Experimental Gene Therapy Utilizing a Skin Transplant

大多数肽激素是作为前肽产生的，并在从高尔基体转运到分泌颗粒的过程中转化为生物活性肽。从前肽到生物活性肽的转化是内分泌细胞的独特功能。包括人胰岛素原cDNA在内的许多前肽激素cDNA已被引入内分泌和非内分泌细胞，在内分泌细胞中表达的cDNA通常被正确处理，而在非内分泌细胞中表达的其他cDNA则组成性地以非裂解前肽的形式分泌。然而，非内分泌细胞不能将前肽转化为生物活性肽并不意味着它们不能将前肽加工成活性肽。非内分泌细胞（包括成纤维细胞、肝细胞和淋巴细胞）产生无生物活性的前肽，并通过切割一个独特的一致序列-Arg^<-4>-X^<-3>- lys /Arg^<-2>-Arg^<-2>@*X^<+1…More >-因此，我们以胰岛素原作为其在非内分泌细胞中表达的模型前肽，构建了突变体胰岛素原DNA，其肽结构由B链和a链组成，通过一对四碱基残基依次与C肽相连：B链- arg - arg - lys - arg -C肽- arg - lys - arg - a链，而天然胰岛素原结构为B链- arg - arg -C肽- lys - arg - a链。突变型和天然胰岛素原均在猴肾源性细胞系COS-7中表达，该细胞系仅具有组成性分泌途径，被认为不会将天然胰岛素原加工成成熟胰岛素。当表达突变型胰岛素时，培养基中大约60%的免疫反应性胰岛素出现为成熟胰岛素。此外，突变体胰岛素原通过与枯草杆菌样内蛋白酶furin共表达，完全转化为成熟的胰岛素原。通过将3- o -[^3]甲基- d -葡萄糖结合到脂肪细胞中，COS细胞中产生的胰岛素具有与合成人胰岛素相同的生物活性。我们证明了在加工位点有一对四基残基的突变胰岛素原可以在非内分泌细胞系COS-7细胞中转化为完全生物活性的胰岛素。在未来，这种类型的突变胰岛素原DNA结构可能用于杂交型人工胰岛或基因治疗。少

Most of peptide hormones are produced as a propeptide and converted to a biologically active peptide during their transport from the Golgi apparatus to secretory granules. The conversion from propeptide to biologically active peptide is a unique function of endocrine cells. A number of propeptide hormone cDNAs, including a human proinsulin cDNA, have been introduced into both endocrine and non-endocrine cells, and those expressed in endocrine cells were generally processed correctly, while others expressed in non-endocrine cells were secreted constitutively as non-cleaved propeptides. However, inability of non-endocrine cells to convert propeptides to biologically active peptides does not mean their inability to process propeptides to active peptides. Non-endocrine cells including fibroblasts, hepatocytes, and lymphocytes produce biologically inactive propeptides and convert them to bioactive peptides by cleaving a unique consensus sequence -Arg^<-4>-X^<-3>-Lys/Arg^<-2>-Arg^<-2>@*X^<+1 … More >-. Thus, we took proinsulin as a model propeptide for its expression in non-endocrine cells, and constructed a mutant proinsulin DNA where peptide structure was comprized of B and A chains linked to C peptide by a pair of tetrabasic residues in the following order : B chain-Arg-Arg-Lys-Arg-C peptide-Arg-Arg-Lys-Arg-A chain, while the native proinsulin structure was B chain-Arg-Arg-C peptide-Lys-Arg-A chain. Both mutant and native proinsulin were expressed in a monkey kidney derived cell line, COS-7 cells that possess only a constitutive secretory pathway and is thought not to process native proinsulin to mature insulin. When mutant insulin was expressed, approximately 60% of the total immunoreactive insulin appeared as mature insulin in the culture medium. Moreover, mutant proinsulin was completely converted to mature one by co-expressing it with the subtilisin-like endoprotease, furin. The insulin produced in COS cells presented an identical biological activity to a synthetic human insulin by the capability of incorporating 3-O-[ ^3] methyl-D-glucose into adipocytes. We demonstrated that the mutated proinsulin with a pair of tetrabasic residues at the processing sites can be converted to fully bioactive insulin in a non-endocrine cell line, COS-7 cells. In the future this type of a mutant proinsulin DNA construct may be utilized for a hybrid type artificial islet or for gene therapy. Less