Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer.
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.
Auricchio
A
A
Institute for Human Gene Therapy, The Wistar Institute, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Gao
G-P
GP
Yu
Q C
QC
Raper
S
S
Rivera
V M
VM
Clackson
T
T
Wilson
J M
JM
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
D007328:Insulin; D011384:Proinsulin; D020123:Sirolimus