Experimental Gene Therapy Utilizing a Skin Transplant

大多数多肽激素是以前肽的形式产生的，并在从高尔基体到分泌颗粒的运输过程中转化为生物活性多肽。前肽向生物活性多肽的转化是内分泌细胞的独特功能。在内分泌细胞和非内分泌细胞中表达的一些前肽激素cDNAs，包括人胰岛素原cDNAs，一般都被正确处理，而在非内分泌细胞中表达的cDNAs则以非裂解的前肽的形式被结构性地分泌。然而，非内分泌细胞不能将前肽转化为生物活性多肽并不意味着它们无法将前肽转化为活性多肽。包括成纤维细胞、肝细胞和淋巴细胞在内的非内分泌细胞产生生物活性前肽，并通过切割唯一的共有序列-Arg^&lt；-4&gt；-X^&lt；-3&gt；-Lys/Arg^&lt；-2&gt；-Arg^&lt；-2&gt；@*X^&lt；+1…将其转化为生物活性多肽。更多&>-。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，构建了一个突变的胰岛素原DNA，它的结构是由一对四碱基残基以如下顺序连接到C肽上的：B链-Arg-Arg-Lys-Arg-C肽-Arg-Arg-Lys-Arg-A链，而天然的胰岛素原结构是B链-Arg-Arg-C肽-Lys-Arg-A链。突变的和天然的胰岛素原都在猴子肾脏来源的细胞系COS-7中表达，该细胞只有一个结构性的分泌途径，被认为不能将天然的胰岛素原加工成成熟的胰岛素。当表达突变胰岛素时，约60%的免疫活性胰岛素在培养基中以成熟胰岛素的形式出现。此外，突变的胰岛素原与枯草杆菌素样内切酶呋喃共表达，胰岛素原完全转化为成熟胰岛素。通过将3-O-甲基-D-葡萄糖掺入脂肪细胞，COS细胞产生的胰岛素具有与合成的人胰岛素相同的生物活性。我们证明，在非内分泌细胞系COS-7细胞中，突变的胰岛素原在加工部位带有一对四碱基残基，可以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构建可能被用于杂交类型的人工胰岛或用于基因治疗。较少

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