Complete Correction of Hemophilia A with Adeno-Associated Viral Vectors Containing a Full-Size Expression Cassette

Abstract

Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a ␤-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3-to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3-to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

“…However, progress has been hampered by low levels of FVIII transgene expression and the large size of the FVIII cDNA. 16,17,36 In this study, we show that a single administration of rAAV that contains a codop hFVIII construct that includes the proximal 226 aa of the hFVIII B domain resulted in therapeutic expression of hFVIII in murine and non-human primate models. For a given dose of vector, expression mediated by rAAV-HLP-codophFVIII-N6 was almost 10-fold higher than observed with rAAV containing wild-type hFVIII sequences for the BDD or N6 variants (Table 1).…”

Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia

“…Specifically, human gene therapy trials using AAV2 and AAV8 serotypes for liver-directed correction of hemophilia are being performed, and preclinical approaches have used AAV2, AAV5 and AAV8 in animal models of hemophilia, directing therapy to the liver and to the joints. 3,23,24,26,[30][31][32] In addition, the onset of the most common complications of hemophilia (for example, inhibitor antibody formation and hemophilic joint disease) most commonly occur in childhood, so that gene therapy approaches to avoid or treat these complications ideally would be delivered to boys at this age. Boys entered the JOS trial at a very young age (o30 months of age) and regular prospective sample collection was performed for years during early childhood.…”

Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection

“…[1][2][3][4][5] The presence of smaller and fragmented genomes in oversized rAAV vector preparations can be tolerated because such vectors support the production of active FVIII protein and are efficacious in animal models of hemophilia A. [6][7][8][9][10] However, to improve oversized vector quality, the number of incomplete genomes should be minimized. Strategies that have been proposed to overcome the limitation of oversized rAAV vectors include the use of a two-vector system with separate rAAV vectors engineered to express the FVIII heavy and light chains and the use of split (transsplicing) AAV vectors.…”