Gene Therapy for diabetes mellitus with non-endocrine cells

点击翻译按钮获取中文摘要

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.