Ligand-mediated, vehicle-free delivery of small RNAs

项目摘要 microRNAs（miRNAs）已经成为基因组的强大调控因子，通过共同努力， 鉴定它们功能并评估它们在体外和体内改变细胞生长的能力，其中一些已经获得青睐 作为潜在的治疗方法。虽然这些基于miRNA的方法可以彻底改变肿瘤的方式， 在诊断和治疗的过程中，必须解决关于miRNA的体内递送的挑战。 为了应对这些挑战，我们开创了一种强大而有效的方法，用于配体介导的 miRNAs（FolamiRs），它已经去除了有毒的传递载体。在本申请中，我们建议增强 并通过使用该方法促进细胞内核内体介导的释放来表征FolamiR靶向。 小分子离子载体尼日利亚菌素缀合到FolamiRs上，并通过表征细胞间 叶酸受体阳性和叶酸受体阴性细胞之间的旁观者效应。为了实现这些目标， 提出了以下三个具体目标：通过合成促进FolamiRs的内体释放 替代Nig-FolamiR化学，II.评价Nig-FolamiRs的体内功效和安全性，和III. 评价FolamiR治疗后观察到的旁观者效应的机制。 我们广泛的初步证据支持所有三个目标。我们发现，临床相关的 miRNA，miRNA-34 a（miR-34 a）选择性靶向肿瘤，进入致瘤细胞，下调 靶向基因，并防止体内肿瘤生长。成功取决于快速的肿瘤吸收， 通过将miR-34 a标记到相关配体（在这种情况下为叶酸（FolamiR-34 a））介导。事实上，叶酸受体 在各种肿瘤中特异性上调，包括乳腺癌、肺癌、卵巢癌、肾癌和结肠癌， 可以促进FolamiR的肿瘤特异性摄取。此外，使用小分子离子载体尼日利亚菌素，我们 显示FolamiR的细胞内浓度可由于内体释放而增强。临床 将在体内评价尼日利亚菌素的效用和安全性。尼日利亚菌素-FolamiR的其他化学成分 将合成和评估缀合物，包括每个FolamiR包含多个尼日利亚菌素分子 以增强内体肿胀和破裂，以及防止尼日利亚菌素从 Folamir在循环中。最后，将确定参与细胞间旁观者效应的机制。 总的来说，这种创新的递送方法的意义远远超出了miRNA的递送。人能 想象一下，将任何小RNA连接到叶酸上，并实现快速和肿瘤特异性摄取。同样，小 正在测试内体介导的逃逸的分子离子载体有可能对许多 被隔离到内体中的治疗剂。再次强调了广泛而强大的影响， 这些创新的研究将有。

PROJECT SUMMARY MicroRNAs (miRNAs) have emerged as powerful regulators of the genome and, through concerted efforts to identify their function and evaluate their ability to alter cell growth in vitro and in vivo, some have gained favor as potential therapeutics. Although these miRNA-based approaches can revolutionize the way that tumors are diagnosed and treated, the challenges that remain with regard to in vivo delivery of miRNAs must be tackled. To address these challenges, we have pioneered a robust and efficient method for ligand-mediated delivery of miRNAs (FolamiRs), which has removed the toxic delivery vehicle. In this application we propose to enhance and characterize FolamiR targeting through facilitating intracellular endosomal-mediated release using the small molecule ionophore nigericin conjugated onto the FolamiRs, and through characterizing an intercellular bystander effect between folate receptor positive and folate receptor negative cells. To achieve these goals, the following three Specific Aims are proposed: I. Facilitate endosomal release of FolamiRs through synthesis of alternative Nig-FolamiR chemistries, II. Evaluate in vivo efficacy and safety of Nig-FolamiRs, and III. Evaluate the mechanism involved in the observed bystander effect following FolamiR treatment. Our extensive preliminary evidence supports all three Aims. We show that the clinically relevant miRNA, miRNA-34a (miR-34a) is selectively targeted to the tumor, enters the tumorigenic cells, downregulates target genes, and prevents growth of tumors in vivo. Success is dependent on rapid tumor uptake that is mediated by tagging miR-34a to a relevant ligand, in this case folate (FolamiR-34a). Indeed, the folate receptor is specifically upregulated on various tumors including breast, lung, ovarian, kidney, and colon cancers, which can facilitate tumor specific uptake of FolamiRs. Moreover, using a small molecule ionophore, nigericin, we show that intracellular concentrations of FolamiRs can be enhanced due to endosomal release. The clinical utility and safety profile of nigericin will be evaluated in vivo. Additional chemistries of nigericin-FolamiR conjugates will be synthesized and evaluated, including inclusion of multiple nigericin molecules per FolamiR to enhance endosomal swelling and rupture, and chemistries to prevent nonspecific release of nigericin from FolamiRs in circulation. Finally, the mechanism involved in the intercellular bystander effect will be determined. Overall, this innovative delivery method has implications well beyond miRNA delivery. One could imagine attaching any small RNA onto folate and achieving fast, and tumor-specific uptake. Similarly, the small molecule ionophore being tested for endosomal mediated escape has the potential to positively impact many therapeutics that are sequestered into the endosomes. Again, highlighting the broad, and powerful impact that these innovative studies will have.