression and nuclear targeting of the DNases in those cells resulted in destruction of their genomes and led to their death, as validated by labeling with the molecular death tags. In healthy cells, which did not over-express EGFR, no changes were recorded. Targeted expression of the recombinant DNASE1, DNASE1L3, DNASE2, DFFB in the ovarian cancers in vitro resulted in their complete eradication, but had no effects upon the healthy cells. This novel therapeutic strategy has a potential for streamlining it into in vivo trials, as personalized, targeted therapy of ovarian and other cancers.