血友病B基因治疗临床试验的安全性研究

Safety assessments required for animal test and preclinical trial of hemophilia B gene therapy

报道了血友病Ｂ基因治疗临床Ⅰ期试验的安全性研究，包括反转录病毒基因的ＤＮＡ聚合酶链式反应（ＰＣＲ）、反转录ＰＣＲ（ＲＴ／ＰＣＲ）、Ｓｏｕｔｈｅｒｎ杂交以及ｎｅｏ基因和ｌａｃＺ基因的补救分析（ｒｅｓｏｕｅａｓｓａｙ）．从ＤＮＡ水平。ＲＮＡ水平和病毒活体水平对野生型病毒进行了检测，没有检测到反转录病毒，并对兔和人转基因细胞进行形态学观察、染色体核型分析、软琼脂实验、裸鼠接种实验、兔和裸鼠的病理检测及电镜分析以检测恶性细胞的存在与否，同时进行支原体检测、内毒素和过敏原测定，结果均为阴性．建立了有复制能力的野生型反转录病毒（ＲＣＲ）的检测系统，为基因治疗临床试验的安全性提供了保证．

This paper deals with safety assessments required for animal test and clinical trial in haemophilia B gene therapy. Based on ourprevious works on gene transfer therapy for hemophilia B, a series of safety assessments were complered before clinical trial. Firstly, the existence of replication-completent retrovirus (RCR)were sensitively detected by Neo gene and lac Z gene rescue assay on the cellular level, and PCR, RT/PCR with Southern blot and dot blot on DNA and RNA level,results were found to be negative. Secondly, malignant transformation was followed on cells transduced with F ac cDNA by various techipues including cell morphology, histochemistry, chromsome karyotyping, soft-agar test, nude mice test and electromicroscopy, no abnormal changes were observed. Lastly, presence of bacteria, mold, endotoxin, toxin, allergen was rotinely done for culture medium and collagen and others used in clinical trial. All these safety assessments demonstrate that the safety of implantion of transduced cells is guaranteed.