Gene Therapy for diabetes mellitus with non-endocrine cells

我们一直致力于产生能够分泌成熟人胰岛素的非内分泌细胞系。如果人成熟胰岛素在异位细胞或非内分泌细胞中产生，则应加工生物合成的胰岛素原。为此目的，改变人胰岛素cDNA的BC和CA连接处的C肽结构域的核苷酸序列，以获得激素原转化酶、弗林蛋白酶、识别位点。将修饰后的cDNA与重组逆转录病毒载体连接，构建小鼠L1前脂肪细胞和C2C12成肌细胞的间充质祖细胞系，用人特异性免疫分析法检测其培养液中人胰岛素的表达，并用特异性抗人胰岛素抗体进行免疫细胞化学染色。使用反相HPLC和质谱（MALDI）的生化分析显示，从转化细胞系分泌的胰岛素与天然人胰岛素相同。当将细胞移植到具有用于评估分泌的胰岛素的生物活性的由半透膜组成的免疫隔离室的糖尿病小鼠中时，小鼠的血糖水平恢复到接近正常范围，而非工程化细胞的小鼠的血糖水平没有变化，证明了工程化细胞移植的生物学效力。在对工程化前体细胞胰岛素分泌的观察中，胰岛素分泌速率随着细胞分化的进行而增加。这一发现对于基于替代细胞的治疗来说非常重要。我们目前的研究表明，新技术可以使替代细胞治疗严重的人类糖尿病。

We have focused on generation of non-endocrine cell lines that could secret mature human insulin. If human mature insulin would be generated in an ectopic cell, or a non-endocrine cell, biosynthesized proinsulin Should be processed. For this purpose, nucleotide sequences of C-peptide domain at the BC and CA junctions of human insulin CDNA were changed for the prohormone convertase, furin, recognition site. Murine mesenchymal progenitor celllines including LI preadipocytes and C2C12 myoblasts were then engineered by the modified CDNA with a recombinant retroviral vector.Human insulin was detected by a human-specific immunoassay in culture media of the engineered cells, and the cells could be stained immunocytochemically by specificanti-human insulin antibody. Biochemical analysis using reversephase HPLC and mass spectrometry (MALDI) revealed that the insulin secreted from the transformed cell lines was identical to native human insulin. When the cells were transplanted to diabetic mice with immunoisolating chamber consisted of semipermeable membrane for evaluation of biological activity of secreted insulin, blood glucose level of the mice were recovered to near normal range while that of mice with non-engineered cells showed no change, demonstrating the biological potency of transplantation of the engineered cells. In the observation of insulin secretion from the engineered precursor cells, insulin secretion rate is increased according to the differentijation of the cells would proceeds. This finding should be extremely important for surrogate cell-based therapy in general. Our present study suggests that the novel technologies could enable surrogate cell therapy for severe form of human diabetes mellitus.