Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing

Abstract

Background Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). Methods We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. Results Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 108–3.6 × 109 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. Conclusions Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

Label free metabolic imaging to enhance the efficacy of chimeric antigen receptor T cell therapy

“…3E). These findings align with prior research showing that genome editing during mitosis increases transgene incorporation efficiency (11,15). Additionally, our data indicate the potential of OMI to identify gene transfer conditions that maximize CAR T cell yield.…”

“…Second, while viral transduction is the common CAR gene transfer method used in all six FDA-approved products, it suffers from batchto-batch variability (6,7) and safety concerns due to random transgene insertion (8). Meanwhile, despite promising in vivo potency (9,10), CRISPR-edited CAR T cells face challenges such as low transgene incorporation efficiency and viability (11). Extended ex vivo culture is thus needed to reach the desired CAR T dosage, but comes with the risk of inducing terminal differentiation that decreases potency (12,13) while delaying patient treatments.…”

“…Extended ex vivo culture is thus needed to reach the desired CAR T dosage, but comes with the risk of inducing terminal differentiation that decreases potency (12,13) while delaying patient treatments. Considering remarkable metabolic changes throughout the cell cycle, T cell metabolic states following activation can potentially indicate cell cycle stage and, thus, the optimal CAR gene transfer timeframe (11,14,15). Identifying this optimal time for CAR gene transfer could maximize CAR transgene incorporation efficiency and, hence, minimize the expansion time to reach the desirable dosages.…”

Label free metabolic imaging to enhance the efficacy of Chimeric Antigen Receptor T cell therapy

“…Second, while viral transduction is the common CAR gene transfer method used in all six FDA-approved products, it suffers from batch-to-batch variability (6,7) and safety concerns due to random transgene insertion (8). Meanwhile, despite promising in vivo potency (9,10), CRISPR-edited CAR T cells face challenges such as low transgene incorporation efficiency and viability (11). Extended ex vivo culture is, thus, needed to reach the desired CAR T dosage, but comes with the risk of inducing terminal differentiation that decreases in vivo potency (12,13) while delaying patient treatments.…”

“…Identifying the optimal time for CAR gene transfer could maximize yield while minimizing expansion time to increase CAR T potency and patient access. Considering remarkable metabolic changes throughout the cell cycle, T cell metabolic states following activation can potentially indicate cell cycle stage and the optimal CAR gene transfer timeframe (11,14,15). Third, media composition and cytokines used during expansion can impact CAR T cell phenotype, fitness and clinical outcomes (16).…”