Choosing the Right Tool for Genetic Engineering: Clinical Lessons from Chimeric Antigen Receptor-T Cells

T cell modification with genes that encode chimeric antigen receptors (CAR-T cells) has shown tremendous promise for the treatment of B cell malignancies. The successful translation of CAR-T cell therapy to other tumor types, including solid tumors, is the next big challenge. As the field advances from second- to next-generation CAR-T cells comprising multiple genetic modifications, more sophisticated methods and tools to engineer T cells are being developed. Viral vectors, especially γ-retroviruses and lentiviruses, are traditionally used for CAR-T cell engineering due to their high transduction efficiency. However, limited genetic cargo, high costs of production under good manufacturing practice (GMP) conditions, and the high regulatory demands are obstacles for widespread clinical translation. To overcome these limitations, different nonviral approaches are being explored at a preclinical or clinical level, including transposon/transposase systems and mRNA electroporation and nonintegrating DNA nanovectors. Genome editing tools that allow efficient knockout of particular genes and/or site-directed integration of the CAR and/or other transgenes into the genome are also being evaluated for CAR-T cell engineering. In this review, we discuss the development of viral and nonviral vectors used to generate CAR-T cells, focusing on their advantages and limitations. We also discuss the lessons learned from clinical trials using the different genetic engineering tools, with special focus on safety and efficacy.