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Updated: May 5, 2026
Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice
Published on: July 11, 2016
A universal platform for simultaneous TCRα/β removal enables safer and more potent TCR therapies and autoimmune
Giorgia Zanetti1, Mateusz Legut2, Austin Chen1
1Columbia Center for Translational Immunology, Department of Medicine, Columbia University Medical Center, Columbia University, New York, NY, USA.
Abstract:
Adoptive T-cell therapies using tumour-specific T-cell receptors (TCRs) are limited by competition with endogenous receptors, which impairs efficacy and poses risks of off-target autoreactivity. Here we present a CRISPR-based platform that completely and selectively eliminates both endogenous TCR-α and -β chains without affecting introduced transgenic TCRs, irrespective of codon optimization. This approach achieves >90% deletion efficiency in Jurkat and primary human T cells, markedly enhancing the expression, pairing fidelity, and functional potency of transgenic receptors. Using a clinically relevant HLA-A*02:01-restricted DMF5 TCR, we show that dual TCR ablation boosts antigen-specific activation and cytotoxicity in vitro and significantly enhances tumor clearance in vivo in human immune system (HIS) mice, while preventing graft-versus-host disease (GVHD). Targeted locus amplification revealed that CRISPR-induced double-strand breaks did not alter lentiviral integration profiles, confirming genomic safety. Extending this approach to four insulin-reactive TCRs demonstrated that removal of endogenous receptors increased transduction efficiency and functional activity, with one (1E6) showing selective activation and infiltration of stem cell-derived islet grafts (SC-islets) in vivo. This study establishes a universal, safe, and scalable genome-editing platform for generating functionally precise human T cells. By integrating cancer immunotherapy and autoimmune disease modelling within a single framework, it provides a strong preclinical rationale for dual endogenous TCR removal as a route to improved specificity, safety, and therapeutic efficacy in TCR-based cell therapies.
Insights
This study introduces a CRISPR-based method to eliminate endogenous T-cell receptors (TCRs), enhancing adoptive T-cell therapy efficacy and safety. This approach improves transgenic TCR function and reduces risks like graft-versus-host disease (GVHD).
Area of Science:
- Immunology
- Gene Editing
- Cell Therapy
Background:
- Adoptive T-cell therapies are hindered by endogenous T-cell receptor (TCR) competition, leading to reduced efficacy and potential off-target effects.
- Existing methods struggle to completely remove endogenous TCRs without impacting therapeutic efficacy.
Purpose of the Study:
- To develop a CRISPR-based platform for complete and selective elimination of endogenous TCR-α and -β chains.
- To enhance the expression, pairing fidelity, and functional potency of introduced transgenic TCRs.
- To improve the safety and efficacy of T-cell therapies, including cancer immunotherapy and autoimmune disease treatments.
Main Methods:
- Utilized CRISPR-Cas9 technology to target and delete endogenous TCR-α and -β chain genes in Jurkat and primary human T cells.
- Assessed deletion efficiency, transgenic TCR expression, pairing, and function.
- Evaluated *in vitro* and *in vivo* efficacy using a clinically relevant DMF5 TCR in human immune system (HIS) mice, including assessment of graft-versus-host disease (GVHD).
- Confirmed genomic safety through targeted locus amplification to analyze lentiviral integration profiles.
Main Results:
- Achieved >90% deletion efficiency of endogenous TCR chains in human T cells.
- Demonstrated enhanced expression, pairing fidelity, and functional potency of transgenic TCRs.
- Showed improved antigen-specific activation, cytotoxicity, and tumor clearance *in vivo* with the DMF5 TCR, while preventing GVHD.
- Confirmed no alteration in lentiviral integration profiles, indicating genomic safety.
- Observed increased transduction efficiency and functional activity in insulin-reactive TCRs, with one showing selective islet graft infiltration.
Conclusions:
- Established a universal, safe, and scalable genome-editing platform for precise human T-cell generation.
- Dual endogenous TCR removal offers a promising strategy to improve specificity, safety, and therapeutic efficacy in TCR-based cell therapies.
- This platform has implications for both cancer immunotherapy and autoimmune disease modeling and treatment.
