TABLE 1.
Virion properties of hybrid vectors based on AAV1 and AAV2a
| Vector | % Factor IX secretion
|
||
|---|---|---|---|
| Heparin | Anti-AAV2 | Anti-AAV1 | |
| AAV1 | 51.70 | 11 | 12.90 |
| AAV2 | 7.90 | 4.90 | 85.70 |
| pH221-1-112 | 5.70 | 4.30 | 70.80 |
| pH221-113-155 | 5.80 | 18.90 | 83.20 |
| pH221-156-212 | 21.30 | 14.70 | 83.60 |
| pH221-213-423 | 13.50 | 38 | 81.10 |
| pH221-670-763 | 13.30 | 16.90 | 85.80 |
Heparin and NABs against AAV were assayed in the following way. COS cells (105 cells/well) were seeded in 24-well plates and infected with AAV-CMV-human factor IX vectors (multiplicity of infection, 20,000) at 37°C for 1 h in serum-free Dulbecco's modified Eagle's medium containing 25 mM HEPES buffer. At 1 h postinfection, cells were washed with PBS and incubated in Dulbecco's modified Eagle's medium with 10% fetal bovine serum for 48 h and medium was assayed directly in an ELISA for human factor IX expression. To study the effect of heparin on the infectivity of AAV vectors, AAV vectors were preincubated with heparin (Sigma) at a final concentration of 200 μg/ml in serum-free Dulbecco's modified Eagle's medium containing 25 mM HEPES buffer at 37°C for 1 h before they were applied to the cells. To obtain NABs against AAV1 and AAV2, mouse plasma samples were collected 4 weeks after AAV1 or AAV2 vectors were administered to C57BL/6 mice intravenously. The effects of NABs against AAV1 and AAV2 on the hybrid vectors were assayed by preincubating the hybrid vectors with mouse sera containing NABs which were diluted 1:5 in Dulbecco's modified Eagle's medium containing 25 mM HEPES buffer. After the vectors and mouse sera were incubated for 30 min at room temperature, the vectors were applied to target cells. Supernatants were harvested at 48 h postinfection, and human factor IX in the samples was assayed in duplicates. The secretion of human factor IX into the medium was determined by ELISA. AAV1 and AAV2 values are shown as controls. The data are shown as the percentiles of factor IX secretion compared to the values in the absence of any inhibition (incubation with PBS) because of variations in expression for the individual vectors. The two vectors pH221-565-669 and pH221-481-564 were not included because their titers were very low.