Article Text
PDF
Abstract
Background Chimeric Antigen Receptor T-cell (CAR-T) therapy has shown remarkable effectiveness and transformational impact in treating hematological malignancies and solid tumors. Site-specific nucleases such as TALENs and CRISPR/Cas9 have been applied in CAR-T therapies to enhance its cellular potency and functions. However, Cas9 cleavage might lead to chromosomal aberrations, raising potential safety issues in CAR-T therapy.
Methods In this study, we developed epigenetic modulation technology (EPIREG) wherein a DNA cleavage-free domain is fused with epigenetic modulation effectors to manipulate disease-causing genes through the endogenous epigenetic regulation pathway. Utilizing computational biology approach in combination with high-throughput screening, we have optimized and selected single-guide RNAs (sgRNAs) for precise, efficient, and sustainable gene modulations. Then the EPIREG mRNA and sgRNAs were delivered into CAR-T cells using electroporation method.
Results Delivery efficiency into CAR-T cells can reach over 96% (shown in figure 1), while maintaining high cell viability. Consequently, a high level of gene silencing (up to 90% for target gene A, B, and C, respectively, shown in figure 2) was observed. Furthermore, this gene silencing effect was sustained for more than 2 weeks, sufficient manufacturing time for CAR-T cell products. EPIREG also showed capabilities to simultaneously manipulate multiple target genes (efficiency shown in figure 3), thereby strengthening the performance of CAR-T cells. These cells with multiplex gene modifications displayed improved cytotoxicity, increased cytokine production, and sustained presence both in vitro and in vivo. Importantly, EPIREG demonstrated minimal off-target effects and circumvented the risks of chromosomal aberrations associated with Cas9.
Conclusions EPIREG demonstrated superior potency, durability, and safety for gene modifications in CAR-T cells. Without Cas9-related risks, EPIREG offers a potential and promising strategy in future CAR-T cell therapies with enhanced efficacy and improved genomic integrity.
Efficient electroporation of EPIREG mRNA into T cells.
EPIREG achieves efficient and sustained editing effects on multiple targets.
EPIREG enables simultaneous and efficient editing of three targets.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.