Novel bioengineered microRNA therapeutics for lung cancer

肺癌仍然是美国女性和男性癌症死亡的主要原因。有 显然需要开发新的和更有效的治疗肺癌的疗法，特别是 最常见的亚型非小细胞肺癌（NSCLC）。作为转录组的主要调节者， 蛋白质组动力学，微RNA（miRNA或miR）控制许多关键的癌症细胞过程，包括 增殖、侵袭和干性。因此，恢复肿瘤抑制性miRNA（例如，miR-34a 和miR-124）在NSCLC细胞中丢失代表了一种新的治疗策略。然而，目前的miRNA模拟物 用于研究和开发是由化学合成和装饰各种和广泛的 人工修改。这与活细胞中产生的miRNA分子形成鲜明对比， 携带任何修饰或仅仅一些必需的转录后修饰。的确， 记载了化学工程化/合成的寡核糖核苷酸（例如，miRNA模拟物）很容易 被识别为外源RNA分子并因此引起免疫原性。为了打破这一障碍，我们必须 致力于开发大规模生产生物miRNA试剂的新方法 （BERA）在活细胞中。我们鉴定了在大肠杆菌中稳定表达的杂交tRNA/pre-miRNA分子。杆菌 为RNA生物工程开辟了一条新的途径。此外，我们已经证明，靶向miRNAs (e.g., miR-34 a）选择性地从人细胞中的BERA“前药”释放，并因此调节 靶向基因表达，抑制NSCLC细胞增殖，并抑制异种移植肿瘤生长， 不会引起严重的免疫反应此外，我们的研究发现，脂质体-聚乙烯亚胺 (LPP)纳米复合物增加了BERA在血清中的稳定性，并提高了向肺组织的递送效率。 鉴于这些令人兴奋的初步发现，我们假设BERA可以在更高水平上进行工程设计 并且完全人源化的BERA/miR-34 a和BERA/miR-124可以通过LPP以 用于治疗NSCLC的新疗法。为了验证这一假设，我们建议建立更稳定的 ncRNA载体，并产生一组完全人源化的即用型BERA（目标1），描述了分子生物学特性。 BERA在控制对治疗至关重要的人NSCLC细胞过程中的药理作用 结果（目标2），并定义LPP负载的BERA/miR-34 a和 转移性NSCLC异种移植物和患者来源的异种移植物（PDX）小鼠模型中的miR-124（目的3）。的 拟议的研究将建立一种生产完全人源化生物miRNA的新技术 药物，并为生物RNA疗法的发展开辟了新的方向。

Lung cancer remains a leading cause of cancer death in both women and men in the United States. There is a clear need for developing new and more effective therapeutics for the treatment of lung cancer, especially the most common subtype non-small cell lung cancer (NSCLC). As master regulators of transcriptome and proteome dynamics, microRNAs (miRNAs or miRs) govern many critical cancer cellular processes including proliferation, invasion and stemness. Therefore, restoration of tumor suppressive miRNAs (e.g., miR-34a and miR-124) lost in NSCLC cells represents a new therapeutic strategy. However, current miRNA mimics for research and development are made by chemical synthesis and decorated with various and extensive artificial modifications. This is in sharp contrast to miRNA molecules produced in living cells that do not carry any modifications or just a few necessary posttranscriptional modifications. Indeed it has been well documented that chemically-engineered/synthesized oligoribonucleotides (e.g., miRNA mimics) are readily recognized as foreign RNA molecules and thus cause immunogenicity. To break this barrier, we have made large efforts to develop novel approach for large-scale production of biologic miRNA agents (BERAs) in living cells. Our identification of hybrid tRNA/pre-miRNA molecules stably expressed in E. coli opens up a new avenue for RNA bioengineering. Furthermore, we have demonstrated that target miRNAs (e.g., miR-34a) are selectively released from BERA “prodrugs” in human cells, and consequently regulate target gene expression, inhibit NSCLC cell proliferation, and suppress xenograft tumor growth while they do not induce severe immune responses. In addition, our studies have found that liposome-polyethylenimine (LPP) nanocomplex increases BERA stability in serum and improves delivery efficiency to lung tissues. Given these exciting preliminary findings, we hypothesize that BERAs can be engineered at higher levels and on large scale; and fully-humanized BERA/miR-34a and BERA/miR-124 may be delivered by LPP as novel therapeutics for the treatment of NSCLC. To test the hypothesis, we proposed to establish more stable ncRNA carriers and produce a set of full-humanized ready-to-use BERAs (Aim 1), delineate the molecular pharmacological actions of BERAs in the control of human NSCLC cellular processes critical for therapeutic outcomes (Aim 2), and define the effectiveness and safety profiles of LPP-loaded BERA/miR-34a and miR-124 in metastatic NSCLC xenograft and patient-derived xenograft (PDX) mouse models (Aim 3). The proposed research will establish a novel technology for the production of fully-humanized biologic miRNA agents and open up new directions for the development of biologic RNA therapeutics.