Efficient Production of Dual Recombinant Adeno-Associated Viral Vectors for Factor VIII Delivery
Efficient Production of Dual Recombinant Adeno-Associated Viral Vectors for Factor VIII Delivery
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DOI：
10.1089/hgtb.2014.093
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发表时间：
2014-08-01
影响因子：
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通讯作者：
Xiao, Weidong
中科院分区：
文献类型：
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作者：
Wang, Qizhao;Dong, Biao;Xiao, Weidong
重组腺相关病毒（rAAV）载体因其高安全性和临床疗效而在人类基因治疗中受到关注。对于因子 VIII 基因递送，将编码区拆分为两个 AAV 载体仍然是避免包装容量限制（类似于 5.0 kb）的可行策略。然而，分批生产两个 rAAV 载体既费时又费力。在这里，我们展示了在单一制剂中成功生产用于血友病 A 基因治疗的双 rAAV 载体。当携带人因子VIII重链(hHC)和轻链(hLC)表达盒的AAV载体质粒与AAVrep&cap和微型腺病毒辅助质粒一起共转染到293细胞中时，rAAV-hHC和rAAV-hLC都以所需的比例和高滴度产生。有趣的是，由于 rAAV-hHC 基因组的复制更有效，rAAV-hHC 载体总是比 rAAV-hLC 载体产生更高的滴度。所得载体可有效地体外转导组织培养细胞。当将这些载体给予A型血友病小鼠时，通过活化部分凝血活酶时间测定和酶联免疫吸附测定在小鼠血浆中检测到因子VIII。分泌因子VIII的功能活性以及抗原水平与传统方法产生的载体相似。双载体生产方法已成功扩展到AAV2和AAV8血清型。总之，载体质粒的共转染提供了一种以显着降低的成本和劳动力生产双或多 AAV 载体的有效方法。
Recombinant adeno-associated viral (rAAV) vectors have gained attention for human gene therapy because of their high safety and clinical efficacy profile. For factor VIII gene delivery, splitting the coding region between two AAV vectors remains a viable strategy to avoid the packaging capacity limitation (similar to 5.0 kb). However, it is time-consuming and labor-intensive to produce two rAAV vectors in separate batches. Here we demonstrated successful production of dual rAAV vectors for hemophilia A gene therapy in a single preparation. When the AAV vector plasmids carrying the human factor VIII heavy chain (hHC) and the light chain (hLC) expression cassettes were cotransfected into 293 cells along with the AAV rep&cap and mini-adenovirus helper plasmids, both rAAV-hHC and rAAV-hLC were produced at the desired ratio and in high titer. Interestingly, the rAAV-hHC vectors always yielded higher titers than rAAV-hLC vectors as a result of more efficient replication of rAAV-hHC genomes. The resulting vectors were effective in transducing the tissue culture cells in vitro. When these vectors were administered to hemophilia A mice, factor VIII was detected in the mouse plasma by both the activated partial thromboplastin time assay and enzyme-linked immunosorbent assay. The functional activity as well as the antigen levels of secreted factor VIII were similar to those of vectors produced by the traditional method. The dual-vector production method has been successfully extended to both AAV2 and AAV8 serotypes. In conclusion, cotransfection of vector plasmids presents an efficient method for producing dual or multiple AAV vectors at significantly reduced cost and labor.