在基于 CRISPR/Cas9 的 LRP2 基因敲除人肾细胞模型中，由 megalin（LRP2 受体）介导的放射性标记 15-mer 肽内化作用

IF 4.4 Q1 CHEMISTRY, INORGANIC & NUCLEAR EJNMMI Radiopharmacy and Chemistry Pub Date : 2024-04-18 DOI:10.1186/s41181-024-00262-2

Anna Durinova, Lucie Smutna, Pavel Barta, Rajamanikkam Kamaraj, Tomas Smutny, Bernhard Schmierer, Petr Pavek, Frantisek Trejtnar

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引用次数: 0

摘要

背景Megalin（LRP2受体）介导放射性标记肽内吞进入近端肾小管细胞，这可能会因放射性示踪剂的积累而导致肾毒性。本研究旨在利用 CRISPR/Cas9 技术开发 LRP2 基因敲除（KO）的人类肾脏 HK2 细胞模型。该模型用于测定 68Ga 和 99mTc 标记的 15-mer 多肽介导的巨肽积累，这些多肽是针对肿瘤放射诊断中的血管内皮生长因子（VEGF）受体而开发的。结果通过测试两种著名的巨球蛋白配体，验证了 LRP2 KO 模型中的基因编辑，在细胞毒性浓度的庆大霉素处理后，观察到 KO 细胞的存活率更高，而且在蓄积研究中检测到 KO 细胞的 FITC-白蛋白内化率更低。荧光激活细胞分拣技术用于分离基因修饰的 LRP2 KO 细胞亚群。此外，使用针对巨球蛋白的特异性抗体进行的流式细胞术证实了 LRP2 基因敲除。经验证的 KO 模型确定 68Ga- 和 99mTc 放射性标记的 15-mer 肽是积累研究中的megalin 配体。我们发现，与亲代细胞相比，两种放射性标记的 15-mer 进入 LRP2 KO HK2 细胞的程度较低。没有观察到放射性标记不同导致的galin介导的细胞摄取差异。结论在人肾HK2细胞中CRISPR/Cas9敲除LRP2是测定由megalin介导的放射肽内化的有效方法。这种体外方法为细胞通过megalin吸收放射性标记的抗血管内皮生长因子受体15-mer肽提供了直接的分子证据。

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Radiolabeled 15-mer peptide internalization is mediated by megalin (LRP2 receptor) in a CRISPR/Cas9-based LRP2 knockout human kidney cell model

Background

Megalin (LRP2 receptor) mediates the endocytosis of radiolabeled peptides into proximal tubular kidney cells, which may cause nephrotoxicity due to the accumulation of a radioactive tracer. The study aimed to develop a cellular model of human kidney HK2 cells with LRP2 knockout (KO) using CRISPR/Cas9 technique. This model was employed for the determination of the megalin-mediated accumulation of ⁶⁸Ga- and ^99mTc-labeled 15-mer peptide developed to target the vascular endothelial growth factor (VEGF) receptor in oncology radiodiagnostics.

Results

The gene editing in the LRP2 KO model was verified by testing two well-known megalin ligands when higher viability of KO cells was observed after gentamicin treatment at cytotoxic concentrations and lower FITC-albumin internalization by the KO cells was detected in accumulation studies. Fluorescent-activated cell sorting was used to separate genetically modified LRP2 KO cell subpopulations. Moreover, flow cytometry with a specific antibody against megalin confirmed LRP2 knockout. The verified KO model identified both ⁶⁸Ga- and ^99mTc-radiolabeled 15-mer peptides as megalin ligands in accumulation studies. We found that both radiolabeled 15-mers enter LRP2 KO HK2 cells to a lesser extent compared to parent cells. Differences in megalin-mediated cellular uptake depending on the radiolabeling were not observed. Using biomolecular docking, the interaction site of the 15-mer with megalin was also described.

Conclusion

The CRISPR/Cas9 knockout of LRP2 in human kidney HK2 cells is an effective approach for the determination of radiopeptide internalization mediated by megalin. This in vitro method provided direct molecular evidence for the cellular uptake of radiolabeled anti-VEGFR 15-mer peptides via megalin.