247. Characterization of Oversized rAAV Vectors for Human FVIII Gene Transfer

Severe hemophilia A patients with 4 years and consequent reduction or elimination of the need for frequent factor infusions. Similar rAAV-mediated gene transfer for hemophilia A patients is challenging due to the size of the FVIII cDNA that when combined with the necessary transcriptional elements exceeds the rAAV packaging limit of 4.7 kb. To test the feasibility of rAAV-mediated gene therapy for hemophilia A, we generated several oversized FVIII expression cassettes of different lengths and containing human B-domain deleted FVIII cDNA. These constructs were used to produce rAAV vectors by triple transfection and virus quality and yield were analyzed. Characterization of the packaged vector genomes (VGs) by Southern blot showed that the majority of genomes were 4.7 kb in size (with some being larger) and that the vector yield was affected by VG size. The vectors were also administered intravenously into C57BL/6 mice to test in vivo potency. Plasma FVIII levels were measured for 7 months post-treatment and at the end of the study, levels of FVIII mRNA and liver VG copies were quantitated. The data showed intact expression cassettes and comparable number of VG copies in liver among the vectors. Also, the higher liver mRNA levels correlated with higher plasma FVIII levels indicating that expression levels were a limiting factor. Lastly, the rAAV vector ranking was similar to ranking of plasmid expression cassettes (hydrodynamic injections) in vivo. These studies confirmed the viability of oversized rAAV vectors as an approach for the treatment of severe hemophilia A.