Previously, we used a lentiviral vector to deliver furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes using intervallic infusion in full flow occlusion (FFO), with resultant reversal of diabetes, restoration of glucose tolerance and pancreatic transdifferentiation (PT), due to the expression of beta (β)-cell transcription factors (β-TFs). The present study aimed to determine whether we could similarly reverse diabetes in the non-obese diabetic (NOD) mouse using an adeno-associated viral vector (AAV) to deliver INS-FUR ± the β-TF Pdx1 to the livers of diabetic mice. The traditional AAV8, which provides episomal expression, and the hybrid AAV8/piggyBac that results in transgene integration were used. Diabetic mice that received AAV8-INS-FUR became hypoglycaemic with abnormal intraperitoneal glucose tolerance tests (IPGTTs). Expression of β-TFs was not detected in the livers. Reversal of diabetes was not achieved in mice that received AAV8-INS-FUR and AAV8-Pdx1 and IPGTTs were abnormal. Normoglycaemia and glucose tolerance were achieved in mice that received AAV8/piggyBac-INS-FUR/FFO. Definitive evidence of PT was not observed. This is the first in vivo study using the hybrid AAV8/piggyBac system to treat Type 1 diabetes (T1D). However, further development is required before the system can be used for gene therapy of T1D.

These data show that human hepatocytes can be induced to undergo partial pancreatic transdifferentiation in vivo, indicating that the technology holds promise for the treatment of type 1 diabetes.

Previously we introduced furin-cleavable human insulin (INSFUR) to the livers in several animal models using a lentiviral vector. Reversal of diabetes with normal glucose tolerance and pancreatic transdifferentiation was seen. The aim of the present study was to ascertain if we could similarly reverse diabetes in the non-obese diabetic (NOD) mouse using a clinically-applicable AAV system to deliver INSFUR ± the β-cell transcription factor (TF) Pdx1 to the livers of the diabetic mice. The traditional AAV8 that allows predominantly episomal expression, and the hybrid AAV8/piggyBac that results in transgene integration into the host genome were used. Weight and blood glucose levels were monitored. Intraperitoneal glucose tolerance tests (IPGTTs) were performed. Immunofluorescence was carried out to monitor transduction utilising marker genes Venus and mCherry. RT-PCR was used to identify expression of pancreatic genes. Diabetic mice that received intraperitoneal (IP) injections of AAV8-INSFUR-mCherry (2.3x 1010-4.5x1010vg) became hypoglycaemic with abnormal responses to IPGTTs. No β-cell TF could be detected in the livers. Reversal of diabetes could not be achieved in the mice (n=8) that received combinations of AAV8-INSFUR-venus (5x1010 or 7.5x1010vg) and AAV8-Pdx1-mCherry (7.5x1010 vg). Both INSFUR and Pdx1 were detected using RT-PCR and immunofluorescence showed the expression of both marker genes. IPGTTs were abnormal; however, the β-cell TF MafA was detected by RT-PCR. Euglycaemia and normal IPGTTs were achieved in mice (n=4) that received AAV8/piggyBac-INSFUR-mCherry (transposase 4x1010 vg and transposon 3.5x1010 vg). The INSFUR gene and the β-cell TFs NKx6.1 and MafA were detected in the livers, however wider evidence of pancreatic transdifferentiation was not seen. This is the first in vivo study using the hybrid AAV8/piggyBac system to treat type 1 diabetes (T1D). The AAV system, with further development, may hold promise for gene therapy of T1D. Disclosure Q.T. La: None. B. Ren: None. G.J. Logan: None. S.C. Cunningham: None. N. Khandekar: None. N.T. Nassif: None. B.A. O'Brien: None. I.E. Alexander: Other Relationship; Self; LogicBio. A.M. Simpson: Consultant; Self; Pharmacyte Biotech.