Extended Data Fig. 5: CymR cell lines improves AAV9 vector yield when packaging additional UGA-sup-tRNAArg genes and a UAA-sup-tRNA gene.
From: An engineered UGA suppressor tRNA gene for disease-agnostic AAV delivery
(a) Assessment of the readthrough capabilities of three UGA-sup-tRNAArg designs (hNFS-R8, hNFS-tRT5, OptACE-tRNAArgUGA) using the digenic PTC reporter system in HEK293 cells. Each dot represents a biological repeat (n = 3). Data are presented as mean and standard deviation. (b) Assessment of readthrough capabilities using the dual luciferase (dual-luc) PTC reporter system in HEK293 cells. Each dot represents a biological repeat (n = 3). (c) Bar graph showing the crude lysate titers of rAAV9 vectors packaging various UGA-sup-tRNAArg genes without (-) or with (+) CuO, in unmodified HEK293 cells (HEK) or the M20 cell line. Each vector genome contains one copy of the indicated sup-tRNA cassette. Data are normalized to the titer of rAAV9.mCherry produced with 100% pCis in unmodified HEK293 cells in parallel (defined as 100%) and presented as percentages. Each dot represents a biological repeat (n = 3). Data are presented as mean and standard deviation. (d) Schematics showing the tandem (2x) UAA-sup-tRNA gene construct with its mouse natural flanking sequence (mNFS). (e) Bar graph showing the rAAV9.2xCuO-mNFS-UAA-sup-tRNA titers in the crude lysates of HEK293 or various CymR monoclonal cell line cultures in the absence or presence of the CymR plasmid. Data are normalized to the titer of rAAV9.CMV/CB-CuO-mCherry produced with 100% pCis in parallel (defined as 100%) and presented as percentages. Each dot represents a biological repeat (n = 2).
