Background Chimeric antigen receptor (CAR) T-cells have achieved unprecedented success treating various cancers, with complete responses exceeding 90% in several hematological malignancies. However, these remarkable outcomes have not been replicated in other types of cancers, particularly solid tumors. In fact, solid tumors present additional layers of complexity that significantly hamper the effectiveness of immune cell therapies. Consequently, further advancements are necessary in CAR T-cells to enhance their persistence, overcome exhaustion and enable them to cope with the immunosuppressive tumor microenvironment. Gene-editing technologies, especially CRISPR/Cas9, have proven effective in introducing a limited number of edits in T-cells. Nevertheless, the success of CAR T-cell therapies for solid tumors require the simultaneous engineering of multiple cellular features. Achieving this level of multiplexing with gene-editing technologies has proven challenging in terms of scalability and manufacturability. Moreover, the inherent mechanism of action of gene-editing technologies carries the risk of introducing unintended, irreversible genomic alterations. Preclinical data convincingly demonstrate that undesired mutations and chromosomal aberrations are not merely hypothetical concerns. Non-gene editing technologies are a viable alternative capable of circumventing many of these issues. In this context, we describe an effective, safe and scalable non-gene edited approach with a high degree of multiplexing capability for the engineering of CAR T-cells.

Methods miRNA scaffolds highly expressed in T-cells were redirected against genes of interest using custom-designed, shRNA-based guide sequences and were tested for their knock-down efficiency. The best performing scaffolds were then combined into a chimeric miRNA cluster able to target up to five genes of interest. The cluster knock-down efficiency and stability with a wide variety of guide sequences were validated against functionally relevant target genes in primary T-cells.

Results Previously, we introduced a miRNA-based shRNA platform capable of targeting up to four genes simultaneously. In this study, we further advanced this technology by expanding the platform to a 5-plex system. The novel chimeric cluster demonstrated high efficiency in knocking down five highly relevant genes in CAR T-cells simultaneously. Notably, our non-gene editing technology enabled independent modulation of each target gene to achieve the desired expression levels, thus fine-tuning the functional outcomes based on the specific biology of each target.

Conclusions Our data validate the efficacy of our technology for introducing up to five functionally relevant edits in CAR T-cells simultaneously. The 5-plex miRNA-based shRNA platform stands out as an easy, safe, and effective strategy for multiplex engineering of CAR T-cells.