Figure 6.

Testing of an updated version of the 4-plex miRNA-based shRNA platform with true plug-and-play capability

As an alternative to the unstable sc106a-sc20a-sc93-sc20b configuration of the 4-plex, different designs were tested, in which sc20b was substituted by sc106b or sc17, in the context of an anti-CD19 CAR. The clusters carried validated shGuide sequences against β2M in position 1, CD28 in position 3, and CD95 in position 4. In position 2, where sc20b originally laid, validated shGuide sequences against CIITA (A) and LAG-3 (B) were introduced. KD efficiency was assessed for all targets by monitoring the surface expression of HLA ABC, HLA DR (respectively controlled by β2M and CIITA), LAG-3, CD28, and CD95. As LAG-3 is expressed at low levels in resting T cells, but is induced following T cell activation, surface LAG-3 expression was assessed upon co-culture for 24 h with NALM-6, a human pre-B ALL cell line positive for CD19. In the upper part of the panels, MFIs are depicted as relative to the no-shRNA control. Bars represent mean ± SD for three independent biological replicates. Each symbol superimposed to the bars represents a different PBMC donor. All p values refer to the comparison with the no-shRNA control. In the lower part of the panels, representative dot plots showing multiple simultaneous KD are depicted for the no-shRNA control and for the sc106a-sc17-sc93-sc20b 4-plex configuration. The absolute MFI values for the no-shRNA control samples are reported in Table S2.