Researchers Develop One-Step, High-Efficiency Large-Fragment Site-Specific Integration Platform for CAR-T Cell Preparation

Chimeric Antigen Receptor T-cell (CAR-T) therapy has achieved revolutionary success in treating hematological malignancies. However, mainstream CAR-T products currently rely on lentiviral or retroviral vectors to randomly insert CAR genes into the genome, posing risks such as insertional mutagenesis, gene silencing, and high product heterogeneity. While CRISPR technology offers the potential for site-specific integration, achieving large-fragment knock-in (such as CAR constructs exceeding 3 kb) in primary T cells remains hindered by low efficiency, significant cytotoxicity, and complex manufacturing processes, which severely limit its clinical application.

Recently, researchers published a study titled "BASIC Enables Selection-Free Efficient Knock-In of Large DNA in Primary Human T Cells" online in the journal Molecular Therapy. The study proposes and validates a novel immune cell genetic engineering system named BASIC (BaEVshort-AAV6 Site-specific Integration for CAR-T). This platform allows for the rapid and efficient generation of site-specific integrated CAR-T cells without any selection process, offering enhanced safety and superior cytotoxic function, thereby providing a new technical pathway for immune cell therapy.

Virus-like particles (VLPs) are viral assemblies that possess the external structure of a virus but lack genetic material. Through genetic engineering, they can be repurposed as delivery tools for gene-editing nucleases. The research team engineered a more compatible envelope protein, BaEVshort (a truncated form of the Baboon Endogenous Virus envelope), for the VLPs. Compared to traditional VSV-G enveloped VLPs, BaEVshort significantly increases packaging titers and drastically improves delivery efficiency to T cells and NK cells.

To address the tumorigenic risks associated with random integration by retroviruses/lentiviruses and the cytotoxicity caused by electroporation, the team utilized AAV6 as the CAR donor template in combination with VLPs. This "one-step transduction" method achieves site-specific integrated CAR-T cells. The platform was named BASIC: BaEVshort-AAV6 Site-specific Integration for CAR-T.

Figure 1. BASIC: BaEVshort-AAV6 Site-specific Integration for CAR-T. (Wang K, et al., 2026)

With a single viral transduction step, BASIC simultaneously achieves high-efficiency TCR knockout (>95%) and precise CAR gene knock-in at the TRAC locus (>85%). The entire process requires no electroporation or drug selection while maintaining high levels of cell viability and proliferative capacity. CAR-T cells prepared via the BASIC platform demonstrated exceptional anti-tumor capabilities both in vitro and in vivo.

In vitro, these cells showed stronger killing capacity against CD19+ tumor cells, higher cytokine secretion, and sustained function without easy exhaustion even after multiple tumor challenges. Phenotypic analysis revealed highly uniform CAR expression and significantly lower expression of exhaustion markers (such as PD-1 and TIM-3), indicating superior persistence and safety. In vivo, the BASIC CAR-T cells achieved 100% tumor clearance and long-term survival in mouse models, significantly outperforming traditional lentiviral methods (which showed survival rates of only 37.5-62.5%).

Beyond T cells, the study found that the BaEVshort envelope protein is also highly effective for VLP transduction of NK cells. It enables high-efficiency multi-gene editing (60-80% efficiency) in both T and NK cells, laying the foundation for the development of "off-the-shelf" universal CAR-T/NK products. The platform can flexibly target multiple gene loci for CAR integration to meet diverse therapeutic needs.

The BASIC platform utilizes a transient delivery strategy, greatly enhancing safety. Its single-step viral transduction workflow significantly simplifies the production process, reduces the requirement for initial cell numbers, and is compatible with existing GMP manufacturing systems, demonstrating strong clinical scalability and industrial potential.

Reference

Wang K, et al. BASIC Enables Selection-Free Efficient Knock-In of Large DNA in Primary Human T Cells. Molecular Therapy, 2026.