Sustained production of human transferrin by transduced fibroblasts implanted into athymic mice: a model for somatic gene therapy.
Sustained production of human transferrin by transduced fibroblasts implanted into athymic mice: a model for somatic gene therapy.
复制标题
通过植入无胸腺小鼠的转导成纤维细胞持续产生人转铁蛋白：体细胞基因治疗模型。
DOI：
10.1111/1523-1747.ep12612734
复制
发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Krueger,GG
中科院分区：
文献类型：
--
作者：
Petersen,MJ;Kaplan,J;Jorgensen,CM;Schmidt,LA;Li,L;Morgan,JR;Kwan,MK;Krueger,GG
体细胞基因治疗被认为是一种治疗遗传性疾病的手段，包括血浆蛋白缺陷或缺失、病毒疾病和癌症。将目的基因导入成纤维细胞并使用皮肤等效系统植入这些转基因成纤维细胞可能是一种有吸引力的基因治疗模式，因为皮肤成纤维细胞很容易获得并在培养中繁殖。本研究通过体外和体内转基因成纤维细胞表达人转铁蛋白基因。将编码人转铁蛋白基因的逆转录病毒载体导入裸鼠非转移性肿瘤NIH 3T3成纤维细胞中。用有限稀释法克隆转导细胞，并测定克隆细胞的HTF产量。用两个克隆的高表达HTF的细胞接种于包被胶原的尼龙基质，培养长达53d。接种的基质在体外22 d后保持恒定的HTF合成速率。将克隆的转导细胞接种于7只裸鼠皮下，每两周检测血浆HTF水平。在所有动物中，在植入后6周可检测到血浆HTF水平。在动物体内，HTF水平持续升高，直到第12周植入物被取出。在第10周，血浆中HTF水平与小于2000mm3的肿瘤体积相关。HTF在小鼠体内的半衰期为39.5h。在该模型中，基因表达在12周的观察期内没有下降。
Somatic gene therapy has been proposed as a means of treating inherited diseases involving defective or absent plasma proteins, viral diseases, and cancer. Introduction of the gene of interest into fibroblasts and implantation of these genetically modified fibroblasts using a skin equivalent system may be an attractive model for gene therapy because skin fibroblasts are easily obtained and propagated in culture. This study evaluated expression of the gene for human transferrin (hTf) by genetically modified fibroblastsin vitroandin vivo.NIH 3T3 fibroblasts, which form non-metastasizing tumors in athymic mice, were transduced with a retroviral vector encoding hTf. The transduced cells were cloned by limiting dilution and hTf production by the cloned cells measured. Two clones of cells producing high levels of hTf were used to seed collagen-coated nylon matrices, which were maintained in culture for up to 53 d. The rate of synthesis of hTf by the seeded matrices was constant after 22 din vitro.Matrices seeded with cloned, transduced cells were implanted subcutaneously into seven athymic mice, and plasma levels of hTf were assessed biweekly. In all animals, the plasma level of hTf was detectable at week 6 after implantation. Levels of hTf remained elevated in the animals until the implants were removed at week 12. At week 10, the level of hTf in the plasma correlated with tumor volume in tumors less than 2000 mm3 in size. The half-life of hTf in the mice was 39.5 h. In this model, gene expression did not decline for the 12-week observation period.