Review: Sustainable Clinical Development of CAR-T Cells - Switching From Viral Transduction Towards CRISPR-Cas Gene Editing

Abstract

T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first gene-modified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as "off-the-shelf" products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

Keywords: CAR-T; CRISPR-Cas; GMP; gene editing; lentiviral; mouse models; retrovirus.

Figure 1

Comparison between retroviral vector and lentiviral vector (RV/LV) gene delivery systems with CRISPR-Cas gene editing for production of chimeric antigen receptor (CAR)-T cells. (A) Scheme of T cell transduction with RV/LV (left) and cell transfection with ribonucleoprotein (RNP, Right). (B) Schematic representation of genetic structures. Upper structure: Displays an integrated prototypic LV gene transfer vector encoding a CAR, not to scale. LTR: Long terminal repeats; HIV: Human immune deficient virus U5: Untranslated region in the 5’ side; Ψ: encapsidation signal; RRE, Rev responsive element; cPPT, polypurine tract; EF1α, Elongation factor 1 α. Lower structure: Represents a prototypic integrated CAR generated by gene editing. TRAC, Locus of T cell receptor alpha chain; HDR, Homology-directed recombination.