重组AAV2/hFⅨ病毒制备及其基因治疗血友病B的实验研究  

Preparation of rV2/hFⅨ and experimentally application to gene therapy for hem ophilia B

作　　者：彭建强[1] 董小岩[2] 彭忞[2] 陈立[3] 谭淑萍[2] 袁洪[4] 陈方平[1] 薛京伦[3] 吴小兵[2] 

机构地区：[1]中南大学湘雅医院血液科,长沙410078 [2]国家863计划生物领域病毒基因载体研发基地 [3]复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室 [4]中南大学湘雅三医院

出　　处：《中华血液学杂志》2004年第9期513-518,共6页Chinese Journal of Hematology

基　　金："八六三"高科技发展计划基因治疗重大关键技术资助项目 (863 BH0 3 0502 )

摘　　要：目的　制备携带人凝血因子Ⅸ (hFⅨ )基因的重组AAV2病毒 (rAAV2 /hFⅨ ) ,并对用rAAV2 /hFⅨ肌肉注射治疗血友病B模型小鼠的疗效进行评价。方法　通过“一株载体细胞 /一株辅助病毒”的双因素包装策略制备出rAAV2 /hFⅨ ,体外转导BHK 2 1、C2C12细胞后 ,检测细胞培养上清中hFⅨ的表达量 ;肌肉直接注射血友病B模型小鼠后 ,检测其血浆中hFⅨ的抗原水平和凝血活性等指标。结果　转导 2 4h后在细胞上清中即可检测到hFⅨ ,连续检测 12 0h都有表达 ,BHK 2 1、C2C12细胞 2 4h最高表达量分别达到 (5 1.0± 6 .5 )ng/ 10 5细胞和 (6 8.0± 7.2 )ng/ 10 5细胞。rAAV2 /hFⅨ经肌肉直接注射后 ,高、中、低三个剂量组均能检测到小鼠体内高效表达hFⅨ ,在给药后第 3周达到高峰 ,小鼠血浆中hFⅨ的表达量与对照组比较差异有显著性 (P <0 .0 1) ,之后缓慢下降 ,到第 10周仍可检测到低水平hFⅨ表达 ;取第 3周小鼠血浆样品检测凝血功能 ,高、中、低剂量组FⅨ活性均得到明显改善 ,小鼠的割尾实验出血时间明显缩短 ,5min失血量也相应显著减少 ,其中高剂量组hFⅨ最高表达量达到 (387.0± 12 .5 )ng/ml血浆 ,FⅨ活性达到正常水平的 (30 .0± 5 .5 ) % ;给药后第 10周 ,除在注射点外 ,其它主要脏器均未检测到AAV载体DNA。Objective To prepare the rV2/hFⅨ and evaluate the efficiency of the preparation on gene therapy of hemophilia B model mice. Methods The rV-2/hFⅨ was prepared by 'one helper virus-one vector cell line' strategy and transfected both BHK-21 and C2C12 cells in vitro. The hFⅨ antigen level in ce ll culture supernatant was assayed. The rV-2/hFⅨ was injected into muscles o f hemophilia B model mice and assayed the serum hFⅨ levels, hFⅨ clotting acti vity, bleeding time, 5 min bleeding volume. Results The hFⅨ antige n could be detected from 24 h till 120 h after BHK-21 and C2C12 cells were t ransfected with highest levels at 24 h reaching (51.0±6.5)ng/10 5 cells a nd ( 68.0±7.2)ng/10 5cells, respectively. The rAAV2/hFⅨ injected mice could efficiently express hFⅨ and peaked at three weeks after injection, th en slowly decreased but low level hFⅨ antigen was still detectable till 10 week s after injection. There were significant differences between the high?middle and low dose groups of rAAV2/hFⅨ and the control group (P<0.01), t he plasma FⅨ clotting activities in the model mice were improved remarkably, b leeding time was greatly shortened and bleeding in 5 min was decreased. The hF Ⅸexpression level and FⅨclotting activity of the high dose of rAAV2/hFⅨ group (1.6×10 13v.g./kg) reached about (387.0±12.5)ng/ml plasma in contrast w ith the normal levels of ( 30.0±5.5)% at the third week after injection. No rAAV2 vector DNA was detected in the organs except for injected muscle tissue. Conclusion The rAAV2/hFⅨ transfected BHK-21 and C2C12 cells coul d efficiently express hFⅨ antigen and was of therapeutic effects for the hemop hilia B model mice by intramuscularly injection.The results provide the basis fo r clinical trial of rAAV2 gene therapy for hemophilia B.

关 键 词：血友病B 血浆 模型小鼠 基因治疗 表达量 C2C12细胞 转导 高效表达 凝血因子Ⅸ 高表达 

分 类 号：R554.1[医药卫生—血液循环系统疾病] R285.5[医药卫生—内科学]