Diabetes reversal via gene transfer: building on successes in animal models

Figures & data

Table 1 Animal models of T1D

Mechanism of induction	Model	Uses/studies
Chemically induced	High-dose STZ Multiple-dose STZ Alloxan	Drug discovery Models of transplantation Preventatives of β-cell destruction Restoration of normoglycemia
Spontaneous diabetes	NOD mice BB rats LEW.IARI/Ztm-iddm rat Keeshond dog Celebes black ape (Macaca nigra)	TID genetics and pathogenesis analyses Preventatives of β-cell destruction Immunomodulation studies Restoration of normoglycemia
Breeding/genetically selected	AKITA mice LETL rats New Zealand white rabbit Chinese hamster	Drug discovery Models of transplantation Preventatives of β-cell destruction
Pancreatectomized	Westran pigs Dogs	Restoration of normoglycemia β-cell regeneration
Virally induced	Coxsackie B virus Encephalomyocarditis virus Kilham rat virus	Role of viruses in the induction of TID

Note: This table describes the most commonly used animal models for the study of Type 1 diabetes.

Abbreviations: BB, biobreeding; LETL, Long-Evans Tokushima Lean; NOD, nonobese diabetic; STZ, streptozotocin; T1D, Type 1 diabetes.

Table 2 Insulin used for the reversal of T1D in animal models

Insulin used	Targetcell/tissue	Vector used	Animal model	Outcome	References
INS-FUR	Liver (in vivo)	pPAX2 (lentivirus)	LEW.IAR1/Ztm-iddm rats	Blood glucose concentrations were normalized in the treated animals, no transdifferentiation or expression of β-cell transcription factors, constitutive insulin expression, and no development of insulin secretory granules	71
	Liver (in vivo)	HMD (lentivirus)	NOD mice, STZ-rats, pancreatectomised westran pigs	Spontaneous expression of key β-cell transcription factors (Pdx-1, Neurog3, and NeuroD1), expression of pancreatic hormones, development of insulin secretory granules and normal intravenous glucose tolerance, permanent reversal of diabetes	18–20
	Murine and porcine BMSC (ex vivo)	pTopo3EGRIchINS (plasmid)	NOD/Scid mice	Transduced cells were capable of restoring normoglycemia, glucose tolerance, and body weight in a dose-responsive manner	91
	Liver (in vivo)	Ad/(GIRE)3BP-I 2xfur (adenovirus)	STZ-rats	Hepatic production of human insulin produced near normal glycemia, and weight gain without exogenous insulin, and without lethal hypoglycemia	88
	Vascular smooth muscle (in vivo)	LhI*TFSN (retrovirus)	BB rats	Major reduction in insulin requirement to as low as 25% of pretreatment level for up to 3 months, characteristic decline in blood glucose after IPGTT; hypoglycemic episodes	66
Insulin cDNA	GFP-mMSC (ex vivo)	MSCV-Ins (retrovirus)	STZ-C57BL/6J mice	Diabetes could be relieved effectively for up to 6 weeks by intrahepatic transplantation of GFP-mMSC-MSCV-Ins stem cells expressing human insulin in the liver. Cells were not glucose responsive; constitutive release of insulin	54
	Huh7 cells (ex vivo)	pRcCMV (plasmid)	STZ-NOD/Scid mice	Developed insulin storage granules and exhibited regulated secretion of insulin in response to increasing concentrations of glucose. After transplantation of Huh7ins into NOD/Scid mice, diabetes was reversed	16
	Skeletal muscle (in vivo)	AAVI-Ins+GK (adeno-associated virus)	STZ-mice	Mice restored and maintained normoglycemia in fed and fasted conditions for >4 months after STZ administration. Mice showed normalization of metabolic parameters, glucose tolerance, and food and fluid intake	67

Notes: This table describes a number of studies utilising in vivo or ex vivo insulin gene transfer for the reversal of T1D in animal models. It does not list all studies performed in these scenarios, nor does it describe any in vitro insulin gene transfer studies.

Abbreviations: BB rats, biobreeding rats; BMSC, bone marrow-derived MSC; MSC, mesenchymal stem cells; GFP-mMSC, green fluorescent protein murine mesenchymal stem cells; INS-FUR, furin-cleavable insulin; Ins, insulin; NOD, nonobese diabetic; STZ, streptozotocin; HMD, human immunodeficiency virus, murine stem cell lentiviral vector; T1D, Type 2 diabetes; Scid, severe combined immunodeficiency; GK, glucokinase; GIRE, glucose-responsive element; IPGTT, intraperitoneal glucose tolerance test; MSCV, mouse stem cell virus; AAVI, adeno-associated viral vector containing insulin and glucokinase.

Figure 1 Plasma glucose levels following an IVGTT in NOD mice treated with INS-FUR in a lentiviral vector (HMD).

Notes: An IVGTT was performed on NOD (12–16 weeks) and NOR mice, as well as HMD-treated and HMD/INS-FUR-treated NOD mice, 5 months after reversal of diabetes. (n=5, data were examined by one-way analysis of variance after log transformation of data and expressed as the mean ± SEM). Modified from Ren B, O’Brien BA, Byrne MR, et al. Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy. J Gene Med. 2013;15(1):28–41.¹⁹ Copyright © 2013 John Wiley & Sons, Ltd.
Abbreviations: IVGTT, intravenous glucose tolerance test; INS-FUR, furin-cleavable insulin; HMD, human immunodeficiency virus, murine stem cell lentiviral vector; NOD, nonobese diabetic; NOR, nonobese resistant; SEM, standard error of mean.

Figure 2 β-cell transcription factor hierarchy.

Notes: Pancreatic hormone-producing cell differentiation and function is governed by the temporal and spatial expression patterns of the pancreatic transcription factors. The differentiation of insulin-producing β-cells is directed by the expression of Pdx-1, Neurog3, Neurod1, Pax4, Nkx2.2, Nkx6.1, and MafA.

Table 3 β-cell transcription factors used for the reversal of T1D in animal models

Transcription factor used	Target cell/tissue	Vector used	Animal model	Outcome	Reference
Pdx-1	Liver (in vivo)	Recombinant-adenovirus	STZ-Balb/c and STZ-C57BL/6	Expression of Pdx-1 resulted in an increase in hepatic immunoreactive insulin content and an increase of 300% in plasma immunoreactive insulin levels, compared with that in mice treated with control adenovirus. Hepatic immunoreactive insulin induced by Pdx-1 was processed to mature mouse insulin 1 and 2 and was biologically active; it ameliorated hyperglycemia in diabetic treated mice	17
	Liver (in vivo)	HDAD	STZ-mice	Produced hypoglycemia that lasted only about a week, exocrine differentiation in the liver resulting in fulminant hepatitis	97
	Hepatocytes (ex vivo)	Lentivirus	STZ-Sc/d mice	Transduced cells expressed insulin at both mRNA and protein level, and showed glucose-responsive production of insulin with expression of a number of β-cell transcription factors. Upon transplantation in STZ-Sc/d mice, reduced blood glucose levels for up to 2 months	37
	Liver (in vivo)	Recombinant- adenovirus	STZ-Balb/c mice	Hepatic insulin production that reversed diabetes for up to 8 months; normal hepatic function. Transdifferentiation of liver tissue characterized by persistent expression of endogenous β-cell transcription factors	118
	Liver (in vivo)	AAv and plasmid/unrelated adenovirus cotransfection	STZ-C57BL/6 mice	AAv-mediated transfer of Pdx-1 did not result in any improvement in hyperglycemia in the diabetic mice. However, cotransfection of plasmid containing the β-cell transcription factor with an unrelated adenovirus corrected diabetes for up to 2 months. No deleterious effects on liver function	119
	Hepatocytes (ex vivo)	Recombinant adenovirus	STZ-NOD/Sdd mice	Pdx-1 treated human liver cells expressed and stored insulin in defined granules, and secreted the hormone in a glucose-regulated manner. when transplanted under the renal capsule of diabetic NOD/Sc/d mice, the cells ameliorated hyperglycemia for prolonged periods of time	120
	BMSC (ex vivo)	Retrovirus	STZ-Sc/d mice	Expression of all four islet hormones and transcription factors except NeuroDI. Significant insulin content, as well as glucose-stimulated insulin release. Cell transplantation into STZ-Sc/d mice resulted in further differentiation, including induction of NeuroDI, and reduction of hyperglycemia	35
	MSC (ex vivo)	Recombinant- adenovirus	STZ-mice	Transduced MSCs expressed multiple islet-cell genes including Neurog3, insulin, GK, GLUT2, and glucagon. Produced and released insulin/C-peptide in a weak, glucose-regulated manner. Upon transplantation into STZ- mice, euglycemia was maintained for at least 42 days	129
	AMSC (ex vivo)	Retrovirus	STZ-mice	Stable expression of Pdx-1 in AMSCs did not induce the pancreatic phenotype in vitro. Upon transplantation, STZ- mice showed significantly decreased blood glucose levels and increased survival. Transplanted stem cells became engrafted in the pancreas, wherein they expressed insulin and C-peptide	133
Neurog3	Liver (in vivo)	Adeno-associated virus and plasmid/unrelated adenovirus cotransfection	STZ-C57BL/6 mice	AAv-mediated transfer of Neurog3 did not result in any improvement in hyperglycemia in the diabetic mice. However, co-transfection of plasmid containing the β-cell transcription factor with an unrelated adenovirus corrected diabetes for up to 2 months. The livers from mice treated with Neurog3 and AdvhFIX exhibited cystic lesions and enlarged nuclei	121
	Liver (in vivo)	Recombinant-adenovirus	STZ-C57BL/6 mice	Pdx-1/VP16 expression, together with NeuroDI or Neurog3, markedly induced insulin gene transcription and ameliorates glucose tolerance	138
	Pancreas (in vivo)	Adenovirus	RagI^–/–INOD mice	Combination of three transcription factors Neurog3, Pdx-1, and MafA reprogrammed differentiated pancreatic exocrine cells in adult mice into cells that closely resemble β-cells. The induced β-cells were indistinguishable from endogenous islet β-cells in size, shape and ultrastructure. They expressed genes essential for β-cell function and ameliorated hyperglycemia by remodeling local vasculature and secreting insulin	125
NeuroD1	Liver (in vivo)	HDAD	STZ-mice	Diabetes was partially reversed by a combination of NeuroD1 and betacellulin, without producing hepatitis. Treated mice were healthy and normoglycemic for the duration of the experiment (> 120 days). Detected insulin and other islet-specific transcripts, including proinsulin- processing enzymes, β-cell transcription factors Neurog3, Pax6, Pax4, Nkx2.2, and Nkx6.1. Immuno-electron microscopy showed typical insulin-containing granules	97
	H4IIE cells (in vitro + in vivo)	Retrovirus	STZ-NOD/Scid mice	Following transduction, cells were able to synthesize, store and secrete insulin within storage granules. Expressed Pdx-1, NeuroD1, Pax6, Nkx2.2, and Nkx6.1, in addition to rat insulin 1 and 2, glucagon, somatostatin, proconvertase 1 and 2 (PC1/2), and pancreatic polypeptide. Upon transplantation in NOD/Scid mice, the cells secreted insulin in response to increasing concentrations of glucose and restored normoglcyemia	141

Notes: This table describes a number of studies utilising in vivo or ex vivo β-cell transcription factor gene transfer for the reversal of T1D in animal models. It does not list all studies performed in these scenarios, nor does it describe any in vitro β-cell transcription factor gene transfer studies.

Abbreviations: AAV, adeno-associated virus; AMSC, adipose-derived MSC; BMSC, bone marrow-derived MSC; HDAD, helper-dependent adenovirus; MSC, mesenchymal stem cells; T1D, Type 1 diabetes; NOD, nonobese diabetic; STZ, streptozotocin; Scid, severe combined immunodeficiency; GK, glucokinase.

Ren B, O’Brien BA, Byrne MR, et al. Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy. J Gene Med. 2013;15(1):28–41.

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