Author + information
- Liao Haihan and
- Qizhu Tang
Recombinant adeno-associated virus (serotype9, AAV9) has been widely used to direct gene transfer to the heart. It has been demonstrated that AAV9 might be a promising tool for gene therapy of heart diseases. However, the optimal delivery pathway, virus genome particles and expression time point of sufficient target protein expression remain unclear. We intended to investigate these perplexities in mice heart.
We delivered the AAV9 containing the green fluorescent protein (GFP) through four different jugular vein, tail vein, orbital vein and direct injection into myocardium. The administration of AAV9 vectors were performed at different titers, varying from 1.25×1011 to 5 ×1011 viral genome particles (vg) through vein pathway, 1×1011 to 2×1011 for direct myocardium injection respectively. For jugular vein and myocardium injection, mice were anesthetized with 3% pentobarbital sodium (90mg/kg, ip) and mechanically ventilated. Mice prepared to orbital vein were anesthetized with 1.5% isoflurane. Conscious mouse were injected through tail vein with fixing device. We harvested the heart at 0 week (without injection), 1week, 2week, 4week, 6week and 8week. Fluorescence intensity was detected with frozen sample tissue and the expression level of GFP was determined by immunohistochemical analysis and Western blot.
Through intravenous method, the expression of target gene could be detected at 2 week after injection, but the maximum and stable expression was present from 4 week. However, myocardium around the injection point had GFP expression at 1 week and presented the highest expression at only 2 week after direct injection. We found that injection of 2.5×1011Vg AAV9 is sufficient to 2-3 fold over-expression of target protein through intravenous routes. At the same time, we analyzed the liver and lung frozen sections, a strong ubiquitous expression of GFP was observed in the liver and a small proportion in the lung. Although the direct injection of AAV9 in cardiac tissue was more efficient, needed less genomic particles and showed less expression of GFP in the liver and lung, the expression of GFP in the heart showed extremely uneven distribution and localized high transgene expression around the needle. Besides, compared to other delivery methods, injection via tail vein on the mice is not easy to operate because of the small lumen of vein and deep color of the tail. Injection via jugular vein and direct injection of heart tissue had a relative higher mortality for the reason of bleeding and tissue injury.
Our data suggest that a dose of more than 2.5×1011Vg systematic administration allowed for a highly efficient cardiomyocyte transduction. Direct myocardium injection has higher deliver efficiency but uneven distribution. Intravenous methods allowed for successful transfection of global cardiomyocytes but not restricted in the heart. This study also suggested that orbital vein was the optimal delivery method for feasible and effective AAV9-mediated gene transfer.