Site-specific transgene integration in chimeric antigen receptor (CAR) T cell therapies

Abstract

Chimeric antigen receptor (CAR) T cells and natural killer (NK) cells are genetically engineered immune cells that can detect target antigens on the surface of target cells and eliminate them following adoptive transfer. Recent progress in CAR-based therapies has led to outstanding clinical success in certain patients with leukemias and lymphomas and offered therapeutic benefits to those resistant to conventional therapies. The universal approach to stable CAR transgene delivery into the T/NK cells is the use of viral particles. Such approaches mediate semi-random transgene insertions spanning the entire genome with a high preference for integration into sites surrounding highly-expressed genes and active loci. Regardless of the variable CAR expression level based on the integration site of the CAR transgene, foreign integrated DNA fragments may affect the neighboring endogenous genes and chromatin structure and potentially change a transduced T/NK cell behavior and function or even favor cellular transformation. In contrast, site-specific integration of CAR constructs using recent genome-editing technologies could overcome the limitations and disadvantages of universal random gene integration. Herein, we explain random and site-specific integration of CAR transgenes in CAR-T/NK cell therapies. Also, we tend to summarize the methods for site-specific integration as well as the clinical outcomes of certain gene disruptions or enhancements due to CAR transgene integration. Also, the advantages and limitations of using site-specific integration methods are discussed in this review. Ultimately, we will introduce the genomic safe harbor (GSH) standards and suggest some appropriate safety prospects for CAR integration in CAR-T/NK cell therapies.

CAR-T cell immunotherapy for ovarian cancer: hushing the silent killer

“…To overcome the immunosuppressive nature of the TME of ovarian cancer, CAR-Ts could be genetically engineered to be immunosuppression-resistant (using CRISPR-Cas, TALEN, shRNAs, etc.) or secrete anti-immunoinhibitory molecules (such as anti-PD-1 scFvs or VHHs) ( 26 ). To overcome the limitation of poor CAR-T infiltration in the context of ovarian cancer, localized administration of CAR-Ts could be a potent solution instead of systemic administration.…”

“…The genetic manipulation of CAR-Ts usually entails the application of viral gene introduction techniques (such as by the means of lentiviral or retroviral particles) or non-viral gene introduction methods (including transposons, mRNA electroporation, etc.) ( 26 ). In reference to the structural characteristics of CAR molecules, CARs are composed of three topological domains; extracellular domain, transmembrane domain, and intracellular domains ( Figure 4 ).…”

Biomarkers for prediction of CAR T therapy outcomes: current and future perspectives

“…The vector encoding the CAR receptor is integrated semi-randomly into the genome, leading to variable expression and consequently variable efficacy of the CAR T cells. The receptors can also be expressed constitutively for extended periods or inductively for a brief duration (125,126).…”

“…Furthermore, the integration of transgenes via viral vectors raises concerns about potential risks, including insertional oncogenesis, gene inactivation or dysregulation, and impairment of cell functions (126). Early detection of such integration events is crucial for ensuring the safety of CAR T cell therapies.…”

Influencing factors and solution strategies of chimeric antigen receptor T-cell therapy (CAR–T) cell immunotherapy

“…Currently, the following three sites are widely acknowledged as target sites for CAR integration in the context of gene editing: (1) AAVS1; (2) CCR5 gene; (3) Human Rosa26 locus. Hamed Dabiri has reviewed previous studies that suggest integrating CAR coding sequences into TCR sites and placing them under the control of endogenous regulatory elements can reduce tonic signaling, prevent accelerated T cell differentiation and failure, and enhance the therapeutic efficacy of engineered CAR-T cells [233,212]. Kinetic measurements of antigeninduced CAR internalization and degradation suggest that the expression and cell surface dynamics of CAR are contingent upon enhancer/promoter elements [212].…”