Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL

CAR-T cell therapy has rapidly advanced for cancer but faces limitations including complex manufacturing, high cost, long preparation time, and safety concerns linked to viral vectors such as insertional mutagenesis, innate responses to viral or exogenous DNA that can impede CAR expression, and manufacturing cost. Non-viral approaches like transposon systems and mRNA transduction avoid viruses but can suffer from random integration or transient expression, leading to product heterogeneity. Genome editing strategies enabling locus-specific CAR integration, often using AAV donors, and a preferential non-viral genome targeting approach have emerged. To address both viral-use disadvantages and random integration, the authors developed non-viral, gene-specific targeted CAR-T cells via CRISPR-Cas9 and evaluated their safety and effectiveness in relapsed/refractory B cell non-Hodgkin lymphoma.

The paper situates the work within prior advances in CAR-T therapy and its challenges. It cites: risks of insertional mutagenesis and integration biases with retro/lentiviral vectors; innate DNA sensing limiting CAR expression; and high costs of viral manufacture. Alternative non-viral methods such as Sleeping Beauty transposon and mRNA transduction have been explored but introduce random integration or transient expression, reducing product homogeneity. Locus-specific genome editing has previously targeted CARs to safe or functional loci (e.g., TRAC) often using AAV donor templates, and non-viral genome targeting in T cells has been shown for receptor insertions and point corrections. Prior reports indicate that PD1 pathway blockade or disruption can enhance CAR-T antitumor activity. This work builds on these findings by combining non-viral CRISPR-Cas9 HDR with linear dsDNA donors for precise insertion and evaluating PD1 locus integration of an anti-CD19 CAR.

- Non-viral genome targeting: Developed a CRISPR-Cas9 HDR-based method using linear double-stranded DNA (dsDNA) templates with homology arms to integrate an anti-CD19 CAR (19bbz; 4-1BB and CD3ζ) into defined loci. Optimization identified 800-bp homology arms and electroporation of stimulated T cells as yielding high homologous recombination efficiency and viability. - AAVS1 targeting (AAVS1-19bbz): Inserted the 19bbz cassette into the AAVS1 safe harbour. Assessed integration efficiency and indel rates. Compared product characteristics to conventional lentiviral CAR-T (LV-19bbz) including cell expansion, CD4/CD8 balance, activation markers, cytokine secretion, in vitro cytotoxicity (LDH assay) against Raji cells, and in vivo efficacy in xenograft mouse models (ffLuc Raji; 2×10^6 CAR-T cells infused 5 days after tumor injection; bioluminescence imaging). - PD1 locus targeting (PD1-19bbz): Integrated the same CAR cassette into the PD1 gene to disrupt PD1 while expressing CAR. Quantified CAR+ frequency, indel rates, and PD1 protein impairment. Compared to LV-19bbz and to lentiviral CAR-T with PD1 knockout via CRISPR (LV-19bbz_PD1-KO). Assessed activation, cytokines, antigen-independent/dependent tonic signaling, proliferation upon repeated stimulation with PD-L1+ Raji, cytotoxicity against PD-L1^high/low Raji in vitro, and xenograft efficacy (5×10^5 PD-L1+ Raji; 1×10^6 CAR-T cells infused after 5 days). - Clinical trial: Phase 1 study (NCT04213469) in 8 patients with relapsed/refractory aggressive B-NHL, naïve to prior CAR-T therapy. Manufacturing per optimized non-viral protocol produced infusion products with measured CAR integration and PD1 indel rates, viability, and in vitro function. Lymphodepletion with cyclophosphamide and fludarabine followed by single infusion of PD1-19bbz at 0.56×10^6 to 2.35×10^6 cells/kg. Safety monitoring included adverse events, CRS grading, and ICANS. Pharmacokinetics included peripheral blood CAR+ T cell frequency and CAR copy number over time; persistence was tracked. Off-target analysis used iGUIDE (improved GUIDE-seq) to identify potential sites and deep sequencing to validate (PHACTR1 off-target site noted). Efficacy assessed by PET-CT for response and duration of response. - Single-cell analyses: Performed scRNA-seq on CAR-T products made by different methods (non-viral gene-specific targeted PD1-19bbz and AAVS1-19bbz vs lentiviral), as well as on patient samples before and after infusion. Defined clusters based on CD8+ memory vs dysfunction/cytotoxicity gene signatures. Conducted GSEA to compare pathways (e.g., immune response, proliferation) between groups and across timepoints; evaluated expression dynamics of memory, dysfunction, and cytotoxicity genes in CD8+ CAR+ cells post-infusion.

- Manufacturing feasibility: Linear dsDNA HDR with 800-bp homology arms and electroporation of stimulated T cells yielded viable, locus-specific CAR-T cells. - AAVS1-19bbz: Integration efficiency ~10% (up to 19.8%); indel rates 67–87%. Comparable tumor reactivity to lentiviral products; unbiased integration between CD4+ and CD8+; increased CD8/CD4 ratio with electroporation. Robust in vitro cytotoxicity and in vivo tumor clearance in xenografts. - PD1-19bbz: CAR+ frequency about 20% (up to 30.3%); PD1 indels 83–93% with functional PD1 impairment. Exhibited higher proliferation than LV-19bbz upon repeated stimulation with PD-L1+ targets. Maintained activation and cytokine secretion capacity comparable to controls. Demonstrated superior in vitro killing of PD-L1^high and PD-L1^low tumor cells and enhanced in vivo tumor control and mouse survival versus LV-19bbz, LV-19bbz_PD1-KO, and AAVS1-19bbz. - Clinical outcomes (n=8 r/r B-NHL): Lymphodepletion followed by single PD1-19bbz infusion (0.56–2.35×10^6 cells/kg). Infusion products showed ~20% CAR integration and ~60% PD1 indels; viability >90%. Safety: no grade ≥3 non-hematologic adverse events; mild CRS in some patients; no ICANS. Pharmacokinetics: CAR-T cells expanded and persisted in vivo. Efficacy: complete remission in 7/8 patients (87.5%) by PET-CT; durable responses ongoing in 5 at last follow-up; 2 relapses at 6 months; remaining patient achieved partial remission; objective response rate 100%. Effective even with low infusion dose and low CAR+ percentages. Off-target: low-frequency event at PHACTR1 validated; not expected to impact T cells. - Single-cell transcriptomics: Non-viral gene-targeted products (PD1-19bbz and AAVS1-19bbz) had significantly higher proportions of CD8+ memory cells than lentiviral products. PD1 interference enhanced immune response signatures versus AAVS1-19bbz. In patients, infused CAR+ cells showed sustained expression of memory genes and attenuated dysfunction/cytotoxicity gene expression post-infusion. CD8+ CAR+ PD1− cells exhibited higher proliferation and immune response pathway enrichment in vivo. Pre-infusion products with enriched immune-response gene sets correlated with better prognosis.

The study demonstrates that CRISPR-Cas9-mediated, non-viral, gene-specific CAR insertion enables safe and effective CAR-T cell products. Clinically, PD1-targeted anti-CD19 CAR-T cells showed superior safety with only mild CRS and no neurotoxicity, and high efficacy with 87.5% complete remissions and durable responses, including in cases with high PD-L1 expression. The enhanced efficacy likely derives from two features: (1) non-viral dsDNA electroporation yields a higher proportion of memory T cells across targeted-integration products, supporting persistence and potency; and (2) PD1 disruption augments antitumor immune functions, improving proliferation and tumor clearance even when PD-L1 expression is low. The persistence of other inhibitory receptors (e.g., LAG3, TIM3, TIGIT) suggests opportunity for further functional augmentation via multiplex checkpoint interventions. The results align with prior clinical experiences using CRISPR-edited T cells, reinforcing the safety of precise genome editing in T cell therapy.

The authors present an innovative two-in-one manufacturing strategy that combines non-viral production with precise, locus-specific genome editing to generate CAR-T cells. This approach simplifies manufacturing, shortens timelines, reduces costs, and enhances product homogeneity and safety. Bench-to-bedside validation, including a phase 1 trial in r/r B-NHL, shows high safety and efficacy of PD1-integrated anti-CD19 CAR-T cells. The platform holds considerable potential for broader cell therapy applications and for engineering versatile, next-generation CAR-T products.

- Early-phase, small clinical cohort (n=8) with a median follow-up of 12 months limits generalizability and long-term safety/efficacy assessment. - Off-target editing: a low-frequency event at PHACTR1 was detected; while not expected to affect T cells, broader off-target risk requires continued surveillance. - scRNA-seq patient analyses were performed on a subset of samples and timepoints, which may not capture full interpatient heterogeneity. - Despite PD1 disruption, other inhibitory receptors (e.g., LAG3, TIM3, TIGIT) remained elevated post-infusion, indicating residual exhaustion pathways and potential room for improvement.