title: Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer.
abstract: To test whether hepatocytes engineered in vivo can serve as surrogate beta cells by similarly secreting mature insulin in a glucose-sensitive manner, we prepared adenoviral vectors encoding wild-type proinsulin (hIns-wt), a modified proinsulin cleavable by the ubiquitously expressed protease furin (hIns-M3), or each of the two beta cell specific pro-insulin convertases PC2 and PC3. Following a detailed in vitro characterization of the proteins produced by our vectors, we infected the liver and, for comparison, the muscle of a chemically induced murine model of type I diabetes. Insulin expression from the transduced tissues was extensively characterized and showed to be constitutive rather than regulated. To obtain regulated expression, we placed expression of hIns-M3 under the control of the dimerizer-inducible transcription system. Hormone secretion from mouse liver was negligible in the absence of the dimerizer drug rapamycin, was inducible in a dose-dependent manner upon its administration, and reversible following drug withdrawal. These data confirm liver as a promising target for in vivo expression of processed insulin. While suggesting that hepatocytes cannot provide authentic glucose-responsive regulation, these results demonstrate that pharmacological regulation is a promising alternative route to the controlled delivery of insulin following hepatic gene transfer.
authors.affiliation: Institute for Human Gene Therapy, The Wistar Institute, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: A
authors.initials: A
authors.lastname: Auricchio
authors.forename: G-P
authors.initials: GP
authors.lastname: Gao
authors.forename: Q C
authors.initials: QC
authors.lastname: Yu
authors.forename: S
authors.initials: S
authors.lastname: Raper
authors.forename: V M
authors.initials: VM
authors.lastname: Rivera
authors.forename: T
authors.initials: T
authors.lastname: Clackson
authors.forename: J M
authors.initials: JM
authors.lastname: Wilson
mesh_terms: D000256:Adenoviridae; D000818:Animals; D002478:Cells, Cultured; D003131:Combined Modality Therapy; D003921:Diabetes Mellitus, Experimental; D003922:Diabetes Mellitus, Type 1 / Q000628:therapy*; D019281:Dimerization; D005260:Female; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D051379:Mice; D008819:Mice, Nude; D011384:Proinsulin / Q000235:genetics*; D020123:Sirolimus / Q000627:therapeutic use*; D014162:Transfection / Q000379:methods
chemical_list: D007328:Insulin; D011384:Proinsulin; D020123:Sirolimus