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. 2023 Sep 20;34:102038. doi: 10.1016/j.omtn.2023.102038

Figure 7.

Figure 7

Tunability and functional relevance of the KD achieved with the 4-plex miRNA-based shRNA platform

(A) Conversely from gene editing technologies, our shRNA-based approach allows for the fine-tuning of target gene expression. Indeed, shGuide sequences can be designed that lead to varying degrees of KD. The desired level of KD can therefore be achieved by selecting the appropriate shGuide sequence. Representative examples are shown here for CD3ζ, CD95, and β2M. KD efficiency was assessed by the surface expression of TCRa/b (controlled by CD3ζ), CD95, and HLA ABC (controlled by β2M), respectively, in the context of an anti-CD19 CAR. Our miRNA-based shRNA platform allows to modulate the target gene expression to achieve the desired biological outcome. (B) When CD3ζ was knocked down with a highly efficient shGuide sequence (orange bar in A, left panel), the resulting downregulation of the TCRa/b receptor rendered the CAR T cells insensitive to TCR-specific activation by OKT3, a monoclonal antibody against CD3 able to induce T cell activation, as assessed by the measure IFN-γ secretion, mimicking a functional KO. (C) Likewise, a highly effective KD of death receptor CD95 (orange bar in A, middle panel) protected CAR T cells from apoptosis upon incubation with its soluble ligand CD95L, with an effect comparable with that of a CD95 blocking antibody. (D) On the contrary, a shGuide sequence for β2M with less pronounced KD ability could efficiently downregulate HLA ABC (orange bar in A, right panel) while, at the same time, preserving expression levels that protected CAR T cells from NK cells. Indeed, the percentage of CD107a+ NK cells upon co-culture with CAR T cells knocked down for β2M was comparable with that of NK cells in presence of CAR T cells without shRNA. Conversely, β2M KO in CAR T cells led to a substantial increase in the percentage of CD107a+ NK cells upon co-culture (D, left panel). As a consequence, CAR T cells harboring the β2M KD maintained a viability comparable with control CAR T cells without shRNA, whereas the viability of CAR T cells with β2M KO was strongly reduced (D, right panel). Bars represent mean ± SD for three independent biological replicates. In (A), for screening purposes only one PBMC donor was tested, and the bars therefore refer to a single biological replicate.