Department of Urology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands.
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands.
Prostate. 2022 May;82(6):657-665. doi: 10.1002/pros.24309. Epub 2022 Jan 31.
Cell-penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor (AR) pre-mRNA, thereby preventing the generation of the splice variant AR-V7 mRNA. AON-mediated knockdown of AR-V7 resulted in inhibition of androgen-independent cell proliferation. In this study, we evaluated the CPP-mediated delivery of this AON into castration-resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate).
Nanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter-Glo cell viability assay. The effectivity of CPP-mediated delivery of the splice-correcting AON-intronic splicing enhancer (ISE) targeting the ISE in the castration-resistant prostate cancer (CRPC)-derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR-V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 (HP1BP3). Western blot analysis was used to confirm AR-V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy.
The amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine-rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier-associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 μM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON-ISE directed against AR pre-mRNA achieved significant downregulation of AR-V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen-independent cell proliferation of DuCaP and VCaP cells.
This proof-of-principle study constitutes the basis for further development of CPP-mediated delivery of AONs for targeted therapy in prostate cancer.
细胞穿透肽（CPPs）是一种很有前途的传递反义寡核苷酸（AONs）的方法，因为它们通过非共价相互作用形成纳米大小的复合物，从而表现出高效的细胞摄取。以前，我们设计了一种 AON 系统来纠正雄激素受体（AR）前 mRNA 的剪接，从而阻止剪接变体 AR-V7 mRNA 的产生。AON 介导的 AR-V7 敲低导致雄激素非依赖性细胞增殖的抑制。在这项研究中，我们评估了 CPP 介导的这种 AON 递送到去势抵抗性前列腺癌细胞系模型 22Rv1、DuCaP（硬脑膜前列腺癌）和 VCaP（椎体前列腺癌）中的情况。
通过快速混合形成 AON 和 CPP 的纳米颗粒（多聚物）。通过流式细胞术评估肽载体、配方参数和细胞孵育条件对荧光标记 AON 细胞摄取的影响。使用 CellTiter-Glo 细胞活力测定法测量这些制剂的细胞毒性活性。通过测量 22Rv1、DuCaP 和 VCaP 细胞中针对雄激素抵抗性前列腺癌（CRPC）衍生的 22Rv1、DuCaP 和 VCaP 细胞中剪接校正 AON-内含子剪接增强子（ISE）的 AR-V7 mRNA 的水平来确定 CPP 介导的剪接纠正 AON 的有效性。将人异染色质蛋白 1 结合蛋白 3（HP1BP3）归一化后，确定 AR-V7 mRNA 的水平。Western blot 分析用于确认 AR-V7 在蛋白质水平上的下调。通过共聚焦激光扫描显微镜确定由 CPP 或转染试剂递送至荧光标记 AON 的细胞分布。
两亲性和亲脂性 CPP PepFect 14（PF14）比富含精氨酸的 CPP 具有更高的摄取效率。通过调整配方参数、浓度和孵育时间，发现一种由氨基/磷酸比为 3、0.35 μM AON 浓度和 30 分钟的细胞与多聚物孵育时间组成的配方具有最佳的载体相关毒性和传递效率之间的平衡。针对 AR 前 mRNA 的 AON-ISE 的细胞递送可使 22Rv1、DuCaP 和 VCaP 细胞的 AR-V7 分别下调 50%、37%和 59%，并降低 DuCaP 和 VCaP 细胞的雄激素非依赖性细胞增殖。
这项原理验证研究为 CPP 介导的 AON 靶向治疗在前列腺癌中的进一步发展奠定了基础。
