Polymeric Gene Delivery for Diabetic Treatment

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1 Review pissn eissn D I A B E T E S & M E T A B O L I S M J O U R N A L Polymeric Gene Delivery for Dibetic Tretment Sung Wn Kim 1,2 1 Deprtment of Phrmceutics nd Phrmceuticl Chemistry nd Deprtment of Bioengineering, University of Uth, Slt Lke City, UT, USA, 2 Deprtment of Bioengineering, Hnyng University, Seoul, Kore Severl polymers were used to delivery genes to dibetic nimls. Polyminobutyl glycolic cid ws utilized to deliver IL-1 plsmid DNA to prevent utoimmune insulitis of non-obese dibetic (NOD) mouse. Polyethylene glycol grfted polylysine ws combined with ntisense glutmic cid decrboxylse (GAD) MRNA to represent GAD utontigene expression. GLP1 nd TSTA (SP-EX4) were delivered by bioreducible polymer to stop dibetic progression. Fs sirna delivery ws crried out to tret dibetic NOD mice niml. Keywords: Il-1 plsmid DNA; GAD MRNA; SP-Ex4; FsSiRNA INTRODUCTION Somtic gene therpy is defined s the introduction of genetic informtion into cells resulting in the production of proteins which correct or modulte disese. Gene therpy is dvntgeous over conventionl protein delivery becuse it is designed to overcome the limittions due to protein delivery such s low biovilbility, poor phrmcokinetics, nd complex purifiction processes tht result in high mnufcturing costs, unstble biologicl ctivity, ntibody formtion, nd toxicity. In ddition, gene therpy cn be used to restrict the expression of therpeutic protein to specific cells within the body, to control expression levels, nd chieve sustined expression over time s opposed to the burst effect which is the typicl phrmcokinetic profile of protein delivery. There re 2 min types of gene therpy, virl nd non-virl gene delivery. Virl-medited gene therpy is bsed on the use of ttenuted or defective viruses produced by genetic engineering nd includes the use of retroviruses, denoviruses, deno-ssocited viruses, nd herpes viruses. Virl vectors hve much greter trnsfection efficiencies in comprison to non-virl vectors, however, there re mny drwbcks including the induction of immunologicl responses, rndom insertion of virl sequences into the host chromosomes resulting in the ctivtion of oncogenes or the inctivtion of tumor suppressor genes, recombintion events leding to virulent virl prticles, limittions of the DNA size for encpsultion s well s lrge scle production of virl titers [1-5]. Plsmid DNA, which encodes informtion for the expression of 1 or more therpeutic proteins, is highly negtively chrged nd hs reltively high moleculr weight. Hence, the intrcellulr trnsport of plsmid DNA is limited due to the chrge repulsion between the plsmid DNA nd negtively chrged plsm membrne. In ddition, plsmid DNA is rpidly clered from the bloodstrem due to uptke by the liver nd degrdtion due to endonucleses [6]. These limittions cn be overcome vi the use of ctionic polymer-bsed crriers tht cn be utilized to condense plsmid DNA molecules into prticles protecting the plsmid DNA from the degrdtion by nucleses, resulting in delyed clernce time from the bloodstrem, nd promote internliztion of complexes on cellulr surfces due to electrosttic interctions. The positive Corresponding uthor: Sung Wn Kim Deprtment of Phrmceutics nd Phrmceuticl Chemistry nd Deprtment of Bioengineering, University of Uth, Slt Lke City, UT 84112, USA E-mil: SW.Kim@phrm.uth.edu This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Non-Commercil License ( which permits unrestricted non-commercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Copyright 211 Koren Dibetes Assocition

2 Kim SW chrges on the complexes enble stbiliztion in wter or low concentrtion buffers. Systemic gene delivery requires the rtionl design of polymeric gene vehicle with high solubility, low cytotoxicity, serum stbility, efficient endosoml relese, dequte protection ginst endonucleses, nd most importntly, high trnsfection efficiency nd gene expression. Type 1 dibetes ws formerly clled insulin-dependent or juvenile onset dibetes. Type 1 dibetes ccounts for 5% to 1% of ll dibetes cses (17 million people) in the United Sttes [7]. In person with type 1 dibetes, utoimmune processes destroy the pncretic β-cells, thus, no insulin is produced in the pncres. Type 2 dibetes ws formerly clled non-insulindependent or dult-onset dibetes nd rises due to insulin resistnce in which the body cnnot use insulin properly, combined with reltive insulin deficiency. Type 2 dibetes typiclly presents in middle nd lte life, lthough n incresing number of children nd teengers round the world re developing type 2 dibetes due to n increse in obesity. Type 2 dibetes does not exhibit cler set of symptoms. When fully developed, bnormlities in both insulin secretion nd insulin ction re present. Type 2 dibetes is chrcterized by n increse in bsl glucose concentrtions, upon which re superimposed exggerted postprndil glucose excursions induced by combintion of β-cell dysfunction nd impired insulin sensitivity. Type 2 dibetes mellitus occurs when the pncretic β-cells fil to generte n dequte insulin level to mintin euglycemi in the setting of insulin resistnce. The moleculr bsis of insulin resistnce nd β-cell filure in type 2 dibetes remins uncler. Type 2 dibetes constitutes 9% to 95% of ll dibetes cses, nd the incidence of type 2 dibetes is rpidly incresing worldwide [8]. Orl gents necessry to ttin the gol of ner-norml glycemi re needed. Tretment for type 2 dibetes relies on severl pproches minly to reduce the hyperglycemi [9]. The oldest clss of orl ntihyperglycemic gents is the sulfonylure nd remins the most commonly used initil phrmcologic tretment for the disese due to potency, effectiveness, nd low cost. The sulfonylures work by binding to receptors on the surfce of pncretic islet β-cells nd stimulting the relese of endogenous insulin [1]. However, sulfonylure stimultion of insulin secretion is not glucose-dependent, nd hence hypoglycemi is n dverse effect of the tretment. Another problem with sulfonylure is tht fter n extensive tretment, mny ptients hve shown secondry filure nd no longer respond to sulfonylure tretment [11]. There re severl other therpies currently used for type 2 dibetes: metformin, which reduces heptic glucose production, thizolidinediones, which enhnce insulin ction nd α-glucosidse inhibitors which interfere with gut glucose dsorption. These therpies lso hve significnt side effects nd hve limited efficcy [12]. Therefore, newer physiologicl pproches re despertely needed for tretment of type 2 dibetes. The use of hormones, other thn insulin, is reltively new re of clinicl investigtion nd therpy for the tretment of dibetes. Preventing the risk of hypoglycemi by potentiting insulin secretion in glucose-dependent mnner is possible. In this mnuscript, severl works in the re of gene delivery, which hve been performed t the University of Uth, re described. DELIVERY OF IL-1 PLASMID DNA TO PREVENT AUTOIMMUNE INSULITIS OF NOD MOUSE Erly in 2, the uthor of the present study previously reported biodegrdble poly [α-(4-minobutyl)-l-glycolic cid] (PAGA) cn protect nd condense plsmid DNA from DNse I. Whether the systemic dministrtion of pcaggs mouse IL-1 (mil-1) expression plsmid complexed with PAGA cn reduce the development of insulitis in non-obese dibetic (NOD) mice ws investigted. PAGA/mIL-1 plsmid complexes were stble for more thn 6 minutes, lthough DNse I destroyed the nked DNA within 1 minutes. The PAGA/ DNA complexes were injected into the til vein of 3-week-old NOD mice. Serum mil-1 level peked t 5 dys fter injection, nd could be detected for more thn 9 weeks. The prevlence of severe insulitis on 12-week-old NOD mice ws mrkedly reduced by the intrvenous injection of PAGA/DNA complex (15.7%) compred with nked DNA injection (34.5%) nd non-treted controls (9.9%). In conclusion, systemic dministrtion of pcaggs mil-1 plsmid/paga complexes cn reduce the severity of insulitis in NOD mice. The study shows the PAGA/DNA complex hs the potentil for the prevention of utoimmune dibetes mellitus [13]. Two hundred microliters of PAGA/plsmid complexes (2/1, +/-) were injected into the til vein of NOD mice t dose of μg DNA per mouse. At designted times, the nimls were killed by cervicl disloction nd the blood ws collected from the inferior ven cv, centrifuged t 3, rpm for 15 minutes 318

3 Polymeric gene delivery for dibetic tretment nd stored t -7 C (n=2 in ech group, totl n=3). The pncres nd liver were lso hrvested. The mount of serum mil-1 ws determined by using n enzyme-linked immunosorbent ssy (ELISA). For hemtoxylin nd eosin stining, the pncres ws fixed in 2% formlin contining 75 mm phosphte buffer t ph 7., nd embedded in prffin. Fivemicron thick sections were cut nd stined with hemtoxylineosin. More thn 2 islets from ech pncres were exmined using double blind methods from 3 niml groups. To chrcterize the progression of insulitis, ech islet ws ssigned by using the following insulitis grding system: 28 Grde 3 nd 4 were considered s severe insulitis (grde, norml islets; 1, mononucler cell infiltrtion lrgely in the periphery in less thn 25% of the islet; 2, 25% to % of islet showing mononucler infiltrtion; 3, over % of islet showing mononucler infiltrtion; nd 4, smll retrcted islet with few mononucler cells); the results re shown in Fig. 1. In ddition, plsmid in which the expression of interleukin-4 (IL-4) ws driven by the rt insulin promoter ws constructed (prip-il4). Wter soluble lipopolymer (WSLP) prip-il4 complex ws chrcterized by pncres β-cell specific nd glucose responsive expression of IL-4. prip-il4 ws completely retrded t 6:1 or higher N/P (nitrogen tom of WSLP/phosphte of plsmid) rtio in 1% grose gel. In ddition, WSLP protected plsmid DNA from DNse I for more thn 1 hour. In the cytotoxicity ssy, WSLP showed less cytotoxicity thn polyethylenimine (PEI) (25, D) to mouse insulinom (MIN6) cells. ELISA showed prip-il4 expressed much higher levels of IL-4 in MIN6 cells thn in NIH3T3 cells. The expression level of IL-4 by prip-il4 incresed with incresing concentrtion of glucose. Additionlly, IL-4 ws expressed in dose-dependent mnner. The WSLP prip-il4 system could be useful in the development of pncres-specific expression system for the prevention of dibetes without systemic side effects [14]. Studies of nimls with spontneous utoimmune dibetes hve reveled utorective T-cells tht medite islet β-cell destruction belong to the Th1 subset (producing IL-2 nd IFN-γ), wheres regultory T-cells re Th2 type (producing IL-4 nd IL-1). The effect of combined delivery of plsmid DNA encoding IL-4 nd IL-1 ws evluted using the degrdble, ctionic polymeric crrier, PAGA, in NOD mice. In the liver of NOD mice, mouse Il4 nd Il1 mrna ws detected 5 dys fter intrvenous injection of both PAGA-Il4 nd PAGA-Il1 plsmid complexes. Six weeks fter injection, 75% of observed islets were intct compred with less thn 3% in the control group. Furthermore, in the tretment group, only 5% of the islets were severely infiltrted by the lymphocytes compred with over 3% in the control group. Glucose levels were mesured weekly up to 32 weeks of ge, reveling co-injection of PAGA-Il4 nd PAGA-Il1 plsmids prevented the development of dibetes in 75% of the treted nimls. Thus, combined dministrtion of mouse IL4 nd IL1 plsmids prevented the development of utoimmune dibetes in NOD mice [15]. % of islets % of islets % of islets Insulitis grde 4 Insulitis grde 3 Insulitis grde 2 Insulitis grde Age (wk) Insulitis grde Grde D E F Fig. 1. Suppression of insulitis in NOD mice fter til vein injections of PAGA/DNA complexes (2/1, +/-) t the dose of mg pcaggs mil-1 plsmid per mouse. Insulitis ws evluted by hemtoxylin-eosin stining of more thn 2 islets from ech pncres nd evluting the progression of insulitis. (A) PAGA injected group. (B) PAGA/DNA complex injected group. (C) Nked DNA injected group. (D) Insulitis grde. (E) Insulitis grde 2. (F) Insulitis grde 4. A B C 319

4 Kim SW REPRESSION OF GAD AUTOCENTIGEN EXPRESSION IN PANCREATIC β-cell BY DELIVERY OF ANTISENSE PLASMID Silencing the expression of glutmic cid decrboxylse (GAD) in trnsgenic NOD mice ws reported to completely protect islet β-cells ginst the development of dibetes, suggesting the repression of GAD utontigen by somtic gene delivery cn prevent utoimmune destruction of pncretic β-cells. To repress GAD expression in islet β-cells, n ntisense GAD mrna expression plsmid (prip-as-gad) ws delivered using poly (ethylene glycol)-grfted poly-l-lysine (PEG-g-PLL) s gene crrier. In gel retrdtion ssy, the prip-as-gad/ PEG-g-PLL complex ws completely retrded bove weight rtio of 1:1.5 (plsmid:peg-g-pll). PEG-g-PLL protected the plsmid DNA from DNse I for more thn 6 minutes. In reporter gene trnsfection ssy, PEG-g-PLL showed the highest trnsfection efficiency t weight rtio of 1:3. The prip- AS-GAD/PEG-g-PLL complex ws lso trnsfected into GAD-producing MIN6 cell line. The ntisense mrna ws expressed specificlly in β-cells nd expression ws dependent on the glucose level. The repression of GAD fter trnsfection of prip-as-gad ws confirmed by immunoblot ssy. In ddition, in vivo expression of ntisense RNA in the pncres ws confirmed by RT-PCR fter intrvenous injection of the complex into mice. Therefore, the study reveled the prip-as- GAD/PEG-g-PLL system is pplicble for the repression of GAD utontigen expression [16]. An islet cell trgeting polymeric gene crrier ws synthesized by conjugting n nti-gad Fb frgment to PEI vi PEG linker (PEI-PEG-Fb ). The Fb frgment ws prepred from murine monoclonl ntibody ginst GAD, which hs been identified s mjor uto-ntigen expressed in islet cells nd used s trgeting moiety for islet cell trgeting. The elec trophoretic migrtion of plsmid DNA (pcmvluc)/pei-peg- Fb complexes in grose gel ws completely retrded bove the N/P rtio of 2. The complexes demonstrted size of to 275 nm with n lmost neutrl surfce chrge. Confocl microscopy reveled the PEI-PEG-Fb complexes showed much higher cellulr binding nd uptke efficiency compred to PEI-PEG complexes. The PEI-PEG-Fb showed pproximtely 1-fold higher trnsfection efficiency (reltive luciferse ctivity) thn PEI-PEG in GAD-expressing MIN6. However, the trnsfection efficiency of PEI-PEG-Fb reduced the PEI-PEG in GAD-negtive cells (293) nd in the presence of competitive free Fb s. Considering the neutrl surfce chrge of the PEI-PEG-Fb conjugte complexes with DNA nd selectivity towrd the islet cells expressing specific ntigen, PEI-PEG- Fb conjugte could be considered s potentil cndidte of the systemic gene therpy for the tretment of type I dibetes [17]. GLP-1 GENE DELIVERY In 193, L Brre introduced the term incretin to describe the ctivity of the gut tht could enhnce the endocrine secretion of pncres [18]. In response to the ingestion of food, both insulin nd insulin counter-regultory hormones re relesed into the blood strem. Insulin secretion is relted to the serum concentrtion of bsorbed nutrients, such s glucose, mino cids, nd ftty cids. Insulin secretion is lso influenced by incretins. The incretins re intestinl hormones tht ugment insulin secretion in the presence of elevted glucose levels [19]. In humns, 2 gstrointestinl peptide hormones re thought to be responsible for the incretin effect [2]. One is glucosedependent insulinotropic polypeptide (GIP), which ws originlly nmed gstric inhibitory polypeptide [21]. As the cloning of cdnas encoding the preproglucgons of the nglerfish ws ccomplished in the erly 198s, the second incretin hormone ws discovered [22]. Shortly fter cloning the proglucgon cdnas of nglerfish, the proglucgon cdnas of humn nd other mmmls were cloned [23]. Glucgon-like peptide-1 (GLP-1) is synthesized in the intestinl L-cells by posttrnsltionl processing of the glucgon precursor, preproglucgon, nd is relesed into the circultion in response to mel. The GLP-1 isoforms GLP-1 (7-37) nd GLP-1 (7-36) mide re well known s bioctive insulinotropic peptides derived from preproglucgon. Studies of the secretion of GLP-1 nd GIP in type 2 dibetic ptients hve shown tht mel-induced GIP secretion is norml or ner-norml, wheres GLP- 1 secretion is significntly impired. Potentil therpeutic use of GLP-1 for type 2 dibetes ws derived from the study demonstrting the glucose-lowering ctions of GLP-1 re preserved in ptients with type 2 dibetes. To compre insulinotropic ctions of exogenous incretin hormones (GIP nd GLP-1), norml nd type 2 dibetic ptients prticipted in study. The effect of GIP in type 2 dibetic ptients ws significntly lower thn in norml subjects, wheres GLP-1 retined the glucoselowering effects in type 2 dibetic ptients. In other words, GLP-1 ws cpble of restoring insulin responses to glucose in 32

5 Polymeric gene delivery for dibetic tretment type 2 dibetic ptients. GLP-1 binds to G-protein-linked receptor expressed on islet β-cells. Binding of GLP-1 to its receptor stimultes insulin secretion in glucose-dependent mnner [24]. Aprt from insulinotropic ctions nd bsed on number of effects, GLP-1 is extremely desirble s type 2 dibetes tretment. Firstly, GLP- 1 enhnces insulin secretion only when glucose levels re high (>2 mg/dl) [25]. GLP-1 potentites glucose-induced insulin secretion, but does not hve ny effect on unstimulted insulin secretion nd therefore, is unlikely to cuse hypoglycemi. Secondly, GLP-1 stimultes not only insulin gene trnscription, but lso ll steps of insulin biosynthesis. Thus, GLP- 1 helps to provide continuous supply of insulin for secretion. Additionlly, GLP-1 upregultes genes for the cellulr mchinery involved in insulin secretion [26] nd hs been shown cpble of providing new β-cells in subjects with insufficient cells [27]. Thirdly, GLP-1 strongly inhibits glucgon secretion. As result of the studies of GLP-1 infusion in dibetic ptients without ny residul β-cell secretory cpcity, GLP-1 retins glucose-lowering ctivity due to strong inhibition of glucgon secretion. Further effects of GLP-1 include inhibition of gstrointestinl motility, especilly gstric emptying. The slower rte of gstric emptying is desirble in dibetic ptients, becuse postprndil increse of glucose level is reduced. Tken together, these effects render GLP-1 very promising s therpeutic gent for type 2 dibetes. However, problem limiting the usefulness of GLP-1 in tretment exists. The mjor drwbck for GLP-1 use s therpeutic gent is the extremely short hlf-life due to rpid degrdtion. The rpid initil degrdtion is due to the ubiquitously expressed enzyme, dipeptidyl peptidse IV (DPP-IV) [28], which cleves off the 2 N-terminl mino cid residues. The conversion of intct, biologiclly ctive GLP-1 to its metbolites occurs with n pprent hlf-life of 1 to 1.5 minutes. After subcutneous injection of GLP-1, concentrtions pek fter 3 to 6 minutes nd little remins in the circultion fter 1 to 2 hours [29] indicting the mjority of exogenous GLP-1 is present in the circultion s the truncted metbolite. Intrvenous infusions of GLP-1 hve been reported to completely normlize blood glucose in type 2 dibetic ptients [3]. Mny reserchers hve ttempted vrious other types of dministrtion becuse continuous intrvenous infusion is not suitble for routine dministrtion, nd the studies hve shown the effectiveness of GLP-1. In order to dely the degrdtion of GLP-1, the properties of the injectble form hve been modified. Possibilities include the preprtion of GLP-1 with protmine or zinc, s hs been done with insulin [31]. However, the results from using vrious methods hve shown the period before degrdtion is still too short to obtin therpeutic effects from GLP-1 in dibetic subjects. Therefore, multiple injections re required for therpeutic use of GLP-1. Although mny prcticl problems still remin to be solved, therpy bsed on GLP-1 is mong the most promising recent pproches for the tretment of type 2 dibetes. The use of DPP-IV inhibitors ws suggested s result of extreme degrdtion of GLP-1 in type 2 dibetic ptients. Numerous subsequent studies hve indicted dministrtion of DPP-IV inhibitors cn be offered to subjects t risk of developing dibetes. However, whether DPP-IV inhibition will hve long-term effects is uncler. Since GLP-2, n intestinl growth fctor is lso substrte of DPP-IV, DPP-IV inhibition my hve n effect on intestinl prolifertion [32]. Over the lst decde, gene therpy techniques hve been developed for introducing genes into cells tht lter the properties of the cells. As the technology for introducing new genes into cells hs improved, the disese trgets for gene therpy hve expnded beyond trditionl genetic diseses to chronic diseses such s dibetes. If construction of GLP-1 plsmid with successful delivery is possible, GLP-1 cn be produced endogenously. In this cse, frequent injection of GLP-1 would no longer be required. Therefore, developing GLP-1 gene delivery system s tretment of type 2 dibetes could be n ttrctive pproch [33]. In the uthor s first study, the effect of GLP-1 gene delivery both in vitro nd in vivo using new plsmid constructed with modified GLP-1 (7-37) cdna ws evluted. The cdna contined furin clevge site between the strt codon nd the GLP-1 coding region. The expression of the GLP-1 gene ws driven by chicken β-ctin promoter (pβglp1). The GLP-1 mrna level ws evluted by RT-PCR 24 hours fter trnsfection. The in vitro results showed dose-dependent expression of GLP-1. Coculture ssy of the GLP-1 plsmid-trnsfected cells with isolted rt islet cells demonstrted GLP-1 incresed insulin secretion by twofold, compred to controls during hyperglycemic chllenge. A single injection of polyethyleneimine/pβglp1 complex into ZDF rts resulted in incresing insulin secretion nd decresing blood glucose levels tht ws mintined for 2 weeks. This GLP-1 gene delivery system my provide n effective nd sfe tretment modlity for type 2 dibetes [33]. 321

6 Kim SW In second study, the effect of GLP1 gene delivery ws evluted both in vitro nd in vivo using new plsmid constructed with GLP-1 (7 37) cdna. The expression of the GLP-1 gene ws driven by SV4 promoter/enhncer. To increse the expression level of GLP-1, nucler fctor (NF) κb binding sites were introduced. The in vitro results showed expression of GLP-1 nd in vitro ctivity of GLP-1, which is glucose-dependent insulinotropic ction. A single systemic dministrtion of polyethyleneimine/psiglp1nf κb complex into dietinduced obese (DIO) mice resulted in incresing insulin secretion nd decresing blood glucose levels for durtion longer thn 2 weeks [34]. The expressed GLP-1 should be secreted in its ctive form for therpeutic purposes. For ssessing the ctive form of GLP- 1, ELISA ws performed 48 hours fter trnsfection. Fig. 2A shows the mount of GLP-1 in the medium fter trnsfection with ech plsmid. Six μg of plsmid DNA complexed with PEI t N:P rtio of 5:1 ws used to trnsfect the cells in 6-well culture pltes. When 6 μg of psiglp1n κb-trnsfected HepG2 cells for GLP-1 production ws ssyed, the cells produced 1.5-fold more GLP-1 thn 6 μg of psiglp1-trnsfected HepG2 cells (13 ng/l/24 hr vs. 21 ng/l/24 hr, P<.5). Whether the secreted GLP-1 from the trnsfected HepG2 cells cn stimulte secretion of insulin from isolted rt islets ws lso studied. Isolted rt islets were cultured with the psi- GLP-1- nd psiglp1n κb-trnsfected HepG2 cells. No enhncement of insulin secretion under low glucose concentrtions ( mg/dl) ws observed. However, significnt increment of insulin secretion occurred under high glucose concentrtions (3 mg/dl) (Fig. 2B). The trnsfected HepG2 cells in ech well produced 13.2 nd 16.3 μg GLP-1 over 4 hours, respectively. The results showed GLP-1 significntly stimulted the secretion of insulin under high glucose conditions but not under low glucose conditions. The psiglp1/pei nd psiglp1n κb/pei (N:P rtio 5:1) complexes were injected into mice vi the til vein. All mice tolerted the injections well, nd no injection-relted deths occurred. Whether the delivery of psiglp1n κb plsmid cn decrese blood glucose levels in insulin-resistnt DIO mice nd the effect of incorporting NFB binding sites into the plsmid ws studied. After intrvenous dministrtion of 2 μg of the PEI/pSIGLP1N κb complex, the blood glucose levels begn to decrese (Fig. 3A). The decrese continued until the 2nd dy following dministrtion, fter which the blood glucose levels incresed until the 21st dy fter injection. However, the blood glucose levels did not return to the predministrtion bseline until the 17th dy fter injection. The control groups showed no significnt chnge in blood glucose levels during similr time period. The second group of nimls tht received psiglp1/pei showed pttern of glucose level chnge similr to the psiglp1n κb/pei group, but the chnge of blood glucose in the psiglp1 group ws reltively smller thn in the psiglp1n κb group. The plsm level of GLP-1 from ech group ws lso moni- GLP-1 (ng/l) w/o NFκB with NFκB b b Insulin (mg/l) Control psiglp1 psiglp1 NFκB, b 5 pciglp1 pβiglp1 psiglp1 A mg/dl 3 mg/dl Glucose concentrtion B Fig. 2. In vitro trnsfection ssy in HepG2 cells for GLP-1 expression. (A) The GLP-1 levels fter trnsfection of the PEI/pSI- GLP1 complex into HepG2 cells. (B) Insulin production in isolted rt islets cocultured with psiglp1-trnsfected HepG2 cells. The grph represents the SE verges for 6 experiments. P<.5 compred to control, b P<.5 compred to psiglp1. 322

7 Polymeric gene delivery for dibetic tretment tored. In conjunction with the chnges in blood glucose levels, the PEI/pSIGLP1N κb group plsm GLP-1 concentrtion begn to increse t the 2nd dy fter injection (Fig. 3B). The GLP-1 level stedily decresed through the 28th dy of the study. The control groups showed lmost no chnges in GLP-1 concentrtion. Plsm insulin levels were ssyed to evlute the insulinotropic effect of GLP-1. The pttern of the insulin concentrtion chnge in the PEI/pSIGLP1N κb group mirrored the temporl profile of the plsm GLP-1 levels. The PEI/pSIGLP1N κb group s plsm insulin concentrtion vlue incresed 2.5-fold bove the bseline wheres the control groups showed miniml chnges in plsm insulin concentrtion (Fig. 3C). Two dys fter the complex ws injected, plsm GLP-1 vlues incresed drmticlly, nd the vlues grdully returned to bseline levels fter 3 weeks. Blood glucose levels lso decresed nd returned to preinjection levels fter 3 weeks. Plsm insulin levels lso incresed nd grdully returned to bseline 17 dys fter injection. An intrperitonel glucose tolernce test (IPGTT) ws performed to verify the improvement of glucose tolernce. The blood glucose level showed mrked decrese. A third study ws performed for gene delivery of exendin-4 (Ex-4). Ex-4 is n nlogue of GLP-1, which hs resistnce to DPP-IV nd shows longer hlf-life. Therefore, Ex-4 encoding pdna delivery my be more potent for type 2 dibetes. For gene therpy, pdna systems for Ex-4 were constructed, which included pβ-sp-ex4 nd TSTA (SP-Ex4) consisting of pβ-gl- 4p65 nd puas-sp-ex4. Previously, when PEI25k ws used s gene crrier, 3:1 mixing rtio between puas-sp-ex4 nd pβ-gl-4p65 reportedly showed the highest Ex-4 level mong vrious rtios in TSTA (SP-Ex4) systems nd secreted Ex-4 from tht rtio induced the highest insulin level in NIT-1 insulinom cells. Next, Ex-4 levels from pβ-sp-ex4 nd TSTA (SP-Ex4) were mesured nd compred fter trnsfections using PEI25k or ABP in NIH3T3 cells. Before performing trnsfection, the cytotoxicity of PEI25k nd rginine-grfted bioreducible polydisulfide mine (ABP) ws exmined in NI- H3T3 cells by MTT ssy. As shown in Fig. 4, RCV of PEI25k ws bruptly decresed in ccordnce with the concentrtion, Ex4 concentrtion (ng/l) PEI25k pb-sp-ex4 ABP A Fig. 4. Produced EX4 concentrtion with (A) PB-SP-EXP nd (B) TSTA (SP-EX4) using PEI25K nd ABP polymers. Ex4 concentrtion (ng/l) PEI25k ABP TSTA (SP-Ex4) B Glucose (mg/dl) GLP-1 (ng/l) Insulin (mg/l) Time (dys) Time (dys) Time (dys) No injection Polymer only Empty plsmid psiglp1 psiglp NfkB psiglp NfkB psiglp1 Polymer only No injection psiglp NfkB psiglp1 Polymer only No injection A Empty plsmid B Empty plsmid C Fig. 3. Delivery of PEI/pSIGLP1 in DIO mice. (A) Blood glucose level, (B) plsm GLP-1 level, nd (C) plsm insulin concentrtion chnges fter PEI/pSIGLP1 injection. The DIO mice received intrvenous injection of PEI only or PEI with empty plsmid or PEI with psiglp1 or PEI/pSIGLP1. Ech group ws composed of 6 rts, nd the grphs represent the SE verges. P<.5 compred to control. 323

8 Kim SW showing severe cytotoxicity. However, RCV of ABP ws mintined bove % regrdless of the concentrtion, indicting miniml cytotoxicity of ABP. Secretion levels of Ex-4 ws mesured by ELISA in NIH3T3 cells. TATA (SP-Ex-4) pdna system ws prepred t 3:1 mixing rtio between puas-sp-ex-4 nd pβ-gl4-p65 ccording to the previous result. The Ex-4 secretion level from TSTA (SP-Ex-4) ws found to be pproximtely 1 times lower thn from p β-sp-ex-4. The result my be due to some type of TSTA (SP-Ex-4) mlfunction. However, ABP showed 3 to 5 fold higher Ex-4 secretion level thn PEI25k in both pdna systems, displying potency s n efficient Ex-4 gene crrier. Currently, insulin induction ctivity of secreted Ex-4 is being exmined (unpublished dt). PREVENTION OF TYPE-1 DIABETES USING RAE-1 PLASMID AND EPHRIN CONJUGATED POLYETHYLENE GLYCOL TRIETHYLENE TETRA AMINE/CYSTAMINE BISABRYLAMIDE (EPH-PEG-TETA/CBA) COPOLYMER PCMV-REA-1γ ws constructed nd chrcterized. The copolymer of Eph-PEG-TETA.CBA ws synthesized nd its complex with REA ws studied. Mouse islet cells (NIT-1) trnsfected with PCMV-REA-1γ were treted with isolted NKG2D + CD8 + T-cells. Prevention of the T-cell infiltrtion into islet by RAE-1γ ws investigted by dding NKG2D + CD8 + T-cells to mouse islets. The pncretic PAE-12 expression profile ws observed by immunohistochemistry using nti-pae-12 NOD mouse. Animl experiments demonstrted the inhibition of type I dibetic progress (unpublished dt). Fs sirna DELIVERY IN CYCLOPHOSPHAMIDE INDUCED WITH DIABETES IN NOD MICE A membrne receptor, Fs (CD95), nd its lignd FsL hve been considered s key plyers in dibetes pthogenesis nd re known to medite interctions between β-cells nd cytotoxic T-cells, resulting in poptotic cell deth. The uthor of the present study hypothesized the interruption of Fs FsL interctions by suppressing Fs expression in β-cells would ffect the development of dibetes. The effect of Fs-silencing sirna (Fs sirna) on dibetes development ws evluted in cyclophosphmide (CY)-ccelerted dibetes niml model Incidence of dibetes (%) Fig. 5. Dibetes ws induced by injecting single dose of cyclophosphmide (CY) (2 mg/kg body weight) (Dy ) prior to the dministrtion of indicted formultions. Animls with blood glucose level bove 2 mg/dl were considered hyperglycemic (n=15). fter intrvenous dministrtion using the crrier PEI. The systemic non-virl delivery of Fs sirna showed significnt dely in dibetes incidence up to 4 dys, while the control mice treted with nked Fs sirna, scrmbled dsrna, or PBS were fflicted with dibetes within 2 dys. The retrdtion of dibetes incidence fter the tretment of Fs sirna my be due to the delyed progression of the pncretic insulitis. In the study, the potentil use of non-virl crrier-bsed sirna gene therpy for the prevention of type 1 dibetes is demonstrted [35]. The prevention nd effect of Fs sirna/pei complexes on CY-induced dibetes in NOD mice re shown in Fig. 5. CONFLICTS OF INTEREST No potentil conflict of interest relevnt to this rticle ws reported. ACKNOWLEDGMENTS The uthor wishes to thnk K. S. Ko, J. J. Ko, S. Oh, M. Lee, T. Kim, S. Choi, nd J. Jeong. Finncil supports were by NIH DK7773, DK875 nd WCU REFERENCES Fs sipna/pei Nked Fs sirna Sc sirna/pei PBS Dys fter CY injection 1. Rollnd AP. From genes to gene medicines: recent dvnces in nonvirl gene delivery. Crit Rev Ther Drug Crrier Syst 1998; 324

9 Polymeric gene delivery for dibetic tretment 15: Peel D. Virus vectors nd gene therpy: problems, promises nd prospects. MBChB Specil Study Module Project Report. Leicester: Deprtment of Microbiology nd Immunology, University of Leicester; Anchordoquy TJ, Koe GS. Physicl stbility of nonvirl plsmid-bsed therpeutics. J Phrm Sci 2;89: Grnett MC. Gene-delivery systems using ctionic polymers. Crit Rev Ther Drug Crrier Syst 1999;16: Hung L, Hung M, Wgner E. Nonvirl vectors for gene therpy. Sn Diego: Acdemic Press; p Kwbt K, Tkkur Y, Hshid M. The fte of plsmid DNA fter intrvenous injection in mice: involvement of scvenger receptors in its heptic uptke.phrm Res 1995;12: Americn Dibetes Assocition. Avilble from: dibetes.org (cited 211 Jn 12). 8. Anderson R, Jones P. Wht s new in type 2 dibetes? Liverpool: Ntionl Prescribing Centre; Lorenzn GG, Hsi SH. The phrmcotherpy of type 2 dibetes to chieve tight glycemic control. Home Helth Cre Consult 22;9: Mlisse WJ, Lebrun P. Mechnisms of sulfonylure-induced insulin relese. Dibetes Cre 199;13 Suppl 3: Dvies MJ. Insulin secretgogues. Curr Med Res Opin 22:18 (1 Suppl 1): Moller DE. New drug trgets for type 2 dibetes nd the metbolic syndrome. Nture 21;414: Koh JJ, Ko KS, Lee M, Hn S, Prk JS, Kim SW. Degrdble polymeric crrier for the delivery of IL-1 plsmid DNA to prevent utoimmune insulitis of NOD mice. Gene Ther 2; 7: Lee M, Hn S, Ko KS, Kim SW. Cell type specific nd glucose responsive expression of interleukin-4 by using insulin promoter nd wter soluble lipopolymer. J Control Relese 21; 75: Ko KS, Lee M, Koh JJ, Kim SW. Combined dministrtion of plsmids encoding IL-4 nd IL-1 prevents the development of utoimmune dibetes in nonobese dibetic mice. Mol Ther 21;4: Lee M, Hn SO, Ko KS, Koh JJ, Prk JS, Yoon JW, Kim SW. Repression of GAD utontigen expression in pncres bet-cells by delivery of ntisense plsmid/peg-g-pll complex. Mol Ther 21;4: Jeong JH, Lee M, Kim WJ, Yockmn JW, Prk TG, Kim YH, Kim SW. Anti-GAD ntibody trgeted non-virl gene delivery to islet bet cells. J Control Relese 25;17: L Brre J, Still EU. Studies on physiology of secretin. III. Further studies on the effects of secretin on blood sugr. Am J Physiol 193;91: Creutzfeldt W, Ebert R. New developments in the incretin concept. Dibetologi 1985;28: Creutzfeldt W. The incretin concept tody. Dibetologi 1979; 16: Beck B. Gstric inhibitory polypeptide: gut hormone with nbolic functions. J Mol Endocrinol 1989;2: Lund PK, Goodmn RH, Montminy MR, Dee PC, Hbener JF. Anglerfish islet pre-roglucgon II. Nucleotide nd corresponding mino cid sequence of the cdna. J Biol Chem 1983;258: Lopez LC, Frzier ML, Su CJ, Kumr A, Sunders GF. Mmmlin pncretic preproglucgon contins three glucgon-relted peptides. Proc Ntl Acd Sci U S A 1983;8: McDonld PE, El-Kholy W, Riedel MJ, Slptek AM, Light PE, Wheeler MB. The multiple ctions of GLP-1 on the process of glucose-stimulted insulin secretion. Dibetes 22;51 Suppl 3:S Nthn DM, Schreiber E, Fogel H, Mojsov S, Hbener JF. Insulinotropic ction of glucgonlike peptide-i-(7-37) in dibetic nd nondibetic subjects. Dibetes Cre 1992;15: Buteu J, Roduit R, Susini S, Prentki M. Glucgon-like peptide-1 promotes DNA synthesis, ctivtes phosphtidylinositol 3-kinse nd increses trnscription fctor pncretic nd duodenl homeobox gene 1 (PDX-1) DNA binding ctivity in bet (INS-1)-cells. Dibetologi 1999;42: Perfetti R, Zhou J, Doyle ME, Egn JM. Glucgon-like peptide-1 induces cell prolifertion nd pncretic-duodenum homeobox-1 expression nd increses endocrine cell mss in the pncres of old, glucose-intolernt rts. Endocrinology 2;141: Decon CF, Johnsen AH, Holst JJ. Degrdtion of glucgonlike peptide-1 by humn plsm in vitro yields n N-terminlly truncted peptide tht is mjor endogenous metbolite in vivo. J Clin Endocrinol Metb 1995;8: Drucker DJ. Minireview: the glucgon-like peptides. Endocrinology 21;142: Nuck MA, Kleine N, Orskov C, Holst JJ, Willms B, Creutzfeldt W. Normliztion of fsting hyperglycemi by exogenous glucgon-like peptide 1 (7-36 mide) in type 2 (non-insulindependent) dibetic ptients. Dibetologi 1993;36: Pridl L, Agerbek H, Christensen LN, Thomsen K, Kirk O. 325

10 Kim SW Absorption of glucgons-like peptide-1 cn be protrcted by zinc or protmine. Int J Phrm 1996;136: Holst JJ, Decon CF. Inhibition of the ctivity of dipeptidylpeptidse IV s tretment for type 2 dibetes. Dibetes 1998; 47: Oh S, Lee M, Ko KS, Choi S, Kim SW. GLP-1 gene delivery for the tretment of type 2 dibetes. Mol Ther 23;7: Choi S, Oh S, Lee M, Kim SW. Glucgon-like peptide-1 plsmid construction nd delivery for the tretment of type 2 dibetes. Mol Ther 25;12: Jeong JH, Kim SH, Lee M, Kim WJ, Prk TG, Ko KS, Kim SW. Non-virl systemic delivery of Fs sirna suppresses cyclophosphmide-induced dibetes in NOD mice. J Control Relese 21;143: