Long-Term Expression of Human Factor Ⅸ cDNA in Rabbits

In this study, rabbits were used as a model for gene therapy for hemophilia B, Human factor Ⅸ cDNA was transferred to cultured normal rabbit skin fibroblasts (RSF) by a recombinant plasmid (pCMVIX) or retrovirus(XL-IX or N2CMVIX) constructed in our laboratoy. Infected fibroblasts capable of synthesizing and secreting high levels of biologically active human factor Ⅸ protein were selected and embedded in a collagen matrix. The latter was surgically implanted into rabbits as autografts or allografts. Human factor Ⅸ protein was detected in the plasma of all the grafted rabbits, and its expression has been maintained for more than 10 months at the time of writing. In addition, we have improved and simplified the method of implantation from surgically grafting the tissue-like matrix to the injection of the infected cell-collagen mixture subcutaneously. Using the latter method, human factor Ⅸ in rabbits injected with RSF-N2CMVIX reached a peak of 480 ng/ml plasma, and its expression has continued for more     

带有人凝血因子Ⅸ cDNA的反转录病毒载体的构建及其在离体细胞中的高效表达

报道了带有人凝血因子IX cDNA的高滴度高表达安全性反转录病毒载体的构建。采用LNL6反转录病毒载体为骨架,构建了由人巨细胞病毒启动子(hCMV)驱动的反转录病毒载体LNCIX,由反转录病毒LTR启动子驱动的反转录病毒载体LIXSN,以及由LTR和CMV启动子共同控制转录的反转录病毒载体LCIXSN,分别用电穿孔方法转导PA317辅助细胞株。LNCIX和LIXSN反转录病毒载体能在离体细胞中表达人IX因子蛋白,而LC'IXSN反转录病毒载体转移到离体细胞中没有检测到人IX因子蛋白。PA317/INCIX细胞的产病毒滴度为8×10~5CFU/ml,该重组病毒感染人纤维肉瘤细胞HT1080及血友病B患者皮肤成纤维细胞(HSF),用ELISA方法分别测定这些细胞的IX因子蛋白产量,LNCIX载体在HT-1080细胞中的人IX因子平均表达量为3.3μg/10~6细胞·d~(-1);在HSF细胞中的平均表达量为2.5μg/10~6细胞·d~(-1),其中80％以上的IX因子具有凝血活性,与过去相比,提高了产病毒滴度,增加了人IX因子的平均表达量,病毒载体骨架的设计更完善,降低了产生野生型病毒的概率,提高了安全性,对于进一步开展血友病B的基因治疗具有重要的意义。     

HIGH EFFICIENT TRANSFER AND EXPRESSION OF HUMAN CLOTTING FACTOR Ⅸ cDNA IN CULTURED HUMAN PRIMARY SKIN FIBROBLASTS FROM HEMOPHILIA B PATIENT BY RETROVIRAL VECTORS

To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 cfu/ml. The factor Ⅸ proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor Ⅸ at the rate of 584 rig/10~6 cells/24h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor Ⅸ than LTR promoter. The highest expressed retroviral vector XL-Ⅸ was used to infect a line of f     

Expression of Human Factor Ⅸ cDNA in Mice by Implants of Genetically Modified Skin Fibroblasts From a Hemophilia B Patient

Double-copy retroviral vector containing human factor Ⅸ cDNA driven by human cytomegalovirus enhancer-promoter was constructed. The vector was introduced into the amphotropic packaging cell line PA317. The recombinant virus produced in PA317 was used to transduce skin fibroblasts from a hemophilia B patient. The infected cells produced high levels of biologically active human factor Ⅸ at a rate of 3420 ng/10~6 cells/24 h. These cells were embedded in a collagen matrix and implanted into the peritoneal cavity or subcutaneous space of mice. It was demonstrated that human factor Ⅸ was produced by the implants for at least 12 days in vivo, reaching a peak of 105 ng/ml plasma. Over 90% of the protein was functionally active. This technique has the potential to be developed into a new approach for gene therapy for hemophilia B.     

50例中国人IX因子基因的限制性片段长度多态性

用IX因子基因内探针F9(VⅢ)对TaqI,BamHI和EcoRI酶切的50例中国人基因组DNA进行杂交分析。结果表明,所有个体经TaqI酶切的杂交片段为4.5kb和1.8kb,BamHI和EcoRI酶切的杂交片段分别为23kb和5.0 kb。基因组DNA样本中未发现限制性片段长度多态性(RFLP),这与欧美国家的民族群体中存在着IX因子基因内TaqI和BamHI的RFLP的结论不同。造成不同种族间DNA水平差异的原因,很可能与长期在不同地理环境中的进化适应有关。     

带有XylE基因的EB病毒穿梭质粒pFD891的构建

构建了一个以EB病毒为基础、以邻苯二酚氧化酶(XylE)基因为靶基因、以潮霉素B邻酸转移酶为转化细胞的选择标记基因的穿梭质粒。这种质粒既能在细菌中复制,又能在真核细胞中自主复制,并有较高的拷贝数,因而能很容易地从真核细胞中分离出来,并可用来研究基因突变和突变类型的形成机制,建立一种化学物质诱变性的分子检测系统。     

人凝血Ⅸ因子乳腺生物反应器的研制

为了建立人凝血Ⅸ因子乳腺生物反应器,以ＭＣＫ增强子,β－ａｃｔｉｎ启动子控制ｈＦＩＸ小基因表达顺序,反向插入Ｇ１Ｎａ框架,构建复制缺陷反转录病毒载本,并制备重组反围录病毒颗粒;     

REGIONAL ASSIGNMENT OF HUMAN ALCOHOL DEHYDROGENASE (ADH) GENE TO 4pter→4q21

The hybrid clone FD1 constructed by fusion of Chinese hamster cell line Wg3-h with human lymphocyte was irradiated with X-ray. Fourteen survival clones were isolated and 3 of them, F5B, F52B, F61A were analyzed in detail by cytogenetic and biochemical methods. The results of chromosome Gbanding followed by Giemsa-11 differential staining show that there exists a deleted human chromosome 4 in all of the three hybrids. This deletion of human chromosome 4 in F61A is 4pter→4q21. The results of isozyme analysis of phosphoglucomutase-2 (PGM2) which is located on 4p14→4q21 confirm our cytogenetic conclusion. We used polyaerylamide gel electrophoresis to study the alcohol dehydrogenase (ADH) in human lymphocyte, Wg3-h and hybrid clones. Their electrophoretic pattern showed that human ADH isozyme did express, in the peripheral blood lymphocyte, hybrids FSB, F52B, F61A and FD1. According to these results, we suggest that one of the Class 1 ADH structural genes is located on the human chromosome 4pter→4q21. Rec