Aptamer-siRNA gene knockdown to treat epithelial triple negative breast cancer

 描述(申请人提供)：RNA干扰(RNAi)提供了通过击倒致病基因来治疗疾病的令人兴奋的机会。最近的早期临床试验显示，在少数由肝脏基因表达异常引起的疾病中，有希望和持续的基因敲除和/或临床益处。利用RNAi进行癌症治疗的主要障碍是将小RNAs输送到播散性癌细胞。大多数上皮性癌细胞及其内部的肿瘤起始细胞(T-IC)都高度表达EpCAM，这是第一个被发现的肿瘤抗原。我们测试的所有上皮性乳腺癌细胞株都有明亮的EpCAM染色，而永生化的正常乳腺上皮细胞和成纤维细胞则没有。利用嵌合RNA可以在体外实现上皮癌细胞的靶向基因敲除。嵌合RNA由一种称为适体的结构RNA组成，该适体被选为与EpCAM高亲和力结合，并与siRNA共价连接。这些EpCAM适体-siRNA嵌合体(ASIC)被EpCAM细胞摄取，并选择性地导致上皮性乳腺癌细胞的基因敲除，而不是正常的上皮性细胞。此外，用EpCAM-ASICS敲除PLK1可以抑制上皮性乳腺癌细胞系的集落和乳房形成，体外分析肿瘤启动潜力，以及肿瘤启动。皮下注射PLK1 EpCAM-ASIC可被EpCAM BaseA三阴性乳腺癌(TNBC)原位异种移植特异性摄取，并导致肿瘤快速消退。TNBC是所有乳腺癌中预后最差的，目前还没有针对性的治疗方法。这项建议的目的是评估EpCAM-ASICS是否可以用于靶向基因敲除，以治疗上皮性(基底细胞样)TNBC癌，保留正常细胞，并消除其中的T-IC。一个目标是确定EpCAM-ASICS可以针对哪些乳腺癌亚型，并确定EpCAM水平如何影响摄取和基因沉默。在人类乳腺癌组织外植体中，将评估表达EpCAM的癌细胞和正常上皮的相对摄取/敲除。另一个目标是确定EpCAM-ASICS是否可以靶向乳腺T-IC来干扰肿瘤的启动。一个重要的目标是优化EpCAM-ASICS的类药物特性，以生产出适合临床评估的候选药物。为了实现这一目标，我们将优化EpCAM-ASICS的细胞摄取、内体释放、全身递送和体内基因敲除。在TNBC细胞系异种移植模型中，将使用活体动物成像来评估EpCAM-ASIC摄取、基因沉默和肿瘤抑制的药代动力学(PK)和药效学(PD)。作为原理的证明，我们将评估PLK1基因敲除的抗肿瘤效果，这是细胞增殖所必需的。此外，在小鼠异种移植模型中，将评估在全基因组siRNA筛查中确定的TNBC遗传依赖性的新基因靶点的敲除。在该计划结束时，我们将拥有一个优化的EpCAM-ASIC，并了解其PK、PD和可能的毒性，为启动临床概念验证研究所需的正式毒性和其他临床前研究奠定基础。

 DESCRIPTION (provided by applicant): RNA interference (RNAi) offers the exciting opportunity to treat disease by knocking down disease-causing genes. Recent early phase clinical trials have shown promising and sustained gene knockdown and/or clinical benefit in a handful of diseases caused by aberrant gene expression in the liver. The major obstacle to harnessing RNAi for cancer treatment is delivery of small RNAs to disseminated cancer cells. Most epithelial cancer cells and the tumor-initiating cells (T-IC) within them highly express EpCAM, the first described tumor antigen. All epithelial breast cancer cell lines we tested stain brightly for EpCAM, while immortalized normal breast epithelial cells and fibroblasts do not. Targeted gene knockdown in epithelial cancer cells in vitro can be achieved using chimeric RNAs composed of a structured RNA, called an aptamer, selected for high affinity binding to EpCAM, that is covalently linked to an siRNA. These EpCAM aptamer-siRNA chimeras (AsiC) are taken up by EpCAM+ cells and selectively cause gene knockdown in epithelial breast cancer cells, but not normal epithelial cells. Moreover knockdown of PLK1 with EpCAM-AsiCs suppresses colony and mammosphere formation of epithelial breast cancer lines, in vitro assays of tumor-initiating potential, and tumor initiation. Subcutaneously injected PLK1 EpCAM-AsiCs are taken up specifically by EpCAM+ basal-A triple negative breast cancer (TNBC) orthotopic xenografts and cause rapid tumor regression. TNBC has the worst prognosis of any breast cancer and there is no targeted therapy for it. The goal of this proposal is to evaluate whether EpCAM-AsiCs can be used for targeted gene knockdown to treat epithelial (basal-like) TNBC cancers, sparing normal cells, and eliminate the T-ICs within them. One goal is to define which breast cancer subtypes can be targeted by EpCAM-AsiCs and determine how EpCAM level affects uptake and gene silencing. Relative uptake/knockdown in cancer cells expressing EpCAM and normal epithelium will be evaluated in human breast cancer tissue explants. Another goal is to determine whether EpCAM-AsiCs can target breast T-ICs to disrupt tumor initiation. An important goal is to optimize the drug-like features of EpCAM-AsiCs to produce a drug candidate suitable for clinical evaluation. To achieve this aim, we will optimize EpCAM-AsiCs for cell uptake, endosomal release, systemic delivery and in vivo gene knockdown. Pharmacokinetics (PK) and pharmacodynamics (PD) of EpCAM-AsiC uptake and gene silencing and tumor suppression will be evaluated using live animal imaging in TNBC cell line xenograft models. As proof of principle, we will evaluate the antitumor effect of knockdown of PLK1, which is needed for cell proliferation. In addition knockdown of novel gene targets identified in a genome-wide siRNA screen for TNBC genetic dependencies will be evaluated in mouse xenograft models. At the end of this program, we will have an optimized EpCAM-AsiC and knowledge of its PK, PD and possible toxicity, to lay the groundwork for formal toxicity and other preclinical studies needed to initiate clinical proof of concept studies.