Adeno‐Associated Viral Vectors for Homology‐Directed Generation of CAR‐T Cells

Abstract

Immunotherapy with T cells expressing chimeric antigen receptors (CAR) is an emerging and promising treatment against refractory cancers. However, the currently adopted methods of modification of T cells pose a risk of insertional oncogenesis because lentiviral and retroviral vectors integrate the CAR transgene in a semi‐random fashion. In addition, this therapy is only available using autologous cells, which create problems in production and limit the access for patients who have their T cells depleted. One modification method that shows the ability to overcome both drawbacks is the knock‐in of the CAR simultaneously knocking‐out genes that prevent allogeneic therapy, such as the endogenous T cell receptor. In this mini‐review, the authors present recent efforts to develop safer universal CAR‐T cells. More specifically, the combined application of target‐directed nucleases, which create a double‐strand break at a specific genome locus, and the delivery of CAR DNA via adeno‐associated viral vectors for subsequent integration via homologous recombination and silencing of the targeted gene is focused on.

Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a

“…However, the stoichiometry of the RNP targeting each locus can be precisely controlled and optimized to achieve a much better overall multiplex genome-editing efficiency. With our protocol, we could achieve locus-specific integration of a CAR gene into the T cell genome with an editing efficiency of >50%, which is one of the highest reported efficiencies for locus-specific CAR integration into T cells ( Moço et al., 2020 ). However, the efficiency is much lower than that of the traditional lentivirus-based CAR-T preparation method, which typically shows an editing efficiency of >90%.…”

Immunotherapy perspectives in the new era of B-cell editing

“…CRISPR tools recently allowed to insert a CD19-specific CAR in the T-cell receptor alpha (TCRA) locus, improving both CAR expression, cytotoxicity, and persistence of functional CAR T cells in a preclinical model. 35 Successful TCR gene replacement was then obtained both with an adeno-associated virus (AAV), which favors HDR, 36 and with a simple naked DNA template. 37 The possibility to simultaneously target several loci with CRISPR also opens the way for multiengineered universal off-the-shelf CAR T cells (designed for tolerance by various recipients and limited graft-versus-host disease).…”

Towards Clinical Implementation of Adeno-Associated Virus (AAV) Vectors for Cancer Gene Therapy: Current Status and Future Perspectives

“…Applications of this technology for other cancer types and solid tumors are under development [ 166 ]. Currently applied methods for the modification of T cells, however, pose at least a theoretical risk of insertional oncogenesis because lentiviral and retroviral vectors integrate the CAR transgene in a semi-random fashion [ 167 ]. From this background, the use of AAV vectors for the generation of CAR-T cells is very promising.…”

“…The CAR-T cell approach currently used in the clinic is only available using autologous cells, which create problems in production and limit the access for patients who have their T cells depleted or stressed due to previous therapies. Techniques to knock-in the CAR into target cells and simultaneously knock-out genes that prevent allogeneic therapy, such as the endogenous T cell receptor, are promising strategies to overcome this barrier [ 167 ].…”