Development of a nanoparticle based anti-tumor gene therapy for treatment of breast cancer

乳腺癌是世界上最常见的癌症之一，也是女性癌症相关死亡的最常见原因。由于三阴性乳腺癌缺血治疗是目前唯一可用的治疗方法，因此需要有针对性的治疗来改善预后和减少副作用。该项目代表了德国(UKER-seon)和以色列(TAU)在中东地区的合作，旨在开发一种基于纳米颗粒的抗肿瘤基因疗法，用于乳腺癌的治疗。毒素的基因治疗是基于毒素基因在癌细胞中的特异性表达，例如白喉毒素A(DTA)，它阻断了细胞的翻译机制。为了限制DTA在肿瘤细胞上的表达，已经开发了一个由两个质粒组成的二元系统(TAU)。质粒1携带DTA基因，该基因通过转录终止子从肿瘤特异性启动子hTERT中分离出来。转录终止子的两侧是AttR和attL重组位点。第二个质粒编码hTERT启动子控制下的位点特异性重组酶(SSR)基因。一旦重组酶在肿瘤细胞中表达，它就会从质粒1中切下重组终止子，DTA就会表达出来。在患有肺癌的小鼠身上，已经证明二元系统在效率和副作用方面优于只有一种质粒的系统。到目前为止，一个问题是将足够数量的质粒带到肿瘤中，因为许多质粒在应用于血液循环后已经丢失。利用超顺磁性氧化铁纳米颗粒(SPION)的磁控传输将提高质粒在肿瘤内的浓度。磁性药物靶向的原理已经成功地应用于化疗药物米托蒽醌在荷瘤兔体内的转运。在拟议的项目中，将开发携带DTA和重组酶质粒的SPION。将对SPIONS进行全面的物理化学和毒理学表征，并在体外测试其在乳腺癌细胞株上的抗肿瘤效果。最后，将研究其在不同乳腺癌小鼠模型中的疗效。在这里，可以通过增强的渗透和滞留效应(EPR)被动地或通过肿瘤中的磁控来主动地丰富SPION。两种方案在药代动力学、生物分布和抗肿瘤疗效方面进行了比较。在项目的每个阶段，两个项目合作伙伴之间会发生反馈循环，从而使粒子合成在项目过程中得到优化，并适应各自的问题。通过以纳米颗粒为基础的表达载体的靶向性，我们期望在提高抗肿瘤效果的同时减少毒副作用。

Breast cancer is one of the most common cancers worldwide and the most common cause of cancer related death in women. Because theonly currently available therapy for triple-negative breast cancer ischemotherapy, targeted therapies are needed to improve prognosis and reduce side effects. The project, which represents a Middle East cooperation between Germany (UKER-SEON) and Israel (TAU), aims to develop a nanoparticle-based anti-tumor gene therapy for the treatment of breast cancer. Gene therapy by toxins is based on the specific expression of a toxin gene in the cancer cells, e.g. Diphteria toxin A (DTA), which blocks the translation machinery of the cell. To limit the expression of DTA on tumor cells, a binary system of two plasmids has been developed (TAU). Plasmid 1 carries the DTA gene, which is separated from its tumor-specific promoter hTERT by atranscription terminator. The transcriptional terminator is flanked by attR and attL recombination sites. A second plasmid encodes thegene for a site-specific recombinase (SSR) under the control of a hTERT promoter. Once the recombinase is expressed in tumor cells, it cuts out the recombination terminator from plasmid 1 and DTA is expressed. In mice with lung carcinomas, the binary system has already been shown to be superior to the system with only one plasmid in terms of efficiency and side effects. A problem so far is to bring the plasmids in sufficient amount to the tumor, because many plasmids are already lost after the application in the blood circulation.The intratumoral concentration of the plasmids will be enhanced bymagnetically controlled transport using superparamagnetic iron oxide nanoparticles (SPIONs). The principle of magnetic drug targeting has already been successfully applied to the transport of thechemotherapeutic agent mitoxantrone in tumor-bearing rabbits (UKER-SEON). In the proposed project SPIONs carrying DTA andrecombinase plasmids will be developed. The SPIONs will be comprehensively characterized physico-chemically and toxicologically and tested for their anti-tumor efficacy in vitro on breast cancer cell lines. Finally, the efficacy in various breast cancer mouse models will be investigated. Here it is possible to enrich the SPIONs either passively via the Enhanced Permeation and Retention Effect (EPR) or actively by means of magnetic control in the tumor. Both options are compared in terms of pharmacokinetics, biodistribution and anti-tumor efficacy. At each stage of the project, feedback loops will take place between the two project partners, so that particle synthesis will be optimized in the course of the project and adapted to the respective question. By nanoparticle-based targeting of expression plasmids we expect an improved anti-tumor effect with simultaneous reduction oftoxic side effects.