Fig. 3: Optimization of rAAV yield, purity, and vector genome homogeneity.
From: An engineered UGA suppressor tRNA gene for disease-agnostic AAV delivery
a, Bar graph showing the rAAV9.2×CuO-hNFS-R8 titers in the crude lysates of HEK293 or various CymR monoclonal cell line 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 = 2). Data are presented as the mean. b, Tabulation of the vector genome abundance and CymR DNA encapsidation levels in purified rAAV9.2×CuO-hNFS-R8 vectors produced with the M20 cell line in the absence or presence of the CymR plasmid. GC, genome copies. c, Image of denaturing alkaline gel electrophoresis and DNA stain of the packaged DNA extracted from indicated vectors. The rAAV9.2×U6-UAG-sup-tRNA was produced using unmodified HEK293 cells as described previously10. The rAAV9.2×CuO-hNFS-R8 was produced using the M20 cell line in the absence or presence of the CymR plasmid as indicated. The black arrow indicates the full-length vector genome. The gray arrow indicates the shortened vector genome generated by recombination between repeating sup-tRNA cassettes. Two independent biological replicates were performed. d, Schematics showing four vector genome designs (VG1–VG4) carrying two CuO-NFS-R8 cassettes. The lengths of gene expression cassettes are labeled but not drawn to scale. ITR, inverted terminal repeat. e, Image of denaturing alkaline gel electrophoresis and DNA stain of the packaged DNA extracted from indicated vectors. The black arrow indicates the full-length vector genome. Gray arrows indicate the shortened vector genomes generated by recombination between repeating sup-tRNA cassettes. A single AAV preparation lot was used for each vector.
