A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications

Abstract

Background aims: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. Methods: The Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a realtime cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. Results: Efficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. Conclusions: The Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.

Engineering Efficient CAR‐T Cells via Electroactive Nanoinjection

“…This is particularly important for generating CAR‐T cells for therapeutic purposes, ensuring that the employed gene delivery approach allows for efficient expansion of transfected T cells. [ 62 ] For this investigation, CellTrace Violet (CTV) was used as the proliferation tracking dye. [ 63 ] The CTV dye is designed to covalently bind to intracellular proteins in the cytoplasm and nucleus.…”

“…This lower proliferation rate in BEP group could be caused by the higher cellular stress, which can lead to retarded cell recovery. [62,64]…”

Precise genome-editing in human diseases: mechanisms, strategies and applications

“…Nonetheless, it’s worth noting that electroporation, while effective for gene editing, has been associated with negative impacts on cell viability, as the stress imposed on cells during the process can lead to alterations in gene expression profiles, potentially diminishing their proliferative capacity and modifying cellular functions. 301 LNP technology stands as an effective method for delivering genome-editing RNAs, offering key benefits such as biodegradability, excellent biocompatibility, structural flexibility, low toxicity and immunogenicity, and high delivery efficiency and robust RNA protection against degradation. 302 – 307 Multiple groups have used LNPs to deliver genome-editing RNAs, such as Cas-mediated HDR components and BEs for both in vitro and in vivo applications.…”

Transcriptomic Approaches in Studies on and Applications of Chimeric Antigen Receptor T Cells

“…Transcriptomic studies were also employed to characterize the CAR-T cells constructed using the Solupore non-viral delivery system [ 49 ]. In contrast to CAR-T cells created using the electroporation method, which caused considerable changes in expression of genes encoding proteins involved in immunological reactions, the newly proposed transfection system did not cause such perturbations in the gene expression regulation [ 49 ]. Another method for modification of CAR-T cells is the use of the CRISPR/Cas system.…”