Secretion of bioactive human insulin following plasmid-mediated gene transfer to non-neuroendocrine cell lines, primary cultures and rat skeletal muscle in vivo.
The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.
Shaw
J A M
JA
Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK. jim.shaw@ncl.ac.uk
Delday
M I
MI
Hart
A W
AW
Docherty
H M
HM
Maltin
C A
CA
Docherty
K
K
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003920:Diabetes Mellitus / Q000628:therapy; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007273:Injections, Intramuscular; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D012867:Skin / Q000166:cytology; D014162:Transfection / Q000379:methods*
D001786:Blood Glucose; D011384:Proinsulin