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ABSTRACT: Column chromatography has been described for purification of recombinant adeno-associated viral vectors (rAAV) serotypes 1, 2, 5, 6 and 8. Some of these purification processes have been used in manufacturing pre-clinical grade and clinical grade rAAV vectors. Recently, recombinant AAV9 has been reported to be highly efficient in transducing cardiac muscle in animal models. Systemic or cardiac gene delivery and other applications may require large quantities of rAAV9 vectors, thus a scalable method supporting large scale purification of rAAV9 is needed for clinical development. However, column chromatography-based purification has not been reported to date for rAAV9. This study reports a polyethylene glycol (PEG) modulated chromatography process for purification of AAV9 vectors. Inclusion of PEG in chromatography buffers modulated rAAV9 elution profiles in a manner that resulted in significantly improved resin binding capacity, vector purity and yield. PEG-modulated methods were developed and optimized for hydroxyapatite and ion exchange chromatography, and shown to result in vectors of high purity and functional activity.
Journal of virological methods 02/2011; 173(1):99-107. · 2.13 Impact Factor
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ABSTRACT: The capsid protein synthesis in targeted tissues resulting from residual contaminating replication-competent adeno-associated virus particles (rcAAV) remains a concern for hazardous immune responses that shut down the factor IX expression in the hemophilia B clinical trial. To systematically reduce/eliminate the effects of potential contaminating rcAAV particles, we designed a novel adeno-associated virus (AAV) helper (pH22mir) with a microRNA binding cassette containing multiple copies of liver-specific (hsa-mir-122) and hematopoietic-specific (has-mir-142-3p) sequences to specifically control cap gene expression. In 293 cells, the rep and cap gene from pH22mir functioned similarly to that of conventional helper pH22. The vector yields and compositions from pH22mir and pH22 were indistinguishable. The performance of vector produced in this new system was comparable to that of similar vectors produced by conventional methods. In the human hepatic cell line, the capsid expression was reduced significantly from cap-mir cassette driven by a cytomegalovirus promoter. In the liver, 99.9% of capsid expression could be suppressed and no cap expression could be detected by western blot. In summary, we demonstrated a new concept in reducing de novo capsid synthesis in the targeted tissue. This strategy may not only help AAV vectors in controlling undesirable capsid gene expression, but can also be adopted for lentiviral or adenoviral vector production.
Human gene therapy 01/2011; 22(5):625-32. · 4.20 Impact Factor
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ABSTRACT: Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a "chain imbalance" problem in which LC secretion is approximately 1-2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC(745)). The optimized HC mutein, HC(HL), including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HC(HL)+AAV8-LC or AAV8-HC(745)+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HC(HL)+AAV8-LC achieved three- to fourfold higher HC expression than AAV8-HC(745)+AAV8-LC, consistent with the FVIII functional assays. HC(HL) should be substituted for HC(745) in a dual AAV vector strategy due to its enhanced expression.
Molecular Therapy 02/2009; 17(3):417-24. · 6.87 Impact Factor
