Stable High Level Coagulation Factor VIII Expression In Vivo Following Gene Transfer Using a Novel Expression Cassette Encoding a More Potent FVIII Variant

Abstract 250

Hemophilia A (HA, or clotting factor VIII deficiency), the commonest inherited bleeding disorder, is a good model for early proof of concept gene therapy studies. This is primarily because its clinical manifestations are entirely attributable to the lack of a single gene product (FVIII) that circulates in minute amounts (200ng/ml) in the plasma. Furthermore, a modest increase in the level of FVIII (>1% of normal) can ameliorate the severe phenotype. Several different gene transfer strategies for FVIII replacement have been evaluated but these have been hampered by the fact that hFVIII protein expression is highly inefficient. In addition the relatively large size of the hFVIII cDNA, (≂f7.0 kb) far exceeds the normal packaging capacity of adeno-associated viral vectors (AAV), which are currently the vectors of choice for the correction of genetic disorders such as hemophilia A. We have begun to address some of these limitations through the development of a 5.7kb AAV expression cassette (rAAV-HLP-codop-hFVIII-N6) which consists of a novel more potent hFVIII (codop-hFVIII-N6) which contains a short 226 amino-acid B-domain spacer, rich in asparagine-linked oligosaccharides, which is currently the most efficiently expressed hFVIII variant. This variant is under the control of a small ≂f200bp liver specific promoter. This expression cassette can be efficiently packaged into a single AAV vector, without significantly compromising vector yields. Transient transfection of this rAAV expression cassette into the HuH7 liver cell-line resulted in hFVIII expression that was between 4 and 8 (0.05±0.02IU/ml/24h) fold higher than that achieved with the B domain deleted (BDD-hFVIII) and N6-hFVIII variants respectively. Tail vein injection of serotype 5 or 8 pseudotyped vector rAAV-HLP-codop-hFVIII-N6 in C57Bl/6 mice resulted in detectable hFVIII within two weeks of gene transfer, reaching steady state levels of 23±6 IU/ml and 54±12 IU/ml respectively by 10 weeks. This level of expression is at least 400-fold greater than required for therapeutic efficacy (0.05IU/ml) and at least 10 fold higher than achieved in mice transduced with a comparable dose of rAAV encoding either the BDD or N6 variant of hFVIII. Southern blot analysis of DNA extracted from the liver of rAAV-HLP-codop-hFVIII-N6 transduced mice revealed head-to-tail and head-to-head concatemer fragments of ≂f5kb and ≂f10kb respectively in the expected ratio of 3:1. Western blot analysis showed that the rAAV-HLP-codop-hFVIII-N6 cassette mediated the synthesis and secretion of a single chain 210kd protein. To confirm correction of the bleeding phenotype, either 4×10¹¹ (low-dose cohort, n=3) or 4×10¹² (high-dose cohort, n=3) rAAV5-HLP-codop-hFVIII-N6 vector genomes were injected into the tail vein of haemophilia A knockout mice. Peak hFVIII levels, as determined by a one-stage clotting assay, were 137±27% and 374±18% of normal levels in the low and high-dose cohorts of F8-/- mice respectively. These levels were sufficient to arrest bleeding in a modified tail clip assay. Anti-hFVIII antibodies were not detected in the rAAV treated HA mice at any stage. Therefore, the higher potency of our novel codop-hFVIII-N6 construct and the ability to package this FVIII variant within AAV virions has substantially improved the prospects of effective gene transfer for Hemophilia A.