Adeno-associated viral vectors are derived from naturally occurring parvoviruses and have been used to direct long-term expression of transgenes ín muscle, liver and CNS.
We have carried out studies to develop an experimental basis for gene transfer for hemophilia E using an AAV vector introduced into hepatocytes.
Using a vector expressing canine F.IX under the control of the human µ1-antitrypsin promoter coupled to 4 copies of the ApoE enhancer, we infused ~1 x 10-12 vg/kg via the portal vein into two different strains of hemophilia B dogs.
In both strains, one with a missense mutation ion G381E in the catalytic domain, the other with a small deletion resulting in a stop codon at amino acid 146, infusion of vector was well-tolerated.
There were minimal elevations of transaminases with the infusion procedure and these rapidly returned to normal.
Plateau Factor IX Levels ranged from 5-14% of normal in all three treated animals, and have now been sustained for a period of 10 months in the longest treated animal.
In parallel safety studies, we infused rats with doses up to 1 x io1 3 vg/kg of a similar AAV-ApoE/hAAT-human F.IX vector via the intrahepatic artery and showed no evidence of toxicity as measured by serial serum chemistries, CBCs, or histologic analysis of tissues from sacrificed animals We carried out long-term safety studies in mice, which showed no evidence of gross pathology in livers 0129 animals sacrificed 12-19 months after vector infusion.
We carried out additional safety studies in normal dogs, these animals were infused with -5 x 1012 vg/kg of an AAV-null vector (non-functional expression cassette) by non-surgical, radiological-guided infusion into the hepatic artery.
No vector-related toxicity was observed.
Serial semen samples collected over a period of 90 days were negative for vector sequences by a sensitive PCR assay capable of detecting as few as 100 copies/300,000 haploid genomes.
This suggests that the risk of germline transmission of vector delivered by an intravascular route is remote. Based on these and other pre-clinical studies we have proposed a clinical trial of AAV-mediated liver-directed gene transfer for hemophilia E.
The proposed starting dose is 50-fold Lower than the highest doses used in the toxicology studies. Pre-clinical studies indicate that intrahepatic delivery of rAAV s safe and likely to be therapeutic for hemophilia B.