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

Figure 2.

Figure 2

Identification of the optimal scaffold and shGuide sequence length

(A) Schematic representation of the deletion strategy used to identify the best length for each of the six candidate scaffolds selected for the multiplex platform development. (B) KD efficiencies of a shGuide sequence against the TCR component CD3ζ, obtained from the sequential deletions of each of the candidate scaffolds, measured in CAR T cells as surface expression of TCRa/b. The orange bars correspond to the full-length scaffolds, ultimately chosen for further experiments. (C) Schematic representation of the shGuide sequence lengths tested (in red) compared with the typical shGuide sequence length in miRNA scaffolds (in gray). (D) KD efficiencies of shGuide sequences spanning between 18 and 25 bases against CD3ζ and the HLA class I component β2M in the context of two different scaffolds (sc106a and sc20b), measured in CAR T cells as surface expression of TCRa/b and HLA ABC, respectively. The orange bars correspond to the shGuide sequence lengths ultimately chosen for further experiments. KD efficiency was measured by flow cytometry as MFI relative to a control without shRNA, using validated shRNA-derived sequences. 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.