Gao Kai, Li Mengxin, Zhong Li, Su Qin, Li Jia, Li Shaoyong, He Ran, Zhang Yu, Hendricks Gregory, Wang Junzhi, Gao Guangping
Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605 ; National Institute for Food & Drug Control, Beijing, China.
Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605 ; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605 ; Department of Microbiology and Physiology Systems, University of Massachusetts Medical School, Worcester, MA 01605.
Mol Ther Methods Clin Dev. 2014;1(9):20139. doi: 10.1038/mtm.2013.9.
Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs). The aforementioned dosing formulations of rAAV8 expressing either cellular ( or nuclear-targeted (n) ) or secreted (human α1-antitrypsin, ) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ATL levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side-effects of rAAV8 transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.
空病毒粒子是重组腺相关病毒（rAAV）包装过程中产生的意外副产物，导致载体批次中含有不同比例的完整病毒粒子和空病毒粒子混合物。空病毒粒子对rAAV转导效率和副作用的影响尚未得到充分表征。在此，我们制备了部分和完全空的AAV8病毒粒子、完全包装的rAAV8批次以及具有不同空病毒粒子比例（REVs）的空病毒粒子与完全包装病毒粒子的混合物。将上述表达细胞（或核靶向（n））或分泌型（人α1-抗胰蛋白酶）报告基因的rAAV8给药制剂静脉注射到两种不同的小鼠品系中，然后在不同时间点分析转基因表达和血清丙氨酸氨基转移酶（ALT）水平。我们发现，在固定剂量的rAAV8制剂中添加空病毒粒子，分别使C57BL/6小鼠的肝脏转导抑制高达64%（血清hA1AT）和44%（nLacZ）。在BALB/c小鼠中观察到了抑制EGFP表达的类似趋势以及血清ATL水平的同时升高，表明空病毒粒子也可能加剧rAAV8转导的副作用。我们的结果表明，从rAAV制剂中去除空病毒粒子可能会提高AAV在临床应用中的疗效和安全性。
