Tan Josephine You Pheng, Thevendran Ramesh, Quek Yek Song, Maheswaran Solayappan
Faculty of Applied Science, AIMST University, Jalan Bukit Air Nasi, Semeling, 08100, Bedong, Kedah, Malaysia.
MOIRAI TECH SDN. BHD., Infinity8 Medini Iskandar, CIMB Leadership Academy, Unit 02-02, Level 2, No 3, Jalan Medini Utara 1, 79200, Iskandar Puteri, Johor, Malaysia.
Mol Biol Rep. 2025 Apr 9;52(1):379. doi: 10.1007/s11033-025-10487-2.
Acute Myeloid Leukemia (AML) is a complex, genetically driven cancer disease with several mutations that complicate therapy regimes. The Tribbles gene family, specifically the TRIB2 gene, has garnered substantial interest as a crucial oncogenic factor of AML progression. Studying cancer through gene expression studies and focusing on the primary oncogenes provide accurate information for future therapies. Here, CRISPR stands as the most exceptional tool used to explore both oncogene functionalities and therapeutic utilities. The ribonucleoprotein (RNP) mode of CRISPR formation and usage are preferred compared to plasmid-mediated CRISPR systems, however, it can lead to complications post-transfection to sensitive cellular entities such as human cancer cells in-vitro, transcribing to similar outcomes in-vivo as well. Therefore, this study describes the use of in-house designed CRISPR-RNP systems targeting the TRIB2 oncogene and evaluates their post-transfection cellular safety and efficacy aspects for future clinical applications.
This study uses a designed guide RNA targeting the TRIB2 gene assembled via In-vitro synthesis. The gRNA with Cas9 protein leads to the formation of CRISPR-RNP structures, which target and cleave the TRIB2 gene. The assembled CRISPR-RNP system is transfected into target AML cell and control cell lines (i.e. HEK cells), and the subsequent gene cleavage and resulting changes to the AML cells in terms of cellular safety/tolerability and gene knockdown efficacy were studied via RT-qPCR, flow cytometry, and cell viability analysis.
The outcome demonstrates the well-tolerated transfection of the in-vitro assembled CRISPR RNP system with no signs of cellular toxicity and disruptions towards the AML cell's metabolic activities, promoting the safety aspects of CRISPR RNP post-transfection to human cells. The study further highlights the in-vitro efficacy of the CRISPR RNP in targeting the TRIB2 oncogene, where a statistically significant gene knockdown of more than 80% was detected via qPCR analysis of TRIB2 gene expression with minimal to no background effects from individual RNP components, equating to their targeted gene cleavage effects. In addition, the CRISPR TRIB2 gene knockdown also indicated the possibilities of induced AML cell death measured via flow cytometry markers, translating to favourable outcomes in eliminating cancerous growths.
This study research contributes to the refinement of the CRISPR-RNP strategy and preliminary evaluation for future clinical uses.
急性髓系白血病（AML）是一种复杂的、由基因驱动的癌症疾病，存在多种突变，这使得治疗方案变得复杂。Tribbles基因家族，特别是TRIB2基因，作为AML进展的关键致癌因素已引起了广泛关注。通过基因表达研究来研究癌症并聚焦于主要致癌基因，可为未来治疗提供准确信息。在此，CRISPR是用于探索致癌基因功能和治疗效用的最卓越工具。与质粒介导的CRISPR系统相比，CRISPR形成和使用的核糖核蛋白（RNP）模式更受青睐，然而，它可能会在转染后对敏感细胞实体（如体外培养的人类癌细胞）产生并发症，在体内也会导致类似结果。因此，本研究描述了使用针对TRIB2致癌基因的内部设计的CRISPR-RNP系统，并评估其转染后细胞安全性和有效性方面，以用于未来的临床应用。
本研究使用通过体外合成组装的靶向TRIB2基因的设计指导RNA。该gRNA与Cas9蛋白导致形成CRISPR-RNP结构，其靶向并切割TRIB2基因。将组装好的CRISPR-RNP系统转染到目标AML细胞和对照细胞系（即HEK细胞）中，并通过RT-qPCR、流式细胞术和细胞活力分析研究随后的基因切割以及AML细胞在细胞安全性/耐受性和基因敲低功效方面产生的变化。
结果表明，体外组装的CRISPR RNP系统转染耐受性良好，没有细胞毒性迹象，也未干扰AML细胞的代谢活动，这促进了CRISPR RNP转染后对人类细胞安全性的考量。该研究进一步突出了CRISPR RNP靶向TRIB2致癌基因的体外功效，通过对TRIB2基因表达的qPCR分析检测到，其基因敲低具有统计学意义，超过80%，且单个RNP组件的背景效应极小或无背景效应，等同于它们的靶向基因切割效应。此外，CRISPR对TRIB2基因的敲低还表明通过流式细胞术标记物测量诱导AML细胞死亡的可能性，这意味着在消除癌性生长方面有良好结果。
本研究有助于完善CRISPR-RNP策略，并为未来临床应用进行初步评估。
