pSTI-A120 plasmid vector質(zhì)粒載體

Plasmid templates for in vitro transcription of antigen-encoding RNAs were based on the pST1-A120 and pST1-MITD vector which feature 5` and 3`UTRs and poly(A) tails pharmacologically optimized for stability and protein translation.17 pST1-MITD features a signal sequence for routing to the endoplasmic reticulum and MHC class I transmembrane and cytoplasmic domains for improved presentation of MHC class I and II epitopes.19 Mutated amino acids were encoded as 27-mers containing the non-synonymous single nucleotide variants (nsSNV) at position 14 and embedded into pST1-MITD vectors as pentatopes, fused to each other by 10 amino acid long glycine-serine linkers or as monotopes. The following antigen-encoding vectors were used: CT26 PME1 (pentatope, encoding the CT26 neoantigens Aldh18a1P154S (ME1), Ubqln1A62V (ME2), Ppp6r1D309N (ME3), Trip12V1328M (ME4), Pcdhgc3E139K (ME5)),13 CT26 ME1 (monotope, encoding the CT26 neoantigen Aldh18a1P154S (ME1)); Pctrl. (pentatope, encoding the 4T1-luc neoantigens GnpatS110R (M53), Isoc1V205L (M54), H2-Q6E179D (M55), Chsy1G459A (M56), Pi4kbT17A (M57)). Antigen-encoding vectors were in vitro transcribed and RNA capped with β-S-anti-reverse cap analog (ARCA) as previously described.17,29 As control RNA, the same vector backbone without an antigen-encoding open reading was used.

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