Efficient shRNA-based knockdown of multiple target genes for cell therapy using a chimeric miRNA cluster platform

Chimeric miRNA cluster enables multiplex and titratable gene inhibition in CAR-T cells

“…The article in the recent of issue of Molecular Therapy Nucleic Acids by Rossi et al. 5 shows a promising approach to integrate multiplex short hairpin RNA (shRNA)-expressing loci into the viral vector used to manufacture CAR-T cells. In particular, the authors screened 48 human microRNA (miRNA) clusters and their miRNA expressing scaffolds and finally developed a chimeric expression cluster with four scaffolds originating from different natural miRNA clusters.…”

“…Although the utility of multiplexing two shRNAs in viral vectors carrying a CAR has been demonstrated before, 6 , 7 Rossi et al. 5 report the first fourplex shRNA expression system. Rossi et al.…”

“…Rossi et al. 5 are also the first to mechanistically analyze and use natural miRNA cluster scaffolds to power shRNA multiplexing. Furthermore, the possibility to fine-tune the silencing potency of each scaffold in the fourplex separately is a biologically meaningful and attractive way to modulate CAR-T cell phenotype and distinguishes the shRNA approach from a gene editing approach the most: target gene expression cannot be precisely titrated using current gene editing technologies.…”

“…Whereas, RNA inhibitors expressed from a genomic locus such as the fourplex shRNA cluster reported by Rossi et al. 5 are expected to exert permanent effects within the CAR-T cell life cycle.…”

“…The article by Rossi et al. 5 represents an important addition to the tool library to be used when choosing a CAR-T cell modification strategy. Multiplexing four shRNAs enables the combination of several modification goals, such as exhaustion prevention, toxicity control, enrichment for memory phenotype, and potency enhancement.…”