Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal.
Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, E-08003 Barcelona, Spain.
J Control Release. 2017 Jan 10;245:127-136. doi: 10.1016/j.jconrel.2016.11.027. Epub 2016 Nov 24.
Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a single gene mutation, a reciprocal translocation that originates the Bcr-Abl gene with constitutive tyrosine kinase activity. As a monogenic disease, it is an optimum target for RNA silencing therapy. We developed a siRNA-based therapeutic approach in which the siRNA is delivered by pepM or pepR, two cell-penetrating peptides (CPPs) derived from the dengue virus capsid protein. These peptides have a dual role: siRNA delivery into cells and direct action as bioportides, i.e. intracellularly bioactive CPPs, targetting cancer-related signaling processes. Both pepM and pepR penetrate the positive Bcr-Abl Cell Line (BV173). Five in silico designed anti-Bcr-Abl siRNA were selected for in vitro analysis after thorough screening. The Bcr-Abl downregulation kinetics (48h to 168h) was followed by quantitative PCR. The bioportide action of the peptide vectors was evaluated by genome-wide microarray analysis and further validated by testing BV173 cell cycle and cell proliferation monitoring different genes involved in housekeeping/cell stress (RPL13A, HPRT1), cell proliferation (ki67), cell apoptosis (Caspase 3 and Caspase 9) and cell cycle steps (CDK2, CCDN2, CDKN1A). Assays with a commercial transfection agent were carried out for comparison purposes. Maximal Bcr-Abl gene knockdown was observed for one of the siRNA when delivered by pepM at 120h. Both pepM and pepR showed downregulation effects on proliferative CML-related signaling pathways having direct impact on BV173 cell cycle and proliferation, thus reinforcing the siRNA effect by acting as anticancer molecules. With this work we show the therapeutic potential of a CPP shuttle that combines intrinsic anticancer properties with the ability to deliver functional siRNA into CML cell models. By such combination, the pepM-siRNA conjugates lowered Bcr-Abl gene expression levels more extensively than conventional siRNA delivery technologies and perturbed leukemogenic cell homeostasis, hence revealing their potential as novel alternative scaffolds for CML therapy.
慢性髓性白血病（CML）是一种由单一基因突变引起的骨髓增生性疾病，这种基因倒位导致 Bcr-Abl 基因具有组成性酪氨酸激酶活性。作为一种单基因疾病，它是 RNA 沉默治疗的最佳靶点。我们开发了一种基于 siRNA 的治疗方法，该方法将 siRNA 递送至 pepM 或 pepR 中，这两种细胞穿透肽（CPP）源自登革热病毒衣壳蛋白。这些肽具有双重作用：将 siRNA 递送至细胞内，并直接作为生物肽发挥作用，即细胞内生物活性 CPP，靶向与癌症相关的信号转导过程。 pepM 和 pepR 都能穿透阳性 Bcr-Abl 细胞系（BV173）。经过彻底筛选，选择了五种针对 Bcr-Abl 的体外分析的 siRNA。通过定量 PCR 跟踪 Bcr-Abl 下调动力学（48 小时至 168 小时）。通过全基因组微阵列分析评估肽载体的生物肽作用，并通过测试 BV173 细胞周期和细胞增殖监测涉及管家/细胞应激的不同基因（RPL13A、HPRT1）、细胞增殖（ki67）、细胞凋亡（Caspase 3 和 Caspase 9）和细胞周期步骤（CDK2、CCDN2、CDKN1A）进一步验证。进行了商业转染剂的测定以作比较。当用 pepM 在 120 小时递送至时，观察到一种 siRNA 的最大 Bcr-Abl 基因敲低。 pepM 和 pepR 均显示出对增殖性 CML 相关信号通路的下调作用，对 BV173 细胞周期和增殖有直接影响，从而通过作为抗癌分子增强 siRNA 的作用。通过这项工作，我们展示了一种 CPP 穿梭物的治疗潜力，该穿梭物将内在抗癌特性与将功能性 siRNA 递送至 CML 细胞模型的能力相结合。通过这种组合， pepM-siRNA 缀合物比传统的 siRNA 递送技术更广泛地降低了 Bcr-Abl 基因表达水平，并扰乱了白血病细胞的内稳态，从而揭示了它们作为 CML 治疗的新型替代支架的潜力。
