A carrier-free multiplexed gene editing system applicable for suspension cells

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• CRISPR/Cas9 gene editing holds promise for cancer immunotherapy.
• Overcoming tumor-induced immune suppression requires multiple genetic alterations.
• Efficiently manipulating genes in suspension cells is technically challenging due to low transfection rates.

Purpose of the Study:

• To develop a carrier-free multiplexed gene editing platform for enhancing cancer immunotherapy.
• To improve the function of cytotoxic CD8+ T cells by modulating suspension cancer cells.
• To overcome the limitations of low transfection efficacy in multiple gene manipulation of suspension cells.

Main Methods:

• Established a simplified, carrier-free multiplexed gene editing platform using multiple Cas9 ribonucleoproteins (RNPs).
• Engineered Cas9 proteins for complexation and cellular penetration to simultaneously disrupt PD-1 ligands (PD-L1 and PD-L2).
• Combined multiplexed Cas9 RNP treatment with electroporation to enhance gene editing efficacy in suspension cells.

Main Results:

• Achieved high gene editing efficiency at multiple loci in suspension cells.
• Demonstrated synergistic cytotoxic effects against cancer by improving Th1-type cytokine production in CD8+ T cells.
• Showcased significant deletion efficacy (70-90%) when targeting PD-L1, PD-L2, and TIM-3 simultaneously.

Conclusions:

• The developed multiplexed gene editing strategy effectively enhances anti-tumor immunity by simultaneous suppression of PD-L1 and PD-L2 on cancer cells.
• This platform offers potential clinical utility in advancing cancer immunotherapy.
• The simplified method overcomes technical difficulties in genetically engineering suspension cells for therapeutic applications.