Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

作者： Meisam Naeimi Kararoudi1, Hamid Dolatshad2, Prashant Trikha1, Syed-Rehan A. Hussain3, Ezgi Elmas1, Jennifer A. Foltz1, Jena E. Moseman1, Aarohi Thakkar1, Robin J. Nakkula1, Margaret Lamb1, Nitin Chakravarti1, K. John McLaughlin3, Dean A. Lee1 1Center for Childhood Cancer and Blood Disease,Nationwide Children's Hospital, 2Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine,University of Oxford, 3Center for Clinical and Translational Research,Nationwide Children's Hospital Research Institute

简介：

Overview

This article presents a protocol for genetically modifying human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). The method allows for the generation of NK cells deficient in TGFBR2 and HPRT1, facilitating research into NK cell biology and immunotherapy.

Key Study Components

Area of Science

• Neuroscience
• Immunology
• Gene Editing

Background

• Natural killer (NK) cells play a crucial role in the immune response.
• Understanding gene functions in NK cells can enhance immunotherapy approaches.
• Current methods for gene editing in NK cells have limitations.
• This protocol aims to improve the efficiency of gene knockout in NK cells.

Purpose of Study

• To establish a reliable method for modifying NK cells.
• To investigate the roles of specific genes in NK cell function.
• To explore implications for immunotherapy targeting solid tumors.

Methods Used

• Isolation and culture of primary NK cells in RPMI medium.
• Transduction of NK cells using IL2 and feeder cells.
• Electroporation for introducing Cas9/RNPs into NK cells.
• Assessment of gene knockout efficiency and functionality.

Main Results

• Successful generation of NK cells deficient in TGFBR2 and HPRT1.
• High success rate of gene knockout compared to traditional methods.
• Potential applications in enhancing NK cell therapy for tumors.
• Insights into the mechanisms of immune escape in tumors.

Conclusions

• The protocol provides a robust approach for NK cell modification.
• Findings contribute to understanding NK cell biology and therapy.
• Future studies can leverage this technique for clinical applications.

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