摘要
目的评价携带人凝血因子FⅧ(hFⅧ)的重组腺相关病毒(AAV)血清型8(AAV8/hFⅧ)病毒治疗血友病A(HA)小鼠效果。方法应用pAAV-CB-EGFP,pH22(AAV2血清型)和pfΔ6(腺病毒辅助质粒)摸索在HEK-293细胞中AAV的包装条件,pH22、pfΔ6及pAAV-CB-EGFP质粒按照1∶1∶1的比例进行转染,免疫荧光显微镜观察四个条件(包括10 cm培养皿转10μg质粒,20 cm培养皿分别转20、30和40μg质粒)的病毒包装效果。应用最佳包装条件在HEK-293细胞中包装AAV2-EGFP,通过冻融法获得AAV2粗提液,感染HEK-293细胞和16095细胞,应用荧光显微镜观察包装效果。应用最佳转染条件将pAAV-TTR-hFⅧ、pH28、pfΔ6按1∶1∶1的比例转染HEK-293细胞获得AAV8/hFⅧ,两轮的氯化铯梯度离心法纯化病毒,以8×10^12 vg/kg剂量经尾静脉注射HA小鼠进行基因治疗,活化部分凝血活酶时间(APTT)测定分析FⅧ的活性。结果20 cm培养皿转染20μg质粒的条件能够在24、48和72 h都达到最佳的转染效果,且该条件包装的AAV2粗提液在16095细胞中具有较高的感染率。HA小鼠体内AAV8/hFⅧ能够在注射后12周仍有维持在治疗水平的FⅧ活性。结论利用优化包装条件制备的AAV8/hFⅧ能够在HA小鼠体内有效维持治疗水平的FⅧ活性达12周。
Objective To evaluate the effects of adeno-associated virus(AAV)carrying hFⅧby serotype 8(AAV8/hFⅧ)on hemophilia A(HA)mice by gene therapy strategy.Methods pAAV-CBEGFP,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/hFⅧin HEK-293 cells using the optimized package condition.The purified AAV8/hFⅧwere intravenously injected into HA mice and the effects of gene therapy were estimated.Results 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 10μg plasmids,20 cm-dish to 20μg,30μg and 40μg plasmids were employed,and the condition of 20 cm-dish to transfect 20μg 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/hFⅧwas packaged and purified based on the optimized transfection condition,and the high purity of AAV8/hFⅧwas detected by Western blot.Fractions of AAV8/hFⅧat 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 FⅧwas measured by aPTT assay.Results showed that the activity of FⅧmaintained at the therapeutic level and lasted up to 12 weeks after injection.Conclusion The purified AAV8/hFⅧbased on the optimized package condition could play a role in HA mice gene therapy,and the long-term therapeutic ef
作者
毛建华
沈艳
王嫱
王韵
阮铮
奚晓东
Mao Jianhua;Shen Yan;Wang Qiang;Wang Yun;Ruan Zheng;Xi Xiaodong(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)
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2020年第1期34-39,共6页
Chinese Journal of Hematology
基金
国家自然科学基金(81970112、81670127、81101721)
上海市浦江人才计划(16PJ1406100)
上海市卫生健康委员会面上项目(201940342)
中国诺和诺德血友病基金。
