Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising treatment for hematological malignancies. However, current CAR-T cell therapies face limitations, including complex manufacturing processes, high costs, long preparation times, and safety concerns associated with viral vectors. Viral vectors can lead to insertional mutagenesis, immune responses that impair CAR expression, and high manufacturing costs. While alternative approaches like transposon systems and mRNA transduction exist, these methods suffer from issues like low homogeneity due to random integration and transient CAR expression. Genome editing technologies, particularly CRISPR-Cas9, offer a solution by enabling locus-specific integration of CAR cassettes. This study aimed to overcome the limitations of existing CAR-T cell therapies by developing a non-viral, gene-specific targeted approach using CRISPR-Cas9, focusing on the safe and effective treatment of relapsed/refractory B-cell non-Hodgkin lymphoma (r/r B-NHL). The researchers hypothesized that this approach would result in safer and more effective CAR-T cells with improved clinical outcomes.

The authors reviewed existing literature on CAR-T cell therapy, highlighting its promise while acknowledging limitations like insertional mutagenesis from viral vectors, immune responses against viral DNA, and high manufacturing costs. They discussed existing non-viral approaches such as transposon systems and mRNA transduction, highlighting their shortcomings in terms of homogeneity and transient expression. The literature review also covered previous studies utilizing genome editing technologies for locus-specific integration of CARs, often using AAV vectors. The authors cite studies demonstrating the use of genome editing for precise insertion of TCR elements, laying the groundwork for their innovative two-in-one approach combining non-viral manufacturing and precise genome editing using CRISPR-Cas9. This approach aimed to address the limitations of existing methods by achieving both non-viral production and targeted integration, leading to more homogeneous and effective CAR-T cells.

The study involved optimizing a protocol for generating non-viral, gene-specific integrated T cells. Researchers used a homology-directed repair (HDR) template in the form of linear double-stranded DNA (dsDNA) to achieve high homologous recombination efficiency and cell viability. They optimized electroporation parameters, including the use of stimulated T cells and 800-bp homology arms, for efficient gene integration. An anti-CD19 CAR sequence (19bbz) was targeted to the AAVS1 safe harbor locus. The integration efficiency and indel percentages were assessed. AAVS1-integrated (AAVS1-19bbz) and lentivirus-produced (LV-19bbz) anti-CD19 CAR-T cells were compared for expansion, activation, cytokine secretion, and cytotoxicity in vitro and in vivo. To enhance anti-tumor activity, an anti-CD19 CAR sequence was also integrated into the PD1 gene (PD1-19bbz). The PD1-19bbz cells were compared to LV-19bbz and LV-19bbz_PD1-KO cells for proliferation, activation, cytokine secretion, and cytotoxicity. A phase 1 clinical trial (NCT04213469) was conducted to evaluate the safety and efficacy of PD1-19bbz cells in treating r/r B-NHL patients. Patients received lymphodepleting chemotherapy followed by a single infusion of PD1-19bbz cells. The percentage of CAR integration, indels, cell viability, and off-target effects were assessed. Treatment response, adverse events (AEs), and persistence of CAR-T cells were monitored. Single-cell RNA sequencing (scRNA-seq) was performed on CAR-T cells generated by different methods to analyze cell characteristics and gene expression profiles. This comprehensive methodology combined in vitro experiments, in vivo studies in mouse models, and a clinical trial to thoroughly evaluate the safety and efficacy of the novel CAR-T cell approach.

The optimized protocol for producing non-viral, gene-specific integrated T cells using linear dsDNA and electroporation yielded high homologous recombination efficiency and cell viability. Targeting the CAR cassette to the AAVS1 safe harbor locus resulted in approximately 10% integration efficiency. AAVS1-19bbz cells exhibited similar functional characteristics to LV-19bbz cells in terms of tumor cell eradication in vitro and in vivo. Integration of the anti-CD19 CAR into the PD1 gene (PD1-19bbz) resulted in superior anti-tumor activity compared to LV-19bbz cells, particularly against PD-L1-expressing tumor cells. In the phase 1 clinical trial (NCT04213469), eight patients with r/r B-NHL achieved a remarkable 87.5% complete remission (CR) rate after infusion of PD1-19bbz cells. The treatment demonstrated durable responses in five patients, with only mild cytokine release syndrome (CRS) observed as an adverse event, and no immune effector cell-associated neurotoxicity syndrome (ICANS). Single-cell analysis revealed a significantly higher percentage of memory T cells in non-viral, gene-specific targeted CAR-T cells (both AAVS1-19bbz and PD1-19bbz) compared to lentiviral counterparts. PD1 disruption enhanced the anti-tumor immune function of PD1-19bbz cells. ScRNA-seq analysis showed that infused PD1-19bbz cells exhibited sustained memory gene expression and attenuated dysfunction/cytotoxicity gene expression, correlating with patient prognosis and indicating a high proliferative and immune response capability in vivo.

The findings of this study demonstrate that CRISPR-Cas9-mediated non-viral gene-specific targeted CAR-T cell therapy offers a significant advancement in cancer treatment. The high CR rate (87.5%) and durable responses in patients with r/r B-NHL, along with the absence of serious adverse events, highlight the safety and efficacy of this approach. The superior performance of PD1-19bbz cells is attributed to the high percentage of memory T cells generated by the electroporation method and the enhanced anti-tumor immune function due to PD1 disruption. The success of the therapy even with low infusion doses and low percentages of CAR+ cells underscores the potency of the PD1-integrated CAR-T cells. These results are consistent with recent clinical trials using CRISPR-Cas9-edited T cells, further validating the safety of this technology. The study's findings provide a strong rationale for further clinical investigation of this novel CAR-T cell therapy, potentially leading to improved treatment outcomes for a broader range of cancers.

This study successfully developed a non-viral, gene-specific targeted CAR-T cell therapy using CRISPR-Cas9, demonstrating high safety and efficacy in a clinical trial for r/r B-NHL. The approach simplifies manufacturing, reduces costs, and enhances the homogeneity and potency of CAR-T cells. The high complete remission rate, durable responses, and lack of serious adverse events highlight the potential of this technology to revolutionize CAR-T cell therapy. Further research should focus on refining the manufacturing process, exploring its application in other cancers, and investigating the combined targeting of multiple inhibitory pathways to further enhance CAR-T cell efficacy.

While the study demonstrates high efficacy and safety, several limitations should be acknowledged. The relatively small number of patients in the clinical trial limits the generalizability of the findings. The study focused on r/r B-NHL; further investigation is needed to assess the effectiveness of this approach in other cancer types. Long-term follow-up is required to fully assess the durability of responses and potential for late-onset adverse events. The optimization of the manufacturing process for large-scale clinical applications remains an ongoing challenge.