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Background <p>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 <i>in-vitro</i>, transcribing to similar outcomes <i>in-vivo</i> 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.</p> Methods <p>This study uses a designed guide RNA targeting the TRIB2 gene assembled via <i>In-vitro</i> 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.</p> Results <p>The outcome demonstrates the well-tolerated transfection of the <i>in-vitro</i> 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 <i>in-vitro</i> 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.</p> Conclusions <p>This study research contributes to the refinement of the CRISPR-RNP strategy and preliminary evaluation for future clinical uses.</p>

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Studying the cellular efficacy and tolerability of using CRISPR-gRNA ribonucleoprotein (RNP) complex for in-vitro knockdown of TRIB2 in acute myeloid leukaemia (AML) cells as preliminary clinical evaluations

  • 期刊论文

  • 发表日期：

    2025年4月9日

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  • Josephine You Pheng Tan,
  • Ramesh Thevendran,
  • Yek Song Quek,
  • Solayappan Maheswaran

摘要

Background

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.

Methods

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.

Results

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.

Conclusions

This study research contributes to the refinement of the CRISPR-RNP strategy and preliminary evaluation for future clinical uses.