Mao J H, Shen Y, Wang Q, Wang Y, Ruan Z, Xi X D
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Center of experimental medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Department of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Zhonghua Xue Ye Xue Za Zhi. 2020 Jan 14;41(1):34-39. doi: 10.3760/cma.j.issn.0253-2727.2020.01.007.
To evaluate the effects of adeno-associated virus (AAV) carrying hFⅧ by serotype 8 (AAV8/hFⅧ) on hemophilia A (HA) mice by gene therapy strategy. pAAV-CB-EGFP, pH22 (serotype 2) and pfΔ6 (adenovirus helper) were used to package AAV into HEK-293 cells in different conditions (ratios of cells to plasmids). The efficiency of transfection and infection were evaluated using immunofluorescence microscope to seek an optimized package condition. pAAV-TTR-hFⅧ, pH 28 (serotype 8) and pfΔ6 were applied to package AAV8/hFⅧ in HEK-293 cells using the optimized package condition. The purified AAV8/hFⅧ were intravenously injected into HA mice and the effects of gene therapy were estimated. The efficiency of package was evaluated according to the amount and intensity of enhanced green fluorescent protein (EGFP) under immunofluorescence microscope. Four package conditions including 10 cm-dish to transfect 10 μg plasmids, 20 cm-dish to 20 μg, 30 μg and 40 μg plasmids were employed, and the condition of 20 cm-dish to transfect 20 μg plasmids reached the highest transfection efficiency at 24 h, 48 h and 72 h after transfection. The small scale AAV-EGFP was packaged using the optimized condition and an AAV crude extract was harvested by a freeze-thaw method. HEK-293 and 16095 cells were infected by the AAV crude extract, and the preferential infection efficiency was recognized in 16095 cells under immunofluorescence microscope. Then, AAV8/hFⅧ was packaged and purified based on the optimized transfection condition, and the high purity of AAV8/hFⅧ was detected by Western blot. Fractions of AAV8/hFⅧ at the dose of 8×10(12) vg/kg were injected into HA mice through tail vein, an eye-bleeding was performed at every two weeks, and the activity of FⅧ was measured by aPTT assay. Results showed that the activity of FⅧ maintained at the therapeutic level and lasted up to 12 weeks after injection. The purified AAV8/hFⅧ based on the optimized package condition could play a role in HA mice gene therapy, and the long-term therapeutic effects of AAV8/hFⅧ were observed in vivo.
通过基因治疗策略评估携带8型血清型人凝血因子Ⅷ（AAV8/hFⅧ）的腺相关病毒（AAV）对甲型血友病（HA）小鼠的影响。使用pAAV-CB-EGFP、pH22（血清型2）和pfΔ6（腺病毒辅助质粒）在不同条件下（细胞与质粒的比例）将AAV包装到HEK-293细胞中。使用免疫荧光显微镜评估转染和感染效率，以寻找优化的包装条件。使用优化的包装条件，将pAAV-TTR-hFⅧ、pH 28（血清型8）和pfΔ6应用于在HEK-293细胞中包装AAV8/hFⅧ。将纯化的AAV8/hFⅧ静脉注射到HA小鼠体内，并评估基因治疗的效果。根据免疫荧光显微镜下增强绿色荧光蛋白（EGFP）的量和强度评估包装效率。采用4种包装条件，即10 cm培养皿转染10 μg质粒、20 cm培养皿转染20 μg、30 μg和40 μg质粒，其中20 cm培养皿转染20 μg质粒的条件在转染后24 h、48 h和72 h达到最高转染效率。使用优化条件包装小规模AAV-EGFP，并通过冻融法收获AAV粗提物。用AAV粗提物感染HEK-293和16095细胞，免疫荧光显微镜下观察到16095细胞具有更高的感染效率。然后，基于优化的转染条件包装并纯化AAV8/hFⅧ，通过蛋白质免疫印迹法检测到高纯度的AAV8/hFⅧ。将剂量为8×10(12) vg/kg的AAV8/hFⅧ组分通过尾静脉注射到HA小鼠体内，每两周进行一次眼部出血实验，并通过活化部分凝血活酶时间（aPTT）测定法测量FⅧ活性。结果显示，注射后FⅧ活性维持在治疗水平并持续长达12周。基于优化包装条件纯化的AAV8/hFⅧ可在HA小鼠基因治疗中发挥作用，并在体内观察到AAV8/hFⅧ的长期治疗效果。
