in molecular medicine GLP-1-mediated gene therapy approaches for diabetes treatmenttip.akdeniz.edu.tr/wp-content/uploads/2016/10/GLP-1... · 2020-02-26 · GLP-1-mediated gene therapy

GLP-1-mediated gene therapyapproaches for diabetes treatment

Mukerrem Hale Tasyurek1,2, Hasan Ali Altunbas1,3, Halit Canatan4,Thomas S. Griffith5 and Salih Sanlioglu1,2,*

Glucagon-like peptide (GLP)-1 is an incretin hormone with several antidiabeticfunctions including stimulation of glucose-dependent insulin secretion,increase in insulin gene expression and beta-cell survival. Despite the initialtechnical difficulties and profound inefficiency of direct gene transfer into thepancreas that seriously restricted in vivo gene transfer experiments withGLP-1, recent exploitation of various routes of gene delivery and alternativemeans of gene transfer has permitted the detailed assessment of thetherapeutic efficacy of GLP-1 in animal models of type 2 diabetes (T2DM).As a result, many clinical benefits of GLP-1 peptide/analogues observedin clinical trials involving induction of glucose tolerance, reduction ofhyperglycaemia, suppression of appetite and food intake linked to weightloss have been replicated in animal models using gene therapy. Furthermore,GLP-1-centered gene therapy not only improved insulin sensitivity, but alsoreduced abdominal and/or hepatic fat associated with obesity-inducedT2DM with drastic alterations in adipokine profiles in treated subjects. Thus,a comprehensive assessment of recent GLP-1-mediated gene therapyapproaches with detailed analysis of current hurdles and resolutions, isdiscussed.

1Human Gene and Cell Therapy Center, Akdeniz University Hospitals, Antalya 07058, Turkey2Department of Medical Biology and Genetics, Akdeniz University Faculty of Medicine, Antalya07058, Turkey3Department of Internal Medicine, Division of Endocrinology and Metabolism, Akdeniz UniversityFaculty of Medicine, Antalya 07058, Turkey4Genome and Stem Cell Research Center, Department of Medical Biology, Erciyes University Facultyof Medicine, Melikgazi, Kayseri 38039, Turkey5Department of Urology, University of Minnesota, Minneapolis, MN 55455, USA

*Corresponding author: Professor Dr. Salih Sanlioglu VMD, PhD, Human Gene and Cell TherapyCenter, Akdeniz University Hospitals and Clinics, B Block, 1st floor, Campus, Antalya 07058,Turkey. E-mail: [email protected]

expert reviewshttp://www.expertreviews.org/ in molecular medicine

1Accession information: doi:10.1017/erm.2014.7; Vol. 16; e7; March 2014

© Cambridge University Press 2014

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IntroductionAn incretin effect is defined as a biologic processwhere orally taken carbohydrates induce therelease of intestinal hormones augmentinginsulin secretion more than what could beachieved with intravenous glucose delivery(Ref. 1). These hormones are released from theintestinal mucosa to orchestrate glucose-inducedinsulin secretion (insulinotropic effect) frompancreatic beta cells. Therefore, incretin hormonesare crucial in the maintenance of postprandialglucose levels by facilitating glucose transportinto peripheral tissues (Ref. 2). Gastric inhibitorypolypeptide/glucose-dependent insulinotropicpolypeptide (GIP) and glucagon-like peptide-1(GLP-1) are the two incretin hormones withinsulinotropic effect in humans, which isresponsible for 70% of postprandial glucose-dependent insulin secretion (Ref. 3). Some of theremaining insulinotropic activity can beattributed in part to neurotransmitters, such asvasoactive intestinal peptide (VIP) and pituitaryadenylate cyclase-activating peptide (PACAP)(Refs 4, 5).GLP-1 is one of the two essential gut-derived

incretin hormones involved in the modulation ofglucose homoeostasis (Fig. 1). Its insulinotropicactivity has been demonstrated both inpreclinical and clinical studies (Refs 6, 7). Afteringestion of a meal, GLP-1 is released into thebloodstream where it stimulates glucose-dependent insulin release and insulin biosynthesisin pancreatic beta cells (Ref. 8) through aG-protein-coupled receptor (GLP-1R) (Ref. 9).While carbohydrates are the most effective agentcausing GLP-1 secretion, proteins and fat alsocontribute to the secretion of GLP-1 (Refs 10, 11).Apart from its insulinotropic action, GLP-1interferes with glucagon release (Ref. 12) andimproves age-related glucose intolerance (Ref. 13).In addition, GLP-1 possesses mitogenic effectsresulting in cellular differentiation (Ref. 14) andincreased beta-cell mass (Ref. 15). Weight lossdue to reduced appetite and food intake(Ref. 16) is also observed as a result ofsuppression of gastrointestinal motility andsecretion (Ref. 17). Lastly, GLP-1 displayedbeneficial effects in patients with myocardialischaemia and heart failure (Ref. 18).Isoglycaemic glucose tolerance tests

demonstrated that type 2 diabetes (T2DM)patients manifested a 50% reduction in the

incretin effect, despite a 300% increase inglucose-induced insulin secretion of healthycontrols (Ref. 19). Thus, the loss of incretinresponse certainly results in glucose intolerancein patients with T2DM, since incretins are themain modulators of postprandial glucoseexcursions. Interestingly, meal-stimulated GLP-1response, but not postprandial GIP secretion,was severely reduced in patients with T2DM(Ref. 20). Moreover, GLP-1 retained itsinsulinotropic effect in T2DM patients, while noincretin response was obtained with GIPadministration (Ref. 21). Because GLP-1infusions restored down-regulated beta-cellresponse to glucose in T2DM patients (Ref. 22),GLP-1 has been considered a therapeutic agentfor the treatment of T2DM.

GLP-1 is initially synthesised as part ofproglucagon, a prohormone consisting of 180amino acids (Ref. 23). Besides GLP-1, severalother small peptides glucagon, GLP-2, glicentinand oxyntomodulin are also encoded withinproglucagon fragment (Fig. 2). GLP-1 andglucagon are generated as a result of thedifferential post-translational processing ofpreproglucagon in the intestine and pancreas,respectively (Ref. 24). Therefore, the post-translational process is carried out by twodistinct prohormone convertases specificallyexpressed in two different tissues, PC2 inpancreas (Ref. 25) and PC3 in intestinal L cells(Ref. 26). In addition, GLP-1 is produced in thehindbrain, primarily in the nucleus of thesolitary tract (NTS) to regulate food motivation/reward (Refs 27, 28). It is the central GLP-1production from brainstem neurons, which isresponsible for the appearance of meal-relatedbenefits of GLP-1 involving reduction in mealsize, meal frequency, food motivation andreward (Refs 28, 29, 30, 31). GLP-1 productionfrom preproglucagon in non-endocrine tissues isimpractical, although, without the expressionof the specific prohormone convertase (Ref. 32).Nonetheless, proglucagon is intracellularlytransported to the regulated secretory pathwaywhere it is processed into the smaller peptides.

GLP-1 is a potent stimulator of glucose-induced insulin release without causing reactivehypoglycaemia (Ref. 33). However, GLP-1 has ashort biological half-life (2–3 min) due to rapidtruncation by the ubiquitous serine proteasedipeptidyl peptidase-4 (DPP-4), which limits its




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therapeutic use (Ref. 3). While frequent injectionsor larger quantities are needed to compensate forthe short biological half-life of GLP-1, viral or non-viral vector gene delivery technologies weredeveloped to provide a constant bioactive GLP-1production and secretion (Ref. 34). Becauseutilisation of the preproglucagon transgene mightlead to unpredictable production of glucagon,or other processed peptides with unknownfunction, gene transfer experiments involvingGLP-1 encoding sequence normally is restrictedto GLP-17–37 transfer rather than the entirepreproglucagon cDNA (Fig. 3). In addition, since

the first two amino acids of GLP-1 are essentialfor its receptor binding, constructs encodingGLP-17–37 from a methionine start codon need tobe synthesised using a DNA synthesiser. A furinrecognition site (RGRR) is introduced into theGLP-1 cDNA following the start codon tofacilitate removal of the preceding amino acidsby furin endopeptidases to generate the activeform of the peptide before secretion. Lastly, asecretory signal peptide is needed to target GLP-1 to the constitutive secretory pathway (CSP) toallow post-translational processing by a signalpeptidase facilitating its production and secretion

Reduction of appetiteSlowing down of gastric emptying

Insulin synthesis and secretionBeta cell proliferation,differentiation & protection

Increase in cardiac output

& cardioprotection

Inhibition ofglucose production

GLP-1

Major antidiabetic properties of GLP-1Expert Reviews in Molecular Medicine © 2014 Cambridge University Press

Figure 1. Major antidiabetic properties of GLP-1. GLP-1 is released from intestinal L cells located in thelower intestine (ileum). Target organs include, but not limited to, pancreas, liver, stomach, muscle, adiposetissue and brain. GLP-1 also suppresses glucagon secretion from alpha cells, and stimulates somatostatinsecretion from pancreatic delta cells. In addition, GLP-1 reduces gastric acid secretion. The effects ofGLP-1 on adipose and muscle tissue (enhancement of glucose update and glycogen synthesis) are omittedfor clarity.




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in non-endocrine tissues. Consequently, currentprogress in gene therapy approaches involvingGLP-1 cDNA transfer for diabetes treatment willbe highlighted in this manuscript.

Non-viral gene delivery approachesPlasmidsA plasmid-based gene delivery method involvinga modified GLP-17–37 cDNA with a furincleavage site between the start codon andGLP-1 coding region was developed to evaluatethe consequence of in vivo GLP-1 gene deliveryin diabetic animals (Ref. 35). A single intravenous

injection of polyethylenimine (PEI)/pGLP1complex into Zucker diabetic fatty (ZDF) ratsresulted in an increase in glucose-inducedinsulin secretion with a reduction in bloodglucose level for 2 weeks. To increase GLP-1expression, an SV40 promoter with NF-κB-binding sites was incorporated into the plasmidcarrying GLP17–37 cDNAwith furin cleavage site(Ref. 36). A single systemic administration ofPEI/pGLP1 complex into the diet-induced obese(DIO) mice resulted in increased insulinsecretion and decreased blood glucose longerthan 2 weeks.

Differential proglucagon processing in the intestine versus pancreas

INTESTINES

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Expert Reviews in Molecular Medicine © 2014 Cambridge University Press

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Figure 2. Differential proglucagon processing in the intestine versus pancreas. Proglucagon is processedto generate glicentin, GLP-11–37 and/or GLP-11–36 amide, Intervening Peptide 2 (IP2) and GLP-2 by the actionof PC1/3 in the intestine. PC1/3 can further process glicentin and GLP-1 to produce oxyntomodulin andGLP-17–37 and/or GLP-17–36 amide. PC2 processing of proglucagon fragment in pancreatic alpha cellsyields Glicentin-related pancreatic polypeptide (GRPP), glucagon, IP-1 and the major proglucagon fragmentrather than GLP-1.




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Because GLP-1 must be delivered through aparenteral route and has a short lifespan, afusion protein consisting of an active humanGLP-1 and mouse IgG1 heavy chain constantregions (GLP-1/Fc) was generated to prolongand enhance the therapeutic potency of GLP-1(Ref. 37). IgG–Fc homodimerisation wouldresult in the formation of bivalent GLP-1peptide ligands with longer half-life comparedto native GLP-1, since the formation of largemolecular weight homodimers slows renalclearance and reduces degradation of theconjugated peptide. The anti-diabetic effects ofthe GLP-1/Fc plasmid injection took time todevelop, as delivery of GLP-1/Fc fusion proteinnormalised fasting blood glucose levels three

months after the first injection in db/db miceresulting in augmented glucose-induced insulinsecretion and reduced glucagon release.However, the GLP-1/Fc fusion protein could notpenetrate through the blood–brain barrier, sobody weight and peripheral insulin sensitivitieswere not affected by this treatment.

A chitosan-based gene delivery system wasconstructed by taking advantage of the naturalability of cationic polymers to condense plasmidDNA through electrostatic interaction to protect itfrom a nuclease attack (Ref. 38). In addition,nanoparticles made of chitosan are small enoughto pass through intercellular tight junctions togain entry into cells to deliver GLP-1-encodingplasmid DNA (Ref. 39) The therapeutic efficacy of

Gene therapy vector design encoding GLP-1Expert Reviews in Molecular Medicine © 2014 Cambridge University Press

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Figure 3. Gene therapy vector design encoding GLP-1. A cell-type-specific promoter (e.g., insulin promoter)restricts transgene expression in target tissues. Epitope targeting by way of pseudotyping or use of alternativeserotypes of viral vectors is also employed to achieve tissue specificity. A variety of signal peptides areemployed to direct GLP-1 into secretory pathways. Furin cleavage is necessary to remove GLP-1 fromthe signal peptides. The Ala-to-Gly substitution in GLP-1 provides resistance to DPP-4 cleavage.Alternative routes of gene delivery through celiac artery by transient blockage of splenic and hepaticarteries also provide efficient islet transduction by viral vectors.




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chitosan-based nanocomplexes containing GLP-1-encoding plasmid DNA with a furin recognitionsite and cytomegalovirus (CMV) promoter wasassessed in 12-week-old ZDF rats with overtdiabetes mellitus (Ref. 40). A significant increasein the amount of plasma GLP-1 was detected atday 49 after five injections of chitosan–GLP-1nanoparticles. In spite of the improvement inglucose tolerance and reduced weight gain in thetreated rats, the increase in circulating insulin wastransient and only lasted 14 days following thelast injection. Intriguingly, subcutaneous (s.c.)injection of the nanocomplexes was more efficientthan intramuscular (i.m.) gene deliverypresumably due to an inflammatory reaction atthe injection site that interfered with vectordistribution. The ability of specific chitosanformulations to deliver native GLP-1, DPP-4-resistant GLP-1 analogues and siRNA-targetingDPP-4 mRNA were investigated in a recent invitro study (Ref. 41). Chitosan formulationseffectively delivered nucleic acid into cell linesresulting in a fivefold increase in DPP-4-resistantGLP-1 analogues compared to native GLP-1. Inaddition, a DPP-4 gene-silencing approach usingchitosan formulation was successful and exhibitedreduced toxicity compared to commerciallyavailable lipoplex, DharmaFECT.Because Exendin-4 (Exenatide) is an effective

GLP-1R agonist (50% sequence homology) withlonger half-life, an Exendin-4 expression systemwas designed using the two-step transcriptionamplification method consisting of a dualplasmid, where one plasmid encoded a potenttranscription factor and the second plasmidcontained a promoter driving the Exendin-4encoding sequence (Ref. 42). An arginine-graftedcyctaminebisacrylamide–diaminohexane polymer(ABP) was chosen as a gene carrier, sinceit exhibited minimal toxicity and highertransfection efficiency compared to PEI. Asingle intravenous administration of Exendin-4polyplex with ABP polymer resulted inincreased Exendin-4 expression and enhancedglucose-induced insulin secretion associatedwith decreased blood glucose in C57BL/6J micefed with high-fat diet (HFD).

Viral gene delivery approachesAdenovirusTo increase the efficacyof gene delivery, adenoviralexpression vectors encoding GLP-17–37 wereconstructed and systemically injected into db/db

mice and ZDF diabetic rats (Ref. 43). Sustainedhigh levels of circulating active GLP-17–37expression that led to a reduced hyperglycaemiawere obtained by linking the modified GLP-1-coding region (A8 G substitution to render thepeptide resistant to DPP-4 cleavage) to a leadersequence and a furin recognition site. Systemicinjection of the adenovirus-GLP-1 vectorimproved glucose tolerance and reduced foodintake generating weight loss in ZDF diabetic rats.

In another approach, DPP-4 resistant GLP-17–37linked to the mouse growth hormone (mGH)secretory sequence with the furin cleavage sitewas cloned into the adenovirus vector for GLP-1expression in submandibular glands in mice(Ref. 44). Delivery of Ad-GLP-1 resulted in 3times higher serum GLP-1 levels associated withfaster blood glucose clearance and reduction inalloxan-induced hyperglycaemia compared tomice injected with a control vector. Although,GLP-1 released from exocrine cells of thesalivary glands could be modified for secretioninto the circulatory system to alter blood glucoselevels, retroductal infusion of adenovirus-mediated GLP-1 gene delivery into salivaryglands resulted in an inflammatory reaction dueto viral backbone-limiting therapeutic efficacy ofGLP-1 peptide.

An adenoviral vector carrying GLP-1 cDNA(rAd-GLP-1) driven by a CMV promoter-enhancer and albumin leader sequence wasconstructed to determine the extent to whichcontinuous GLP-1 expression in vivo couldstimulate beta-cell regeneration in mice (Ref. 45).A single i.v. administration of rAd-GLP-1 intostreptozotocin (STZ)-induced diabetic non-obesediabetic–severe combined immunodeficient(NOD/SCID) mice demonstrated that remissionof diabetes could be achieved within 10 daysand normoglycaemia could be maintained atleast 20 days. In addition, rAd-GLP-1-treatedmice manifested a higher number of insulin-positive cells in the pancreas leading tohigh levels of insulin secretion compared toSTZ-induced diabetic mice infected withcontrol adenovirus. Thus, regeneration ofinsulin-producing pancreatic beta cells byGLP-1-mediated gene therapy might be apotential therapeutic strategy for the treatmentof diabetes. A new GLP-17–37 encodingrecombinant adenovirus (Ad-ILGLP-1) with aCMV promoter and insulin leader sequence wasconstructed and injected i.v. into 12-week-old




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ZDFratswithovertT2DM(Ref.46),resultinginhighlevels of circulatingGLP-1 andnormoglycaemia for3weeksand improvedglucose tolerance.Although,both pre-diabetic and diabetic ZDF rats respondedsimilarly to adenovirus-mediated GLP-1 genedelivery, the protection only lasted 21 days due totransient nature of gene expression induced byadenovirus vectors.Obesity and insulin resistance are linked to low-

gradechronic inflammation (Ref. 47).Since,adiposetissue is a source of inflammation contributing toinsulin resistance, a recombinant adenovirusproducing GLP-1 (rAd-GLP-1) was generated andadministered into ob/ob mice to test the extent towhich GLP-1 had anti-inflammatory effects onadipose tissue (Ref. 48). rAd-GLP-1-treated ob/obmice demonstrated significant reductions in fatmass, adipocyte size and lipogenic mRNAexpression compared to untreated mice. Areduction in abdominal fat, but not s.c. fat, isconsistent with previous observations indicatingthat abdominal fat deposition is associated withinsulin resistance (Ref. 49). Direct inhibition ofinflammatory pathways in adipocytes (as well asmacrophages) suggests that insulin sensitivitywas improved by GLP-1.A helper-dependent adenoviral (HDAd) vector

was produced to evaluate the long-term effects ofelevated Exendin-4 expression in vivo in a HFD-induced obesity mouse model (Ref. 50). Thismodel was used instead of the genetic rodentmodels of extreme obesity to better mimic themetabolic changes that occur in obese patients.A single HDAd-Ex4 injection of HFD miceimproved glucose homoeostasis with decreasedgluconeogenic enzyme activity. However, thistreatment did not lead to increased circulatinginsulin levels, but it reduced hepatic fat andimproved adipokine profile of treated animals.The decreased weight gain observed in HFD micewas attributed to increased energy expenditure,and not a result of a change in food intake.

Adeno associated virus (AAV)A double-stranded AAV serotype 8 vector(dsAAV8) containing enhanced green-fluorescentprotein (eGFP) driven by the mouse insulin-IIpromoter (MIP) resulted in a tissue specifictransduction of pancreatic beta-cells (Ref. 51).Considering these data, a DsAAV8–MIPconstruct with GLP-1 instead of eGFP was usedto assess therapeutic efficacy of GLP-1 viaintraperitoneal injection into diabetic mice with

beta-cell damage induced by multiple low-doseSTZ administration (Ref. 52). Despite protectionfrom hyperglycaemia, GLP-1 expression in thebeta cells was not high enough to increasecirculating GLP-1 levels due to insufficienttransduction with dsAAV (27%). The localisedintraislet GLP-1 production did, though,significantly improve islet function and survival.

Hepatocyte growth factor (HGF) has alsobeen considered as a therapeutic agent fordiabetes (Ref. 53) because adenovirus delivery ofHGF prevented pancreatic beta-cell death andminimised islet cell mass necessary fortransplantation (Ref. 54). Intriguingly, combinedtreatment of GLP-1 and HGF enhanced insulinsensitivity, and reduced body weight in obesepatients (Ref. 3). Thus, dsAAV vectors wereconstructed to test the therapeutic efficacy ofbeta-cell growth factors, GLP-1 and HGF, fordiabetes treatment using a gene therapyapproach. Due to the limited capacity of AAVvectors for transgene insertion (∼2.5 kb), onlythe N and K1 domains of HGF (HGF/NK1)with partial activation potential of HGF receptorwere cloned into dsAAV vector (Ref. 55). dsAAVvector-mediated delivery of GLP-1 and HGF/NK1 fragment delayed diabetes onset in db/dbmice inducing pancreatic islet cell proliferation.Failure to improve insulin resistance and weightgain in db/db mice was presumably due to theuse of partial HGF fragment (NK1) because ofthe limited transgene capacity of AAV vectors.

A GLP-17–37-encoding dsAAV vector with themurine Ig ∣ chain leader sequence and a furincleavage site was constructed to evaluate long-term antidiabetogenic effects of GLP-1 genetransfer in db/db obese mice (Ref. 56). A CMVenhancer/chicken®-actin promoter was used forstable liver transduction. A single injection ofdsAAV GLP-1 vector resulted in 4–10-foldincrease in circulating GLP-1, leading to reducedblood glucose levels up to four months. Despite18 weeks of sustained GLP-1 expression, noeffect on body weight was observed.

Salivary glands are considered a suitable depotorgan in gene therapy with high levels of proteinproduction and secretion into the bloodstream(Ref. 57). Since AAV serotype 5 (AAV5) hasexhibited enhanced gene transfer into rodentsalivary glands (Ref. 58), metabolic effects ofAAV5-mediated Exendin-4 gene delivery in twodifferent animal models of T2DM (Zucker fa/farats and HFD) were examined (Ref. 59). The




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Exendin-4 coding sequence was linked to thesecretory signal peptide from nerve growth factor(NGF) that also contained a furin cleavage site.Following per cutaneous injection of AAV5into the salivary glands, sustained Exendin-4expression at pharmacological levels wasdetected in blood and salivary glands of diabeticanimals leading to improved glycaemic controland insulin sensitivity associated with weight loss.

Critical evaluation of GLP-1-mediatedgene therapy approaches

Vector choiceInitial plasmid-based gene delivery techniquesinvolving PEI–plasmid DNA complexes onlymediated transient effects on insulin secretionand blood glucose levels. This was mainlyattributed to the inherent nature of plasmid-based gene delivery method providing short-term gene expression along with an absence of asecretory signal within GLP-1 encodingsequence (Refs 35, 36). Similarly, chitosan-mediated gene delivery systems yieldedtransient GLP-1 gene expression requiringrepeated administration of the chitosan–DNAcomplex to retain insulinotropic activity.Nevertheless, the specific chitosan formulationsmight be more effective when used incombination with siRNA-targeting DPP-4(Ref. 41). Lastly, experiments conducted with aGLP-1/Fc fusion protein encoding plasmiddemonstrated that this bivalent GLP-1 peptideligand should be evaluated as a structurallystable GLP-1 analogue in future studies (Ref. 37).Although numerous non-viral gene delivery

systems have been tested for GLP-1 gene delivery,viral vectors are currently the best for genetransfer. Among the viral vectors tested,adenoviral vectors are very efficient in transducinga wide range of tissues with an ability to infectboth dividing and non-dividing cells, to producehigh titre yield and accommodate large transgenes(Ref. 60). However, adenovirus-transduced cellsare quickly cleared by the immune system due toantigenicity to adenovirus encoded viral peptidesseverely limiting the longevity of transgeneexpression (Ref. 61). Furthermore, systemicdelivery of adenovirus vectors at high doses mightresult in severe adverse effects (Ref. 62), andrepeated administration of the vector is notfeasible due to the presence of neutralisingantibodies. Contrary to first-generation adenovirusvectors, helper-dependent (gutless) adenoviral

(HDAd) vectors encode no viral proteins due todeletion of almost all viral genes except ITRs(Refs 63, 64), resulting in negligible toxicity(Ref. 65) and sustained (even lifelong) transgeneexpression (Ref. 66). Consequently, HDAd-mediated Exendin-4 gene delivery into DIO miceallowed investigators to follow long-termmetabolic changes including decreased weightgain – something that was not possible using firstgeneration of adenovirus vectors (Ref. 50). Despitethis, the therapeutic efficacy of HDAd-mediatedGLP-1 gene delivery remains to be tested inanimal models of diabetes.

Similar to adenovirus vectors, AAV-basedvectors can infect both dividing and non-dividing cells. Since the AAV genome is single-stranded DNA (ssDNA), the conversion todouble-stranded DNA (dsDNA) in transducedcells appeared to be the rate-limiting step inrAAV-mediated gene delivery (Refs 67, 68). Toincrease transduction efficiency, dsAAV vectorswere developed by generating mutations at theITR resulting in the preferential packaging ofdouble-stranded, hairpin-like DNA dimers intoAAV capsids (Ref. 69). Because intrapancreasinjection of AAV vectors with conventionalssDNA vector genomes resulted in transductionof limited number of islet cells (Ref. 70),different serotypes of AAV vectors coupled withvarious routes of gene delivery were exploredfor pancreatic gene transfer in vivo to achievewidespread, robust, and stable transgeneexpression (Ref. 51). Consequently, a self-complimentary dsAAV vector serotype 8(dsAAV8) provided long-term, stable genetransfer and expression in pancreatic beta cellsof C57BL/6 and BALB/c mice. Despite thesedata, AAV vectors have very limited transgenecapacity, low transduction efficiency andproduce low titre yields (Ref. 71). Moreover,while wild-type AAV integrates into the humangenome at a specific site on chromosome-19(AAVS1), recombinant AAV (rAAV) lacks the repprotein required for integration into the hostchromosome (Ref. 72). AAV vectors remainepisomal in slowly dividing cells and are lessimmunogenic compared to adenovirus vectors(Refs 73, 74), suggesting they can provide long-term stable gene expression in pancreatic beta cells.

Neither adenovirus nor adeno-associated virusselectively infect pancreas. Both of these genedelivery vectors manifest broad tissue tropism,with the liver being their prime target organ




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following systemic delivery (Ref. 70). Thus, directinjection into the pancreas (Ref. 70) or the celiacartery (Ref. 75) is needed to target the desiredcell type in pancreas. Insulin promoters can alsobe used to provide beta-cell-specific geneexpression during systemic injection (Ref. 52).Infection of cells outside the pancreas has thepotential to yield adverse effects (Refs 76, 77).Consequently, natural pancreatropic virusescould be used to resolve these problems. Forexample, group B coxsackieviruses (CVBs)manifest extraordinary strong tissue tropism forexocrine cells and islets in pancreatic tissue(Ref. 78). Since CVBs also target heart and livertissues, a unique pancreatropic strain wasdeveloped by introducing two novel mutationsfrom an attenuated CVB vaccine candidate(vCVB(dm)) to direct CVB to pancreas (Ref. 79).Injection of GLP-1-expressing vCVB(dm)(vCVB(dm)GLP-1) reduced STZ-inducedhyperglycaemia in diabetic Balb/c mice throughenhancement of pancreatic insulin content andstimulation of beta-cell neogenesis (Ref. 80).However, vCVB(dm)GLP-1 could provide onlytransient gene expression lasting 4–7 days due tolow transduction rate, inflammatory responseand inability to integrate into the host genome.Because of its past association with T1DM, theclinical use of such vectors has not been advised.Integration of viral vectors into the genome is

required to achieve long-term gene expression invivo. Among the integrating vectors, lentiviralvectors appear to be vector of choice becausethey infect both dividing and non-dividing cells,possess little-to-no immunogenicity and do notcause deleterious mutations (Refs 81, 82).Lentiviral vectors do not mobilise even afterinfection with wild-type HIV-1, such that theyare regarded as safe for clinical applications.Pseudotyping of lentiviral vectors with vesicularstomatitis virus-G protein (VSV-G) is alsoimportant to obtain high titre yield with broadtissue tropism (Refs 83, 84). Since no dose-limiting toxicities have been reported withlentiviral vectors, there is minimal toxicityconcern even after multiple injections.Consequently, lentivirus-mediated GLP-1 genedelivery targeting pancreatic islets eitherthrough pseudotyping and/or using tissue-specific promoters might be necessary toimprove the therapeutic efficacy and safety ofGLP-1-mediated gene therapy approach.

Promoter selectionA number of reports involving GLP-1 genedelivery using viral vectors initially usedconstitutive promoters such as CMV, chicken β-actin or ubiquitin to provide strong butunregulated GLP-17–37 expression. Althoughthese studies clearly demonstrated increasedplasma GLP-1 levels that resulted in lowering ofblood glucose with improved insulin sensitivity,the site of transgene expression remainedelusive. The liver, spleen, heart, pancreas andother tissues were suspected to express thetransgene, but the possibility of transgeneexpression in any given tissue would raise aconcern about the long-term safety of thistherapeutic approach. In addition, it remainsunknown to what extent constant GLP-1expression would desensitise the GLP-1 receptorcreating a GLP-1-resistant status in vivo or causeother side effects. In this sense, the use of tissue-specific promoters might resolve issues withsafety and toxicity of gene delivery. Clearly, thechoice of promoter is determined by the targettissues in which the transgene expressionis desired for. For example, liver-specificexpression of GLP-1 gene has successfully beenachieved using the L-pyruvate kinase (LPK)promoter (Ref. 85). This promoter has theadditional benefit of supplying regulatedpromoter function to the transgene of interest insuch a way that promoter activity is onlyelevated when blood glucose increases, such asafter meals. This would not only be importantfor mimicking physiological secretion of GLP-1,but also essential for avoiding possible side effects.

The insulin promoter with glucoregulatoryactivities is very effective in providing transgeneexpression specifically in pancreatic betacells (Ref. 51). Intraperitoneal delivery ofDsAAV8–MIP–GLP-1 provided both localisedGLP-1 expression in pancreatic beta cells andprotection against the development of STZ-induced diabetes in mice. However, localisedGLP-1 expression in pancreatic beta cells wasnot sufficient to increase plasma GLP-1 levels,which limited the broad therapeutic efficacy ofGLP-1 relevant for modulating insulinsensitivity, food intake and weight loss.Consequently, the inability to increase theamount of circulating GLP-1 with localisedgene expression in the pancreas may limit theinteraction of GLP-1 with glucoregulatory




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tissues involved in the generation of insulinsensitivity and weight loss such as adipocytes,muscle and liver. Since GLP-1 is secreted byintestinal endocrine L cells and acts locally inthe gut by inhibiting gastric emptying andgastric acid secretion that leads to decreasedfood intake and weight loss, restricting GLP-1expression to pancreatic beta cells may interferewith its beneficial gastrointestinal effects (Ref. 52).

Site-specific integrationConsidering the oncogenic potential of retrovirusvectors (Ref. 86), site-specific integration of genetherapy vectors is necessary to avoid the risk ofinsertional mutagenesis. The human parvovirusAAV preferentially integrates into humanchromosome 19 (Ref. 87). The AAV genome hastwo major open reading frames encoding repand cap that are flanked by two invertedterminal repeats (ITRs). Rep is required for thesite-specific integration into the AAV targetsequence (AAVS1) present on chromosome 19.Since a 16 bp Rep-binding element (RBE) issufficient for mediating Rep-dependentintegration into AAVS1, plasmids carrying RBEsequences have been explored to delivertherapeutic genes into the AAVS1 site in vivousing transgenic mice (Ref. 88). Hydrodynamicinjection of plasmids encoding human bloodcoagulation factor IX (hFIX) with the 16 bp RBEand a Rep protein resulted in successful deliveryof hFIX to AAVS1 loci. Similarly, a non-viralGLP-1/Fc gene therapy strategy was tested byi.m. injection of two plasmids, one harbouringthe GLP-1/Fc cassette flanked by the 16 bp RBE-ITR (RBE/GLP-1/Fc) and the second carrying acopy of AAV Rep (Rep78) to facilitate theintegration of the GLP-1-encoding sequence intothe AAVS1 locus (Ref. 89). Persistent expressionof GLP-1/Fc proteins was achieved followingthe site-specific integration of RBE/GLP-1/Fcinto AAVS1 resulting in reduced weight gainand improved insulin sensitivity without anydetrimental effects in mice fed with HFD.Together, these results demonstrated that thesite-specific integration of AAV vectors is afeasible approach for experimental delivery ofgene therapy vectors without the risk ofinsertional mutagenesis.

Route of gene deliveryNumerous recent gene therapy approachesconducted for diabetes mainly addressed ex vivo

modification of pancreatic islets fortransplantation (Refs 90, 91). Non-pancreatictissues, such as liver and muscle, were chosen astarget organs to express therapeutic genes ofinterest to induce beta-cell differentiation orinsulin gene expression (Ref. 60). Severalstrategies are available for gene transfer intopancreatic beta cells. While non-viral vectordelivery systems, such as lipofection andelectroporation, produced low transductionlevels in pancreatic islets (Ref. 92), viral vectorstransduced islets very effectively (Ref. 90).Adenoviral vectors are relatively easy toconstruct, can be produced at high titres, andhave high transduction rates, but adenovirus-mediated gene delivery to pancreatic isletsremains difficult due to the clusteredarchitecture of endocrine cells in islets (Ref. 93).In vivo gene transfer to pancreatic islets usingadenovirus vectors administered via thecommon bile duct (Refs 94, 95) or a distal bloodvessel from the pancreas (Ref. 96) resulted ininefficient gene delivery to pancreatic islets.Moreover, host immune response to adenovirusvectors resulted in pancreatitis leading totransient gene expression. Only injectionthrough the celiac artery combined with theligation of the hepatic artery, portal vein and thesplenic artery resulted in uniform and hightransduction of pancreatic islets (Ref. 75).

Direct injection of ssAAV into the pancreastransduced an insufficient number ofpancreatic islets to yield a therapeutic effect(Ref. 70). Conversely, dsAAV vectors have hightransduction efficiency compared to ssAAVvectors. To achieve long lasting and strong geneexpression in pancreatic islets of C57BL10 mice,three different delivery routes (intraperitoneal,intraductal and intravenous) were comparedusing dsAAV vectors (Ref. 51). Intravenousdelivery of dsAAV vectors resulted in 5–10-foldless efficient pancreatic transduction comparedto intraperitoneal injection since most of thedsAAV were filtered by the liver. Despite this,intraperitoneal delivery primarily transducedcells on the islet peripheral zone compared tocells in central zone due to limited diffusion ofviral particles into the islets. Intriguingly, use ofa retrograde pancreatic intraductal deliveryapproach, which is similar to the commonlyused clinical technique known as endoscopicretrograde cholangiopancreatography, providedbetter transduction rates in pancreatic islets




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compared to intraperitoneal and intravenousdelivery. It is difficult to perform this techniquein small animals like mice, such that rats mightbe a better option to experimentally testretrograde pancreatic intraductal gene deliverydue to feasibility and clinical relevancecompared to intraperitoneal injection. Whiletopical delivery reduced gene transfer to non-pancreatic tissues (liver, heart, testis, andmuscles), transduced cells were still mostlylocated on the peripheral zone of the islets.Regardless of the route chosen for gene delivery,the insulin promoter was still required forhighly specific transgene expression in insulin-producing pancreatic beta cells in vivo,particularly to exclude transgene expression inacinar cells of pancreas. Lastly, the efficacy ofdsAAV vector gene delivery to pancreatic isletswas tested in C57BL10 mice by way ofintravenous delivery in combination with atransient blockade of the liver circulation(Ref. 51). Although intravenous gene transfer byitself to the pancreatic islets was undetectableusing dsAAV vectors, gene transfer to thepancreatic islets was increased 15-fold usingthe liver blockage approach leading to uniformtransgene expression in most of the islets. In thisstudy, different serotypes of AAV vectors werealso tested exhibiting various levels of tissuetransduction in pancreas depending on thedsAAV serotype.

Depository tissuesDue to destruction of pancreatic beta cells duringdisease progression, ectopic expression of GLP-1outside the pancreas is another option fortherapy. Among the potential sites of expression,the liver initially appeared to be the best targetorgan for GLP-1 gene delivery due to easeof access, high transduction rates and release oflarge amount of therapeutic proteins intocirculation. Moreover, the enzymes responsiblefor glucose sensitivity and response, Glucosetransporter 2 (GLUT2) and glucokinase,are mainly synthesised in liver, which gives theliver the ability to detect circulating glucoselevels in our body (Ref. 97). This characteristicalso made the liver a preferred target organ inislet cell transplantation (Ref. 98). However, theliver lacks the regulated secretory systemnecessary for GLP-1 secretion that is present inendocrine tissues such as intestinal L cells,making it necessary for further modifications to

GLP-1 for successful synthesis and secretionfrom liver. Inclusion of a signal peptide to directGLP-1 into secretory pathways following post-translational modifications solved one of theproblems associated with GLP-1 synthesis inliver. The liver is also one of the two organs inwhich furin endopeptidases are synthesised, soinclusion of a furin recognition sequence afterthe secretory signal peptide to liberate GLP-1from its secretory signal increased GLP-1production by the liver (Ref. 99). While most ofthe recent experimental gene therapy studiesincluded vectors with some or all the featuresmentioned above (Fig. 3), utilisation of thehidden Marcov model might be useful indesigning of the most efficient human secretorysignal peptides for GLP-1 secretion (Ref. 100).

Salivary glands are also becoming a frequentlytargeted depository organ to synthesise GLP1,since they have the capacity to synthesise largeamount of protein that can be secreted into thebloodstream. One potential benefit of targetingthe salivary gland is that it is surrounded with acapsule, which would restrict vector distributionminimising side effects of the treatment. Ifadverse events were to occur following genedelivery, removal of the salivary glands wouldbe easy and safe since they are not essentialfor life. Because of these characteristics, salivaryglands have been evaluated as a surrogateendocrine gland using gene therapy for thecorrection of many inherited monogeneticendocrine disorders (Ref. 57). Endocrine,neuroendocrine, and exocrine cells contain aregulated secretory pathway (RSP) in whichpeptide hormones are released upon stimulationwhile a CSP present in all cell types (Ref. 101).In this regard, salivary glands are exocrineglands with both secretory pathways. Whilesecreted proteins are released through the CSPmainly into the bloodstream (endocrine),the RSP releases proteins into the saliva(exocrine). However, synthesis of a prohormone(proglucagon) in cells with CSP without properprocessing enzymes, such as prohormoneconvertases, would lead to constant secretion ofunprocessed prohormone devoid of biologicactivity (Ref. 102). Consequently, a secretorysignal with a protease cleavage site is requiredfor the secretion of the bioactive peptide (GLP-17–37) through the CSP for therapeutic efficacy.Adenoviral delivery of GLP-1 with the signalsequence of the mGH followed by a furin




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cleavage site resulted in reduction of alloxan-induced hyperglycaemia in diabetic animals(Ref. 44). A threefold increase in the amount ofcirculating GLP-1 was achieved using thisapproach compared to mice transduced with thecontrol vector, despite the localised delivery ofthe vector into salivary glands. While adenoviralvectors have been tested in the delivery of genesinto salivary glands, AAV2 vectors are alsoamenable for gene delivery into salivary glands(Ref. 57), as an AAV2 vector encoding adrug inducible form of GLP-1 under thecontrol of glucose-regulated promoters hasbeen investigated to improve the safety andtherapeutic efficacy of GLP-1 gene delivery(Ref. 103).

Molecular alterations concerning GLP-1gene therapy targeting pancreasThe mechanism of GLP-1-mediated islet cellprotection was investigated by global geneexpression profiling of pancreatic islets isolatedfrom STZ-induced diabetic mice (Ref. 104).There was strong induction of p53-responsivegenes and suppression of a wide range ofdiabetes-related genes with short-term low-doseSTZ treatment. REG3 family proteins, likeGLP-1, act as a beta-cell trophic factorwith the potential to reverse STZ-inducedhyperglycaemia through islet neogenesis(Ref. 105). An AAV9-based beta-cell-targetedgene transfer system involving REG3B-GLP-1fusion protein expression was designed toachieve maximum beta-cell trophic effect indiabetic mice. Overexpression of REG3B-GLP-1preserved beta-cell mass and protected micefrom STZ-induced diabetes (Ref. 104). REG3B-GLP-1 gene therapy did not drastically alterSTZ-induced changes in the islets as defined bythe global gene expression profile, but theREG3B-GLP-1 gene therapy was able tosuppress islet cell apoptosis by enhancingexpression of genes involved in beta-cellsurvival. Intriguingly, most of the STZ down-regulated genes were related to genes involvedin beta-cell function and development – not thehousekeeping genes.

Animal modelsMost of the experimental gene therapy studiesinvolving GLP-1 gene delivery have beenperformed in genetically controlled rodentobesity models, such as ob/ob, db/db and Zucker

diabetic rats (Refs 35, 37, 43, 106). Since thesemodels carry mutations in genes that affectappetite (leptin (ob/ob) or leptin receptors (db/db, zucker diabetic fa/fa rats)), the use ofgenetically modified animal models of diabetesmay not be ideal for long-term studies ofmetabolic changes (Ref. 107). The mostappropriate experimental animal model of ahuman disease is the model that can best mimicthe pathophysiology of a human disease ofinterest. In addition, model standardisation,continuity, cost, clinical benefits and widespreadutilisation by scientists are among the factorsinfluencing the decision-making process. SinceT2DM in humans arises from the interactionbetween genes and the environment, DIOcombined with low-dose STZ injection (toinduce beta-cell loss and hyperglycaemia)appears to be the best model to mimic the actualdisease process (Fig. 4). Thus, the realantidiabetic potential of GLP-1-mediated genetherapy approach may require testing theefficacy of treatment with a third generationHIV-based lentiviral vector to deliver GLP-1in a HFD, low-dose STZ-induced diabetesmodel. However, additional doses of virusmight be needed to transduce target tissues to

Induction of obesity in C57BL/6 miceExpert Reviews in Molecular Medicine © 2014Cambridge University Press

Figure 4. Induction of obesity in C57BL/6 mice.Eight-week-old C57BL/6 male mice were fed astandard diet (SD-left panel) or high-fat diet (HFD-right panel) where 60% of the total calories comefrom fat. After glucose tolerance and insulinsensitivity tests, low-dose STZ injection is neededto induce diabetes. Arrows indicate abdominal fatdeposition.




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Table1.

Non

-viral

andviralG

LP-1-m

ediatedge

nede

liverymetho

ds

Gen

ede

livery

metho

d

Rou

teof

deliv

ery

Target

tissu

eSerum

leve

lsTh

erap

eutic

effects

Ons

etDuration

Animal

mod

elRefs

PEI-plasm

idIV

Circ

ulation,

liver

2×

Insu

linotropic

1wee

k2wee

ksZD

Frats

(Ref.3

5)

PEI-plasm

idIV

Circ

ulation,

liver

2.5×

Insu

linotropic,

Ano

rectic

2da

ys3wee

ksDIO

-C57

BL/

6J(Ref.3

6)

GLP

-1/F

cplas

mid

IMCirc

ulation

3×

Insu

linotropic

3 mon

ths

NA

db/d

bmice

(Ref.3

7)

Chitosa

n/plas

mid-D

NA

SC,IM

Circ

ulation

5×

Insu

linotropic,

Ano

rectic

NA

24da

ysZD

Frats

(Ref.4

0)

Exen

din-4or

GLP

-1po

lyplex

IVCirc

ulation

2–4×

Insu

linotropic

3da

ys1wee

kDIO

-C57

BL/

6J(Ref.4

2)

Ade

novirus

IVCirc

ulation,

liver

25×

Insu

linotropic,

Ano

rectic,

redu

ctionin

HbA

1c1da

y6wee

ksdb

/db

mice,

ZDF

rats

(Ref.4

3)

Ade

novirus

SG

Circ

ulation

3×

Insu

linotropic

1da

y2da

ysAllo

xan-

BALB

/c(Ref.4

4)

Ade

novirus

IVCirc

ulation,

liver

NA

Insu

linotropic

10da

ys20

days

STZ

-NOD/

SCID

(Ref.4

5)

Ade

novirus

IVCirc

ulation,

liver

10×

Insu

linotropic,

insu

linse

nsitivity,A

norectic

4da

ys3wee

ksZD

Frats

(Ref.4

6)

Ade

novirus

IVCirc

ulation,

liver

25×

Insu

linotropic,

insu

linse

nsitivity,A

norectic,

redu

cedfatmas

s

4da

ys2mon

ths

ob/o

bmice

(Ref.1

06)

(con

tinue

don

next

page

)




GLP

-1-m

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Table1.

Non

-viral

andviralG

LP-1-m

ediatedge

nede

liverymetho

ds(con

tinue

d)

Gen

ede

livery

metho

d

Rou

teof

deliv

ery

Target

tissu

eSerum

leve

lsTh

erap

eutic

effects

Ons

etDuration

Animal

mod

elRefs

HDAd-Ex

4IV

Circ

ulation,

liver

5–10

×Insu

linotropic,

insu

linse

nsitivity,A

norectic,

redu

cedhe

patic

fat,

improv

edad

ipok

ine

profile

NA

15wee

ksHFD

C57

/BL6

(Ref.5

0)

dsAAV

8IP

Pan

crea

s1×

Protectionag

ains

thy

perglyca

emia

2wee

ksNA

STZ

-Balb/

c(Ref.5

2)

dsAAV

IPPan

crea

s1×

Insu

linotropic

2wee

ks4wee

ksdb

/db

mice

(Ref.5

3)

dsAAV

PV

Live

r4–

10×

Insu

linotropic

3wee

ks4mon

ths

db/d

bmice

(Ref.5

6)

AAV

5-Ex

4SG

Circ

ulation

140–

240pm

ol/l

Insu

linotropic,

insu

linse

nsitivity,A

norectic,

improv

edad

ipok

ine

profile

4wee

ksNA

ZDFrats,

HFD

CD1

mice

(Ref.5

9)

db/d

b:diab

etic/d

iabe

ticLe

ptinrece

ptor

defic

ient

mice;

DIO:d

iet-indu

cedob

esity

;dsA

AV:d

ouble-strand

edad

eno-as

sociated

virus;HDAd:

helper-dep

ende

ntad

enov

iral;HFD

:high-fatdiet;IV:

Intrav

enou

s;IM

:intramus

cular;IP:intrape

riton

eal;NA:d

atano

tava

ilable;

ob/o

b:ob

ese/

obes

eLe

ptin-deficient

mice;

SC:

subc

utan

eous

;SG:s

alivaryglan

d;STZ

:strep

tozo

tocin;

PEI:p

olye

thylen

imine;

PV:

portal

Vein;Z

DF:

Zuck

erdiab

etic

fattyrats.




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compensate for increased body weight in obesemice (Ref. 55).

Concluding remarksAlthough there are several successful clinical genetherapy applications against genetic diseasessuch as Leber’s congenital amaurosis, X-linkedSCID, ADA-SCID, adrenoleukodystrophy, chroniclymphocytic leukaemia, acute lymphocyticleukaemia, multiple myeloma, haemophilia,Parkinson’s disease and thalassaemia (Ref. 108);Alipogene tiparvovec (Glybera) became thefirst gene therapy treatment approved forthe treatment of familial lipoprotein lipasedeficiency in Europe after its endorsement bythe European Commission (Refs 109, 110).Interestingly, there have been few genetherapy clinical trials for diabetes, mainly dueto concern for the need to treat diabetes relatedcomplications to improve wound healing(NCT00065663) and diabetic neuropathy(NCT01002235-NCT00056290).Incretinswere first proposed for the treatment of

T2DM in 1992, but the first incretin mimetic(Exenatide) for commercial use was approved bythe U.S. Food and Drug Administration (FDA) in2005 (Refs 111, 112). In addition Liraglutide wasthe first long-acting GLP-1 analogue approvedby the US FDA in 2010 (Ref. 113). Several otherincretin mimetics have reached to market since2010, and there are even more incretin-baseddrugs under development. As with any drug,there are some risks associated with the benefitsof using incretin-based treatments. For example,Exenatide and Liraglutide have been reported tocause significant gastrointestinal discomfort inT2DM patients (Refs 114, 115), but it remains tobe determined the extent to which they areassociated with increased risk for pancreatitis orpancreatic cancer (Ref. 116). Nevertheless, thesedrugs require daily s.c. injections (once/day forLiraglutide and twice/day for Exenatide) to beeffective. Thus, experimental viral or non-viralgene delivery methods have been underdevelopment to supply a constant GLP-1production and secretion for the treatment ofdiabetes (Table 1). Even though gene therapyappears to be a promising technique forachieving a long-term increase in GLP-1synthesis and secretion, the most effective genedelivery method has yet to be identified.Protocols using dsAAV vectors have producedsome successful results, similar or enhanced

results are expected using lentivirus vectorstargeting pancreas with glucoregulatoryfunction. This is especially true when thelong-term beneficial neuroprotective and/orcardioprotective effects of GLP-1 are expected.GLP-1 gene delivery has produced favourableresults in both pre-diabetic and fully diabeticanimals, suggesting that a GLP-1 gene therapyapproach may be a reasonable alternative toconstant infusions or daily injections of GLP-1peptide. It is important to keep in mind, though,that many of these published results showingthe benefits of GLP-1 gene therapy wereconducted in small rodent models of T2DM,making it crucial to continue testing of thistherapy in larger animal models (such as cats,dogs, pigs and even primates) to increase theclinical relevance of experimental findings anddesign future clinical trials.

AcknowledgmentsThis work is supported by grants from AkdenizUniversity Scientific Research AdministrationDivision and the Scientific and TechnologicalResearch Council of Turkey (TUBITAK-112S114).The authors declare that there is no duality ofinterest associated with this manuscript.

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Further reading, resources and contacts

Bryant, L.M. et al. (2013) Lessons learned from the clinical development and market authorization of Glybera.Human Gene Therapy Clinical Development 24, 55-64

Tibaldi, J.M. (2014) Incorporating incretin-based therapies into clinical practice for patients with type 2 diabetes.Advances in Therapy doi:10.1007/s12325-014-0100-5

Features associated with this article

FiguresFigure 1. Major antidiabetic properties of GLP-1.Figure 2. Differential proglucagon processing in the intestine versus pancreas.Figure 3. Gene therapy vector design encoding GLP-1.Figure 4. Induction of obesity in C57BL/6 mice.

TableTable 1. Non-viral and viral GLP-1-mediated gene delivery methods.

Citation details for this article

Mukerrem Hale Tasyurek, Hasan Ali Altunbas, Halit Canatan, Thomas S. Griffith and Salih Sanlioglu (2014) GLP-1-mediated gene therapy approaches for diabetes treatment. Expert Rev. Mol. Med. Vol. 16, e7, March 2014,doi:10.1017/erm.2014.7




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in molecular medicine GLP-1-mediated gene therapy approaches for diabetes treatmenttip.akdeniz.edu.tr/wp-content/uploads/2016/10/GLP-1... · 2020-02-26 · GLP-1-mediated gene therapy

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