Engineered Polycistronic Peptide Expression

DNA encoding a codon-optimized polycistronic peptide consisting of RQR8 and anti-human HER2 (clone 4D5) interspersed with picornavirus T2A and furin cleavage sequences was synthesized by GeneScript. RQR8, used as the transduction marker, is a chimeric surface protein consisting of domains from CD34 (for detection and purification with clone QBEND/10), CD8 (for anchoring at the cell surface) and CD20 (for depletion in vivo with the anti-CD20 monoclonal antibody rituximab)^{64 (link)}. shRNA sequences corresponding to the best specificity score for each target were retrieved from the RNAi Consortium library. For coexpression of protein and shRNA, shRNA hairpins were flanked with an optimized sequence of miR-30 (refs. ^{65 (link),66 (link)}). 97-mer oligonucleotides (IDT Ultramers) coding for the respective shRNAs^{67 (link)} were PCR-amplified using 10 µM of the primers miRE-XhoI-fw (5′- TGAACTCGAGAAGGTATATTGCTGTTGACAGTGAGCG-3′) and miRE-EcoRI-rev (5′-TCTCGAATTCTAGCCCCTTGAAGTCCGAGGCAGTAGGC-3′), a 0.5-ng oligonucleotide template and the Q5 High-Fidelity 2X Master Mix (New England Biolabs) and cloned into HER2 CAR vectors containing the miRE scaffold sequence. All coding sequences were cloned into pCDCAR1 (Creative Biolabs). Third-generation lentiviral transfer helper plasmids were obtained from Biocytogen. All sequences are available in Supplementary Table 3.^{65 (link),67 (link)} The following lentiviral vectors purchased from Creative Biolabs were used: a truncated form of epidermal growth factor receptor (vector control) and anti-CD19 CAR with a 4-1BB endodomain.