Ligand-mediated, vehicle-free delivery of small RNAs

项目摘要 就像十多年前抗体治疗领域面临的挑战和怀疑一样，RNA 治疗学也面临着同样的问题。而且，就像抗体治疗领域一样，我们开始意识到临床 RNA治疗的影响忽略了这些挑战。这一点在最近批准的 两种用于预防SARS-CoV-2的mRNA疫苗和前三种FDA批准的RNAi药物， 肝脏不幸的是，靶向癌细胞的基于RNA的药物是落后的，即使与无数年 这项研究揭示了利用RNAi治疗肿瘤疾病的力量。在这方面缺乏成功 空间的限制是由于不能安全和有效地递送RNAi。我们以前开发了一种方法， 安全地将治疗性microRNAs（miRNAs）递送到过表达叶酸受体的肿瘤中。但 由于miRNA的不稳定性和不良的药代动力学， 频繁给药。为了克服这些不相容性，需要保持靶向活性的稳定的miRNA。 最近，我们筛选了一组完全修饰的miR-34 a（FM-miR-34 a），并鉴定了一种具有>400- 当与叶酸盐缀合时，稳定性增加了10倍，并且具有突出的体内功效。植入小鼠的治疗 用叶酸-FM-miR-34 a进行乳腺癌异种移植，6只小鼠中有2只完全治愈， 其余4例肿瘤明显消退。基于这些令人兴奋的数据，我们建议推进FM- miR-34以两种方式表达。在目标1中，我们将评估FM-miR-34 a在人乳腺癌中的活性、疗效和安全性。 肺癌和前列腺癌的体内模型。FM-miR-34 a将缀合至：i）叶酸以递送至肺癌， 和ii）PSMA-617，靶向前列腺特异性膜抗原（PSMA）以递送至前列腺的配体 癌在目标2中，我们提出利用FM-miR-34提供的稳定性来增加循环1/2。 叶酸-FM-miR-34和PSMA-617-FM-miR-34 a通过掺入白蛋白结合部分（ABM）的时间 进入配体。使用这些配体，我们将评估血清白蛋白结合和新配体的稳定性。我们 还将验证与ABM的缀合不会改变miR-34 a的活性，也不会改变细胞结合和内化 动力学最后，我们将评估配体-ABM-miR-34 a缀合物的体内分布。 在这项工作完成后，我们希望有一个包罗万象的miRNA运载工具，可以 将稳定的肿瘤抑制RNA特异性靶向NSCLC和前列腺癌。我们还将有新的 具有增加的循环半寿命的配体。所获得的数据最终将在癌症方面产生重大影响 通过提供新的机会来推进下一阶段的基于miRNA的治疗。而 基于miR-34 a用于治疗其他癌症的效用， 叶酸受体在许多上皮癌中单独过表达，包括卵巢癌、肾癌和结肠癌 癌症，成功完成这项研究可能会产生深远的积极影响。

PROJECT SUMMARY Like the challenges and skepticism that faced the antibody therapeutics field over a decade ago, RNA therapeutics is facing the same. And, like the antibody therapeutics field, we are beginning to realize the clinical impact of RNA therapeutics amiss these challenges. This is most clearly highlighted with the recent approval of two mRNA vaccines to prevent against SARS-CoV-2 and the first three FDA approved RNAi drugs targeted to the liver. Unfortunately, RNA-based drugs targeted to cancer cells is lagging behind, even with countless years of work that has revealed the power of using RNAi for treating oncological diseases. Lack of success in this space is attributed to inability to deliver RNAi safely and effectively. We previously developed a method that can safely deliver therapeutic microRNAs (miRNAs) to tumors that overexpress the folate receptor. However, the anti-tumor response was short-lived due to instability of the miRNA and poor pharmacokinetics, necessitating frequent dosing. To overcome these insufficiencies requires a stabilized miRNA that retains targeting activity. Recently we screened a panel of fully modified versions of miR-34a (FM-miR-34a) and identified one with >400- fold increased stability and outstanding in vivo efficacy when conjugated to folate. Treatment of mice implanted with breast cancer xenografts with folate-FM-miR-34a resulted in complete cures in two out of six mice and significant tumor regression in the remaining four. Based on this exciting data, here we propose to advance FM- miR-34 forward in two ways. In Aim 1 we will evaluate the activity, efficacy, and safety profile of FM-miR-34a in in vivo models of lung and prostate cancer. FM-miR-34a will be conjugated to: i) folate for delivery to lung cancer, and ii) PSMA-617, a ligand that targets prostate specific membrane antigen (PSMA) for delivery to prostate cancer. In Aim 2 we propose to capitalize on the stability afforded by FM-miR-34 to increase the circulation ½ time of folate-FM-miR-34 and PSMA-617-FM-miR-34a though incorporating an albumin binding moiety (ABM) into the ligands. Using these ligands we will evaluate serum albumin binding and stability of the new ligands. We will also verify that conjugation to ABM does not alter the activity of miR-34a nor cell binding and internalization kinetics. Finally, we will assess in vivo distribution of ligand-ABM-miR-34a conjugates. At the completion of this work we expect to have an all-encompassing miRNA delivery vehicle that can target a stabilized tumor suppressive RNAs specifically to NSCLC and prostate cancer. We will also have new ligands with increased circulation ½ life. The data obtained will ultimately have a significant impact in cancer treatment by providing new opportunities to advance the next phase of miRNA-based therapeutics. While proposed for NSCLC and prostate cancer, based on the utility of miR-34a for treating other cancers and overexpression of the folate receptor alone on many epithelial cancers, including ovary, kidney, and colon cancers, successful completion of this study could have far-reaching positive consequences.