Multiplex Gene Editing in Primary T Cells: A Non-Viral Approach to CAR Expression
Multiplex Gene Editing in Primary T Cells: A Non-Viral Approach to CAR Expression
Fereshteh Arefi,1,*
1. Biology Department, Faculty of Biosciences, Tehran North Branch, Islamic Azad University, Tehran, Iran
Introduction: CAR T-cell therapy has revolutionized cancer treatment for blood cancers, relying on viral vectors to deliver genetic material to T-cells. However, these vectors can cause secondary cancers through random genome integration, and while successful in blood cancers, they have yet to achieve similar results with solid tumors due to factors such as limited tumor homing, persistence, and exhaustion.
To improve CAR T cell therapy, researchers have investigated methods like preventing T cell exhaustion and creating allogeneic CAR T cells. Strategies to prevent exhaustion include blocking inhibitory receptors or using CRISPR/Cas9 to alter T cell genes. Allogeneic CAR T cells provide convenience as off-the-shelf products but face issues like graft-versus-host disease (GVHD) and immune rejection.
In this study, researchers aimed to tackle these issues by creating CAR T cells with multiple genetic modifications. They used CRISPR/Cas9 to insert a GD2-CAR transgene into the TRAC locus while disrupting the TRAC, β2M, and PDCD1 loci. This approach was intended to reduce GVHD and T-cell rejection, prevent CAR exhaustion, and enhance T-cell persistence by promoting stem-cell memory phenotypes.
Methods: T cells were isolated from leukopaks, activated, and cultured with specific cytokines. For genetic modification, plasmids carrying a GD2-tNGFR-CAR sequence were prepared for CRISPR/Cas9 insertion, followed by nucleofection of T cells with the RNP mixtures and HDR template. The modified T cells were then expanded, cryopreserved, and later thawed. Flow cytometry was used to analyze CAR, TCR, and other markers. In vitro cytotoxicity was assessed using GD2+ neuroblastoma cells and AkaLUC-GFP CHLA-20 cells. ddPCR quantified translocations, and GUIDE-seq assessed off-target effects. Data were analyzed with Prism, Excel, FlowJo, and Illustrator.
Results: Manufacturing of Non-Viral, TRAC-B2M-PD1 Triple-Knockout GD2 CAR T Cells: Multiplex-edited CAR T cells were designed targeting TRAC, B2M, and PD1. A HDR donor template containing a third-generation anti-GD2 CAR transgene and a tNGFR tag was integrated into the TRAC locus. Human primary T cells were isolated, activated, and nucleofected with RNPs knocking out TRAC, B2M, and PD1 and knocking-in the dsDNA CAR donor template. TRAC-B2M-PD1 triple-knockout GD2 CAR T cells were expanded and cryopreserved.
Low Translocation Rate and Off-Target Editing: Simultaneous multiplex editing of T-cells can introduce chromosomal abnormalities. ddPCR and GUIDE-seq were employed to assess the frequency of these events. Low rates of translocations and off-target editing were observed, indicating high efficacy and fidelity in more than 99% of T cells.
Favorable Memory Phenotypes: Higher amounts of naive and central memory T-cells in pre-infusion CAR T products have been correlated with increased persistence and potency. This phenotype can be characterized by the expression of surface markers like CD45RA and CCR7. Over 50% of TRAC-B2M-PD1 triple-knockout GD2 CAR T cells had a naïve or central memory phenotype.
High in Vitro Potency: The potency of triple-knockout GD2-CAR T cells was investigated by measuring cytotoxicity after co-culture with the GD2+ neuroblastoma cell line, CHLA-20. TRAC-B2M-PD1 triple-knockout GD2 CAR T cells showed high cytotoxicity against CHLA-20 target cells, suggesting that the knockout of PD-1 may increase the potency for GD2 CAR T cells against neuroblastoma.
Conclusion: This study demonstrates the feasibility and safety of using multiplex editing with CRISPR/Cas9 to manufacture allogeneic GD2 CAR T cells with minimal chromosomal abnormalities and off-target editing. The engineered T cells exhibited high cytotoxicity against GD2+ human neuroblastoma cells in vitro and displayed favorable memory phenotypes.
The simultaneous disruption of TRAC, B2M, and PD-1 in CAR T cells effectively prevents exhaustion, enhances anti-tumor efficacy, and limits GVHD and host rejection. The non-viral gene delivery strategy used in this study offers potential advantages in terms of scalability and manufacturing efficiency.
Future research should focus on further optimizing the editing process to minimize off-target effects and translocations, exploring alternative editing strategies, and investigating the in vivo potency of these engineered T cells in various GD2-expressing indications. Additionally, efforts to improve the stem cell memory profile and optimize cryopreservation protocols are essential for the successful translation of allogeneic CAR T cell therapies.