Gene Editing of Checkpoint Molecules in Cord Blood-Derived Dendritic Cells and CD8⁺ T Cells Using CRISPR-Cas9

Area of Science:

• * Immunotherapy and gene editing in oncology.
• * Development of allogeneic cell therapies.

Background:

• * Cancer immunotherapies like checkpoint inhibition and cell therapies show promise but have limitations in patient response and resistance.
• * Combining these approaches and utilizing gene editing, such as CRISPR-Cas9, can potentially overcome these limitations.
• * CRISPR-Cas9 enables the genetic removal of checkpoint molecules to enhance immune cell persistence and efficacy.

Purpose of the Study:

• * To optimize a CRISPR-Cas9 protocol for depleting checkpoint molecules from cord blood (CB)-derived immune cells.
• * To assess the feasibility of applying this protocol to CB-dendritic cells (DCs) and CB-CD8+ T cells for allogeneic cell therapy.

Main Methods:

• * Utilized CRISPR-Cas9 ribonucleoprotein complex electroporation for genomic checkpoint molecule depletion.
• * Applied the protocol to CB-derived dendritic cells (DCs) and CD8+ T cells under good manufacturing practice conditions.
• * Assessed knockout efficiency, cell viability, and functional impact on immune cells.

Main Results:

• * Successful genomic knockout of checkpoint molecules in both CB-DCs (50-95% reduction) and CB-CD8+ T cells (25-45% reduction) without compromising cell viability.
• * The procedure did not impair the stimulatory function of CB-DCs or the response (proliferation, TNF-α production) of CB-CD8+ T cells.
• * Demonstrated a protocol easily translatable to clinical settings.

Conclusions:

• * An optimized CRISPR-Cas9 protocol effectively eliminates checkpoint molecules from CB-derived DCs and CD8+ T cells.
• * This method enhances the potential of these cells for allogeneic cell therapy applications in cancer treatment.
• * The findings pave the way for improved and more effective cancer immunotherapies.