TNBC Ligand-displaying Exosomes Using RNA Nanotechnology for Targeted Cytosol Delivery of RNAi without Endosome Entrapment

TNBC 配体展示外泌体，利用 RNA 纳米技术实现 RNAi 的靶向胞质溶胶递送，无需内体截留

• 批准号: 10404055
• 负责人: Daniel W Binzel
• 金额: $ 32.46万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-11 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10404055
• 关键词: Antibodies; Apoptosis; Biodistribution; Breast Cancer Cell; Breast Cancer Patient; Breast Cancer Treatment; Breast Cancer therapy; Caveolae; Cell Proliferation; Cells; Chemicals; Clathrin; Clinic; Clinical Treatment; Clinical Trials; Complex; Cytosol; Data; Drug Delivery Systems; Drug resistance; Dyes; ERBB2 gene; Endocytosis; Endosomes; Estrogen Receptors; Evaluation; Gene Silencing; Genes; Goals; Growth; Homing; Hydrophobicity; Image; In Vitro; Investigation; Investigational New Drug Application; KB Cells; Label; Lead; Ligand Binding; Ligands; Malignant Neoplasms; Membrane; Membrane Fusion; Methods; Methotrexate; MicroRNAs; Nanotechnology; Oncogenic; Organ; Organelles; Pathway interactions; Patient-derived xenograft models of breast cancer; Phagocytosis; Progesterone Receptors; Prognosis; Proteins; RNA; RNA Interference; RNA Interference Therapy; Receptor Cell; Recurrence; Reporting; Safety; Site; Small Interfering RNA; Surface; System; Technology; Testing; Therapeutic; Toxic effect; Translations; Treatment Efficacy; Tumor Burden; Yin; aggressive breast cancer; aptamer; cancer cell; cancer subtypes; cancer therapy; chemotherapy; clinical translation; exosome; hormone therapy; imaging agent; immunogenicity; in vitro Model; in vivo; inhibitor; knock-down; lead candidate; malignant breast neoplasm; mortality; mouse model; nano; nanocarrier; nanoparticle; novel; patient derived xenograft model; pharmacokinetics and pharmacodynamics; pre-clinical; preclinical study; siRNA delivery; side effect; survivin; targeted delivery; therapeutic RNA; therapeutic candidate; therapeutic miRNA; therapeutic siRNA; trafficking; triple-negative invasive breast carcinoma; tumor; tumor growth; uptake

SUMMARY Triple negative breast cancer (TNBC) is a heterogeneous, complex, and aggressive breast cancer subtype. TNBC patients respond poorly to chemotherapy, leading to high mortality rates and a worsening prognosis. RNAi therapeutics, including siRNA and miRNA, have shown tremendous potential for TNBC cancer therapy. Developing a safe targeted delivery system with endosomal avoidance of the payload is crucial in terms of realizing the full potential of RNAi therapeutics and could revolutionize clinical treatment of TNBC. Our group has demonstrated that delivery of anti-miR21 to TNBC can efficiently inhibit TNBC cell proliferation (Shu D, et al. ACS Nano. 2015, 27:9731; Yin H, et al. Shu D. Mol Therapy. 2019, 27:1252). We have also recently reported the use of RNA nanoparticle orientation for decorating exosome surfaces with targeting ligands to deliver siRNA loaded exosomes for TNBC treatment. We proved that exosomes can be utilized as nanocarriers to deliver siRNA to TNBC cancer cells very efficiently to inhibit cancer growth (Pi F, Binzel D et al. Nat. Nanotechnol. 2018;13:82). Preliminary data from the investigation with KB cell models in vitro has shown the mechanism behind the high efficiency of cancer inhibition to be the cytosolic delivery of siRNA via exosomes without endosomal trapping (Zhen Z, et al. J Control Release. 2019,311:43). In this study, we will investigate the targeting and delivery mechanisms of RNAi to TNBC cells by exosomes displayed with TNBC specific ligands for targeted delivery. It’s our goal to select a lead TNBC therapeutic candidate to move towards potential clinical translation. We will combine the targeting and drug delivery capabilities of RNA nanotechnology and exosomes for targeted delivery of RNAi to cell cytosols without endosomal entrapment. We will construct and evaluate the multi-functional exosomal RNA nanoparticle complexes harboring targeting ligands including RNA aptamers (EGFRapt, or alternatively, CD133apt, CD44apt, and nAHRsapt) or chemical ligand (Methotrexate) and tumor suppressing RNAi therapeutics (siRNA, suppressor miRNAs and anti- oncogenic miRNA). We will further investigate the pathways of internalization and intracellular trafficking in addition to investigating whether the payloads in the exosome are delivered to cytosol via fusion or to the endosome via endocytosis. In depth studies of the release and cellular processing of RNAi cargo loaded into exosomes will be completed. We will investigate the PK/PD parameters, delivery mechanisms, antitumor efficacy, and safety of the therapeutic RNA nanoparticles in order to select a lead candidate for preclinical translation. The preclinical studies will give a clear understanding of the viability of exosome-RNA nanoparticle complexes for TNBC therapy and provide data to move towards an Investigational New Drug (IND) application that will facilitate advancement to clinical trials.

总结 三阴性乳腺癌（TNBC）是一种异质性、复杂性和侵袭性乳腺癌亚型。 TNBC患者对化疗反应不佳，导致高死亡率和预后恶化。 RNAi疗法，包括siRNA和miRNA，已经显示出用于TNBC癌症治疗的巨大潜力。 开发具有有效载荷的内体回避的安全靶向递送系统在以下方面至关重要： 实现RNAi疗法的全部潜力，并可能彻底改变TNBC的临床治疗。 我们的小组已经证明，将抗miR 21递送至TNBC可以有效地抑制TNBC细胞增殖 (Shu D等人ACS Nano. 2015，27：9731; Yin H等人Shu D.摩尔疗法2019，27：1252）。我们还 最近报道了使用RNA纳米颗粒取向来用靶向配体装饰外泌体表面 以递送装载siRNA的外来体用于TNBC治疗。我们证明了外泌体可以被用作 纳米载体非常有效地将siRNA递送至TNBC癌细胞以抑制癌症生长（Pi F，Binzel D et al. Nat.纳米技术。2018;13：82）。体外KB细胞模型研究的初步数据显示， 高效率的癌症抑制背后的机制是通过外泌体的siRNA的胞质递送 不存在内体捕获（Zhen Z等人，J Control Release. 2019，311：43）。在这项研究中，我们将调查 通过展示有TNBC特异性的外泌体，RNAi对TNBC细胞的靶向和递送机制 用于靶向递送的配体。我们的目标是选择一种领先的TNBC治疗候选药物， 临床翻译我们将联合收割机结合RNA纳米技术的靶向和药物输送能力， 本发明涉及用于将RNAi靶向递送至细胞胞质溶胶而没有内体截留的外泌体。 我们将构建和评估多功能外泌体RNA纳米颗粒复合物， 靶向配体，包括RNA适体（EGFRapt，或者CD 133 apt、CD 44 apt和nAHRsapt）或 化学配体（甲氨蝶呤）和肿瘤抑制RNAi治疗剂（siRNA、抑制性miRNA和抗- 致癌miRNA）。我们将进一步研究内化和细胞内运输的途径， 除了研究外泌体中的有效载荷是否通过融合递送到胞质溶胶或递送到细胞外膜外， 胞内体通过胞吞作用。在深入研究的释放和细胞加工的RNAi货物加载到 exosomes将完成。我们将研究PK/PD参数、给药机制、抗肿瘤作用， 有效性和安全性的治疗性RNA纳米颗粒，以选择一个领先的候选人， 翻译.临床前研究将使我们对外泌体-RNA纳米颗粒的可行性有一个清晰的认识 用于TNBC治疗的复合物，并提供数据以推动研究性新药（IND）申请 这将有助于推进临床试验。