Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy.
OBJECTIVE
Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral vectors are promising tools for liver-directed gene therapy. However, to date, transduction rates in vivo remain low in hepatocytes, without the induction of cell cycling. We investigated long-term transgene expression in quiescent hepatocytes in vitro and determined whether the lentiviral delivery of furin-cleavable insulin to the liver could reverse diabetes in rats.


METHODS
To improve transduction efficiency in vitro, we optimised hepatocyte isolation and maintenance protocols and, using an improved surgical delivery method, delivered furin-cleavable insulin alone or empty vector to the livers of streptozotocin-induced diabetic rats by means of a lentiviral vector. Rats were monitored for changes in body weight and blood glucose, and intravenous glucose tolerance tests were performed. Expression of insulin was determined by RT-PCR, immunohistochemistry and electron microscopy.


RESULTS
We achieved long-term transgene expression in quiescent hepatocytes in vitro (87 +/- 1.2% transduction efficiency), with up to 60 +/- 3.2% transduction in vivo. We normalised blood glucose for 500 days-a significantly longer period than previously reported-making this the first successful study using a lentiviral vector. This procedure resulted in the expression of genes encoding several beta cell transcription factors, some pancreatic endocrine transdifferentiation, hepatic insulin storage in granules, and restoration of glucose tolerance. Liver function tests remained normal. Importantly, pancreatic exocrine transdifferentiation did not occur.


CONCLUSIONS
Our data suggest that this regimen may ultimately be employed for the treatment of type 1 diabetes.
Ren
B
B
Department of Medical and Molecular Biosciences, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW, 2007, Australia.
O'Brien
B A
BA
Department of Medical and Molecular Biosciences, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW, 2007, Australia.
Swan
M A
MA
Anatomy and Histology, University of Sydney, Sydney, NSW, Australia.|Bosch Institute, University of Sydney, Sydney, NSW, Australia.
Koina
M E
ME
Department of Medical and Molecular Biosciences, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW, 2007, Australia.|Department of Anatomical Pathology, Canberra Hospital, Canberra, ACT, Australia.
Nassif
N
N
Department of Medical and Molecular Biosciences, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW, 2007, Australia.
Wei
M Q
MQ
Gene Therapy Unit, University Department of Medicine, Prince Charles Hospital, Brisbane, QLD, Australia.
Simpson
A M
AM
Department of Medical and Molecular Biosciences, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW, 2007, Australia. Ann.Simpson@uts.edu.au.
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D003001:Cloning, Molecular; D003921:Diabetes Mellitus, Experimental / Q000235:genetics* / Q000628:therapy*; D015316:Genetic Therapy; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D007328:Insulin / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001786:Blood Glucose; D007328:Insulin