In vitro production of biologically active human and canine FVIII using a dual vector approach
Vector . | LC ELISA, ng/mL ± SD . | HC ELISA, ng/mL ± SD . | Activity, mU/mL ± SD . |
|---|---|---|---|
| AAV-hFVIII-HC + AAV-hFVIII-LC | 92 ± 8 | 28 ± 4 | 220 ± 8 |
| AAV-hFVIII-HC | 0 | 9 ± 1 | 0 |
| AAV-hFVIII-LC | 287 ± 22 | 0 | 0 |
| AAV-cFVIII-HC + AAV-cFVIII-LC | 150 ± 9 | Not done | 1966 ± 37 |
| AAV-cFVIII-HC | 0 | Not done | 0 |
| AAV-cFVIII-LC | 104 ± 3 | Not done | 0 |
| Media | 0 | 0 | 0 |
Vector . | LC ELISA, ng/mL ± SD . | HC ELISA, ng/mL ± SD . | Activity, mU/mL ± SD . |
|---|---|---|---|
| AAV-hFVIII-HC + AAV-hFVIII-LC | 92 ± 8 | 28 ± 4 | 220 ± 8 |
| AAV-hFVIII-HC | 0 | 9 ± 1 | 0 |
| AAV-hFVIII-LC | 287 ± 22 | 0 | 0 |
| AAV-cFVIII-HC + AAV-cFVIII-LC | 150 ± 9 | Not done | 1966 ± 37 |
| AAV-cFVIII-HC | 0 | Not done | 0 |
| AAV-cFVIII-LC | 104 ± 3 | Not done | 0 |
| Media | 0 | 0 | 0 |
FVIII expression in vitro. HepG2 cells were infected with each vector at a dose of 1 × 104 vg/cell. The following vectors were tested: AAV-hFVIII-HC, AAV-hFVIII-LC, both AAV-hFVIII-HC and AAV-hFVIII-LC, AAV-cFVIII-HC, AAV-cFVIII-LC, and both AAV-cFVIII-HC and AAV-cFVIII-LC, or no vector. At 48 hours after infection, the media were harvested and analyzed for LC antigen expression using an hFVIII-LC ELISA that crossreacts with cFVIII. The hFVIII-HC ELISA is specific only for hFVIII-HC. FVIII activity was determined by the Coatest assay. All wells were transduced in duplicate and assayed in quadruplet.