Objective:

Develop and evaluate a system for generating CAR T cells directly in the body using CRISPR–Cas9 gene editing.

Approach:

• Delivery system: Researchers combined enveloped delivery vehicles carrying CRISPR–Cas9 ribonucleoproteins with AAVs carrying DNA templates for gene insertion.
• Targeting and insertion: The system targeted CD3-expressing T cells and inserted the CAR transgene into the TRAC locus. The team engineered AAV-hT7 to improve human T-cell uptake and resist pre-existing neutralizing antibodies.
• Preclinical testing: The platform was evaluated in humanized mouse models of B-cell acute lymphoblastic leukemia, multiple myeloma, and sarcoma.

Key Findings:

• In humanized mice, CAR-expressing cells reached up to 19.7% of splenic T cells, with complete B-cell depletion.
• In a B-cell acute lymphoblastic leukemia model, 18 of 20 mice achieved complete responses across four human immune-cell donors. In a direct comparison, all six mice treated with the platform had complete tumor control, exceeding the activity of in vivo lentivi…
• All eight mice with multiple myeloma achieved complete responses; three of four maintained disease control after a subsequent tumor challenge.
• In a sarcoma model, complete responses occurred in five of six mice from one donor and three of eight from another.

Interpretation:

The study reports a preclinical approach for generating CAR T cells in vivo through targeted CRISPR-mediated gene insertion, rather than conventional ex vivo cell manufacturing.

Limitations:

• The immunodeficient mouse models limited assessment of immunogenicity, biodistribution, and safety.
• The authors state that further studies in larger, immunocompetent animal models, including nonhuman primates, are needed before clinical translation.

Conclusion:

The platform showed antitumor activity in several mouse models, but remains at a preclinical stage.

Sources:

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