Stanford researchers have developed a new method for integrating DNA sequences into the genome of human T cells that overcomes the limitations of current approaches, which primarily target exonic regions. This innovative technique, known as nonviral intron knock-in, allows for the precise insertion of synthetic exons into endogenous introns, enabling efficient gene targeting and selective gene knockout. A Nature Biomedical Engineering study described the method as successfully integrating a chimeric antigen receptor (CAR) into the T cell receptor alpha constant (TRAC) locus, achieving over 90% CAR+ T cells through a streamlined selection process in primary human T cells.
The approach is scalable, compatible with various genomic sites, and capable of incorporating large synthetic exons while preserving endogenous gene expression. By expanding the range of targetable genomic locations and simplifying cell selection, this technique offers a powerful new tool for high-throughput gene editing in T-cell therapies.
Challenges of exon targeting in gene editing
CRISPR-based gene editing tools like Cas9 and Cas12a have revolutionized genetic engineering by enabling precise DNA insertions through homology-directed repair (HDR). These targeted integrations have broad applications, from correcting disease-causing mutations to inserting synthetic genes such as CARs in T cells for immunotherapy. However, most current methods integrate genes within exons, disrupting endogenous gene function, and rely on complex selection strategies to isolate successfully edited cells. While positive selection methods using antibodies or drugs are effective, they introduce foreign elements that can affect cell function.
A promising alternative is “touchless” negative selection, which eliminates unedited cells without direct manipulation but requires a way to distinguish edited from nonedited cells. Traditional gene-insertion techniques struggle to achieve this distinction, as non-homologous end joining (NHEJ) mutations often lead to gene disruptions similar to HDR edits. Developing more precise strategies for targeted gene integration and selection is crucial for advancing gene therapy and cell-based treatments.
Boosting CAR T cell purity with intron knock-ins
Using CRISPR/Cas9 and Cas12a, scientists successfully inserted a CAR into the TRAC locus. This process yielded over 90% pure CAR+ T cells through negative selection, a streamlined method that eliminates unedited cells without requiring additional selection markers. The approach worked across 18 TRAC intron sites and was effective in T cells from multiple donors. CAR T cells produced through this method demonstrated robust tumor-killing ability, comparable to those generated using traditional viral-based techniques.
By engineering splice donor and acceptor sites, researchers programmed alternative splicing, allowing cells to either knock out or retain endogenous gene function alongside the synthetic gene. This fine-tuned control over gene expression expands the potential for customized cell therapies. Unlike previous methods limited to short DNA insertions, intron knock-ins successfully integrated large synthetic exons up to 5.3 kb in size, and even with larger sequences, CAR T cells retained their ability to recognize and destroy leukemia cells efficiently. Among four selection methods tested, negative selection (CD3 depletion) yielded the purest CAR T cell populations, removing unedited cells while avoiding additional modifications that could interfere with cell function.
Beyond the TRAC locus, the technique enabled gene edits in highly expressed surface receptors, including CD3E, B2M, and CD47, achieving high efficiency across multiple T cell types. These results suggest that intron knock-ins could be widely applicable across the genome. Researchers optimized splicing silencers and enhancers to control whether the inserted synthetic gene would replace or coexist with the endogenous gene. This ability to adjust gene expression levels could be valuable for tailoring cell therapies. Edited T cells retained high viability, activation potential, and standard proliferation rates, ensuring the process did not compromise their therapeutic efficacy.
This nonviral, highly precise, and scalable gene editing strategy significantly advances T-cell engineering. By expanding the range of editable genomic locations and simplifying cell selection, intron knock-ins could transform gene therapy and immunotherapy applications.
