Current Non-Viral-Based Strategies to Manufacture CAR-T Cells

Abstract

The successful application of CAR-T cells in the treatment of hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers of registered CAR-T cell clinical trials, efforts are being made to streamline and reduce the costs of CAR-T cell manufacturing while improving their safety. To date, all approved CAR-T cell products have relied on viral-based gene delivery and genomic integration methods. While viral vectors offer high transfection efficiencies, concerns regarding potential malignant transformation coupled with costly and time-consuming vector manufacturing are constant drivers in the search for cheaper, easier-to-use, safer, and more efficient alternatives. In this review, we examine different non-viral gene transfer methods as alternatives for CAR-T cell production, their advantages and disadvantages, and examples of their applications. Transposon-based gene transfer methods lead to stable but non-targeted gene integration, are easy to handle, and achieve high gene transfer rates. Programmable endonucleases allow targeted integration, reducing the potential risk of integration-mediated malignant transformation of CAR-T cells. Non-integrating CAR-encoding vectors avoid this risk completely and achieve only transient CAR expression. With these promising alternative techniques for gene transfer, all avenues are open to fully exploiting the potential of next-generation CAR-T cell therapy and applying it in a wide range of applications.

Keywords: CAR-T cells; CRISPR; gene transfer; non-integrating; non-viral; piggybac; programmable endonuclease; sleeping beauty; transient; transposase.

Figure 1

Overview of different strategies for gene transfer. Viruses, transposases, and programmable endonucleases mediate stable integration of the GOI into the genome, and therefore, stable CAR expression. Non-integrating vectors do not induce gene integration and thus induce transient CAR expression as long as the vector is present in the cell. The respective mechanisms and methods of delivery are depicted in a generalized but not necessarily inclusive manner. Transposase protein (blue ellipses); transposon ITRs (red DNA); CAR/GOI (green RNA/DNA/protein); genomic DNA (violet).

Figure 2

Transposon-based cut-and-paste gene transfer. Transposon and Transposase are encoded separately. The transposase can be delivered as DNA, mRNA, or protein. A transposon carrying the GOI requires delivery as circular DNA. The SB protein binds to the ITR region of the transposon vector and forms a synaptic complex, in which both ends of the transposon are held together and excised from the DNA vector. For SB, the transposon is integrated at a random TA target site in the host cell genome, resulting in stable expression of the GOI. SB protein (blue); transposon ITRs (red); GOI (green).

Figure 3

Targeted transgene integration using double-strand break induction via programmable nucleases: Genomic DNA containing the targeted sequence is cleaved by protein-DNA interactions or RNA-guided endonucleases. The resulting double-strand break (DSB) is repaired either by the error-prone non-homologous end-joining (NHEJ) pathway or by homology-directed repair (HDR). This results in correct repair or insertions and deletions (INDELs). Supplying a single- or double-stranded DNA donor template carrying homologous sequences can facilitate precise integration of the GOI at the target locus. Concurrent delivery and cleavage of a non-homologue DNA donor template can facilitate non-directional targeted integration. Targeted genomic DNA sequence (violet); GOI (green); INDEL (red with halo).