Table 2. Titration of AAV RSMs using ITR2 or target sequences specific to sample.
| Sample | Template | Run 1 (vg/ml) | Run 2 (vg/ml) | Run 3 (vg/ml) | Average (vg/ml) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| AAV2 RSM (published range for SV40 pA: 2.70E+10 to 4.75E+10 vg/ml; 95% CI) | ITR2 psub201/HindIII |
1.42 ± 0.09 E+11 |
1.68 ± 0.17 E+11 |
1.53 ± 0.30 E+11 | 1.54 ± 0.19 E+11 (P < 0.001) | ||||||
| Free ITR psub201/PvuII |
3.83 ± 0.15 E+10 |
3.70 ± 0.11 E+10 |
4.21 ± 0.17 E+10 | 3.91 ± 0.14 E+10 (N.S.) | |||||||
| Target: SV40 polyA pTR-UF11 |
4.27 ± 0.42 E+10 |
3.00 ± 0.28 E+10 |
3.88 ± 0.42 E+10 | 3.72 ± 0.37 E+10 | |||||||
| AAV8 RSM (published range for SV40 pA: 3.05E+11 to 1.09E+12 vg/ml; 95% CI) | ITR2 psub201/HindIII |
7.92 ± 0.73 E+11 |
6.87 ± 0.45 E+11 |
7.41 ± 0.24 E+11 | 7.40 ± 0.47 E+11 (P < 0.001) | ||||||
| Free ITR psub201/PvuII |
1.96 ± 0.05 E+11 |
1.42 ± 0.13 E+11 |
1.75 ± 0.27 E+11 | 1.71 ± 0.15 E+11 (N.S.) | |||||||
| Target: SV40 polyA pTR-UF11 |
2.77 ± 0.48 E+11 |
1.69 ± 0.31 E+11 |
2.69 ± 0.60 E+11 | 2.38 ± 0.46 E+11 | |||||||
| Template |
Run 1 |
Run 2 |
Run 3 |
||||||||
| r-squared | Slope | Efficiency | r-squared | Slope | Efficiency | r-squared | Slope | Efficiency | |||
| ITR2 psub201/HindIII | 99.9% | −3.30 | 100.5% | 99.9% | −3.26 | 101.4% | 99.6% | −3.36 | 99.3% | ||
| Free ITR psub201/PvuII | 99.9% | −3.47 | 97.0% | 99.9% | −3.26 | 101.4% | 99.3% | −3.06 | 106.2% | ||
| Target: SV40 polyA pTR-UF11 | 99.5% | −3.16 | 103.50% | 99.6% | −3.20 | 102.80% | 99.7% | −3.19 | 102.80% | ||
ITR2 qPCR was performed using linearized plasmid (psub201/HindIII) or double digested plasmid with free ends at ITR sequences (psub201/PvuII) as standard curve. The standard deviations listed for AAV RSMs in each run is from three replicate samples. P values were calculated between ITR2 and free ITR versus SV40 and considered statistically significant when P < 0.05.
AAV, adeno-associated virus; ITR, inverted terminal repeats; N.S., nonsignificant; RSM, reference standard materials; qPCR, quantitative polymerase chain reaction.