CMV-b-Actin Promoter Directs Higher Expression from an Adeno-Associated Viral Vector in the Liver than the Cytomegalovirus or Elongation Factor 1a Promoter and Results in Therapeutic Levels of Human Factor X in Mice

Although AAV vectors show promise for hepatic gene therapy, the optimal transcriptional regulatory elements have not yet been identified. In this study, we show that an AAV vector with the CMV enhancer/chicken b -actin promoter results in 9.5-fold higher expression after portal vein injection than an AAV vector with the EF1a promoter, and 137-fold higher expression than an AAV vector with the CMV promoter/enhancer. Although induction of the acute-phase response with the administration of lipopolysaccharide (LPS) activated the CMV promoter/enhancer from the context of an adenoviral vector in a previous study, LPS resulted in only a modest induction of this promoter from an AAV vector in vivo. An AAV vector with the CMV-b -actin promoter upstream of the coagulation protein human factor X (hFX) was injected intravenously into neonatal mice. This resulted in expression of hFX at 548 ng/ml (6.8% of normal) for up to 1.2 years, and 0.6 copies of AAV vector per diploid genome in the liver at the time of sacrifice. Neonatal intramuscular injection resulted in expression of hFX at 248 ng/ml (3.1% of normal), which derived from both liver and muscle. We conclude that neonatal gene therapy with an AAV vector with the CMV-b -actin promoter might correct hemophilia due to hFX deficiency. 563 OVERVIEW SUMMARY Optimization of gene expression from an AAV vector might allow higher levels of expression to be achieved, which will be necessary for effective gene therapy for some genetic deficiencies. It might also allow a lower dose of vector to be administered, which would reduce the risk of insertional mutagenesis or germ line transmission. Neonatal gene transfer might reduce the chance of inducing an immune response, and would lead to a more immediate correction of a genetic disease than would transfer into adults. We demonstrate here that the CMV-b -actin promoter is expressed well from an AAV vector in the liver. Neonatal intravenous administration of an AAV vector that expresses the coagulation protein factor X from this promoter results in therapeutic levels of factor X for more than 1 year in mice. Neonatal gene therapy with an AAV vector may allow effective gene therapy to be achieved for hemophilia.