Gene Therapy (2007) 14, 38–48 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt ORIGINAL ARTICLE Ectopic expression of -like 1 for gene therapy of type II diabetes

GB Parsons, DW Souza, H Wu, D Yu, SG Wadsworth, RJ Gregory and D Armentano Department of Molecular Biology, Genzyme Corporation, Framingham, MA, USA

Glucagon-like peptide 1 (GLP-1) is a promising candidate for lowered both the fasting and random-fed hyperglycemia the treatment of type II diabetes. However, the short in vivo present in these animals. Because the insulinotropic actions half-life of GLP-1 has made peptide-based treatments of GLP-1 are glucose dependent, no evidence of hypogly- challenging. Gene therapy aimed at achieving continuous cemia was observed. Improved glucose homeostasis was GLP-1 expression presents one way to circumvent the rapid demonstrated by improvements in %HbA1c (glycated he- turnover of GLP-1. We have created a GLP-1 minigene that moglobin) and in glucose tolerance tests. GLP-1-treated can direct the of active GLP-1 (amino acids 7–37). animals had higher circulating levels and increased Plasmid and adenoviral expression vectors encoding the 31- insulin immunostaining of pancreatic sections. GLP-1-treated amino-acid peptide linked to leader sequences required for ZDF rats showed diminished food intake and, in the first few secretion of GLP-1 yielded sustained levels of active GLP-1 weeks following vector administration, a diminished weight that were significantly greater than endogenous levels. gain. These results demonstrate the feasibility of gene Systemic administration of expression vectors to animals therapy for type II diabetes using GLP-1 expression vectors. using two diabetic rodent models, db/db mice and Zucker Gene Therapy (2007) 14, 38–48. doi:10.1038/sj.gt.3302842; Diabetic Fatty (ZDF) rats, yielded elevated GLP-1 levels that published online 24 August 2006

Keywords: glucagons/metabolism; diabetes mellitus; type II/therapy; blood glucose/analysis; rats; zucker; mice; obese

Introduction intestine and brain, GLP-1 acts as an ileal brake by slowing the rate of gastric emptying, promoting feelings Type II diabetes is characterized by two principal defects: of satiety and diminishing food intake.9–14 There are also insulin resistance in peripheral tissues and defective reports of GLP-1 acting to decrease glucagon secretion, insulin secretion from beta cells.1 These two pathologies promote glucose uptake in skeletal muscle, and stimulate lead to a progressive beta-cell failure that results in a loss hepatic glycogen synthesis.15–20 of beta-cell mass and the need for hypoglycemic agents The combined properties of GLP-1 action make it an used alone, or in combination with insulin replacement attractive candidate for treatment of type II diabetes but therapy, to control blood glucose. Although many drugs its actions on the beta-cell render it unique for potentially are initially adequate as monotherapies, their effectiveness changing the course of disease. In addition, treatments wanes over time and polypharmacy is often required. such as insulin secretagogues or parenteral administra- This is most likely due to the polygenic and progressive tion of insulin are associated with significant risks of nature of type II diabetes and that current treatments do hypoglycemia. Because GLP-1 increases insulin secretion not arrest the deterioration in beta-cell function. only in the presence of elevated glucose, the risks of Glucagon-like peptide-1 (GLP-1) is a 30 amino-acid hypoglycemia are low. As a consequence, several GLP-1 peptide that is secreted from the L-cells of the intestine analogs are being evaluated pre-clinically and clinically in response to meal ingestion.2,3 GLP-1 has a variety of as a treatment for type II diabetes.21 However, use and effects related to nutrient digestion and acts primarily in development of GLP-1 as a therapeutic agent is the pancreas, intestine, and brain as a prandial agent that hampered by its short half-life owing to degradation by dampens blood glucose excursions. In the pancreas, dipeptidyl peptidase IV (DPP-IV) and neutral endopep- GLP-1 has been shown to stimulate glucose-induced tidase and clearance by the kidney.22,23 One approach insulin secretion as well as insulin production.4,5 GLP-1 to extend serum half-life utilizes analogs of GLP-1 that has also been shown to preserve beta-cell function and to maintain potent blood glucose lowering effects but are stimulate beta-cell replication and neogenesis.6–8 In the peptidase resistant. Exendin-4 (EX4) is a GLP-1 homolog found in the venom of the Gila monster. It has a high affinity for the GLP-1 receptor and is a poor substrate for Correspondence: Dr GB Parsons, Department of Molecular Biology, DPP-IV and neutral endopeptidase, which results in a Genzyme Corporation, 31 New York Avenue, Framingham, MA greatly extended half-life.24 Another approach is based 01701, USA. E-mail: [email protected] on leveraging the long half-life of serum albumin. This Received 10 April 2006; revised 19 June 2006; accepted 28 June 2006; has been accomplished either by direct conjugation of published online 24 August 2006 GLP-1 analogs to albumin25 or by attachment of an GLP-1 gene therapy for type II diabetes GB Parsons et al 39 albumin-binding fatty acid.26 A recombinant albumin- containing a variety of leader sequences was generated, GLP-1 fusion is also under investigation.27 including signal from SEAP and , and However, even these stabilized forms require daily leaders from EX4, glucose-dependent insulinotropic injection for full efficacy. Patient compliance is a key peptide (GIP), and helodermin (Hel). component of diabetes management and once or twice Expression vectors containing these hybrid mini-genes daily injection may present a significant hurdle for were transfected into 293 cells and the cell supernatants adoption of any therapeutic. were assayed for the presence of GLP-1 3 days following Ectopic expression of GLP-1 in vivo represents an transfection. The use of radioimmunoassays specific for alternate strategy for peptide delivery. Cells transduced the N- and C-termini of GLP-1 confirmed the presence with a GLP-1 could serve as a depot of significant amounts of processed GLP-1 in the cell for continuous production of GLP-1 and because the supernatants. The data from a representative experiment insulinotropic actions of GLP-1 are glucose dependent, using the N-terminal radioimmunoassay (RIA) is shown constitutively elevated GLP-1 should not cause hypo- in Figure 1b and demonstrates that constructs containing glycemia. Previously published studies on plasmid- a furin-cleavable leader sequence (solid bars) produce mediated GLP-1 gene delivery used a form of GLP-1 significantly more GLP-1 than constructs containing just that was not engineered for secretion and achieved only a signal peptide (striped bars). minimal elevations in circulating GLP-1 with modest and In order to confirm that the constructs are capable transient effects on blood glucose in a rat model of type II of producing elevated circulating GLP-1 levels in vivo, diabetes or mouse model of diet-induced obesity.28,29 plasmid DNA was administered to BALB/c mice via Here, we describe the construction and analysis of novel high-volume tail vein injection. The next day following GLP-1 plasmid and adenoviral expression vectors that vector administration, high levels of GLP-1 could be generate significantly elevated circulating peptide levels detected in the plasma of injected mice (Figure 1c). The in vivo. We demonstrate efficacy of these vectors in two RIA is specific for the C-terminus of GLP-1 (7–37) and well-studied rodent models of type II diabetes, db/db thus measures GLP-1 (total), the combined amount of mice and Zucker Diabetic Fatty (ZDF) rats. These results active- and DPP-IV-cleaved GLP-1. As endogenous GLP- establish proof of concept for a gene therapy of type II 1 is predominantly in the 7–36 amidated form, there was diabetes using ectopic expression of GLP-1. no detectable GLP-1 in the control vehicle-injected mice. Circulating GLP-1 levels in the mice injected with a GLP- 1 expression vector were several orders of magnitude Results greater than the reported endogenous levels of 10– 20 pM. Consistent with the transient transfection experi- GLP-1 is encoded by the preproglucagon gene. Differ- ments, constructs containing a furin-cleavable leader ential post-translational processing of preproglucagon in yielded significantly higher GLP-1 levels than a construct the intestine and pancreas results in the secretion of GLP- employing just a signal peptide. The construct containing 1 and glucagon, respectively. This processing is due to the modified exendin leader and the Gly8 mutation, the actions of distinct prohormone convertases that are termed EX4GLP1Gly8, yielded the highest GLP-1 levels differentially expressed in these two tissues. GLP-1 is and was chosen for further experiments. Additional generated in the L-cells of the gut by the action of the experiments (see below) with the N-terminal RIA prohormone convertase PC3, while glucagon is gener- confirmed correct processing of GLP-1. ated in pancreatic alpha-cells by PC2.30 These proteases We next wished to determine whether GLP-1 ex- are restricted to a subset of endocrine and neuroendo- pressed in vivo would be effective in improving glycemic crine cell types and thus are not practical for generating control in a model of type II diabetes. Diabetic db/db GLP-1 from preproglucagon expressed in non-endocrine mice are deficient in signaling and are obese and tissues. Furthermore, use of a preproglucagon transgene hyperphagic. They are also severely insulin resistant and could result in the unpredictable production of glucagon, start developing hyperglycemia by 4 weeks of age. GLP- its C-terminally extended forms, or other processed 1 linked to the EX4 leader sequence was cloned into an peptides with unclear function. We therefore constructed expression vector harboring the CMV enhancer linked to a GLP-1 minigene encoding only the 31 amino acids the promoter and intron (CUbi). The CUbi present in GLP-1 (7–37). Because the active form of GLP- vector is capable of directing expression for more than 1 1 (7–37 or 7–36 amide) is rapidly cleaved to an inactive month in the liver.32 Third-generation adenoviral vectors form (9–37 or 9–36 amide) by DPP-IV,22 the alanine at provide a simple and efficient tool for achieving position 8 was mutated to glycine to render the peptide sustained, high levels of . Therefore, an DPP-IV resistant and thus prolong its circulating half- adenoviral vector containing the CUbi-EX4GLP1Gly8 life.31 To facilitate its production and secretion in ectopic, transcription cassette was constructed for use in experi- non-endocrine tissues, it was necessary to attach a signal ments in diabetic animal models. The vector backbone peptide to GLP-1 that would target the peptide to the contains deletions in the E1, E3B, and E4 regions but constitutive secretory pathway and allow post-transla- retains E4 ORF6.33,34 The vector was administered to the tional processing of the hybrid peptide by signal tail vein of db/db mice at a dose of 1 Â1011 viral particles peptidase, a ubiquitous enzyme. The signal peptide from per mouse. Control mice received adenovirus devoid secreted alkaline phosphatase (SEAP) was linked to the of a transgene (empty vector (EV)). The blood glucose GLP-1-coding region (Figure 1a). We also tested another of Ad2/GLP-1-treated db/db mice declined to a level means of processing the hybrid peptide by fusing GLP-1 equivalent to that of lean mice within 1 day following to a furin protease recognition sequence that was then vector administration (Figure 2a). Glucose levels rose out linked to the leader sequence from EX4 (Figure 1a). of the euglycemic range over the next week but even at 6 Using this strategy, a panel of GLP-1 expression vectors weeks post-vector administration, glucose levels were

Gene Therapy GLP-1 gene therapy for type II diabetes GB Parsons et al 40 a

b 1000 c 100.000

10.000

100 1.000

0.100 10 pg GLP-1/ mU SEAP nM, GLP-1Gly8(total) nM, 0.010

1 0.001 Clusterin SEAP Helodermin GIP Exendin-4 SEAP Exendin-4 Helodermin GIP mock Figure 1 GLP-1 expression constructs. (a) Sequence of GLP-1 (7–37)Gly8 mini-genes. The top sequence contains an example of a leader sequence comprising just a signal peptide, the bottom sequence contains an example of a leader sequence containing both a signal peptide as well as a furin recognition sequence (boxed). SPase designates the site of predicted cleavage by . The solid line above the sequences designates amino-acid sequences from the SEAP (top) and EX4 (bottom) genes. Sequences coding for GLP-1Gly8 are underlined. Restriction sites used for cloning different leader sequences are shown. (b) GLP-1Gly8 secretion from 293 cells. Black bars show amount of GLP-1 in supernatants as determined by an N-terminal-specific RIA from cells transfected with constructs coding for furin-cleavable GLP-1, whereas the striped bars show results for constructs containing just a signal peptide. Data are mean values7s.d. of two independent transfections. (c) Expression of GLP-1Gly8 in vivo. Expression plasmids (10 mg) were administered to BALB/c mice by high-volume tail vein injection. Plasma levels of GLP-1Gly8 (total) were determined by a C-terminal-specific RIA. Data are mean values7s.d. of circulating GLP- 1Gly8 24 h following plasmid injection (n ¼ 4).

still significantly (Po0.001) below the level of the Ad2/EV- Diabetic db/db mice are severely insulin resistant and treated mice. GLP-1 vector administration to non-diabetic thus have greatly elevated circulating insulin levels. On lean mice resulted in a small but statistically significant the C57BLKS/J (black Kaliss) background, db/db mice (P ¼ 0.02) decline in blood glucose without evidence of experience a progressive beta-cell failure that eventually hypoglycemia. The improved blood glucose was also results in a large reduction in circulating insulin levels reflected in a lower %HbA1c (glycated hemoglobin) for the and a dramatic rise in blood glucose. db/db mice treated treated mice (Table 1), 42 days post-treatment. with Ad2/GLP-1 experienced only a small, statistically GLP-1 levels were measured using an RIA specific for insignificant, reduction in plasma insulin levels, whereas the N-terminus of GLP-1 (7–37) and thus detected active the Ad2/EV-treated db/db mice showed a greater than GLP-1 and not the inactive DPP-IV cleavage product. 60% decline in circulating insulin (Table 1). Thus, GLP-1 Plasma levels of active GLP-1Gly8 (7–37) in db/db mice treatment resulted in a delay of the appearance of insulin were 41nM 6 weeks after vector administration (Figure deficiency in this strain. In addition, a glucose tolerance 2b). Despite an equivalent dose of Ad2/GLP-1, GLP-1 test performed at 2 weeks post-vector administration levels were not as high in treated lean animals. In also showed that the GLP-1-treated db/db mice had addition, elevated GLP-1 levels were not sustained in improved glucose homeostasis (Figure 2c). Although the Ad2/GLP-1-treated lean mice. Several of the animals two groups had similar peaks of blood glucose excur- showed a reduction to undetectable levels by 6 weeks sion, the GLP-1-treated animals had significantly lower post-vector administration. The reason for this is unclear glucose levels at 120 and 180 min time points (Po0.02, but could be owing to an immune response to the Gly8 Po0.01, respectively). At the last time point, the glucose version of GLP-1. Endogenous GLP-1 levels in control levels of the treated db/db mice were not significantly animals were in the normal range. different from the lean controls (P ¼ 0.14).

Gene Therapy GLP-1 gene therapy for type II diabetes GB Parsons et al 41 Ad2/GLP-1 db/db We investigated whether the maintenance of circulat- 700 Ad2/EV db/db a Ad2/GLP-1 Lean ing insulin levels was reflected in a preservation of beta- Ad2/EV Lean cell mass. In a separate study, db/db mice were injected 600 with Ad2/CUbiEX4GLP-1Gly8 or Ad2/EV. At 28 days after vector administration, pancreatic sections were 500 * immunostained for insulin. Representative images are shown in Figure 3a–c. Lean mice showed small, densely 400 * staining, islets (Figure 3a), whereas db/db mice had larger lightly staining islets (Figure 3b and c). GLP-1- 300 treated db/db mice tended to have larger islets (Figure 3c) than control db/db mice. This increased islet size 200 resulted in a significant (Po1 Â10À4) increase in the

100 overall fraction of the pancreas staining positive for insulin (Figure 3d). 0 We evaluated GLP-1 gene therapy in another model -100 1020304050of type II diabetes, the ZDF rat. Similar to db/db Days Post-vector Administration mice, these obese animals carry a mutation in the b 10000.0 , are hyperphagic, insulin resistant, and Day 21 Day 42 experience a progressive decline in beta-cell function that leads to severe hyperglycemia. Eight-week-old male 1000.0 ZDF rats received a tail vein injection of Ad2/ CUbiEX4GLP-1Gly8 at a dose of 1 Â1012 viral particles.

100.0 The hyperglycemia was corrected in these animals within a few days of vector administration; the blood

pM, GLP-1(active) glucose levels of the Ad2/GLP-1-treated ZDF rats 10.0 declined to the level of the lean controls, whereas the blood glucose of the Ad2/EV-treated ZDF rats continued to increase (Figure 4a). As was seen in lean 1.0 mice, there was a small but statistically significant db/db GLP-1 Lean GLP-1 (Po1 Â10À4) decrease in the blood glucose of the Ad2/ c 1000 GLP-1-treated lean rats, but no hypoglycemia was observed. The differences in blood glucose in the lean GLP-1 db/db groups were significant only until 1-week post-vector EV db/db 800 administration. The Ad2/GLP-1-treated ZDF rats Lean EV remained euglycemic for the next 3 weeks and then glucose levels rose slowly over the final 3 weeks of the 600 study. At the end of the study, most of the Ad2/GLP-1- treated animals were modestly hyperglycemic (Po0.04) * with only one of the animals showing blood glucose 400 ** greater than 300 mg/dl. ZDF rats typically develop fasting hyperglycemia by 11 weeks of age. At 5 weeks * post-vector administration (13 weeks of age), the GLP-1- ** 200 treated ZDF rats still had not developed fasting hyperglycemia, whereas the empty vector-treated ani- mals had fasting glucose levels greater than 200 mg/dl 0 (Table 2). The improved blood glucose of the Ad2/GLP- 0 20 40 60 80 100 120 140 160 180 200 Time, minutes 1-treated animals is also reflected in a lower %HbA1c, o6% for Ad2/GLP-1-treated animals versus 412% for Figure 2 Effect of Ad2/GLP-1Gly8 in db/db mice. (a) Random-fed Ad2/EV-treated animals (Table 2). Both of the lean blood glucose values following vector administration. Values for db/db mice that received Ad2/CUbiEX4GLP-1Gly8 (n ¼ 10) are groups had a %HbA1c of o5%. shown as black diamonds, db/db mice that received empty vector Circulating levels of active GLP-1Gly8 (7–37) were (n ¼ 10) are shown as open diamonds. Lean mice that received B1nM and persisted with only a slight drop (P ¼ 0.05) Ad2/CUbiEX4GLP-1Gly8 or empty vector are shown as closed or during the entire 6 weeks of the study (Figure 4b). open circles, respectively (n ¼ 5). Data are mean values7s.d. In contrast to what was observed with lean mice, *Po0.001. (b) Plasma levels of GLP-1Gly8 in Ad2/CUbiEX4GLP- lean control rats treated with Ad2/GLP-1 maintained 1Gly8-treated animals. Data are mean values7s.d. of circulating GLP-1Gly8 detected with an RIA specific for the amino terminus of elevated plasma GLP-1 levels. GLP-1. The inactive, DPP-IV cleaved, form of GLP-1 is not detected Progressive beta-cell failure is a key feature of the ZDF by this assay. The black bars show plasma levels at 3 weeks rat model. As expected, plasma insulin declines in these following vector administration and the white bars show levels at 6 animals as diabetes progresses35 (Figure 4c). Interest- weeks. The striped band shows the endogenous range detected in ingly, insulin levels were not statistically different in the control animals treated with empty vector. (c) Ad2/GLP-1 treatment Ad2/GLP-1-treated group compared to the Ad2/EV improves glucose tolerance. Blood glucose was measured following an i.p. glucose injection (0.8 g/kg). Ad2/GLP-1-treated db/db mice group in the first week following vector administration. (n ¼ 4) are shown as closed diamonds, Ad2/EV-treated db/db mice However, by the end of the study, the Ad2/GLP-1- (n ¼ 2) are shown as open diamonds. Lean Ad2/EV-treated mice treated group had significantly higher (P ¼ 0.01) plasma (n ¼ 2) are shown as open circles. *Po0.02, **Po0.01. insulin levels than the Ad2/EV group. As was observed

Gene Therapy GLP-1 gene therapy for type II diabetes GB Parsons et al 42 Table 1 Plasma analyses: db/db mice

db/db GLP-1 db/db EV Lean GLP-1 Lean EV

Day 0 Day 42 Day 0 Day 42 Day 0 Day 42 Day 0 Day 42

%HbA1c ND 5.5370.72a ND 8.8270.99a ND 3.0270.04 ND 3.2270.49 Insulin (ng/ml) 11.0777.95 8.3374.84b 10.8577.07 3.5673.21b 0.2470.09 0.1770.06c 0.2470.11 0.24740.03c Glucose (mg/dl) 460.0754.2 374.5785.1d 488.4784.8 506.9750.5d 142.4710.0 108.2715.5e 150.4712.4 141.8712.9e

Data are provided as mean7s.d. Adenoviral vectors were administered to mice on day 0. For db/db mice, n ¼ 10 for each group, for lean mice n ¼ 5 for each group. aPo2 Â 10À6, bPo0.02, cPo0.04, dPo0.001, ePo0.006. Abbreviation: ND: not determined.

ab

c d * 6

4

* 2 Insulin/Total Pancreas (%) Pancreas Insulin/Total

0 db/db Ad2/GLP-1 db/db Ad2/EV Lean Ad2/EV

Figure 3 Insulin immunostaining (brown) of pancreatic sections. (a) Ad2/EV-treated lean, (b) Ad2/EV-treated db/db, and (c) Ad2/GLP-1- treated db/db. (d) Quantitation of insulin staining as a percentage of total tissue area. Magnification 10-fold. Data are mean values7s.d. from three slides per db/db mouse. Each group contains four animals. Data for the lean group is derived from three animals, seven slides total. *Po1 Â10À5.

in db/db mice, Ad2/GLP-1 treatment appeared to Ad2/GLP-1-treated ZDF rats actually showed an significantly delay the onset of beta-cell failure. increased body weight gain when compared with the As GLP-1 has been shown to delay gastric emptying Ad2/EV-treated controls (Po0.04). This differential and promote feelings of satiety, we wanted to determine weight gain is expected35 and is probably due to the whether Ad2/GLP-1 treatment would result in a progressive decline in beta-cell function and insulin decrease in food intake and consequently impact body levels that result in poor glucose utilization and weight in the ZDF rats. Ad2/GLP-1-treated ZDF rats glucosuria in Ad2/EV-treated animals. The GLP-1- consumed less food throughout the 6 weeks of the study treated animals did not show as large a decline in (Figure 5a) and, early on, showed a diminished body circulating insulin and were able to continue gaining weight gain (Figure 5b). However, by three weeks post- weight. Thus, during the first half of the study, the Ad2/ vector administration, the Ad2/EV control group had GLP-1-treated group showed a diminished weight gain stopped gaining weight and by the end of the study the when compared with the Ad2/EV group, most likely

Gene Therapy GLP-1 gene therapy for type II diabetes GB Parsons et al 43 ZDF GLP-1 600 (Figure 5c), the Ad2/GLP-1-treated ZDF rats showed a ZDF EV Lean GLP-1 a normal change in body weight. Lean EV 500 Similar to the ZDF rats, the lean rats treated with Ad2/ GLP-1 showed decreased food intake throughout the 400 course of the study and a small decrease in body weight during the first week following vector administration 300 (Figure 5c). Subsequent to this initial weight loss, the two lean groups showed a similar rate of weight gain. In 200 contrast to the obese animals, the two lean groups * showed no change in circulating insulin levels and the 100 * Ad2/GLP-1-treated animals showed significantly dimin- ished weight gain, despite a similar rate of increase 0 -50 5 10 15 20 25 30 35 40 45 during the last 5 weeks of the study. Thus, the weight Day loss that occurred during the first week of the study was b 10000.0 maintained over the subsequent weeks. Day 5 Day 28 Day 41 1000.0 Discussion We have created a GLP-1 minigene that can direct the 100.0 secretion of active GLP-1 (7–37). Transient transfection experiments demonstrated that the most efficient pro-

pM, GLP-1(active) pM, duction of GLP-1 (7–37) was achieved by linking the 10.0 GLP-1-coding region to a leader sequence containing a signal peptide as well as a furin recognition site. A mutation of the alanine at position 8 to glycine was 1.0 GLP-1 ZDF GLP-1 Lean included to render the peptide resistant to DPP-IV cleavage. Administration of GLP-1 expression vectors c 25 to animals either as naked plasmid DNA or as an adenoviral vector demonstrated that GLP-1 gene deliv- 20 Day 0 ery can yield sustained levels of circulating active GLP-1 Day 5 (7–37) that are several orders of magnitude greater than Day 28 15 Day 41 endogenous levels and are significantly greater than the circulating levels that have been reported to be effica- cious in patients in clinical trials of GLP-1 and EX4 10

Insulin, ng/ml Insulin, peptide therapy. For reasons that are not clear, GLP-1

* levels were lower and not sustained in treated lean mice 5 compared to db/db mice. This could be owing to * different transduction efficiency of the db/db liver or 0 to generation of an immune response to GLP-1 carrying ZDF GLP-1 ZDF EV Lean GLP-1 Lean EV the Gly8 mutation. As this phenomenon was not Figure 4 Effect of Ad2/GLP-1Gly8 in ZDF rats. (a) Random-fed observed in lean Zucker rats, it was not pursued further. blood glucose values following vector administration. Values for This demonstrates that production of therapeutic ZDF rats that received Ad2/CUbiEX4GLP-1Gly8 (n ¼ 6) are shown amounts of GLP-1 via gene therapy is feasible in these as black diamonds, ZDF rats that received empty vector (n ¼ 6) are rodent models of diabetes. A previously published shown as open diamonds. Lean rats that received Ad2/CU- 29 biEX4GLP-1Gly8 or empty vector are shown as closed or open attempt at GLP-1 gene therapy achieved only a partial, circles, respectively (n ¼ 6). Data are mean values7s.d. *Po0.04, transient effect on blood glucose. This limited effect is Ad2/GLP1 ZDF vs Ad2/EV lean. (b) Plasma levels of GLP-1Gly8 in probably due to poor secretion of GLP-1 from the Ad2/CUbiEX4GLP-1Gly8-treated animals. Data are mean va- transduced cells, because the transgene did not include lues7s.d. of circulating GLP-1Gly8 detected with an RIA specific a signal peptide. for the amino terminus of GLP-1. The black, white, and striped bars As the insulinotropic actions of GLP-1 are glucose show plasma levels at 5, 28, and 41 days following vector administration, respectively. The striped band shows the endogen- dependent, no evidence of hypoglycemia was found, ous range detected in control animals treated with empty vector. (c) even in lean non-diabetic animals. Both db/db mice and Random-fed plasma insulin levels. Data are mean values7s.d. of ZDF rats showed dramatic improvements in glucose circulating insulin detected by RIA. The black bars show circulating homeostasis as a result of GLP-1 vector administration, levels before vector administration. The white, gray, and striped which was evident in fed and fasting blood glucose bars show plasma levels on day 5, 28, and 41 following vector measurements, %HbA1c, and glucose tolerance tests. administration, respectively. *P ¼ 0.01. These animal models experience rapid beta-cell failure that results in severe insulin deficiency. Constitutive GLP-1 expression resulted in a significant delay of this because food intake was reduced. By the second half of decline in beta-cell function and enabled the mainte- the study, severe insulin deficiency in the Ad2/EV- nance of effective insulin levels. The exact mechanism of treated group resulted in a diminished weight gain due this delay is not known as GLP-1 has been demonstrated to poor energy utilization in the diabetic state. In fact, to have a number of effects that serve to improve insulin when compared to the growth rate of lean controls production and secretion. This includes stimulation of

Gene Therapy GLP-1 gene therapy for type II diabetes GB Parsons et al 44 Table 2 Plasma analyses: ZDF rats

ZDF GLP-1 ZDF EV Lean GLP-1 Lean EV

Day 0 Day 41 Day 0 Day 41 Day 0 Day 41 Day 0 Day 41

%HbA1c 4.5070.46 5.8870.75a 4.3770.59 12.1370.82a 3.3070.21 4.4770.52 3.2870.13 4.7770.3 Insulin (ng/ml) 14.278.6 4.171.9b 16.373.5 1.770.4b 0.7 0.970.6 1.371.0 0.8

Day 26 Day 38 Day 26 Day 38 Day 26 Day 38 Day 26 Day 38

Fasting glucose (mg/dl) 93.5712.0c 96.378.3d 211.3783.5c 203.37108.1d 82.877.1 76.573.5 83.577.0 83.3710.3

Data are provided as mean7s.d. Adenoviral vectors were administered to rats on day 0. For all groups, n ¼ 6. aPo1 Â10À7, bP ¼ 0.01, cPo0.007, dPo0.04.

insulin transcription, translation, and glucose-dependent understood44 but it is clear that they are dependent on secretion.4,5 Furthermore, GLP-1 has been reported to the genetic background. For example, the db mutation have positive effects on beta-cell mass through stimula- in C57/B6 mice results in severe insulin resistance and tion of beta-cell replication, beta-cell neogenesis, and obesity but without the beta-cell failure that is observed inhibition of apoptosis.7,8,36 In addition to these direct on the C57BLKS/J background.45 As a result, the C57/B6 actions of GLP-1, correction of hyperglycemia is pre- mice do not experience a decline in insulin, only develop dicted to diminish beta-cell death owing to glucotoxi- mild hyperglycemia, and continue gaining weight city.37 Consistent with other published reports,36 we throughout their life. observed a greater than twofold increase in the beta-cell GLP-1 gene therapy caused a decrease in food intake. mass of GLP-1-treated db/db mice compared to control This effect is most pronounced in the first few days empty vector-treated db/db mice. Multiple kinetic following vector administration, when food intake was studies will be required to determine whether this lowered to approximately 10% of that prior to treatment increase is owing to changes in rates of beta-cell (data not shown). Food intake eventually rose to about replication, neogenesis, or inhibition of apoptosis. Alter- 80% of pre-administration levels. The cause of the acute natively, increases in insulin transcription or translation effects are not known but may be related to the nausea may facilitate greater detection of beta-cells. that is a common side effect reported in clinical trials of Whereas the insulinotropic properties of GLP-1 treat- GLP-1 and EX4 peptide therapy. Although the nausea ment are well studied, the effects of GLP-1 on insulin was usually reported as mild and transient, it was severe sensitivity are less clear. Shortly after vector administra- enough in some cases to cause patient withdrawal from tion to ZDF rats, GLP-1 treatment resulted in a normal- the trial. GLP-1 gene therapy caused a reduction in body ization of blood glucose levels as well as a lowering weight during the first week following treatment, a (although not significantly) of circulating insulin levels. period that coincided with the greatest effect on food This implies an improvement in insulin sensitivity. intake. It is not clear whether the more modest effect on However, GLP-1 treatment has also been shown to blunt food intake was sufficient to impact body weight as the postprandial glucose excursions by delaying gastric control obese animals become so insulin deficient that emptying, and a blunted glucose excursion would result they ceased to gain weight. Our results are consistent in a decreased insulin requirement. In fact, studies in with the idea that GLP-1 treatment, through its insulino- humans have shown that a delay in gastric emptying tropic actions, restored adequate energy utilization, results in a smaller peak of insulin secretion following resulting in continued weight gain. GLP-1 vector meal ingestion.38 In addition, GLP-1 has been proposed administration to lean Zucker rats also had significant to directly stimulate glucose uptake in muscle and fat by effects on both food intake and body weight. Lean Ad2/ acting through a putative, yet uncharacterized, GLP-1 GLP-1-treated rats showed diminished food intake that receptor.39 However, as GLP-1 and EX4 do not have resulted in a reduced weight gain. In the lean animals, glucose lowering effects in GLP-1 receptor knockout GLP-1 treatment had minimal impact on plasma insulin mice, it would seem that this mechanism plays a minor levels. Weight loss occurred during the first week role in the action of GLP-1.40 Furthermore, since following vector administration and this difference in hyperglycemia itself can cause insulin resistance,41 body weight was maintained for the remainder of the correction of blood glucose levels should have positive study. Treatment with NN2211, a long-acting derivative effects on insulin sensitivity. In previous studies with of GLP-1, has previously been observed to result in long- EX4, improvements in insulin sensitivity were ob- term weight loss in rats.46 It is also possible that GLP-1 served.42,43 Hyperinsulinemic/euglycemic clamp studies may have effects on body weight that extend beyond its will be required to definitively demonstrate whether anorectic effects, as was observed for , GLP-1 gene therapy has an effect on insulin sensitivity. a peptide that is also produced from preproglucagon GLP-1 gene therapy does not halt the rapid beta-cell and is thought to act through the GLP-1 receptor.47 failure present in these diabetic rodent models but does These studies show that a single administration of a result in a significant delay in its progression. The GLP-1 expression vector can yield significant, prolonged decline in beta-cell function is much more severe than improvements to glucose homeostasis in animals with that found in patients with type II diabetes. The causes of severe diabetes. Such a treatment presents an alternative the beta-cell defect in these animals are not entirely to continuous infusion needed for GLP-1 peptide therapy

Gene Therapy GLP-1 gene therapy for type II diabetes GB Parsons et al 45 a 3000 or to the daily injections required for stabilized forms of * GLP-1 or EX4. Patient compliance is a major component

2500 GLP-1 ZDF of successful diabetes management and it has long been EV ZDF understood that the need for injection presents a GLP-1 Lean 2000 EV Lean significant obstacle to the adoption of a therapy. There- * fore, it is reasonable to assume that this route of 1500 ** administration will represent a hurdle to the adoption of GLP-1 peptide therapy. There may also be physiologic 1000 ** advantages to GLP-1 gene therapy compared to a peptide therapy. It has been shown that continuous 500 infusion of GLP-1 has a greater impact than bolus injection on first-phase insulin secretion.48 As the 0 constitutively elevated levels present with GLP-1 gene 0 10 20 30 40 therapy are analogous to a continuous infusion, there Days Post-vector Administration may be additional benefits to GLP-1 gene therapy b 60% beyond improved patient compliance. Systemic administration of adenoviral and plasmid vectors results primarily in transduction of the liver. However, administration of gene therapy vectors need * 40% not be limited to delivery to the liver. A broad range of * tissues could be used for production of GLP-1. Whereas adenoviral vectors provide a simple and efficient tool for achieving high levels of gene expression, alternative 20% vector systems are currently being explored for delivery of a GLP-1 transgene. ZDF GLP-1

ZDF EV 0% Materials and methods Plasmid vector constructions A nucleotide sequence encoding the signal peptide from -20% 010203040human-secreted alkaline phosphatase (SEAP) linked to Days Post-vector Administration GLY-8-modified GLP-1 (GLP-1Gly8) was generated by c 80% ligation of overlapping synthetic oligonucleotides. The codon-optimized sequence was cloned into the EcoRI and KpnI sites of the expression vector pCI (Promega, 60% ** Madison, WI, USA) to create pCISEAPGLP-1Gly8. To create additional expression vectors, pCISEAPGLP- 1Gly8 was cut with EcoRI and BtrI, which removes the 40% SEAP signal peptide and a portion of the GLP-1 ** sequence. The sequences were replaced with fragments 20% generated by overlapping oligonucleotides containing the signal peptide for clusterin (amino acids 1–21) or the Lean GLP-1 leaders for proexendin-4 (amino acids 1–42), pro-helo- 0% Lean EV dermin (amino acids 1–41), or pro-glucose-dependent insulinotropic polypeptide (GIP) (amino acids 1–46) and -20% the missing portion of GLP-1. The sequence from proexendin-4 that was used to create pCIEX4GLP- 1Gly8 is shown in Figure 1a. Sequences for additional -40% constructs spanning EcoRI to BtrI are as follows: clusterin 0 10 20 30 40 Days Post-vector Administration GAATTCGCCGCCACCATGATGAAGACTCTGCTGCT GTTTGTGGGGCTGCTGCTGACCTGGGAGAGTGGGC Figure 5 Effect of Ad2/GLP-1Gly8 on food intake and body AGGTCCTGGGGCACGGCGAGGGCACCTTCACCAG weight in ZDF rats. (a) Total food intake during the study interval in CGAC; pro-helodermin GAATTCCGC-CCACCATGAA ZDF rats treated with Ad2/GLP-1 (closed diamonds) or Ad2/EV (open diamonds), and lean controls treated with Ad2/GLP-1 GAGCATCCTGTGGCTGTGTGTGTTTGGCCTGCTGAT (closed circles) or Ad2/EV (open circles). Data are mean TGCCACCCTGTTCCCTGTGAGCTGGCAGATGGCCA values7s.d. of three cages of two animals each. Food intake was TCAAGAGCAGACTGTCCTCTGAGGACTCTGAGACA measured by subtraction. *Po0.003, **Po0.005. (b) Body weight in GACCAGAGACTGAAGCGCATCAAGCGCCACGGCG ZDF rats treated with Ad2/GLP-1 (closed diamonds) or Ad2/EV AGGGCACCTTCACCAGCGAC; pro-GIP GAATTCCGC (open diamonds). *Po0.04. (c) Body weight in lean controls treated CC-ACCATGGTGGCCACCAAGACCTTTGCCCTGCTG with Ad2/GLP-1 (closed circles) or Ad2/EV (open circles). **Po1 Â10À5. CTCCTGAGCCTCTTCCTGGCTGTGGGACTGGGCGAG AAGAAGGAAGGCCACTTCAGCGCCCTGCCCAGCCT GCCAGTGGGCAGCCATGCCAAGGTGAGCTCCCCAC AGAAGCGCATCAAGCGCCACGGCGAGGGCACCTTC ACCAGCGAC.

Gene Therapy GLP-1 gene therapy for type II diabetes GB Parsons et al 46 The vector pGZCUbiEX4GLP-1Gly8 was constructed using a One Touch glucose meter (LifeScan, Milpitas, by excising the EX4GLP-1Gly8 gene from pCIEX4GLP- CA, USA). 1Gly8 as an ScaI–NotI fragment and ligating into 0 pGZCUbi digested with NotI and blunted on the 5 end. Plasma analyses Blood glucose values were determined in the morning Recombinant adenovirus from tail vein blood using a One Touch glucose meter Adenovirus expressing EX4GLP-1Gly8 was generated by (LifeScan). %HbA1c was determined on 10 ml of whole excising the GLP-1 expression cassette from pGZCU- blood using an A1cNow test kit (Metrika, Sunnyvale, biEX4GLP-1Gly8 as an SphI (blunt)/MfeI fragment and CA, USA). For the expression study, plasma GLP-1Gly8 ligating into the EcoRV and MfeI sites of pSV2-ICeu1.49 (total) levels were determined in BALB/c mice using an The GLP-1 cassette was then excised by ICeu1 digestion RIA with an antibody specific for the C-terminus of GLP- and ligated into ICeu1 restricted D1.6 E3/orf6 to create 1 (Peninsula Labs, Belmont, CA, USA). In experiments pAd2\CUbiEX4GLP-1Gly8D1.6E3/orf6. Virus was gen- examining efficacy in diabetic animals, GLP-1Gly8 erated by transfection of 293 cells with an SnaBI (active) levels were quantitated using an RIA containing partial restriction of pAd2\CUbiEX4GLP-1Gly8D1.6E3/ an antibody specific for the amino terminus of GLP-1 orf6.49 Viruses were propagated and purified and the (Linco Research). The RIA assay was not sensitive titers of the virus were determined, all as previously enough to accurately determine endogenous GLP-1 described.34 levels. In control animals, GLP-1 was measured using a GLP-1 ELISA (Linco Research). Unlike the RIA, this Transient transfection assay did not detect the GLP-1Gly8 mutation. Plasma Transfections were carried out using the calcium insulin levels were determined by RIA (Linco Research). phosphate method (MBS Transfection, Stratagene, La Significance of observed differences between groups was Jolla, CA, USA) in 293 cells in Dulbecco’s modified tested by single factor analysis of variance (ANOVA). Eagle’s medium (DMEM) with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Transfection efficiency Insulin immunohistochemistry was normalized using a co-transfected, secreted alkaline Upon harvest, pancreata were fixed for 24 h in zinc phosphatase (SEAP) reporter construct. Cells (1 Â106) formalin (EMS, Hartfield, PA, USA) and embedded in were transfected with mixtures containing 5 mg of GLP-1 paraffin for histological studies. The slides were depar- vector and 0.5 mg of SEAP vector. GLP-1 concentrations affinized by immersion in three baths of Hemo-De in cell supernatants were determined using a radio- (Scientific Safety Solvents, Keller, TX, USA) and re- immunoassay (RIA, Linco Research, St Charles, MO, hydrated in two baths of 100% ethanol, two baths of 95% USA) specific for the amino terminus of GLP-1. SEAP ethanol, one bath of 70% ethanol, one bath of 50% activity was measured using the ALP-50 alkaline ethanol, and three baths of distilled water. Endogenous phosphatase kit purchased from Sigma Diagnostics (St peroxidase activity was destroyed by 30 min room Louis, MO, USA). temperature incubation in 3% hydrogen peroxide. Slides were incubated with 5% goat serum (Jackson Immuno- Animal experiments Research, West Grove, PA, USA) for 10 min, washed in All animal experiments were carried out in accordance PBS and then incubated overnight at 41C with a with protocols and guidelines established by the Gen- polyclonal guinea-pig anti-insulin antibody (Dako, Car- zyme IACUC. GLP-1 expression constructs were tested pinteria, CA, USA) diluted 1:1500 in PBS+1% bovine in 6- to 8-week-old male BALB/c mice (Jackson Labs, Bar serum albumin (BSA). After several PBS washes, slides Harbor, ME, USA). Mice were housed at four animals per were incubated for 1 h at 371C with a peroxidase-labeled group. High volume tail vein injections were performed goat anti-guinea pig IgG (Jackson ImmunoResearch, as described.50 Plasma samples were obtained from West Grove, PA, USA) diluted 1:500 in PBS. Slides were retro-orbital bleeds of isoflorane-anesthetized animals washed once in PBS and once in 50 mM Tris-HCl pH 7.5. 24 h after plasmid administration. Signal was detected using DAKO liquid DAB Substrate- Female db/db mice, 7–8 weeks of age, were obtained Chromogen System (Dako, Carpinteria, CA, USA). from Jackson Laboratories and housed in groups of five. Reaction was stopped by washing in water. Slides were Mice received a tail vein injection (150 ml) containing then counterstained with Blue Mayer’s hematoxylin. 1 Â1011 viral particles diluted in phosphate-buffered saline (PBS). In the study examining insulin staining of Quantitation of islet area pancreatic sections, 7- to 8-week-old female db/db mice Three sections of whole pancreas from each animal were were housed in groups of four. Mice received a tail vein imaged following insulin immunohistochemistry. The injection (150 ml) containing 1.2 Â 1011 viral particles relative islet area was determined by quantification of diluted in PBS. the cross-sectional area occupied by beta-cells and the Male ZDF rats, 6 weeks of age, were obtained from total pancreatic cross-sectional area with Metamorph Charles River Labs (Wilmington, MA, USA), housed in (Universal Imaging, Dowington, PA, USA) image analy- groups of two, and fed a diet of Purina 5008. The rats sis software. received a tail vein injection (1 ml) containing 1 Â1012 viral particles diluted in PBS. Glucose tolerance tests were performed on mice Acknowledgements following an overnight fast. The animals were injected i.p. with 0.2 ml of a 200 mg/ml glucose solution. We are grateful to W Weber for help with insulin immuno- Subsequent glucose measurements were performed histochemistry quantitation, to A Sullivan, M Joseph, and

Gene Therapy GLP-1 gene therapy for type II diabetes GB Parsons et al 47 SBoule´ for expert technical assistance, and to C Arbeeny perfused canine and rat pancreases. Endocrinology 1989; 124: for helpful suggestions. 1768–1773. 18 Nauck MA, Heimesaat MM, Behle K, Holst JJ, Nauck MS, Ritzel R et al. Effects of glucagon-like peptide 1 on counterregulatory References hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experi- 1 Ferrannini E. Insulin resistance versus insulin deficiency in non- ments in healthy volunteers. J Clin Endocrinol Metab 2002; 87: insulin-dependent diabetes mellitus: problems and prospects. 1239–1246. Endocr Rev 1998; 19: 477–490. 19 Villanueva-Penacarrillo ML, Alcantara AI, Clemente F, Delgado 2 Mojsov S, Heinrich G, Wilson IB, Ravazzola M, Orci L, Habener E, Valverde I. Potent glycogenic effect of GLP-1 (7–36) amide in JF. Preproglucagon gene expression in pancreas and intestine rat skeletal muscle. Diabetologia 1994; 37: 1163–1166. diversifies at the level of post-translational processing. J Biol 20 Lopez-Delgado MI, Morales M, Villanueva-Penacarrillo ML, Chem 1986; 261: 11880–11889. Malaisse WJ, Valverde I. Effects of glucagon-like peptide 1 on the 3 Elliott RM, Morgan LM, Tredger JA, Deacon S, Wright J, Marks kinetics of glycogen synthase a in hepatocytes from normal and V. Glucagon-like peptide-1 (7–36) amide and glucose-dependent diabetic rats. Endocrinology 1998; 139: 2811–2817. insulinotropic polypeptide secretion in response to nutrient 21 Deacon CF. Therapeutic strategies based on glucagon-like ingestion in man: acute post-prandial and 24-h secretion peptide 1. Diabetes 2004; 53: 2181–2189. patterns. J Endocrinol 1993; 138: 159–166. 22 Mentlein R, Gallwitz B, Schmidt WE. Dipeptidyl-peptidase IV 4 Drucker DJ, Philippe J, Mojsov S, Chick WL, Habener JF. hydrolyses gastric inhibitory polypeptide, glucagon-like pep- Glucagon-like peptide I stimulates insulin gene expression and tide-1 (7–36) amide, peptide histidine methionine and is increases cyclic AMP levels in a rat islet cell line. Proc Natl Acad responsible for their degradation in human serum. Eur J Biochem Sci USA 1987; 84: 3434–3438. 1993; 214: 829–835. 5 Fehmann HC, Habener JF. Insulinotropic hormone glucagon-like 23 Hupe-Sodmann K, McGregor GP, Bridenbaugh R, Goke R, Goke peptide-I (7–37) stimulation of gene expression and B, Thole H et al. Characterisation of the processing by human proinsulin biosynthesis in insulinoma beta TC-1 cells. Endocri- neutral endopeptidase 24.11 of GLP-1 (7–36) amide and nology 1992; 130: 159–166. comparison of the substrate specificity of the enzyme for other 6 Holz GG, Kuhtreiber WM, Habener JF. Pancreatic beta-cells are glucagon-like peptides. Regul Pept 1995; 58: 149–156. rendered glucose-competent by the insulinotropic hormone 24 Edwards CM, Stanley SA, Davis R, Brynes AE, Frost GS, Seal LJ glucagon-like peptide-1 (7–37). Nature 1993; 361: 362–365. et al. Exendin-4 reduces fasting and postprandial glucose and 7 Xu G, Stoffers DA, Habener JF, Bonner-Weir S. Exendin-4 decreases energy intake in healthy volunteers. Am J Physiol stimulates both beta-cell replication and neogenesis, resulting Endocrinol Metab 2001; 281: E155–E161. in increased beta-cell mass and improved glucose tolerance in 25 Kim JG, Baggio LL, Bridon DP, Castaigne JP, Robitaille MF, Jette diabetic rats. Diabetes 1999; 48: 2270–2276. L et al. Development and characterization of a glucagon-like 8 Stoffers DA, Kieffer TJ, Hussain MA, Drucker DJ, Bonner-Weir S, peptide 1-albumin conjugate: the ability to activate the gluca- Habener JF et al. Insulinotropic glucagon-like peptide 1 agonists gon-like peptide 1 receptor in vivo. Diabetes 2003; 52: 751–759. stimulate expression of homeodomain protein IDX-1 and 26 Agerso H, Jensen LB, Elbrond B, Rolan P, Zdravkovic M. The increase islet size in mouse pancreas. Diabetes 2000; 49: 741–748. pharmacokinetics, pharmacodynamics, safety and tolerability of 9 Wettergren A, Schjoldager B, Mortensen PE, Myhre J, Chris- NN2211, a new long-acting GLP-1 derivative, in healthy men. tiansen J, Holst JJ. Truncated GLP-1 ( 78–107-amide) Diabetologia 2002; 45: 195–202. inhibits gastric and pancreatic functions in man. Dig Dis Sci 27 Baggio LL, Huang Q, Brown TJ, Drucker DJ. A recombinant 1993; 38: 665–673. human glucagon-like peptide (GLP)-1-albumin protein (albu- 10 Willms B, Werner J, Holst JJ, Orskov C, Creutzfeldt W, Nauck gon) mimics peptidergic activation of GLP-1 receptor-dependent MA. Gastric emptying, glucose responses, and insulin secretion pathways coupled with satiety, gastrointestinal motility, and after a liquid test meal: effects of exogenous glucagon-like glucose homeostasis. Diabetes 2004; 53: 2492–2500. peptide-1 (GLP-1)-(7–36) amide in type 2 (noninsulin-depen- 28 Choi S, Oh S, Lee M, Kim SW. Glucagon-like peptide-1 plasmid dent) diabetic patients. J Clin Endocrinol Metab 1996; 81: 327–332. construction and delivery for the treatment of type 2 diabetes. 11 Toft-Nielsen MB, Madsbad S, Holst JJ. Continuous subcutaneous Mol Ther 2005; 12: 885–891. infusion of glucagon-like peptide 1 lowers plasma glucose and 29 Oh S, Lee M, Ko KS, Choi S, Kim SW. GLP-1 gene delivery for reduces appetite in type 2 diabetic patients. Diabetes Care 1999; the treatment of type 2 diabetes. Mol Ther 2003; 7: 478–483. 22: 1137–1143. 30 Rouille Y, Westermark G, Martin SK, Steiner DF. Proglucagon is 12 Flint A, Raben A, Astrup A, Holst JJ. Glucagon-like peptide 1 processed to glucagon by prohormone convertase PC2 in alpha promotes satiety and suppresses energy intake in humans. J Clin TC1–6 cells. Proc Natl Acad Sci USA 1994; 91: 3242–3246. Invest 1998; 101: 515–520. 31 Burcelin R, Dolci W, Thorens B. Long-lasting antidiabetic effect 13 Turton MD, O’Shea D, Gunn I, Beak SA, Edwards CM, Meeran K of a dipeptidyl peptidase IV-resistant analog of glucagon-like et al. A role for glucagon-like peptide-1 in the central regulation peptide-1. Metabolism 1999; 48: 252–258. of feeding. Nature 1996; 379: 69–72. 32 Yew NS, Przybylska M, Ziegler RJ, Liu D, Cheng SH. High and 14 van Dijk G, Thiele TE, Seeley RJ, Woods SC, Bernstein IL. sustained transgene expression in vivo from plasmid vectors Glucagon-like peptide-1 and satiety. Nature 1997; 385: 214. containing a hybrid ubiquitin promoter. Mol Ther 2001; 4: 75–82. 15 Creutzfeldt WO, Kleine N, Willms B, Orskov C, Holst JJ, Nauck 33 Scaria A, St George JA, Jiang C, Kaplan JM, Wadsworth SC, MA. Glucagonostatic actions and reduction of fasting hypergly- Gregory RJ. Adenovirus-mediated persistent cystic fibrosis cemia by exogenous glucagon-like peptide I (7–36) amide in type transmembrane conductance regulator expression in mouse I diabetic patients. Diabetes Care 1996; 19: 580–586. airway epithelium. J Virol 1998; 72: 7302–7309. 16 Komatsu R, Matsuyama T, Namba M, Watanabe N, Itoh H, Kono 34 Armentano D, Sookdeo CC, Hehir KM, Gregory RJ, St George N et al. Glucagonostatic and insulinotropic action of glucagon- JA, Prince GA et al. Characterization of an adenovirus gene like peptide I-(7–36)-amide. Diabetes 1989; 38: 902–905. transfer vector containing an E4 deletion. Hum Gene Ther 1995; 6: 17 Kawai K, Suzuki S, Ohashi S, Mukai H, Ohmori H, Murayama Y 1343–1353. et al. Comparison of the effects of glucagon-like peptide-1-(1–37) 35 Peterson R. The Zucker Diabetic Fatty (ZDF) Rat. Smith-Gordon: and -(7–37) and glucagon on islet hormone release from isolated London, 1995.

Gene Therapy GLP-1 gene therapy for type II diabetes GB Parsons et al 48 36 Farilla L, Hui H, Bertolotto C, Kang E, Bulotta A, Di Mario U et Zucker rats independent of glycemia and body weight. al. Glucagon-like peptide-1 promotes islet cell growth and Endocrinology 2005; 146: 2069–2076. inhibits apoptosis in Zucker diabetic rats. Endocrinology 2002; 44 Johnson JH, Ogawa A, Chen L, Orci L, Newgard CB, Alam T

143: 4397–4408. et al. Underexpression of beta cell high Km glucose transporters 37 Unger RH, Grundy S. Hyperglycaemia as an inducer as well as a in noninsulin-dependent diabetes. Science 1990; 250: 546–549. consequence of impaired islet cell function and insulin resis- 45 Hummel KP, Coleman DL, Lane PW. The influence of genetic tance: implications for the management of diabetes. Diabetologia background on expression of mutations at the diabetes locus in 1985; 28: 119–121. the mouse. I. C57BL-KsJ and C57BL-6J strains. Biochem Genet 38 Nauck MA, Niedereichholz U, Ettler R, Holst JJ, Orskov C, Ritzel 1972; 7: 1–13. R et al. Glucagon-like peptide 1 inhibition of gastric emptying 46 Larsen PJ, Fledelius C, Knudsen LB, Tang-Christensen M. outweighs its insulinotropic effects in healthy humans. Am J Systemic administration of the long-acting GLP-1 derivative Physiol 1997; 273: E981–E988. NN2211 induces lasting and reversible weight loss in both 39 Valverde I, Villanueva-Penacarrillo ML, Malaisse WJ. Pancreatic normal and obese rats. Diabetes 2001; 50: 2530–2539. and extrapancreatic effects of GLP-1. Diabetes Metab 2002; 28: 47 Dakin CL, Small CJ, Batterham RL, Neary NM, Cohen MA, 3S85–3S89; discussion 83S108–83S112. Patterson M et al. Peripheral oxyntomodulin reduces food 40 Hansotia T, Baggio LL, Delmeire D, Hinke SA, Yamada Y, intake and body weight gain in rats. Endocrinology 2004; 145: Tsukiyama K et al. Double incretin receptor knockout (DIRKO) 2687–2695. mice reveal an essential role for the enteroinsular axis in 48 Quddusi S, Vahl TP, Hanson K, Prigeon RL, D’Alessio DA. transducing the glucoregulatory actions of DPP-IV inhibitors. Differential effects of acute and extended infusions of glucagon- Diabetes 2004; 53: 1326–1335. like peptide-1 on first- and second-phase insulin secretion 41 Yki-Jarvinen H. Glucose toxicity. Endocr Rev 1992; 13: 415–431. in diabetic and nondiabetic humans. Diabetes Care 2003; 26: 42 Young AA, Gedulin BR, Bhavsar S, Bodkin N, Jodka C, Hansen B 791–798. et al. Glucose-lowering and insulin-sensitizing actions of 49 Souza DW, Armentano D. Novel cloning method for recombi- exendin-4: studies in obese diabetic (ob/ob, db/db) mice, nant adenovirus construction in Escherichia coli. Biotechniques diabetic fatty Zucker rats, and diabetic rhesus monkeys (Macaca 1999; 26: 502–508. mulatta). Diabetes 1999; 48: 1026–1034. 50 Hodges BL, Taylor KM, Joseph MF, Bourgeois SA, Scheule RK. 43 Gedulin BR, Nikoulina SE, Smith PA, Gedulin G, Nielsen LL, Long-term transgene expression from plasmid DNA gene Baron AD et al. (exendin-4) improves insulin therapy vectors is negatively affected by CpG dinucleotides. sensitivity and {beta}-cell mass in insulin-resistant obese fa/fa Mol Ther 2004; 10: 269–278.

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