Adenoviral vector-mediated Glucagon-like

peptide 1 gene therapy to normalize glucose

levels in type ⅡⅡⅡⅡ diabetic models

Mi Kyong Kwon

Department of Medicine or Medical Science

The Graduate School, Yonsei University

Adenoviral vector-mediated Glucagon-like

peptide 1 gene therapy to normalize glucose

levels in type ⅡⅡⅡⅡ diabetic models

The Master's Thesis submitted to the

Department of Medicine

the Graduate School of Yonsei University

in partial fulfillment of the requirements for the

degree of Master of Medical Science

Mi Mi Mi Mi Kyong Kyong Kyong Kyong KwonKwonKwonKwon

December December December December 2006200620062006

This This This This certifies certifies certifies certifies that that that that the the the the Master's Master's Master's Master's Thesis Thesis Thesis Thesis

of of of of Mi-Kyong Mi-Kyong Mi-Kyong Mi-Kyong Kwon Kwon Kwon Kwon is is is is approved.approved.approved.approved.

Thesis Thesis Thesis Thesis Supervisor Supervisor Supervisor Supervisor : : : : Hyun Hyun Hyun Hyun chul chul chul chul Lee, Lee, Lee, Lee, M.D., M.D., M.D., M.D., Ph.D.Ph.D.Ph.D.Ph.D.

Yong-ho Yong-ho Yong-ho Yong-ho Ahn, Ahn, Ahn, Ahn, M.D., M.D., M.D., M.D., Ph.D.Ph.D.Ph.D.Ph.D.

Chul-woo Chul-woo Chul-woo Chul-woo Ahn, Ahn, Ahn, Ahn, M.D., M.D., M.D., M.D., Ph.D.Ph.D.Ph.D.Ph.D.

The The The The Graduate Graduate Graduate Graduate SchoolSchoolSchoolSchool

Yonsei Yonsei Yonsei Yonsei UniversityUniversityUniversityUniversity

December December December December 2006200620062006

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

I would like to thank Prof. Hyun Chul Lee, Cheol Won Park

for their criticisms and thoughtful suggestions. Also I would

like to express my appreciation to Young Mi Park, Ji-Young

Moon who are friends of mine. Specially I wish to my

express Hyung-Kwan Kim who was taught and helped the

most of in vivo study. It was a great pleasure to work with

them and many inspiring discussions with them were

encouraging and helpful to my research. Finally, I would like

to express special thanks to my family for their love and

support during the days of intensive work. This work may

not have been completed without their support and

encouragement. This thesis is dedicated to the God and my

lover Un-Yong Hong who had the foresight to encourage me

and create an enviromnemt in which I could devote the many

hours to this work.

Mi-Kyong Mi-Kyong Mi-Kyong Mi-Kyong Kwon Kwon Kwon Kwon

Table of Contents

Ⅰ. Introduction․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․2

Ⅱ. Materials and Methods․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․3

1. Animals․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․3

2. Plasmid construction․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․3

3. Cell coculture and transfection․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․3

4. Measurement of GLP-1 and insulin concentration․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․4

5. Northern blot analysis․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․4

6. Recombinent adenovirus production․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․4

7. Oral glucose tolerance test ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․5

8. GLP-1 immunostaining in liver sections․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․5

9. Statistical analysis․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․5

Ⅲ. Results ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․6

1. In vitro experiments

1) Construction of various GLP-1 plasmids and measurement of GLP-1 secreted

from transfected HEK293 cells

2) Confirmation of expression of GLP-1 mRNA and protein in transfected HEK293 cells

3) Insulinotropic activity of GLP-1 secreted from transfected HEK293 cells

2. In vivo experiments

1) Adenovirus infection into HEK 293 cells and INS-1E cells for

measurement of the expression of GLP-1 and Insulin.

2) Glucose Tolerance Test

3) Serum GLP-1 and Insulin concentration

4) Construction of various GLP-1 plasmids and evaluation secreted

GLP-1 in transfected HEK293 cells

Ⅳ. Disscusion ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․15~17

Reference․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․18~20

Abstract ( in Korean) ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․21

List of Figures

Figure 1. GLP-1 plasmid ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․6

Figure 2. Transient transfection for secreted GLP-1(3-37)․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․7

Figure 3. Northern blot analysis for measurement of the GLP-1 mRNA transcripts in

GLP-1 analog transfected cell․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․8

Figure 4. Secretion of insulin in response to GLP-1․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․9

Figure 5. Secretion of GLP-1 and insulin hormone in response to GLP-1 expressing

Adenovirus․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․10

Figure 6. Glucose tolerance test for adenoviral GLP-1 injection․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․11

Figure 7. Glucose tolerance test for adenoviral LacZ injection․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․12

Figure 8. The profile of GLP-1 and insulin production in serum pAd-ILGLP-1

virus-treated in diabetic rat․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․13

Figure 9. GLP-1 in liver sections․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․14

Abstract

Keywords GLP-1(7-37), Type 2 DM, Gene therapy, Leader sequence, Adenovirus.

- 1 -

Adenoviral vector-mediated Glucagon-like peptide 1 gene therapy to normalize glucose

levels in type ⅡⅡⅡⅡ diabetic models

Mi Kyong Kwon

Department of Medicine or Medical Science The Graduate School, Yonsei University(Directed by Professor Hyun-chul Lee)

Background Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes. However, the short plasma half-life of the active forms of GLP-1 poses an obstacle to the sustained delivery of this peptide.Methods In vitro, we constructed the plasmid, pAAV-ILGLP-1, which is expressing GLP-1(7-37). GLP-1 peptides which secreted in media were evaluated by ELISA. And the mRNA level was measured by northern blot in 48h after transfection in HEK 293 cell. Co-culture assay to evaluate the insulinotropic effect of the GLP-1(7-37) peptide was performed with INS-1E cell and HEK 293 cell(plasmids are transfected). In vivo, we constructed the plasmid, pAd-ILGLP-1, which is expressing GLP-1(7-37). The ILGLP-1 sequence is inserted in pShuttle vector and pShuttle-ILGLP-1 is linked pAdEasy (adenoviral vector). To compare with the pAd-ILGLP-1, we constructed the control vector pAd-LacZ (none of ILGLP-1 sequence). Then we injected the adenovirus which are expressing GLP-1 and LacZ via tail vein or portal vein in the ZDF rats. After injection, we measured the blood glucose level by oral glucose tolerance test (OGTT) and also measured the plasma insulin and GLP-1 by radioimmunoassay. Immunohistochemical stain was performed in liver. Results GLP-1 is secreted into media more effectively through insulin leader sequence. And the blood glucose level was improved and the effect was maintained for three weeks. And also the plasma GLP-1 and insulin concentration was increased in early stage. Finally we proved that much more GLP-1 peptides are expressed in liver. Conclusion In this study, we developed the mass expression system of GLP-1 peptides using adenovirus and also evaluated the effect of GLP-1 gene delivery both in vitro and in vivo. These data demonstrate that the adenovirus-mediated GLP-1 gene therapy was enable to achieve high levels of gene expression and control the blood glucose level more effectively. This GLP-1 gene delivery system might provide an effective and safe treatment modality for type 2 diabetes.

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- 2 -

Adenoviral vector-mediated Glucagon-like peptide 1 gene therapy to normalize glucose

levels in type ⅡⅡⅡⅡ diabetic models

Mi Kyong Kwon

Department of Medicine or Medical Science The Graduate School, Yonsei University(Directed by Professor Hyun-chul Lee)

IntroductionThe proglucagon-derived peptide glucagon-like peptide-1 (GLP-1) is a hormone secreted after meals from enteroendocrine L cells of the gastrointestinal tract, mainly the lower gut . GLP-1 controls glucose homeostasis by exerting a glucose-dependent insulinotropic effect, lowering plasma glucagons level, and delaying gastric emptying: Continuous administration of GLP-1 lowers blood glucose to near normal levels in both the fasting and postprandial state in diabetic human subjects1-3.Diabetes mellitus (DM) is a chronic and metabolic disease caused by autoimmune destruction of pancreatic cells, resulting in insulin deficiency(type 1 DM) or by the body's inability to respond properly to the insulin action (type 2 DM)4-6. These abnormalities lead to an increased concentration of blood glucose, which in turn damages many organs in the body. The majority of DM patients have the type 2 form. However, these patients also require insulin for survival due to the failure of production of insulin from the pancreas at later stages. Previous studies showing the efficacy of GLP-1 gene therapy have mostly involved use of naked DNA and GLP-1 analogues to deliver the GLP-1 gene to target organs. But this adenoviral vector system is more effective because it have the capacity to harbor a relatively large size of foreign DNA and also to transducer non-dividing cells with a much lower risk of insertional mutagenesis of host chromosomes 7-8. In addition, a relatively high titer can be obtained and it caused to overcome the short life of plasma GLP-19 - 12. Administration of GLP-1 expressing adenovirus to animals are demonstrated that GLP-1 gene delivery can yield sustained levels of circulating active GLP-1(7-37) greater than endogenous levels. Thus we demonstrate efficiency of these vectors in type 2 diabetic model, ZDF rats. These results demonstrate the feasibility of gene therapy for type II diabetes using GLP-1 expression vectors.

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Materials and Methods

Animals Male ZDF (Zucker Diabetic Fatty) rats were purchased from Genetic Models (Indianapolis, IN) at 12 weeks of age. Animals were housed in a temperature-, humidity-, and light-controlled room 2123C, 1212 h lightdark cycle). They were fed ad libitum like another normal rats. Experimental protocols concerning the use of laboratory animals were reviewed and approved by the University of Utah Institutional Animal Care and Use Committee, Protocol 01-11013.

Plasmid construction The fragment resulting from digestion of pGLP1 with BamHI and SalI was subcloned into pAAV-MCS (BamHI/XhoI; Stratagene, La Jolla, CA, USA) to generate pAAV-GLP-1. To create pAAV-ILGLP-1, two different polymerase chain reactions (PCRs) with Pyrobest DNA polymerase (Takara Shuzo Co., Ltd., Tokyo, Japan) were carried out in the presence of pAAV-hPPI(F1) and pAAV-GLP-1 as a template and the primers A/B and C/D respectively. The PCR products were purified, phosphorylated in the presence of T4 polynucleotide kinase, and digested with BamHI and HindIII. The resulting PCR products were purified and subcloned into pAAV-MCS. To create pAAV-ILGLP-1, two different PCRs were carried out in the presence of pAAV-hPPI(F1) and pAAV-GLP-1 as a template and the primers A/B and E/D) respectively. The PCR products were further processed identically as described above: A; 5'-CGG GAT CCA TGG CCC TGT GGA TGC GCC TC-3', B; 5'-GGC TGC GGC TGG GTC AGG TCC CCA G-3', C; 5'-ATG CGT CAA CGT CGT CAT GCT GAA GGG-3', D; 5'-CCC AAG CTT CTA TCC TCG GCC TTT CAC-3', and E; 5'-CAT GCT GAA GGG ACC TTT ACC AG-3'. The fidelity of the manipulated nucleotide sequences was confirmed by sequencing.

Cell coculture and transfection HEK-293(Human Embryonic Kidney) cells and INS-1E cell(the rat insulin-producing beta cell line) were maintained in 5% CO2 at 37°C in DMEM (WelGENE Inc., Daegu, Korea) and RPMI1640 respectively. And same were supplemented with 5-10% fetal bovine serum (WelGENE Inc., Daegu, Korea) and 1% Antibiotic-Antimycotic (Invitrogen). Transfections of HEK 293 cells by using Tfx-50 were performed according to the manufacturer's instructions (Promega, Madison, WI, USA) and conventional calcium phosphate method. In brief, the media were aspirated before transfection and the remaining monolayer cells grown to about 50% confluence in a 12-well tissue culture plate. Then same was exposed to the solution containing of DMEM, of plasmids (2g) and of Tfx-50 reagent for 1 h followed by the addition of 1 ml of the culture DMEM media. After a 2-day

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incubation, the media were saved for further experiments. Calcium phosphate methods were performed . The DNA (12 g) was used to transfect cells in a 100-mm culture dish. At day 1 or 2 post-transfection, the media were saved and kept at -80 C until measurement of the amount of secreted GLP-1 and, at the same time, the transfected cells were harvested and resuspended in PBS. After undergoing four freeze/thaw cycles, the cells were further sonicated for 1-2 min for complete breakdown (40 burst, level 2) in a XL-2020 sonicator (Misonix, Farmingdale, New York, USA). Lysates were cleared at 1.5 x104 R.C.F. for 10 min at 4 C and stored at -80 C until use. The protein concentration was determined using the Bradford dye-binding procedure (Bio Rad, Hercules, CA, USA) and BSA (Sigma Aldrich) as a standard.

Measurement of GLP-1 and Insulin Secreted GLP-1 was measured by ELISA and RIA kit (Linco Research Inc., St Charles, MA, USA).

Northern blot analysis The standard procedures involving prehybridization, hybridization and washing were described elsewhere. Briefly, 10 g of the total RNA was electrophoretically fractionated in a 1.5% agarose formaldehyde gel and electro-transferred to a nylon membrane. A random labeling kit (Roche Molecular Biochemicals) was used for synthesis of radioactive probe in the presence of 32P-dCTP. The specific radioactivity was typically ~0.5 x 10^9 cpm/g.

Recombinant adenovirus production For generation of recombinant adenoviral plasmid vectors harboring the ILGLP-1 mini-gene, the AdEasy adenoviral vector system (Stratagene) and the protocol which provided by the manufacturer were used to generate pShuttle-ILGLP-1, pAd-ILGLP-1. For production of primary recombinant adenoviral particles of pAd-ILGLP-1 or empty pAd-LacZ, transfections of HEK293 cells in a 100-mm diameter culture dish with pAd-ILGLP-1 or pAd-LacZ were performed with Tfx-50. After incubating for 7-10 days, the supernatant was saved and added to HEK293 cells in a 150-mm diameter culture dish for adenovirus amplification. After incubating for 23 days, these cells were harvested by centrifugation at 3000 rpm for 10 min, washed in phosphate-buffered saline (PBS) twice and then resuspended in PBS. The harvested cells underwent four freeze/thaw cycles and were spun at 3,800 rpm for 10 min to obtain the cell lysates. The lysates were then loaded onto a CsCl step gradient of 1.45 and 1.25 g/ml and spun in a SW41Ti rotor (Sorvall, Newton, CT, USA) at 30 000 rpm for 2 h. The viral band was extracted and then reloaded onto a CsCl step gradient of 1.6 and 1.4 g/ml and spun for a further 18~20 h. The white turbid region in the tube was collected with a 22-G needle. Recombinant adenovirus solution was desalted using a Sephadex PD-10 column (Amersham Bioscience, Uppsala, Sweden).

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Oral glucose tolerance test and blood sampling After 6 h fasting, rats were orally loaded with glucose (2 g/kg). Blood glucose levels were measured every 30 min for 2 h after glucose loading. Blood sampling for plasma GLP-1 and insulin is performed and the samples were collected on days 0, 1, 3, 8, 15 and 20 (09:00h in fed) from squeezing the cut tail tip. It contains 500ug of total blood and separates serum by centrifuge(1000rpm, 4 C, 5min) for measurement of the plasma GLP-1 and Insulin.

GLP-1 immunostaining in liver sections. Immunohistochemical staining was performed by standard procedures described elsewhere. In briefly the liver was removed from control rats (Injection of Control, pAd-LacZ virus), and ZDF rats13-15 were treated with pAd-ILGLP-1 virus. Standard procedures were followed for fixation, embedding and sectioning. For GLP-1 immunohistochemical staining, an immunohistochemistry kit AEC (Innogenex, San Ramon, CA, USA) was used and all procedures were performed according to the manufacturer's instructions. Briefly, the endogenous peroxidase was inactivated by treatment of cells with 3% hydrogen peroxide and 10% goat serum which was applied to the samples to prevent non-specific antibody labeling. Primary and secondary antibodies were used at the following dilutions: anti-GLP-1 (1:500) and anti-mouse IgG-HRP (1:10000). Anti-GLP-1 and IgG-HRP were purchased from Santa Cruz Biotechnology Inc, respectively. This was followed by sequential labeling with biotinylated anti-mouse immunoglobulin and streptavidin/peroxidase conjugate.16

Statistical analysis Data are shown as means standard deviation (S.D.).For group differences, Student's t-test was used. A P-value less than 0.05 was considered significantly.

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RESULTSConstruction of GLP-1 plasmids and Adenoviral-GLP-1 vectorsIn this plasmid, GLP-1 expression was driven by a potent cytomegalovirus promoter. It linked to insulin leader sequence which required for secretion of GLP-1 and contains a furin cleavage site between the leader sequence and the GLP-1 coding region(figure 1-c). We proved that the level of GLP-1 secretion is promoted comparing with other control plasmids, pAAV-MCS(vector alone, figure 1-a) and pAAV-GLP-1(none of insulin leader, fugure 1-b). For generation of recombinant adenoviral plasmid vector harboring the ILGLP-1 mini-gene, the AdEasy adenoviral vector system (Stratagene) and the protocol provided by the manufacturer were used to generate pShuttle-ILGLP-1, pAd-ILGLP-1.

Figure 1. GLP-1 plasmids(a) The structure of the pAAV-MCS plasmid. (b)The structure of the pAAV-GLP-1 plasmid. The furin cleavage site is located between the start codon and the GLP-1 cDNA(stripped box). (c)Construction of the pAAV-ILGLP-1. Insulin leader sequence(hatched box) is located between the start codon and the furin cleavage site(light shaded box). (d) Structure of adenoviral GLP-1 plasmid, pAd-ILGLP-1.

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Measurement of GLP-1 secreted from transfected HEK293 cells

Recent studies involving GLP-1 gene therapy have demonstrated that the gene encoding GLP-1 devoid of a signal peptide can be successfully used to achieve the secreted form of GLP-1. GLP-1 was hardly secreted from HEK293 cells transfected with the gene encoding only GLP-1, whereas it was significantly secreted from HEK293 cells transfected with the gene encoding GLP-1 fused to the insulin leader peptide.

Expression vectors containing these hybrid mini-genes were transfected into 293 cells and the cell supernatants were assayed for the presence of GLP-1 2 days following transfection. The use of Enzyme-Linked Immunosorbent Assay (ELISA) specific for the N- and C-termini of GLP-1 confirmed the presence of significant amounts of processed GLP-1 in the cell supernatants. The data from a representative experiment using the ELISA is shown in Figure 2 and demonstrates that pAAV-ILGLP-1, containing a furin-cleavable insulin leader sequence (lane 3), produce significantly more GLP-1 than others (lane 1, 2). Secretion of many peptide hormones such as GLP-1 has been established to require hydrophobic leader peptides for directing the nascent amino acid chains into endoplasmic reticulum for secretion.

Figure 2. Transient transfection for secreted GLP-1(7-37)GLP-1 production in HEK293 cells transfected with the plasmid vectors harboring the human GLP-1 analog genes. Simplified structure of the plasmid vectors used in this study; MCS and CMV indicate multiple cloning sites and cytomegalovirus, respectively. HEK293 cells were transfected with pAAV-MCS as a control (lane 1), 1~2 pmol/L, pAAV-GLP-1 (lane 2), 3~5 pmol/L and pAAV-ILGLP-1 (lane 3), 340~350pmol/L. The amount of GLP-1 released from the transfected cells was measured 24 hrs (black bars) or 48 hrs (gray bar) after transfection. Values are means + S.D., n = 3.

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Confirmation of expression of GLP-1 mRNA and protein in transfected HEK293 cellsThe increased level of the GLP-1 secretion was measured by northern blotting. The mRNA level of GLP-1 was detected in 48h after transfection in HEK 293 cell.

Figure 3. Northern blot analysis for detection of the GLP-1 mRNA transcripts in GLP-1 analog transfected cells. Total RNAs were extracted from HEK 293 cells transfected with pAAV-MCS as a control (lane 1), pAAV-GLP-1 (lane 2), pAAV-ILGLP-1 (lane 3). Total RNAs were electrophoresed informaldehyde-containing 1.5% agarose gels, blotted onto a nylon membrane, and probed with the 32P-labeled cDNA probe for human GLP-1 (lower panel). The ethidium bromide-stained form of 28S and 18S ribosomal RNAs in each lane is shown (upper panel).

As shown in figure 3, the GLP-1 gene expression was highly enriched in the cell which is transfected with pAAV-GLP-1 and pAAV-ILGLP-1. However , GLP-1 mRNA transcript was hardly detected under conditions to transfect other plasmid, pAAV-MCS(control vector), as indicated in the lower panel (figure 3). We showed that compare with lane 2 and 3, following the GLP-1 northern blot (lower panel). The mRNA expression level of lane 2 and 3 are same level but the protein expression level(figure 2) is significantly different because the pAAV-ILGLP-1 (lane3) contained a leader sequence.

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Insulinotropic activity of GLP-1 secreted from transfected HEK293 cellsGLP-1 is secreted from multiple cell types which induces glucose-dependent insulin secretion. The insulinotropic effect for the plasmid pAAV-ILGLP-1 improved the insulin secretion in pancreatic beta cell line, INS-1E cell. It was demonstrated by radioimmunoassays with the coculture media of HEK293 cells transfected with those plasmids and INS-1E cell. As shown in figure 4, human insulin was measured in the cell media which is transfected with those plasmids. The control samples were media without glucose (47.8 ulU/ml), media with high glucose(53.9 ulU/ml), pAAV-MCS (62.3ulU/ml) and pAAV-GLP-1(58.3 ulU/ml). These results showed that the plasmids couldn't stimulate of the insulin secretion because those plasmids are not contained the leader sequence. But the pAAV-ILGLP-1 are measured significantly increasing level of insulin (98.6 ulU/ml).

Figure 4 . Secretion of insulin hormone in response to GLP-1 INS-1E cells were treated with the media of HEK 293 cells transfected with media without glucose(lane 1), media with high glucose(lane 2), pAAV-MCS (lane 3), pAAV-GLP-1 (lane 4) and pAAV-ILFGLP-1 (lane 5). The amount of insulin released from the cells was measured 30 min after treatment. There was no enhancement of insulin secretion under no glucose concentrations. However a remarkable increment of insulin secretion occurred under high glucose concentrations (300 mg/dl) with pAAV-ILGLP-1 transfected cell media (land 5). The transfected HEK293 cells in each well produced the GLP-1 peptides by ELISA following 1 pmol/L( pAAV-MCS), 12~13 pmol/L(pAAV-GLP-1) and 75~82 pmol/L(pAAV-ILGLP-1). These results showed that GLP-1 significantly stimulated the secretion of insulin under high glucose conditions but not under low glucose conditions.

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Adenovirus infection into HEK 293 cell and INS-1E cell for detection GLP-1 and Insulin expression.To examine the effects of pAd-ILGLP-1 virus used on the quality of in vivo GLP-1 gene therapy, we administrated the GLP-1 expressed adenovirus into the HEK293 cells and INS-1E cells for measurement of secreted GLP-1 and Insulin concentration. As shown in Figure 5-(a), concentration of GLP-1 is (pmol/l) evaluated by ELISA, it infected virus as doses 100ul, 1000ul into the HEK293 cell and obtained the media which secreted GLP-1 peptide. Likely we injected the adenovirus into the INS-1E cell and evaluated the Insulin concentration(ulU/ml) by RIA. It was demonstrated in Fig 5-(b). The GLP-1 and insulin concentration is increasing as doses 0ul, 100ul, 1000ul.

Figure 5. Secretion of GLP-1 and insulin in response to GLP-1 expressing Adenovirus.(a) HEK 293 cells were treated with the GLP-1 expressing adenovirus as doses 0 ul, 100ul, 1000ul and evaluated 1 PM(lane 1), 38 PM(lane 2), 57 PM(lane 3). (b) INS-1E cells were treated with the GLP-1 expressing adenovirus as same methods and detected the insulin concentration 82.8 ulU/ml( lane 1), 111.8 ulU/ml(lane 2) and 137.34 ulU/ml(lane 3). The amount of GLP-1 and insulin released from the media was measured 24~48 hrs after treatment. Values are means + S.D., n = 3.

Human GLP-1 and insulin are measured in HEK 293 cells and INS-1E cells transfected with GLP-1 expressed adenovirus which produced GLP-1 peptides. The results of figure 5 are significantly effective. Next we prepared pAd-ILGLP-1(GLP-1 expressed adenovirus) and pAd-LacZ virus(empty virus) for injection into the type 2 diabetic animal model, ZDF rats.

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Glucose Tolerance Test Subsequently, we prepared pAd-ILGLP-1(GLP-1 expressed Adenovirus ) and pAd-LacZ virus(empty virus) and infused them via the tail vein and portal vein into ZDF rats. As shown in figure 6-(a), the ZDF rats treated with higher doses (2 x 10^12, genomic copies) of expressed GLP-1 peptides exhibited a better hyperglycemic control (100~300 mg/dl) while the ZDF rats treated with pAd-LacZ virus showed consistently hyperglycemic levels (350 to 450 mg/dl). Under our conditions, the ZDF rats treated with 2 x 10^12 genomic copies of pAd-ILGLP-1 virus demonstrated the best result by almost maintaining normoglycemic level (~100 mg/dl) for over 21 days.

Figure 6 . Glucose tolerance test with pAd-ILGLP-1 virus injected in ZDF rats.Blood glucose level of ZDF rat after oral glucose loading. Glucose (2 g/kg) was loaded orally after 6h fasting. (a) At 4th day from administration, blood glucose levels in ZDF rat treated with nothing(black, upper line) or pAd-ILGLP-1 virus(gray, lower line). (b) At 8th day from administration. (c) At 15th day from administration. (d) At 21th day from administration.

The results demonstrated that the blood glucose level decrease due to the effect of GLP-1. It is compared to the control test which is before the virus injection(at 0 day). Until 21 days, the blood glucose levels are significantly improved (100~200 mg/dl) comparing to the control level( 300~400 mg/dl). The blood glucose levels were measured at the time intervals indicated.

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Figure 7. Glucose tolerance test with pAd-LacZ virus injected in ZDF rats.Blood glucose level of ZDF rat after oral glucose loading. Glucose (2 g/kg) was loaded orally after 6 hr fasting. (a) At 4th day from administration, blood glucose levels in ZDF rat treated with nothing(black) or pAd-LacZ virus(gray). (b) At 8th day from administration. (c) At 15th day from administration. (d) At 21th day from administration.The results was demonstrated that the blood glucose level not changed comparing to the control test (before injection). Until 21 days, the blood glucose levels are not varied (300~400 mg/dl) comparing to the control level( 300~400 mg/dl). The glucose tolerance level is significantly effective through pAd-ILGLP-1 (figure 6) but not pAd-LacZ (figure 7) virus in gene therapy.

The plasma GLP-1 and Insulin concentration In order to confirm that the those viruses are capable of producing elevated circulating GLP-1 levels in vivo, the pAd-ILGLP-1 and pAd-LacZ viruses were administrated to ZDF rats via high-volume (2 x 10^12 virus particles) tail vein injection. The next day following the viruses administration, high levels of GLP-1 could be detected in the plasma of injected rats, as shown in figure 8. The RIA is specific for the C-terminus of GLP-1 (7-37) and the combined amount of active- and DPP-IV-cleaved GLP-1. As endogenous GLP-1 is predominant in the 7-37 amidated form, there was measured just endogenous level of GLP-1 in the control vehicle-injected rats. Circulating GLP-1 levels in the rat injected with the pAd-ILGLP-1 virus were several orders of magnitude greater than the reported endogenous levels of 1-2 pM. As shown in figure 8-(a), the plasma GLP-1 levels increased until 3 and 8 days

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following administration, and the effect was maintained for 10 days, the concentrations are on ~25 PM at 3 days and 7.5 PM at 8 days. But the GLP-1 concentration of the pAd-LacZ virus injected rats is kept endogenous level.And we also measured the insulin concentration in plasma. It is compared with pAd-ILGLP-1 and pAd-LacZ virus treated rats at days 4,8,15 and 21 following administration. As a results, the insulin levels are slightly changed in figure 8-(b).

Figure 8. The profile of GLP-1 and insulin production in serum pAd-ILGLP-1 virus-treated in diabetic rats. The plasma GLP-1 and insulin concentration was measured in blood samples obtained from four rats. Figure 8-(a) is GLP-1 concentration. It demonstrated the pick point is 4 days after injection (about 26 pmol/l) and decreased after 8 days (about 8.8 pmol/l). Figure 8-(b) is serum insulin concentration but it is not significantly increasing. Values are means S.D., n = 4

These data shows the serum GLP-1 and insulin concentration of ZDF rats.figure 8-(a) is the profile of GLP-1 concentration the black bar means pAd-ILGLP-1 virus treated ZDF rat and white bar means pAd-LacZ virus (control vector) treated data which showing that 4 days after injection (lane 2) is peak point. And figure 8-(b) is the profile of insulin concentration, also the black bar means pAd-ILGLP-1 virus treated ZDF rat and white bar means pAd-LacZ virus (control vector) treated data. It is not significantly changed but there is a little different pAd-ILGLP-1 and pAd-LacZ data. pAd-ILGLP-1 virus injected ZDF rat is secreting more insulin level. It is influenced due to the GLP-1 gene therapy and the improved insulin level is associated with blood glucose lowering effect. Circulating levels of active

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GLP-1(7-37) were persisted during the entire 3 weeks of the study. Higher expression of insulin level in beta cell is a key feature of the ZDF rat model. As expected, the plasma insulin is slightly increased when the pAd-ILGLP-1 virus was injected into rats but not with the pAAV-LacZ virus.

Figure 9. GLP-1 immunostaining(red) in liver sections. Figure 9-(a) is the pAd-LacZ virus injected into the liver. Figure 9-(b) is the pAd-ILGLP-1 virus injected into the liver. We confirmed the adenoviral tropism effect - flowed from the tail vein to the liver. It shows the liver GLP-1 immunostaining data. The pAd-LacZ virus infected tissue is showed lowering GLP-1 secretion and the pAd-ILGLP-1 virus infected tissue is increased as like normal tissue. Values are means S.D., n = 3.

These studies show that the pAd-ILGLP-1 virus can produce the mass yield of GLP-1 peptides and significantly lower the blood glucose level like normal. The pAd-ILGLP-1 virus was injected into the tail vein and it flowed to the liver through portal vein. GLP-1-treated animals had higher circulating GLP-1 levels and increased GLP-1 immunostaining of liver sections. It was proved by the figure 9. We checked the prolonged blood glucose level up to 3 weeks in ZDF rats with severe diabetes. These results demonstrate the feasibility of gene therapy for type II diabetes using GLP-1 expression vectors.

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DiscussionThe proglucagon-derived peptide glucagon-like peptide-1 (GLP-1) is a hormone secreted after meals from enteroendocrine L cells of the gastrointestinal tract, mainly the lower gut17-18. Biologically active GLP-1 exists as two molecular forms, GLP-1(7-37) and GLP-1(7-36) amide. GLP-1 controls glucose homeostasis by exerting a glucose-dependent insulinotropic effect, lowering plasma glucagon level, and delaying gastric emptying: Continuous administration of GLP-1 lowers blood glucose to near normal levels in both the fasting and postprandial state in diabetic human subjects19-20. Thus GLP-1 has been clearly considered to be a therapeutic candidate for type 2 diabetes mellitus. Previous studies demonstrating the efficacy of GLP-1 gene therapy have mostly involved use of naked DNA or GLP-1 analogues to deliver the insulin gene. 21-22. However, this procedure may not guarantee long-term insulin production23-24. To improve the performance, we prepared adenoviral vectors harboring the gene encoding a furin-modified human GLP-1 under the cytomegalovirus (CMV) promoter (pAAV-ILGLP-1). First of all we have constructed a GLP-1 plasmid that secreted effectively the active GLP-1(7-37). The transfection test demonstrated the effectively secretion of GLP-1(7-37) peptides in media. It flows ER and golgi apparatus and secrets out in plasma. The plasmids type 1 is pAAV-GLP-1 and type 2 is pAAV-ILFGLP-1. The former form contains methanione, GLP-1(7-37) and the latter contains methanione, signal peptide(insulin leader sequence) and GLP-1(7-37). A signal peptide is a short(3-60 amino acids long) peptide chain that directs the post-translational transport of a protein. The amino acid sequences of signal peptides direct proteins(which are synthesized in the cytosol) to certain organelles such as the nucleus, mitochondrial matrix, endoplasmic reticulum, chloroplast, and peroxisome. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported. The combined properties of GLP-1 action make it an attractive candidate for treatment of type II diabetes but its actions on the beta-cell render it unique for potentially changing the course of disease. In addition, treatments such as insulin secretagogues or parenteral administration of insulin are associated with significant risks of hypoglycemia. Because GLP-1 increases insulin secretion only in the presence of elevated glucose, the risks of hypoglycemia are low25-27. As a consequence, several GLP-1 analogs are being evaluated pre-clinically and clinically as a treatment for type II diabetes. However, use and development of GLP-1 as a therapeutic agent is hampered by its short half-life owing to degradation by dipeptidyl peptidase IV (DPP-IV) and neutral endopeptidase and clearance by the kidney. One approach to extend serum half-life utilizes analogs of GLP-1 that maintain potent blood glucose lowering effects but

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are 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 DPP-IV and neutral endopeptidase, which results in a greatly extended half-life. Ectopic expression of GLP-1 in vivo represents an alternate strategy for peptide delivery. Cells transduced with a GLP-1 expression vector could serve as a depot for continuous production of GLP-1 and because the insulinotropic actions of GLP-1 are glucose dependent, constitutively elevated GLP-1 should not cause hypoglycemia. Previously published studies on plasmidmediated GLP-1 gene delivery used a form of GLP-1 that was not engineered for secretion and achieved only minimal elevations in circulating GLP-1 with modest and transient effects on blood glucose in a rat model of type II diabetes or mouse model of diet-induced obesity. Here, we describe the construction and analysis of novel GLP-1 plasmid and adenoviral expression vectors that generate significantly elevated circulating peptide levels in vivo. We demonstrate efficacy of these vectors in two well-studied rodent models of type II diabetes, Zucker Diabetic Fatty (ZDF) rats. These results establish proof of concept for a gene therapy of type II diabetes using ectopic expression of GLP-1. Type 2 diabetes is a chronic and metabolic disesase and characterized by hyperglycemia, insulin resistance, absolute or relative insulin deficiency, hyperglucagonemia, and increased hepatic glucose production. GLP-1 possesses a number of properties that make it a potentially ideal antidiabetic agent. Therefore, we have developed a GLP-1 gene delivery system to treat type 2 diabetics. In this study, we evaluated the effect of GLP-1 expression through adenovirus gene therapy both in vitro and in vivo studies. A key factor in the success of gene therapy is the development of gene delivery systems that are capable of efficient gene transfer in a broad variety of tissues, without causing any pathogenic effect. This involves transfer by vectors of the therapeutic gene encoding hormone GLP-1 to target organs such as the liver and the muscle from which expressed insulin is released for its action. Using this approach, several groups have shown that insulin was properly expressed in such ectopic organs and released to reverse hyperglycemia almost to euglycemia. Thus, the use of adenoviral vectors complicates evaluation of the effectiveness which is required for determining whether insulin gene therapy approaches can be clinically introduced in the future. These studies show that a single administration of a GLP-1 expression vector can yield significant, prolonged improvements to glucose homeostasis in animals with severe diabetes. Such a treatment presents an alternative to continuous infusion needed for GLP-1 peptide therapy. GLP-1 gene therapy does not halt the rapid beta-cell failure present in these diabetic rodent models but does result in a significant delay in its progression. The decline in beta-cell function is much more severe than that found in patients with type II diabetes28-30. The causes of the

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beta-cell defect in these animals are not entirely understood but it is clear that they are dependent on the genetic background. For all groups GLP-1 gene therapy for type II diabetes or to the once injections required for stabilized forms of GLP-1. Patient compliance is a major component of successful diabetes management and it has long been understood that the need for injection presents a significant obstacle to the adoption of a therapy. Therefore, it is reasonable to assume that this route of administration will represent a hurdle to the adoption of GLP-1 peptide therapy. There may also be physiologic advantages to GLP-1 gene therapy compared to a peptide therapy31-34. It has been shown that continuous infusion of GLP-1 has a greater impact than bolus injection on first-phase insulin secretion. In conclusion, we have established that GLP-1 gene delivery significantly decreased blood glucose concentration in a type 2 diabetic animal model. The results revealed that the delivered GLP-1 gene stimulated insulin secretion, indicating that GLP-1 gene delivery represents a promising candidate for treatment of type 2 diabetes.

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Abstract (in Korean)

핵심되는 말 :제2형 당뇨, 인슐린, GLP-1, 아데노바이러스

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아데노바이러스를 아데노바이러스를 아데노바이러스를 아데노바이러스를 이용한 이용한 이용한 이용한 GLP-1 GLP-1 GLP-1 GLP-1 유전자의 유전자의 유전자의 유전자의 발현을 발현을 발현을 발현을

통한 통한 통한 통한 제제제제2222형 형 형 형 당뇨 당뇨 당뇨 당뇨 모델의 모델의 모델의 모델의 유전자 유전자 유전자 유전자 치료치료치료치료

((((지도교수 지도교수 지도교수 지도교수 이 이 이 이 현 현 현 현 철철철철))))

연세대학교 연세대학교 연세대학교 연세대학교 대학원 대학원 대학원 대학원 의과학과의과학과의과학과의과학과

권 권 권 권 미 미 미 미 경경경경

Incretin 호르몬인 GLP-1은 소장에서 분비하는 인슐린 촉진 호르몬으로서 제2

형 당뇨의 새로운 치료약물로 연구 개발 중인 중요한 펩타이드이다. 그러나

Plsma에서의 짧은 half-life를 가지는 한계가 있으므로, 본 실험에서 adenovirus

를 이용하여 GLP-1을 다량 발현시키는 것을 고안하였고 그 결과 혈당 및 인슐린

발현에서의 개선 효과를 확인할 수 있었다. 제2형 당뇨 모델인 ZDF rat 을 이용하

여 in vivo 실험을 하였고, HEK 293 cell 과 INS-1E cell 등을 이용하여 In

vitro 실험을 수행하였다. GLP-1 plasmid는 강력한 CMV 프로모터에 의해 발현

이 유도되고 insulin leader 를 통해 media에 분비된다. 이것을 측정하기 위해

293 cell에 transfection한 뒤 media에 분비된 GLP-1을 ELISA로 확인하였다.

Leader sequence가 없는 군과 비교실험결과 leader sequenc가 GLP-1

peptide의 분비에 결정적인 역할을 한다는 것을 증명하였다. 또한 GLP-1이

Insulin의 분비를 촉진시킨다는 것을 증명하기 위해 INS-1E cell 에 GLP-1

peptide를 투여하여 대조군과 비교 하였고 분비된 Insulin은 RIA로 확인결과 더

많은 insulin이 측정되었고 GLP-1이 insulin 발현을 촉진시킨다는 것을 증명할

수 있었다. 마지막으로 이 plasmid를 이용하여 pAd-ILGLP-1(GLP-1을 발현시

키는 아데노바이러스 유전자)을 제작하였고, 이 plasmid 또한 insulin producing

cell인 INS-lE cell에서 insulin분비는 것을 확인할 수 있었다.

GLP-1의 in vitro 실험을 바탕으로 pAd-ILGLP-1 plasmid를 293 cell에

transfection 하여 adenovirus를 만들었고 그것으로 제2형 동물모델인 ZDF rat

의 tail vein과 portal vein에 투여하여 실험한 결과 제2형 당뇨 개선 효과를 보

았다. 결과적으로 이러한 실험 연구들을 통해 adenovirus를 이용한 GLP-1 유전

자 치료의 효과를 볼 수 있었고, 이 실험에서 GLP-1 유전자치료의 한계인

plasma 에서의 짧은 half-life에 대한 것을 아데노바이러스를 이용한 GLP-1의

대량 발현을 통해 극복할 수 있었다. 결론적으로, 우리는 제 2형 당뇨의 치료 및

개선을 위해 아데노바이러스를 이용한 GLP-1 유전자치료를 실시하였고, 제2형

당뇨모델에서의 혈당 개선 및 인슐린 분비촉진 등의 효과를 증명할 수 있었다.