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

 描述（由申请人提供）：RNA干扰（RNAi）提供了通过敲除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已经显示出在由肝脏中异常基因表达引起的少数疾病中有希望的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RNA递送到扩散的癌细胞。大多数上皮癌细胞和其中的肿瘤起始细胞（T-IC）高度表达EpCAM，EpCAM是第一个被描述的肿瘤抗原。我们测试的所有上皮乳腺癌细胞系对EpCAM染色明亮，而永生化的正常乳腺上皮细胞和成纤维细胞则没有。体外上皮癌细胞中的靶向基因敲低可以使用嵌合RNA来实现，所述嵌合RNA由被称为适体的结构化RNA组成，所述适体被选择用于与EpCAM的高亲和力结合，所述适体与siRNA共价连接。这些EpCAM适体-siRNA嵌合体（AsiC）被EpCAM+细胞摄取，并选择性地引起上皮乳腺癌细胞中的基因敲低，但不引起正常上皮细胞中的基因敲低。此外，用EpCAM-AsiC敲低PLK 1抑制上皮乳腺癌细胞系的集落和乳腺球形成、肿瘤引发潜力的体外测定和肿瘤引发。皮下注射的PLK 1 EpCAM-AsiC被EpCAM+基底细胞-A三阴性乳腺癌（TNBC）原位异种移植物特异性摄取，并引起快速肿瘤消退。TNBC是所有乳腺癌中预后最差的，目前尚无靶向治疗方法。本提案的目的是评估EpCAM-AsiC是否可用于靶向基因敲除，以治疗上皮（基底样）TNBC癌，保留正常细胞，并消除其中的T-IC。一个目标是确定哪些乳腺癌亚型可以被EpCAM-AsiC靶向，并确定EpCAM水平如何影响摄取和基因沉默。将在人乳腺癌组织外植体中评价表达EpCAM的癌细胞和正常上皮中的相对摄取/敲低。另一个目标是确定EpCAM-AsiC是否可以靶向乳腺T-IC以破坏肿瘤起始。一个重要的目标是优化EpCAM-AsiC的药物样特征，以产生适合临床评价的候选药物。为了实现这一目标，我们将优化EpCAM-AsiC的细胞摄取、内体释放、全身递送和体内基因敲除。将在TNBC细胞系异种移植模型中使用活体动物成像来评估EpCAM-AsiC摄取和基因沉默以及肿瘤抑制的药代动力学（PK）和药效学（PD）。作为原理证明，我们将评估PLK 1敲低的抗肿瘤作用，这是细胞增殖所需的。此外，将在小鼠异种移植模型中评价在全基因组siRNA筛选中鉴定的TNBC遗传依赖性的新基因靶标的敲低。在该项目结束时，我们将获得优化的EpCAM-AsiC及其PK、PD和可能的毒性知识，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.