Ligand-mediated, vehicle-free delivery of small RNAs

项目总结 MicroRNAs(MiRNAs)已经成为基因组的强大调节者，通过共同努力 鉴定它们的功能并评估它们在体外和体内改变细胞生长的能力，一些已经获得了青睐 作为潜在的治疗方法。尽管这些基于miRNA的方法可以彻底改变肿瘤的治疗方式 在诊断和治疗方面，必须解决在体内传递miRNAs方面仍然存在的挑战。 为了应对这些挑战，我们开创了一种强大而有效的方法来进行配体介导的递送 MiRNAs(FolamiRs)，它已经移除了有毒运载工具。在本应用程序中，我们建议增强 并通过促进细胞内体介导的释放来表征FolamiR的靶向性 小分子黑素离子载体连接到FolamiRs上，并通过表征细胞间 叶酸受体阳性细胞与叶酸受体阴性细胞的旁观者效应。为了实现这些目标， 提出了以下三个具体目标：一、通过合成促进FolamiRs的内体释放 替代的Nig-FolamiR化学药物，II。在体内评价Nig-FolamiR的有效性和安全性，以及III。 评估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这三个目标。我们证明了临床上相关的 MiRNA，miRNA-34a(miR-34a)是选择性靶向肿瘤，进入致瘤细胞，下调 靶基因，并防止体内肿瘤的生长。成功取决于肿瘤的快速吸收，即 通过将miR-34a标记到相关的配体，在这种情况下是叶酸(FolamiR-34a)。事实上，叶酸受体 对包括乳腺癌、肺癌、卵巢癌、肾癌和结肠癌在内的各种肿瘤都有特异性的上调，这些肿瘤 可促进肿瘤特异性摄取FolamiRs。此外，使用小分子离子载体，黑素，我们 结果表明，FolamiRs的细胞内浓度可以由于内毒素释放而增加。临床部 黑素的实用性和安全性将在体内进行评估。黑素-FolamiR的其他化学成分 将合成和评估结合物，包括每个FolamiR包含多个黑素分子 增强内膜肿胀和破裂，以及防止黑素非特异性释放的化学作用。 Folamir在流通中。最后，细胞间旁观者效应的机制将被确定。 总体而言，这种创新的传递方法的影响远远超出了miRNA传递的范围。一个人可以 想象一下，将任何小的RNA连接到叶酸上，就可以实现快速、肿瘤特异性的摄取。同样，小的 正在测试内体介导的逃逸的分子离子载体有可能对许多 被隔离在内体中的治疗药物。再次强调了广泛而强大的影响 这些创新的研究将会有。

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.