Targeting epigenetic regulators using bacterial macromolecule delivery

描述（由申请人提供）：表观遗传学提供了许多可能彻底改变癌症治疗的靶点。表观遗传抑制剂的全身递送由于对再生组织的有害作用而不可行。用癌症特异性细菌递送抑制剂将通过特异性地集中治疗肿瘤和转移来克服这一关键问题。到目前为止，还没有人生产出调节表观遗传靶点的细菌。细菌递送系统将通过将肽和shRNA转运到癌细胞中来发挥作用。抑制关键靶点EZH 2，NIPP 1和PP 1将破坏基本的癌细胞过程并消除癌症干细胞。这将阻止肿瘤扩散并防止转移形成。拟议的研究有四个具体目标，联合收割机结合了表观遗传学和细菌癌症靶向领域的最新发展。Aim 1将创建一个细菌大分子递送系统; Aim 2将创建一个靶向NIPP 1和EZH 2的细菌shRNA递送系统; Aim 3将创建一个破坏NIPP 1：PP 1复合物的细菌肽递送系统; Aim 4将创建一个故障安全电路，以在治疗后清除细菌。将通过靶蛋白测量、免疫沉淀和磷酸酶磷酸化酶测定来检测基因敲减和复合物破坏的影响。细胞活力和癌症干细胞群将在肿瘤芯片装置、皮下肿瘤和小鼠自发转移中定量。本研究的基础已经通过以下方式建立：1）创建无毒细菌大分子递送系统，2）证明其关键组分（细胞内触发的裂解和细胞质定位）及其递送蛋白质和DNA的能力; 3）验证癌细胞和肿瘤中的NIPP 1和EZH 2敲减;以及4）显示NIPP 1：PP 1解离肽的细菌释放诱导癌细胞死亡。拟议的研究将建立在Neil福布斯（化学工程，马萨诸塞大学阿默斯特分校，美国），Aleyde货车Eynde和Mathieu Bollen（细胞和分子医学，KU Leuven，比利时），细菌癌症治疗和表观遗传调节专家之间的合作基础上。该研究将为可控基因和蛋白质递送到癌细胞中建立新的治疗平台。用细菌瞬时递送基因和蛋白质将能够直接靶向癌细胞中特异性的蛋白质和功能。这项研究将创造一种表观遗传疗法，有可能根除转移并防止其形成，这是两个紧迫的临床问题。拟议的动物实验将是第一个证明蛋白磷酸酶导向疗法的好处，这种疗法破坏了全酶中的蛋白质-蛋白质相互作用。该项目的最终目标是开发原发性癌症和转移性疾病的治疗模式。

DESCRIPTION (provided by applicant): Epigenetics offers many targets that could revolutionize cancer therapy. Systemic delivery of epigenetic inhibitors is unfeasible due to deleterious effects on regenerating tissue. Delivering inhibitors with cancer- specific bacteria would conquer this critical problem by specifically focusing treatment to tumors and metastases. To date, no one has produced bacteria that modulate epigenetic targets. Bacterial delivery systems would function by transporting peptides and shRNA into cancer cells. Inhibiting the key targets EZH2, NIPP1 and PP1 would disrupt essential cancer cell processes and eliminate cancer stems cells. This will stop tumor spreading and prevent metastasis formation. The proposed research has four Specific Aims that combine the newest developments in the fields of epigenetics and bacterial cancer targeting. Aim 1 will create a bacterial macromolecule delivery system; Aim 2 will create a bacterial shRNA delivery system that targets NIPP1 and EZH2; Aim 3 will create a bacterial peptide delivery system that disrupts NIPP1:PP1 complexes; and Aim 4 will create failsafe circuitry to clear bacteria after treatment. The effect of gene knockdown and complex disruption will be tested by target-protein measurement, immunoprecipitation, and phosphatase phosphorylase assay. Cell viability and the cancer stem cell populations will be quantified in a tumor-on-a-chip device, subcutaneous tumors and spontaneous metastases in mice. The groundwork for this study has been established by 1) creating a non-toxic bacterial macromolecule delivery system, 2) demonstrating its key components (intracellularly triggered lysis and cytoplasmic localization) and its ability to delive proteins and DNA; 3) validating NIPP1 and EZH2 knockdowns in cancer cells and tumors; and 4) showing that bacterial release of NIPP1:PP1 dissociative peptides induces cancer cell death. The proposed research will build upon a collaboration between Neil Forbes (Chemical Engineering, UMass Amherst, USA), and Aleyde Van Eynde and Mathieu Bollen (Cellular and Molecular Medicine, KU Leuven, Belgium), experts in bacterial cancer treatment and epigenetic regulators. This research will establish a new therapeutic platform for controlled gene and protein delivery into cancer cells. Transient delivery of genes and proteins with bacteria will enable direct targeting of proteins and functions specifically in cancer cells. This study will create an epigenetic therapy with the potential to eradicate metastases and prevent their formation, two urgent clinical problems. Proposed animal experiments will be the first to demonstrate the benefit of a protein-phosphatase-directed therapy that disrupts protein-protein interactions in holoenzymes. The ultimate goal of this project is development of a treatment modality for primary cancers and metastatic disease.