Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265894
Title: Engineering non-neuroendocrine cell lines to constitutively secrete fully processed insulin
Author: Hart, Alan William
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1998
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Abstract:
Gene therapy, where non-islet cells are transduced to express insulin, encapsulated and implanted into the diabetic patient, is a possible alternative therapy for type I diabetes, which may alleviate some of the long-term complications of the disease. Insulin, however, is synthesised as a precursor, proinsulin, which is proteolytically modified by two endocrine-cell specific proteases, PC2 and PC3. Non-neuroendocrine cells do not express PC2 and PC3, but express a related protease, furin. Furin is unable to process proinsulin to insulin efficiently, so we employed PCR mutagenesis to alter the human preproinsulin cDNA around the normal processing sites, which when transcribed and translated yield cleavage sites recognised by furin. Wild type and mutant preproinsulin cDNAs were cloned into a mammalian expression vector under the control of the CMV promoter, and were expressed in the C2C12 and L6 myoblast cell lines and also the HepG2 liver cell line. Radioimmunoassays using an insulin specific antibody and an antibody that recognises proinsulin and insulin equally revealed that cells transfected with the wild type cDNA secreted 90% proinsulin whereas cells expressing the mutant cDNA secreted 75-90% processed insulin, suggesting more efficient processing of the mutant proinsulin by the endogenous furin. Further manipulation of the cDNAs linking them to the neomycin selection gene via an internal ribosome entry sequence (IRES), gave rise to stably transfected L6ins cell lines, expressing 2-10 ng/ml/24h total insulin-like immunoreactivity (ILI). With stable HepG2ins cells expressing in excess of 2.5 μg/5x107 cells/24h mature, biologically active human insulin. These high expressing HepG2ins cells were implanted into BB/Edinburgh rates and strepozotocin treated nude mice.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.265894  DOI: Not available
Keywords: Diabetes mellitus; Gene therapy; Insulin Medicine Molecular biology Cytology Genetics
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