Poly a polymerase

DNA of a third-generation CAR containing a scFv domain directed against CD19 linked to CD3ζ and 4-1BB intracellular signaling domains was generated, as previously described.^{14 (link),37 (link)} The CD19 CAR DNA was linearized, and then a MEGAscript T7 RNA transcription kit was used to synthesize the RNA. Four different mRNA isolates were generated. To synthesize the mRNA for the first group, the transcription reaction was supplemented with m1Ψ triphosphate (Trilink) in place of UTP. For the second group, the m1Ψ-containing mRNAs were purified by digesting with ribonuclease III (RNase III) (Epicentre), as described below. For the third group, mRNA was transcribed in the presence of UTP using standard methods followed by RNase III digestion. Finally, control mRNA was transcribed in the presence of UTP using standard methods without RNase III purification. MEGAscript T7 RNA transcription kit (Ambion, Thermo Fisher Scientific) was used to generate all RNA. To contain cap1, all mRNA was enzymatically capped with guanylyltransferase and 2′-O-methyltransferase (CellScript), and long polyadenylate tail was added using poly(A) polymerase (CellScript), according to protocols previously described.^{38 (link)} RNA purification with RNase III for the two purified experimental arms was completed before capping and poly(A) tailing, using a protocol described below.