Pre-clinical in vivo evaluation of pseudotyped adeno-associated virus (AAV) vectors for liver gene therapy

We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation Factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6, using efficient large-scale technology for particle production and purification. In immunocompetent mice, the resulting vector particles expressed high hFIX levels ranging from 36% (AAV-4) to over 2000% of normal (AAV-1,-2 and-6), which would exceed curative levels in hemophilic humans. Expression was dose-and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2x10 12 vector particles of AAV-1,-4 or-6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases.