Table 1.
BMSC growth characteristics and implantation of each dog, conditions of gene transfer by electroporation, gene transfer efficiency assayed by GFP plasmid expression and FVIII secretion in vitro
| Dog ID | #5954 | #5811 | #6715 | #5274 |
|---|---|---|---|---|
| Implantation site | IMed | IV | IMus | SC |
| Bodyweight (kg) | 25.5 | 20.2 | 18.0 | 23.5 |
| Number of BMSCs grown from 10 mL marrow aspirate (P0 cells) | 3.33 × 107 | 7.48 × 107 | 4.74 × 107 | 15.6 × 107 |
| Cell expansion between passages (‐fold increase) | 2.9‐4.1 | 2.8‐5.5 | 4.8‐10.2 | 2.5‐8.3 |
| Number of BMSCs electroporated with pFVIIIdonor and pcdualZFN | 2.3 × 108 | 2.3 × 108 | 2.2 × 108 | 3.1 × 108 |
| Gene transfer efficiencya | 41.6% | 57.1% | 84% | 93.2% |
| Cell viability post‐electroporation | 58.0% | 80.5% | 74.6% | 41.3% |
| Number of viable BMSCs implanted | 1.71 × 108 | 1.34 × 108 | 1.55 × 108 | 0.57 × 108 |
| FVIII activity in conditioned media (mIU/106 cells/24 h) | 21.7 mIU | 66.3 mIU | 69.3 mIU | 31.1 mIU |
| Composite therapy indexb | 3711 | 8884 | 10742 | 1773 |
BMSCs from fresh bone marrow were cultured in a 3:1 (v/v) mixture of low‐glucose Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) as previously described.4 BMSCs were expanded in culture for 3 weeks after bone marrow aspiration. The number of mononuclear cells in bone marrow aspirates was 3.54 × 108 ± 1.21 × 108 per mL (mean ± standard deviation; n = 4). The number of adherent cells which grew out initially after 9 days (P0) varied by 4.7‐fold among the dogs (3.33 × 107 to 15.6 × 107).
BMSC, bone marrow‐derived mesenchymal stromal cells; IMed, intramedullary; IV, intravenous; IMus, intramuscular; SC, subcutaneous with Matrigel™ cell encapsulation.
Gene transfer efficiency measured by electroporating GFP (green fluorescent protein)‐expressing plasmid under identical conditions.
Composite therapy index = FVIII activity in conditioned medium × number of viable BMSCs implanted.