SYNTHESIS AND PROCESSING OF GENETICALLY-MODIFIED HUMAN PROINSULIN BY RAT MYOBLAST PRIMARY CULTURES

Rat myoblast primary cultures were tested as a model for proinsulin sy nthesis and processing and unregulated insulin delivery for insulin-de pendent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease c leavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (lIFur) and/or containing a histidine B10 to as partic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The alter ed cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creat es a more stable form of insulin leading to an increase in insulin acc umulation. Myoblast transfected with a proinsulin cDNA construct mutat ed at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mu tation at the B10 position (pCMV.B10) accumulated 852 +/- 16, 150 +/- 13, and 883 +/- 39 mu U (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the cul ture medium within 2 hr post-transfection. Significant (pro)insulin re lease continued for 1 week and gradually diminished over a month. Appr oximately 50% of the proinsulin released from rat myoblasts transfecte d with pCMV.IFur.IIFur.B10 was completely processed into mature insuli n based on densitometric analysis of antoradiographs of gels containin g imuunoprecipitated S-35-Cys-labeled (pro)insulin. However, only a tr ace of the proinsulin encoded by pCMV.B10 was processed. In an isolate d rat adipocyte [C-14]glucose oxidation assay, insulin released from m yoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmi d expression was studied by transfecting myoblasts with the beta-galac tosidase (beta-Gal) gene in pCMV, allowing them to divide and fuse int o multinucleated myotubes, followed by staining for beta-Gal. Approxim ately 80% of myotubes expressed beta-Gal. The results indicate that pr oinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblast s a viable target cell for gene therapy of IDDM.