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a. b. Serum fold dilution. Serum fold dilution. 100. 10. 1. 10. 100. 1000. 10000. 1. PBS. 1000. 10000. Fold increase. Fold increase.
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a b Serum fold dilution Serum fold dilution 100 10 1 10 100 1000 10000 1 PBS 1000 10000 Fold increase Fold increase Supplementary Figure 1. Mouse serum enhances AAV8 transduction in vivo. a. Enhanced AAV transduction after systemic administration. 1x1010 particles of AAV8/luc were incubated with mouse serum at different dilutions for 2hr at 4°C. The mixture was administered into C57BL mice via retro-orbital injection. The imaging was carried out for 5min at day 3 post AAV injection. b. Enhanced AAV transduction after muscular injection. 1x109 AAV8 particles incubated with mouse serum were injected into mouse hind leg muscle. At week 2 after injection, the imaging was taken for 5min. Face up: left leg-AAV8 + human sera, right leg-AAV8 + PBS. The fold increase of transduction was calculated by transduction from HSA incubated AAV to that from PBS treated one. Upper panel: representative imaging. Bottom: Data of enhanced AAV transduction from 3 or 4 mice.
a b c Fold increase Fold increase Fold increase Supplementary Figure 2. Sera from dogs and primates enhance AAV transduction in Huh7 cells. 1x108 particles of AAV/luc vector were incubated with 1:500 diluted sera from 6 dogs (a), or 23 primates (b), or fetal bovines (c), or PBS for 2hr at 4°C. The mixture of AAV vector and sera was applied to transduce Huh7 cells in the presence of adenovirus dl309. After 24 hr, luciferase activity from the cell lysate was analyzed. The fold increase of transgene expression from sera incubation was calculated by comparison to PBS.
Albumin concentration (mg/ml) Supplementary Figure 3. Human albumin concentration in albumin depleted serum.
b a 10 fold PBS 100 fold 100 fold 10 fold 1000 fold 10000 fold 1000 fold 10000 fold Fold increase Supplementary Figure 4. rHSA Enhances AAV8 transduction in vivo. a. Enhanced AAV8 transduction from rHSA after systemic administration. 1x1010 particles of AAV8 pre-incubated with rHSA were administered into C57BL mice via retro-orbital injection. The image was taken at day 3 post injection. b. Enhanced AAV transduction from rHSA after muscular injection. 1x109 particles of AAV8/luc incubated with rHSA were injected into hind leg muscles. At week 2 post injection, imaging was carried out. Upper panel: representative imaging. Bottom: Data of enhanced AAV transduction from 3 or 4 mice. Fold increase
0.1 fold 1 fold 10 fold 100 fold Week 1 Week 2 Fold increase Week 4 Week 7 Supplementary Figure 5. Long-term enhanced AAV transduction with clinical grade HSA. After AAV8 muscular administration, imaging was performed at indicated time points. Left panel: representative imaging. Right panel: Data of enhanced AAV transduction from 3 or 4 mice after muscular injection.
a b Fold increase Fold increase c d HSA/IV PBS/IV IM AAV2 Fold increase AAV9 Supplementary Figure 6. The effect of clinical grade of human albumin on AAV transduction from other serotypes.a. HSA enhances AAV2 transduction in Huh7 cells. 1x108 particles of AAV2/luc were incubated with human serum or 5% clinical grade HSA at different dilution for 2hr at 4°C, and then added to Huh7. 48 hr later, luciferase activity in the cell lysate was detected. b. HSA enhances AAV9 transduction in Huh7 cells. c and d. HSA enhances liver or muscle transduction in C57BL mice from AAV2 and AAV9. The imaging from AAV transduction (c) and quantitation of imaging (d). For liver transduction, 1x1010 particles of AAV/luc incubated with 1 fold of HSA were administered via retro-orbital injection (n=4), the imaging was taken at day 7 (AAV2) or day3 (AAV9) after AAV injection. For muscle transduction, 1x109 particles of AAV/luc incubated with 1 fold of HSA were used (n=3); imaging was performed at day 7 post injection.
Supplementary Table 1: MS result for AAV8 binding serum proteins
