Figure 5.

Assessing the plug-and-play capability of the 4-plex miRNA-based shRNA platform

To test the plug-and-play capability of the 4-plex and its stability irrespective of the target, a fifth and sixth shGuide sequence were substituted in each position in the sc106a-sc20b-sc20a-sc93 cluster carrying validated shGuide sequences against β2M, CD3ζ, CD28, and CD95, in the context of an anti-CD19 CAR. Validated shRNA-derived sequences against CIITA (A), a key coregulator that controls expression of HLA class II genes, and LAG-3 (B) were used for the purpose. The plug-and-play capability of the system was assessed by flow cytometry by monitoring the MFI of all targets, including HLA DR, one of the main MHC class II proteins, as a proxy for CIITA and the immune checkpoint LAG-3, in CAR T cells transduced with the 4-plex constructs, or with a no-shRNA control. 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. As a marked loss in KD efficiency was observed when the shGuide sequence against LAG-3 was in sc20b, to determine whether this is due to a positional effect of this scaffold, a construct with sc20b in position 4 (sc106a-sc20a-sc93-sc20b) was tested in comparison with the original cluster with sc20b in position 2 (sc106a-sc20b-sc20a-sc93) and a no-shRNA control. The comparison was made with both CIITA (C) and LAG-3 (D) shGuide sequences. KD efficiencies were assessed by flow cytometry by monitoring the MFI of HLA DR and LAG-3, the latter upon co-culture for 24 h with NALM-6 cells. 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. The absolute MFI values for the no-shRNA control samples are reported in Table S2.