The convergence of CRISPR/Cas9 gene editing technology with chimeric antigen receptor (CAR)-T cell therapy represents a transformative advancement in cancer immunotherapy, offering solutions to persistent challenges that have limited the broader clinical application of this promising treatment modality.
Addressing CAR-T Cell Limitations Through Precision Gene Editing
CAR-T cell therapy has demonstrated remarkable success in treating certain hematological malignancies, with six FDA-approved drugs currently available including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykti. However, the therapy faces significant obstacles including high manufacturing costs, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and limited effectiveness against solid tumors.
CRISPR/Cas9 technology, first demonstrated for gene editing in prokaryotes in 2012 and subsequently proven effective in human cells, provides researchers with a precise tool to modify CAR-T cells at the genetic level. This approach addresses fundamental limitations that have constrained the therapy's broader application.
Overcoming Immune Checkpoint Inhibition
One of the most promising applications involves eliminating immune checkpoint genes that naturally suppress T cell activity. Research has demonstrated that using CRISPR/Cas9 to delete the PD-1 gene results in enhanced long-term persistence and activity of CAR-T cells in preclinical studies. Similarly, deletion of CTLA-4 improved CAR-T cell proliferation and cytolytic activity.
The technology extends beyond surface checkpoint molecules to target intracellular regulators. Scientists have identified checkpoints within cells, including phosphatase 1B (PTP1B), where CRISPR/Cas9-mediated elimination of PTPN1 induces STAT5 signal activation, facilitating T cell proliferation and activation.
Banta et al. discovered that simultaneous inhibition of PD-1 and TIGIT leads to more pronounced restoration of CD226 co-activation signaling compared to single inhibition, resulting in enhanced immune activity of CD8+ T cells. This finding establishes a theoretical foundation for utilizing dual blockade strategies in cancer immunotherapy.
Enhancing Resistance to T Cell Exhaustion
T cell exhaustion represents a significant barrier to CAR-T cell effectiveness, particularly in solid tumor treatment. CRISPR/Cas9 technology enables targeted modification of genes associated with exhaustion pathways. Research has shown that disrupting PR domain zinc finger protein 1 (PRDM1) enhances persistence by promoting expansion of memory CAR-T cells.
To improve resistance to inhibitory cytokines in the tumor microenvironment, researchers have utilized CRISPR/Cas9 to knock out the TGF-β receptor II (TGβRII) gene in CAR-T cells, resulting in improved resistance to exhaustion and enhanced durability.
Studies have identified transcription factors ID3 and Sox4 as elevated in CAR-T cell dysfunction, controlling exhaustion-related genes. CRISPR/Cas9-mediated knockout of these factors considerably delayed CAR-T cell dysfunction and enhanced tumor-killing ability in preclinical studies.
Developing Universal CAR-T Cells
The current reliance on autologous CAR-T cells creates significant manufacturing challenges, including prolonged production times, limited cell availability based on patient health status, and substantial financial burden. CRISPR/Cas9 technology enables development of universal "off-the-shelf" CAR-T cells by addressing the primary drawbacks of allogeneic cells: graft-versus-host disease (GvHD) and host-versus-graft response (HvGR).
Li et al. employed CRISPR/Cas9 technology to eliminate T-cell receptor (TCR) and human leucocyte antigen (HLA)-I/II genes from CAR-T cells while incorporating exogenous expression of HLA-E. This approach effectively prevented rejection and enhanced CAR-T cell durability in preclinical studies.
An alternative approach utilizes induced pluripotent stem cells (iPSCs) as raw materials for universal CAR-T cell production. Wang et al. employed CRISPR/Cas9 technology to integrate the CAR gene into the endogenous TCRα constant (TRAC) locus, successfully developing CAR-T cells with diminished immunogenicity derived from iPSCs. These cells exhibited enhanced tumor cytotoxicity, prolonged survival duration, and decreased likelihood of eliciting an allogeneic response.
Reducing Manufacturing Costs and Timelines
Traditional CAR-T cell manufacturing requires approximately two weeks, contributing to high treatment costs. CRISPR/Cas9 technology offers potential solutions by streamlining the production process. Zhang et al. used CRISPR/Cas9 technology to introduce CAR sequences through electroporation and homologous recombination repair mechanisms, producing CAR-T cells independent of traditional viral-based transduction approaches.
This method simplifies manufacturing and shortens production time. Advanced platforms like the Novartis T-charge platform and Gracell Biotechnologies' FasTCAR platform can reduce manufacturing time from two weeks to as little as one day while preserving T cell functionality.
Clinical Translation and Safety Considerations
Several clinical trials of CRISPR/Cas9-engineered CAR-T cells are currently underway or completed. June et al. proved for the first time in a phase I clinical trial that modifying T cells with CRISPR/Cas9 technology was feasible. The researchers knocked out the PD-1 gene and two TCR genes (TRAC and TRBC), with results showing that patients did not exhibit cytokine release syndrome while modified T cells demonstrated enduring survival and expansion.
However, safety considerations remain paramount. Research has identified potential concerns including off-target effects, DNA damage, and chromosomal instability. Tsuchida et al. discovered that CRISPR technology disruption of the TRAC gene results in 4%-22% of T cells exhibiting significant chromosome deletion. To address this, researchers modified the manufacturing process by performing Cas9-mediated gene editing before T cell activation, effectively reducing chromosome deletion frequency.
Future Directions and Advanced CRISPR Systems
The field continues evolving with more advanced CRISPR/Cas systems including Cas13, Cas14, Cas12a (Cpf1), and nickase Cas9 (nCas9), which may offer improved efficiency, accuracy, or safety compared to the Cas9 system. The Cas13 system can directly edit single-stranded RNA without transcribing RNA into DNA, simplifying the editing process with high efficiency and specificity.
In 2023, the FDA approved the first gene editing therapy utilizing CRISPR technology, Casgevy, marking a significant advancement in biotechnology. This achievement demonstrates the clinical viability of CRISPR-based therapeutic approaches and provides a foundation for continued development in the CAR-T cell field.
The integration of CRISPR/Cas9 technology with CAR-T cell therapy represents a paradigm shift in cancer immunotherapy, offering solutions to longstanding challenges while opening new avenues for treating previously intractable malignancies. As safety profiles continue to be refined and manufacturing processes optimized, this combined approach holds promise for making effective cancer immunotherapy more accessible and broadly applicable.
