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Introduction: The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV‐based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half‐life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). Aim: Adeno‐associated viral vectors (AAV) mediating expression of hFIX‐Alb and hFIX‐Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half‐life translates to higher plasma levels of FIX. Methods: Single‐stranded cross‐packaged AAV2/8 vectors expressing hFIX‐Alb, hFIX‐Fc and hFIX were evaluated in vitro, and in mice. Results: Both hFIX‐Alb and hFIX‐Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV‐mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX‐fusion proteins were comparable to wild‐type hFIX. However, their expression levels were threefold lower than wild‐type hFIX in vivo most likely due to inefficient secretion. Conclusion: This, the first, evaluation of hFIX‐fusion proteins in the context of AAV gene transfer suggests that the hFIX‐fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches. [ABSTRACT FROM AUTHOR]

Background Copolymers based on poly-[ N-(2-hydroxypropyl) methacrylamide] (HPMA) have been used previously to enable targeted delivery of adenovirus. Here we demonstrate polymer-coating techniques can also be used to modify and retarget adeno-associated virus (AAV) types 5 and 8. Methods Three strategies for modifying transductional targeting of AAV were employed. The first involved direct reaction of AAV5 or AAV8 with amino-reactive HPMA copolymer. The second approach used carbodiimide (EDC) chemistry to increase the number of surface amino groups on the AAV5 capsid, thereby improving coating efficiency. In the third approach, the AAV5 genome was isolated from capsid proteins and delivered in a synthetic polyplex consisting of polyethylenimine (PEI) and HPMA. Results Efficient covalent attachment of HPMA copolymer to AAV5 could only be achieved following modification of the virus with EDC. Coating inhibited sialic acid dependent infection and provided a platform for retargeting via new ligands, including basic fibroblast growth factor. Retargeted infection was shown to be partially resistant to neutralising antisera. Delivery of AAV5 genomes using PEI and HPMA was efficient and provided absolute control of tropism and protection from antisera. In contrast AAV8 could be reacted directly with HPMA copolymer and allowed specific retargeting via the epidermal growth factor receptor, but gave no protection against neutralising antisera. Conclusions Reactive HPMA polymers can be used to ablate the natural tropism of both AAV8 and EDC-modified AAV5 and enable receptor-specific infection by incorporation of targeting ligands. These data show transductional targeting strategies can be used to improve the versatility of AAV vectors. Copyright © 2008 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]