Use of a Hybrid Adeno-Associated Viral Vector Transposon System to Deliver the Insulin Gene to Diabetic NOD Mice

Abstract

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.

Keywords: adeno-associated viral vector; diabetes; gene therapy; liver; transposon.

Figure 1

Immunofluorescence detection of marker gene expression in NOD mouse livers following transduction with AAV vectors. Frozen sections of mouse liver were prepared for immunofluorescence detection and visualised by fluorescence microscopy. (A) m-Cherry-INS-FUR immunofluorescence 9 weeks after intial transduction, (B) DAPI-stained nuclei of A and (C) is a merged image of A and B. (D) Venus-INS-FUR immunofluorescence 9 weeks after INS-FUR and Pdx1 were delivered together. (E) shows the DAPI-stained nuclei of D and (F) is a merged image of D and E. (G) is a normal liver section showing no immunofluorescence for either venus or mCherry and (H) is an image of DAPI-stained nuclei of (G) and (I) is a merged image of (G) and (H). (J) piggyBac-INS-FUR-mCherry immunofluorescence 15 weeks after initial transduction, (K) is the DAPI-stained nuclei of (J) and (L) is a merged image of (J) and (K). Bar = 20 µm.

Figure 2

Blood glucose levels, IPGTTs and AAV8 VCNs of NOD mice after expression of INS-FUR alone, or with Pdx1, using the AAV8 vector. Transduction of NOD mice with AAV8-INS-FUR or AAV8-INS-FUR + AAV8-Pdx1 did not reverse hyperglycaemia in diabetic mice. (A) The mean weekly blood glucose levels of diabetic (n = 7), normal (non-diabetic) (n = 7) and treated diabetic mice that received i.p. injections of either AAV8-INS-FUR-mCherry (n = 4), or AAV8-INS-FUR-venus + AAV8-Pdx1 (n = 5) are shown. (B) Blood glucose levels following an IPGTT of diabetic (n = 3), non-diabetic (n = 7) and treated diabetic mice that received i.p. injections of either AAV8-INS-FUR-mCherry (n = 3) or AAV8-INS-FUR-venus + AAV8-Pdx1 (n = 4). (C) AAV8 vector copy numbers of the diabetic mice transduced by AAV8-INSFUR-mCherry (n = 4) or AAV8-INSFUR-venus + AAV8-Pdx1 (n = 5). Results are expressed as the means ± SEMs. * indicates a significant difference of p < 0.05 when comparing the VCNs.

Figure 3

Blood glucose levels, IPGTT and AAV8 vector copy numbers of NOD mice after AAV8-mediated expression of INS-FUR in combination with the FFO surgical procedure, with or without dual expression of the lentiviral HMD vector. NOD mice were transduced with AAV8-INS-FUR in combination with the FFO surgery, as well as with dual expression of HMD. Blood glucose levels and results of subsequent IPGTT tests of the mice are shown. (A) Mean weekly blood glucose levels of the diabetic (n = 7), normal, non-diabetic (n = 7), and diabetic NOD mice that received i.p. injections of AAV8-INS-FUR-venus + FFO surgery (n = 6) and diabetic NOD mice that received AAV8-INS-FUR-mCherry + HMD-EGFP (n = 6). (B) Blood glucose levels following an IPGTT of diabetic (n = 3), non-diabetic (n = 7) and diabetic mice that received i.p. injections of AAV8-INS-FUR-mCherry (n = 3). The symbols for each group are indicated in the accompanying legend. Results are expressed as the means ± SEMs at each time point. (C) AAV8 vector copy numbers of the diabetic mice that received i.p. injections of AAV8-INS-FUR-venus + FFO surgey (n = 6) and diabetic NOD mice that received AAV8-INS-FUR-mCherry + HMD-EGFP (n = 5). Results are expressed as the means ± SEMs.

Figure 4

Blood glucose levels and AAV8 vector copy numbers of NOD mice after piggyBac/AAV8-mediated expression of INS-FUR and INS-FUR combined with the FFO surgical procedure. Diabetic NOD mice were transduced with either INS-FUR or INS-FUR in combination with the FFO surgical procedure at 7 days after transduction. (A) The mean weekly blood glucose levels of untreated diabetic (n = 7), normal, non-diabetic (n = 7) and treated diabetic NOD mice that received either i.p. injections of AAV8-piggyBac/INS-FUR-mCherry (n = 7) alone, or AAV8-piggyBac/INS-FUR-mCherry with FFO surgery (n = 7). (B) Transposon and transposase copy numbers of diabetic NOD mice that received i.p. injections of AAV8/piggyBac-INS-FUR or i.p. injection of AAV8/piggyBac-INS-FUR and FFO sugery. The results are expressed as the means ± SEMs.

Figure 5

Normalisatiion of glucose tolerance of diabetic NOD mice after expression of INS-FUR by the integrating piggyBac/AAV8 vector combined with the FFO surgical procedure. Diabetic NOD mice were transduced with INS-FUR, or INS-FUR in combination with the FFO surgical procedure at 7 days after transduction, using the integrating piggyBac/AAV8. (A) Blood glucose levels following an IPGTT of diabetic (n = 3), non-diabetic (n = 7) and diabetic NOD mice that received i.p. injections of INS-FUR alone as AAV8-piggyBac/INS-FUR-mCherry (n = 7). (B) Blood glucose levels following an IPGTT of diabetic (n = 3), non-diabetic (n = 7) and diabetic NOD mice that received i.p. injections AAV8-piggyBac/INS-FUR-mCherry combined with the FFO surgery (n = 6). (C) Serum concentration of human insulin following IPGTT of the mice represented in (B), which received i.p. injections of AAV8-piggyBac/INS-FUR-mCherry combined with FFO surgery. Results are expressed as the means ± SEMs.

Figure 6

Expression of β-cell transcription factors and pancreatic hormones in the livers of transduced NOD mice. Standard end point (non-quantitative RT-PCR) analysis was conducted from RNA derived from liver tissues of the NOD mice to detect the expression of INS-FUR, Pdx1, NeuroD1, mouse insulin 2, mouse somatostatin, pancreatic polypeptide, Glut 2, and beta-actin (positive control) in normal mouse liver (lane 1), liver tissue from diabetic NOD mice expressing INS-FUR via i.p. injection of AAV8-INS-FUR-mCherry (lane 2), INS-FUR and Pdx1 via i.p. injection of AAV8-INS-FUR-venus + AAV8-Pdx1 (lane 3), AAV8-INS-FUR-venus + FFO surgery (lane 4), via i.p. injection of AAV8-INS-FUR-venus + HMD/MSCV-EGFP via the portal vein (lane 5), AAV8/piggyBac-INS-FUR-mCherry (lane 6), AAV8/piggyBac-INS-FUR-mCherry + FFO surgery (lane 7) and normal mouse pancreas (lane 8).