1. Volume 20, Issue 5, Pages 918-926 (May 2012)
Reversal of Diabetes Through Gene Therapy of Diabetic Rats by Hepatic Insulin Expression via Lentiviral Transduction 
Matthias Elsner, Taivankhuu Terbish, Anne Jörns, Ortwin Naujok, Dirk Wedekind, Hans-Jürgen Hedrich, Sigurd Lenzen 
Molecular Therapy 
Volume 20, Issue 5, Pages (May 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Reversal of a (a) mild and (b) severe diabetic metabolic state in STZ-diabetic rats through stable transduction of the liver by low titer INS-lentivirus injection. A stable mild diabetic state with blood glucose values in the range of 10 and 12 mmol/l was achieved through a single intravenous STZ injection (35 mg/kg b.wt.) five days before the lentiviral administration to hepatocytes by intraportal injection (day 0). Virus preparations were concentrated to a titer of 5 × 107/ml by ultrafiltration with Amicon Ultra columns. Stable persistent blood glucose normalization during a 1 year observation period was achieved after a single INS-lentivirus injection at day 0 within 5–10 days (closed circles). Administration of control EGFP-lentivirus did not cure diabetic rats (open circles). In comparable experiments with severely STZ-diabetic rats (55 mg/kg b.wt.) reduction of hyperglycemia with values above 20 mmol/l to stable persistent blood glucose values between 10 and 12 mmol/l for 1 year were achieved albeit glycemia was not fully normalized (closed circles). Blood glucose concentrations are presented as mean values ± SEM of six rats. The administration of INS-lentivirus resulted in human C-peptide concentrations of 49 ± 7 pmol/l for the mildly diabetic rats and 46 ± 9 pmol/l for the severely diabetic rats. In control rats treated with EGFP-lentivirus human C-peptide was not detectable. In mildly diabetic animals the rat C-peptide concentration was 54 ± 18 pmol/l. In severely diabetic animals rat C-peptide was below the detection limit. b.wt., body weight; EGFP, enhanced green fluorescent protein; STZ, streptozotocin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Blood glucose and human C-peptide concentrations in STZ-diabetic rats transduced with a low dose of INS-lentivirus or control EGFP-lentivirus after an oral glucose tolerance test (OGTT) or a glibenclamide test. (a,b) An OGTT (2 g/kg b.wt.) was performed 60 days after injection of INS-lentivirus (closed circles) or 10 days after injection of EGFP-lentivirus (open circles) in severely STZ-diabetic (55 mg/kg b.wt.) and mildly STZ-diabetic (35 mg/kg b.wt.) rats. (c,d) Human serum C-peptide was measured in parallel in the animals. (e,f) Glibenclamide (0.5 mg/kg b.wt.) was injected intraperitoneally 30 days after injection of INS-lentivirus (closed circles) or 10 days after injection of EGFP-lentivirus (open circles) in mildly diabetic (35 mg/kg b.wt.) rats. Severely STZ-diabetic (in a, c, and e) rats and mildly STZ-diabetic rats (in b, d, and f) were treated with a low dose of INS-lentivirus 5 × 107 infectious particles. Values are presented as mean values ± SEM of 4–6 rats. b.wt., body weight; EGFP, enhanced green fluorescent protein; n.d., not detectable; STZ, streptozotocin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Reversal of a severe diabetic metabolic state in STZ-diabetic rats through stable transduction of the liver by (a) high titer INS-lentivirus injection and (b) in comparison high titer mut-INS-lentivirus injection. A stable diabetic state was achieved through a single intravenous STZ injection (55 mg/kg b.wt.) 5 days before the lentiviral administration to hepatocytes by intraportal vein injection. Virus preparations were concentrated to a titer of 7 × 109/ml by tangential crossflow ultrafiltration. Persisting stable blood glucose normalization during a 1 year observation period was achieved after a single INS-lentivirus injection at day 0 within 5–7 days (closed circles). Administration of control EGFP-lentivirus did not cure diabetic rats (open circles). Blood glucose concentrations are presented as mean values ± SEM of six rats. The administration of INS-lentivirus or mut-INS-lentivirus resulted in human C-peptide concentrations of 101 ± 11 pmol/l and 101 ± 16 pmol, respectively. In control rats treated with EGFP-lentivirus human C-peptide was not detectable. The rat C-peptide concentration was in all groups of animals below the detection limit. b.wt., body weight; EGFP, enhanced green fluorescent protein; STZ, streptozotocin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Reversal of a severe diabetic metabolic state in autoimmune-diabetic IDDM rats through stable transduction of the liver by high dose INS-lentivirus injection. Severely diabetic IDDM rats were treated 5–7 days after diabetes induction with an intraportal vein injection of lentivirus for the transduction of hepatocytes. Virus preparations were concentrated to titers of 7 × 109/ml by tangential crossflow ultrafiltration. Persisting stable blood glucose normalization during a 1 year observation period was achieved after a single INS-lentivirus injection at day 0 within 5–7 days (closed circles). Administration of control EGFP-lentivirus did not cure diabetic rats (open circles). Blood glucose concentrations are presented as mean values ± SEM of 4–6 rats. The administration of INS-lentivirus resulted in human C-peptide concentrations of 111 ± 11 pmol/l. In control rats treated with EGFP-lentivirus human C-peptide was not detectable. The rat C-peptide concentration was in both groups of animals below the detection limit. EGFP, enhanced green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Blood glucose concentrations in (a) severely STZ-diabetic rats or (b) autoimmune-diabetic IDDM rats transduced with a high dose INS-lentivirus or control EGFP-lentivirus after OGTT. An OGTT (2 g/kg b.wt.) was performed 60 days after injection of INS-lentivirus (closed circles) or 10 days after injection of EGFP-lentivirus (open circles) into diabetic rats. The virus dose in both groups of rats was 7 × 109 infectious particles per rat. Blood glucose concentrations are presented as mean values ± SEM of 4–6 rats. EGFP, enhanced green fluorescent protein; OGTT, oral glucose tolerance test; STZ, streptozotocin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Immunofluorescence staining of liver tissue from IDDM rats transduced with (a,b) INS-lentivirus or (c,d) EGFP-lentivirus. Hepatocytes were positively stained either for insulin (a; red) or EGFP (c; green) in the specifically transduced cells and counterstained with DAPI (blue) in representative areas of paraffin sections. In control sections, in which EGFP-transduced liver tissue was stained for insulin, no immunofluorescence was detectable in d as well as in samples from INS-transduced liver tissue, which were stained for EGFP in b. DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Ultrastructure of liver tissue from STZ-diabetic and IDDM rats transduced with (a) EGFP-lentivirus or (b,d) with INS-lentivirus in comparison to (c) no transduction. EGFP, enhanced green fluorescent protein; G, glycogen; M, mitochondria; STZ, streptozotocin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 8
RT-PCR expression analyses of β-cell transcription factors in liver tissue of diabetic IDDM rats after transduction with INS- or EGFP-lentivirus. One year after transduction of diabetic IDDM rats with INS-lentivirus or 10 days after transduction of diabetic IDDM rats with EGFP-lentivirus RNA was isolated from liver tissue, reverse transcribed, and analyzed for expression of the following genes by PCR: pancreatic and duodenal homeobox 1 (Pdx1), neurogenic differentiation 1 (Neurod1), homeobox protein Nkx-6.1 (Nkx6-1), rat insulin 1 (rIns), human insulin (hIns), or enhanced green fluorescent protein (EGFP), β-actin (Actb). EGFP, enhanced green fluorescent protein; RT-PCR, reverse transcription-PCR.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions