CAR T cell therapy has transformed the treatment of certain hematological cancers, but its wider adoption remains constrained by complicated, time-consuming, and expensive manufacturing processes. Now, researchers at the University of California and the Gladstone-UCSF Institute of Genomic Immunology, San Francisco, have demonstrated a new approach that could overcome some of these limitations by engineering therapeutic T cells directly inside the body.
In the study published in Nature, the US-based team developed a two-vector system capable of delivering CRISPR–Cas9 gene-editing machinery and inserting large DNA sequences into specific genomic locations within human T cells in vivo.
Unlike conventional CAR T cell manufacturing, which requires extracting a patient's immune cells, genetically modifying them in a laboratory, and then reinfusing them back into the patient, the novel approach aims to generate functional CAR T cells following direct administration of the gene-editing vectors.
A more precise approach to gene delivery
The researchers combined two engineered delivery vehicles: enveloped delivery vehicles (EDVs), which transport CRISPR–Cas9 ribonucleoproteins, and adeno-associated viruses (AAVs), which deliver the DNA templates required for gene insertion.
The system integrates the CAR transgene into the T cell receptor alpha constant (TRAC) locus, allowing CAR expression to be regulated by the cell's endogenous machinery rather than relying on random genomic integration.
To improve delivery efficiency and specificity, the team engineered an AAV variant, AAV-hT7, that demonstrated resistance to pre-existing neutralizing antibodies and enhanced uptake by human T cells. They also modified the EDVs to target CD3, a protein expressed on T cells, helping restrict gene editing to the intended cell population.
In humanized mouse models, the optimized system generated CAR-expressing cells representing up to 19.7 percent of splenic T cells, alongside complete depletion of B cells.
Promising results across cancer models
The researchers subsequently evaluated the therapeutic potential of the technology in several cancer models.
In mice bearing B-cell acute lymphoblastic leukemia, a single injection of the treatment produced complete responses in 18 of 20 animals across four human immune-cell donors. The approach also demonstrated greater antitumor activity than in vivo lentiviral CAR T cell engineering, achieving complete tumor control in all six mice in a direct comparison.
The researchers extended their investigations to multiple myeloma, where all eight treated mice achieved complete responses. Following a subsequent tumor challenge, three of four animals maintained disease control.
The platform also showed activity against solid tumors, an area where CAR T-cell therapies have historically faced considerable challenges. In a mouse model of sarcoma, the researchers observed complete responses in five of six animals from one donor and three of eight from another. Although CAR T-cell therapies have previously demonstrated activity against sarcomas in clinical trials, the findings suggest that precisely engineered, tumor-targeting T cells can also be generated directly inside the body, potentially eliminating the need for conventional ex vivo manufacturing.
Toward more Accessible cell therapies
The study findings suggest that precise, in vivo genome editing could offer an alternative to traditional CAR T cell production, potentially reducing manufacturing complexity while providing greater control over transgene expression.
However, the technology remains at a preclinical stage. The study authors acknowledge that their immunodeficient mouse models limit assessments of immunogenicity, biodistribution, and safety. As such, the authors note that further investigations will be needed in larger, immunocompetent animal models (including nonhuman primates) before clinical translation of the platform.
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