Fig. 2: CuO–CymR improves rAAV packaging by suppressing sup-tRNA function.

a, Cartoons showing that a sup-tRNA gene recruits transcription factors (only TFIIIB and TFIIIC are shown for simplicity) and Pol III to initiate transcription (top) and that the operator–repressor binding blocks this process presumably by steric hindrance (bottom). b, Schematics showing the plasmid constructs. c, Representative images of EGFP fluorescence in transfected HEK293 cells (left) and quantification of GLuc activity in culture medium (right) to measure readthrough efficiency following co-transfection of indicated plasmids. When the CymR plasmid was not included (−CymR), a plasmid expressing mCherry was used instead to keep the total amount of DNA in cotransfection the same. Each dot represents a biological repeat (n = 3). Data are presented as the mean and s.d. d, Bar graph showing the sup-tRNA levels expressed from the 2×hNFS-R8 construct or 2×CuO-hNFS-R8 construct in the absence or presence of the CymR plasmid in HEK293 cells. Data are presented as the percentage of sup-tRNA to the parental isodecoder and normalized to the −CymR condition. Each dot represents a biological repeat (n = 3). Data are presented as the mean and s.d. e, Cartoon showing the cotransfection step in rAAV production in the presence or absence of the CymR plasmid. f, Representative western blot images showing the Cap (top), Rep (middle) and GAPDH (bottom; serving as the loading control) protein expression in HEK293 cells undergoing rAAV9 production with indicated plasmid cotransfection. g, Bar graph showing the rAAV9 titers in the crude lysates of HEK293 cell cultures cotransfected with indicated plasmids. Data are normalized to the titer of rAAV9.mCherry produced with 100% pCis in parallel (defined as 100%) and presented as percentages. Each dot represents a biological repeat (n = 4). Data are presented as the mean and s.d. In c,d, statistical analysis was performed using a two-sided unpaired t-test.

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