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. 2008 Nov 21;19(12):1421–1427. doi: 10.1089/hum.2008.118

FIG. 3.

FIG. 3.

The AAV5 P41 promoter allows for more efficient splicing of capsid-encoding AAV pre-mRNA than the CMV promoter, and together with an improved donor, led to the generation of high levels of rAAV. Left: RNase protection assay of CMV- and P41-driven AAV2 capsid-encoding pre-mRNAs in the presence of pHelper. Middle: Quantification of the relative spliced-to-unspliced ratios of capsid-encoding pre-mRNA. Data are from at least three experiments and show standard deviations. The position of the “RP” RNase protection probe is indicated. Right: Relative levels of rAAV production observed in AAV2 and AAV8 with the “better” and consensus donor mutants, in addition to the P41 wild-type and consensus donor mutants, in relation to levels obtained with the CMV-driven wild-type donor capsid-encoding construct (set to a value of 1.00). Titers of rAAVs generated with the CMV-driven wild-type donor construct typically ranged from approximately 9.5 × 109 to 2.0 × 1011 packaged genomes per 100-mm dish. Average titers of DNase-resistant rAAV virions per 100-mm dish (with standard deviations in parentheses) were as follows: CMV-Cap2, 8.59 × 1010 (±3.41 × 1010); CMV-Cap2bD, 3.33 × 1011 (±1.28 × 1011); CMV-Cap2cD, 5.02 × 1011 (±1.72 × 1011); P41-Cap2, 5.05 × 1011 (±2.34 × 1011); P41-Cap2cD, 1.08 × 1012 (±4.40 × 1011); CMV-Cap8, 2.77 × 1011 (±1.57 × 1011); CMV-Cap8bD, 1.10 × 1012 (±6.83 × 1011); CMV-Cap8cD, 2.18 × 1012 (±1.53 × 1012); P41-Cap8, 1.31 × 1012 (±5.89 × 1011); P41-Cap8cD, 4.44 × 1012 (±3.18 × 1012).