CRISPR/methotrexate-integrated strategy for TCR-T cell engineering with reduced chromosome 14 loss

Area of Science:

• Immunology
• Gene Therapy
• Oncology

Background:

• Adoptive T cell therapy, including T cell receptor-engineered T (TCR-T) cell therapy, shows potential for treating various cancers.
• Current methods like lentiviral vectors carry risks of insertional mutagenesis, while CRISPR-based approaches face challenges with efficiency and genomic stability.

Purpose of the Study:

• To develop a safer and more efficient non-viral strategy for TCR-T cell therapy.
• To overcome the limitations of existing viral and CRISPR-mediated gene-editing techniques.

Main Methods:

• A non-viral CRISPR-Cas9 electroporation method combined with methotrexate (MTX) metabolic selection was employed.
• Primary human T cells were engineered for TCR integration into the TRAC locus via homology-directed repair (HDR).
• Optimization of electroporation and MTX treatment achieved high purity and reduced chromosomal abnormalities.

Main Results:

• The non-viral strategy achieved initial TCR integration efficiency of ~20%, enriched to ~70% purity using MTX selection.
• MTX enrichment significantly reduced CRISPR-associated chromosome 14 loss.
• Engineered TRAC-TCR-T cells demonstrated enhanced cytokine secretion and reduced exhaustion markers compared to lentiviral counterparts.

Conclusions:

• This integrated platform offers a safer alternative to viral vectors for TCR-T cell therapy.
• The approach mitigates concerns regarding CRISPR-associated genomic instability.
• The GMP-compatible method facilitates the development of advanced adoptive TCR-T immunotherapies.