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TYPE 1 D IABETES

Insulin B chain 9–23 gene transfer to hepatocytesprotects from type 1 diabetes by inducingAg-specific FoxP3+ TregsMahzad Akbarpour,1,2* Kevin S. Goudy,1* Alessio Cantore,1 Fabio Russo,1 Francesca Sanvito,3

Luigi Naldini,1,2 Andrea Annoni,1† Maria Grazia Roncarolo1,2,4†‡

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Antigen (Ag)–specific tolerance in type 1 diabetes (T1D) in human has not been achieved yet. Targeting lentiviralvector (LV)–mediated gene expression to hepatocytes induces active tolerance toward the encoded Ag. The insulinB chain 9–23 (InsB9–23) is an immunodominant T cell epitope in nonobese diabetic (NOD) mice. To determine whetherauto-Ag gene transfer to hepatocytes induces tolerance and control of T1D, NOD mice were treated with integrase-competent LVs (ICLVs) that selectively target the expression of InsB9–23 to hepatocytes. ICLV treatment inducedInsB9–23–specific effector T cells but also FoxP3+ regulatory T cells (Tregs), which halted islet immune cell infiltration,and protected from T1D. Moreover, ICLV treatment combined with a single suboptimal dose of anti-CD3 monoclo-nal antibody (mAb) is effective in T1D reversal. Splenocytes from LV.InsB9–23–treated mice, but not from LV.OVA(ovalbumin)–treated control mice, stopped diabetes development, demonstrating that protection is Ag-specific.Depletion of CD4+CD25+FoxP3+ T cells led to diabetes progression, indicating that Ag-specific FoxP3+ Tregs mediateprotection. Integrase-defective LVs (IDLVs).InsB9–23, which alleviate the concerns for insertional mutagenesis andsupport transient transgene expression in hepatocytes, were also efficient in protecting from T1D. These data dem-onstrate that hepatocyte-targeted auto-Ag gene expression prevents and resolves T1D and that stable integrationof the transgene is not required for this protection. Gene transfer to hepatocytes can be used to induce Ag-specifictolerance in autoimmune diseases.

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INTRODUCTION

Type 1 diabetes (T1D) is an autoimmune disease resulting in the com-plete destruction of insulin-producing pancreatic b cells (1, 2). In humanT1D and in the nonobese diabetic (NOD)mouse, the spontaneousmu-rinemodel of T1D, autoreactiveCD4+ andCD8+ effector T cells have beenshown to target islet-associated antigens (Ags), including glutamic aciddecarboxylase (GAD), islet-specific glucose 6-phosphotase catalyticsubunit–related protein (IGRP), chromogranin A, zinc transporter 8,and insulin (3–6). Effector T cells express high levels of interferon-g(IFN-g), perforin, and granzyme and kill target b cells (7, 8). In T1D,effector responses are predominant over tolerogenic responses, partlydue to defects in peripheral regulatory T cells (Tregs) of the host. Tregs

keep effector T cells and inflammatory responses in check via cytokine-and cell-to-cell contact–mediatedmechanisms (9). Therefore, a dysfunc-tion in Tregs allows effector T cells to prevail and autoaggression to ensue.Reduced frequency and function of CD4+ FoxP3+ Tregs with expansionof autoreactive effectorT cells have been reported inmouse andhumanswith T1D (10, 11). An attractive approach to restore peripheral toler-ance to islet Ags and prevent T1D onset is to induce and/or expandislet-specific Tregs that can control autoreactive effector T cells (12, 13).

In the past decade, encouraging results showed that Ag-based im-munotherapy could be used to restore tolerance in T1D. Several isletAgs including insulin, GAD, heat shock protein 60, and others have

1San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine,Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy.2Vita-Salute San Raffaele University, Milan 20132, Italy. 3Pathology Unit, Department ofOncology, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy. 4Department ofPediatrics, Stanford School of Medicine, Stanford, CA 94305, USA.*These authors contributed equally to this work.†These authors share senior authorship.‡Corresponding author. E-mail: mg1@stanford.edu

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been administered to prevent disease onset (14, 15). Despite promisingpreclinical data showing disease prevention, the Ag-based therapieshave largely failed to show efficacy in the clinic (16, 17). A definitiveexplanation for these failures in humans is lacking, but it appears thatthe immune response elicited by administration of the auto-Ag is notprotective. The type and robustness of the immune response inducedby auto-Ags depend on the site and context of Ag presentation to theT cells. The tolerogenic versus inflammatory properties of certainorgans make Ag delivery strategies a critical factor in designing effica-cious immunotherapies for the treatment of autoimmune diseases.

One promising approach to induce Ag-specific Tregs is the directdelivery of the Ag to the liver. The tolerogenic properties of the liverhave been extensively demonstrated (18–20). Constitutive productionof immunosuppressive cytokines such as interleukin-10 and transform-ing growth factor–b has been reported (21). In addition, Ag presentationby liver resident cells results in tolerogenic T cell responses (22–25).To explore the possibility of exploiting the liver to induce Tregs specificfor the immunodominant epitope of insulin [insulin B chain 9–23(InsB9–23)], which are capable of controlling T1D development, wedesigned a lentiviral vector (LV), which selectively targets expressionof the transgene to the hepatocytes. This LV includes two regulatoryelements in the expression cassette: hepatocyte-specific promoter [en-hanced transthyretin (ET)] for positive transcriptional regulation, andmicro-RNA142 target sequences (142T) for negative posttranscriptionalregulation in hematopoietic cell lineage (LV.ET.142T) (26, 27). Wepreviously showed that expression of transgenes encoded by this LVdesign in hepatocytes leads to the induction of an active state of immunetolerance mediated by FoxP3+ Tregs specific for a neo-Ag (28–30).

Here, we demonstrate that LV.ET.142T-mediated gene transfer sup-presses ongoing T1D autoimmune responses by expressing an earlyauto-Ag–derived epitope (InsB9–23) in hepatocytes. Reversal of overt

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T1D can be achieved by gene transfercombined with a single suboptimal doseof anti-CD3 monoclonal antibody (mAb)(31, 32). The treatment resulted in enrich-ment of FoxP3+ Tregs in the liver, pancre-atic lymph nodes (PLNs), and pancreaticislets. These InsB9–23–specific FoxP3

+ Tregssuppress T cell–mediated diabetogenicresponses against multiple auto-Ags, ar-resting T1D progression.

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RESULTS

Hepatocyte-targeted InsB9–23

expression blocks T1D progressionWeinjected systemically a series ofLV con-structs encoding for InsB9–23 in 10-week-old NOD mice. Glycemia was monitoredto determine the efficacy in controlling di-abetes progression. Integrase-competentLVs (ICLVs) carrying the ubiquitously ac-tive promoter [phosphoglycerate kinase 1(PGK)] to drive InsB9–23 expression (ICLV.PGK.InsB9–23) in all transduced cell typesfailed to block T1D development (Fig. 1A).An ICLV.PGKvector, which incorporatesmicro-RNA142 target sequences (142T)(ICLV.PGK.InsB9–23.142T) to negativelyregulate transgene expression in hemato-poietic lineage cells, was also unable tostop b cell destruction (Fig. 1B). To selec-tively target InsB9–23 expression to hepa-tocytes, we used an LV design in whichwe combined the ET hepatocyte-specificpromoter and 142T regulatory elements(ICLV.ET.InsB9–23.142T). Ninety per-cent of NOD mice injected with ICLV.ET.InsB9–23.142Twere protected from diseasedevelopment and remained normogly-cemic up to 43 weeks of age (mean bloodglucose levels: 135 ± 14 mg/dl) (Fig. 1C).NOD mice treated with control vectorsencoding for an unrelated Ag, such asovalbumin (OVA) (ICLV.PGK.OVA.142Tand ICLV.ET.OVA.142T), failed toprotect

NOD mice from T1D, although the hepatocyte-targeted OVA vectorinduced some delay in T1D development (Fig. 1, B and C). Overall,these results indicate that the expression of InsB9–23 must be stringentlytargeted to the liver parenchyma for maintenance of normoglycemiaand T1D protection.

Mononuclear cells infiltrating pancreatic islets are responsible for bcell death, and the extent of islet infiltration (insulitis) correlates withthe progression of T1D in NODmice (33). To assess the degree of insu-litis, we performed histological analysis of pancreatic tissue isolated fromcontrol NODmice (either untreated or treated with ICLV.ET.OVA.142T)and from ICLV.ET.InsB9–23.142T–treated NOD mice. Results indicatethat in 10-week-old NOD mice (at the time of LV administration),

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~25% of the islets were heavily infiltrated. The progression of the dis-ease was evident in 16-week-old, untreated NODmice, which displayed~80% of islets severely infiltrated. A comparable infiltration patternoccurred in 16-week-old NOD mice 6 weeks after ICLV.ET.OVA.142Ttreatment, confirming that OVA expression in hepatocytes does not haltb cell death. On the contrary, at 16 weeks of age, the ICLV.ET.InsB9–23.142T–treated mice displayed an insulitis profile similar to that of 10-week-old NOD mice: Only ~20% of islets were heavily infiltrated, where-as the remaining islets showed minimal CD3+ T cell infiltration andnormal insulin production. Comparable data were obtained in ICLV.InsB9–23.142T–treated NODmice at 43 weeks of age (Fig. 1, D and E).These results indicate that InsB9–23 ICLV–mediated expression in

Fig. 1. Liver-directed InsB9–23 expression protectsfromT1D. (A toC) Ten-week-oldNOD females at late pre-diabetic stage (blood glucose 100 ± 23 mg/dl, n = 36)were treated with a single dose of ICLV by systemic in-jection of (A) ICLV.PGK.InsB9–23 (n = 8), (B) ICLV.PGK.InsB9–23.142T (n = 8), ICLV.PGK.OVA.142T (n = 5), and (C)ICLV.ET.InsB9–23.142T (n=10; ****P<0.0001, Kaplan-Meierlog-rank test) and ICLV.ET.OVA.142T (n = 5) or left un-treated as control [n = 7, 10, and 14 for (A), (B), and (C),respectively]. Blood glucose levels were measured tomonitor T1D progression in NOD mice, which were con-sidered diabetic when the glucose levels were above

250 mg/dl. (D) Immunohistochemical analysis of pancreatic is-lets was performed on sections obtained from normoglycemicNOD untreated control mice (n = 4, 10-week-old mice, blood glu-cose 100 ± 23mg/dl, n= 100 islets; n= 6, 16-week-oldmice, bloodglucose 203 ± 15 mg/dl, n = 140 islets) and from NOD mice in-

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jected at 10 weeks of age with ICLV.ET.OVA.142T (n = 4 at 16 weeks of age, blood glucose 205 ± 32 mg/dl,n = 90 islets) or with ICLV.ET.InsB9–23.142T (n = 6 at 16weeks of age, blood glucose 119 ± 15mg/dl, n = 140islets; n = 6 at 43 weeks of age, blood glucose 135 ± 14mg/dl, n = 236 islets; ****P < 0.0001, c2 test versusICLV.ET.OVA.142T-treated at 16 weeks or versus untreated controls) to determine the level of lymphocyticinfiltration. Data are shown as percentage of islet with a given level of infiltration (no infiltration; peri-infiltrated, infiltrated less than 50%, and infiltrated more than 50% in the islet area). (E) Representativeimages of anti-CD3 and anti-insulin immunostaining are reported (scale bar, 50 mm).

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hepatocytes blocks infiltration by diabetogenic effector T cells andallows the maintenance of insulin production.

Hepatocyte-targeted InsB9–23 expression results in long-termtransgene expression despite the presence of Ag-specificeffector CD8+ T cellsTo investigate the effects of ICLV.ET.InsB9–23.142T administration onadaptive immunity in mice with an ongoing autoimmune response, we testedthe presence of InsB9–23–specific cytotoxic CD8

+ T lymphocytes (CTLs).The frequency of CTLs responsive to InsB was quantified in the spleen

and in the liver of ICLV.PGK.InsB9–23– and ICLV.PGK.InsB9–23.142T–treated NOD mice at 16 weeks (fig. S1) and ICLV.ET.InsB9–23.142T–treated NOD mice at 43 weeks of age (Fig. 2, A and B). As control,the InsB-specific response was tested in overt diabetic mice (glucose>300 mg/dl) and in 10-week-old normoglycemic untreated NOD mice.In parallel, OVA-specific responses were investigated in ICLV.ET.OVA.142T- or ICLV.ET.InsB9–23.142T–treated mice, and in overt dia-betic and normoglycemic untreated NOD mice (Fig. 2, A and B). Datashow that InsB9–23 gene transfer induced a significant increase in CD8+

T cells in the liver (Fig. 2A). These cells release IFN-g upon in vitrostimulation with a transgene-expressing cell line (Fig. 2, A and B) andkill InsB15–23–pulsed target cells as much as CTLs derived from diabeticmice (fig. S2), demonstrating their competence in exerting effectorfunctions. The insulin-specific response increased also in the spleenof ICLV.ET.InsB9–23.142T–treated NOD mice, and it was significantlyhigher in comparison to that observed in mice with overt disease or inuntreated mice. On the other hand, the response to OVA in ICLV.ET.InsB9–23.142T–treated NOD mice was comparable to that detected inT1D or untreated mice. In ICLV.ET.OVA.142T-treated mice, a strongCTL response to OVA was observed in the liver and in the spleen,whereas the response to InsB9–23 was very low.

Despite the induction of CTLs directed toward the InsB9–23 trans-gene, high LV genome copies [vector copy number (VCN)] were ob-served in the liver of ICLV.ET. InsB9–23.142T–treated NOD mice,suggesting that LV-transduced cells expressing InsB9–23 are protectedfrom the immune-mediated clearance. Similarly, in ICLV.ET.OVA.142T-treated mice, high VCN was observed in the liver despite astrong response to OVA by CTLs. On the contrary, the injection ofan equal dose of ICLV.PGK.InsB9–23 or ICLV.PGK.InsB9–23.142Tconstructs resulted in a very low VCN in the liver of treated NODmice, although integrated LV genomes were higher in the liver ofICLV.PGK.142T-treated NOD mice (Fig. 2C).

Together, these data show that gene therapy with ICLV.ET.142Tcontaining either InsB or OVA induces and/or boosts CTL responsestoward the transgene. However, transgene expression restricted to he-patocytes prevents clearance of transduced cells.

InsB9–23–specific Tregs are induced after treatmentwith ICLV.ET.InsB9–23.142TTo investigate if active tolerance plays a role in protecting b cells fromclearance mediated by CTLs, we evaluated the frequency of FoxP3+

Tregs in the spleen, liver, and PLNs of mice 6 weeks after ICLV admin-istration (Fig. 3A). The percentage of FoxP3+ Tregs in the PLN ofICLV.ET.InsB9–23.142T–treated mice was significantly higher as com-pared to that of ICLV.ET.OVA.142T-treated or untreated NOD mice(Fig. 3A). Higher frequency of FoxP3+ Tregs was also detected in thespleen of ICLV.ET.InsB9–23.142T–treated mice in comparison to untreatedNODmice (Fig. 3A). On the other hand, the frequency of FoxP3+ Tregs

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in the liver of both ICLV.ET.InsB9–23.142T– and ICLV.ET.OVA.142T–treated NOD mice was significantly increased compared to what weobserved in untreated control mice (Fig. 3A). These data indicate that

Fig. 2. Tolerance to transgene is achieved with liver-directed expres-sion despite induction of transgene-specific CTLs. (A and B) IFN-g–

secreting, InsB/OVA-specific CD8+ T cells in the liver (A) and in the spleen(B) were quantified by ELISPOT (enzyme-linked immunospot) assay in NODmice treated with ICLV.ET.InsB9–23.142T [n = 4 liver, n = 3 spleen; ***P < 0.001,analysis of variance (ANOVA)] and ICLV.ET.OVA.142T (n = 2 liver and spleen;***P < 0.001, ANOVA), or in untreated 10-week-old (n = 6 liver, n = 7 spleen)and fully diabetic (n = 3 liver and spleen, blood glucose >300 mg/dl) NODmice as control. Data are presented as means ± SEM of InsB/OVA-specificCD8+ T cells out of 106 total CD8+ T cells. (C) VCN per diploid genome inthe liver of NOD mice measured by quantitative polymerase chain reaction(**P < 0.01, ANOVA). VCN mean ± SEM is shown.

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ICLV.ET.142T administration expands FoxP3+ Treg population, sug-gesting induction of tolerance to the encoded Ag.

The preferential accumulation of FoxP3+ Tregs in the spleen, liver, andPLN of ICLV.ET.InsB9–23.142T–treated mice correlates with the geneexpression data showing an increased expression of Treg-associatedmarkers and immunoregulatory cytokines in CD4+ splenocytes, liver-infiltrating lymphocytes, and PLN cells (foxp3, ctla4, il10, and tgfb; fig.S3). This can be due to the presence of the relevant auto-Ag in PLNand spleen, in addition to its expression in the liver, whereas expres-sion of OVA is confined to the liver.

To determine whether FoxP3+ Tregs were also increased in the pan-creatic islets, FoxP3+-expressing cells were counted in the infiltratedareas of pancreatic islets obtained from mice treated with eitherICLV.ET.InsB9–23.142T or ICLV.ET.OVA.142T at 6 and 33 weeksafter LV treatment and untreated controls. Immunohistochemical analysisidentified FoxP3+ Tregs as a significantly increased population in theislets with <50 and >50% infiltration in ICLV.ET.InsB9–23.142T–treatedmice at both time points (Fig. 3, B and C).

To evaluate whether ICLV treatment selectively induces FoxP3+

Tregs, we measured the expression of T helper 1 (TH1)–, TH2-, andTH17-specific cytokines and transcription factors (ifn-g, tbet, il4, gata3, il17, rorc) in CD4+ splenic T cells and PLN cells. Results showedthat none of these T cell subsets were preferentially expanded byICLV.ET.InsB9–23.142T or ICLV.ET.OVA.142T treatment (fig. S4).

The suppressive activity of CD4+CD25+FoxP3+ Tregs isolated from16-week-old ICLV.ET.InsB9–23.142T–treated NOD mice was testedin vitro in coculture with CD4+ T effector cells isolated from diabeticmice. Tregs suppressed proliferation of effector T cells more efficientlyafter activation with InsB9–23 peptide than with IGRP195–214 peptide(Fig. 3, D and E).

Overall, the distribution of Tregs within the liver, PLN, and pancre-as after ICLV.ET.142T treatment, together with the ability of the liver

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Fig. 3. FoxP3-expressing Tregs are up-regulated in mice receiving liver-directed LV. (A) The frequency of FoxP3+ Tregs was determined by FACS

(fluorescence-activated cell sorting) in the spleen, liver, and PLN of NODmice at 6 weeks (representative FACS plots for FoxP3 staining in the PLNare shown) after injection with ICLV.ET.InsB9–23.142T (n = 8; **P < 0.01, ***P <0.001, ****P < 0.0001, ANOVA) and ICLV.ET.OVA.142T (n = 5; *P < 0.05, ****P <0.0001, ANOVA) or of untreated 10-week-old NOD mice (n = 5). (B) Immu-nohistochemical analysis of FoxP3+ cells in pancreatic islets was performedon sections obtained from 10-week-old normoglycemic NOD untreatedmice (n = 4, n = 26 islets) and from 16-week-old NOD mice injected at10 weeks of age with ICLV.ET.OVA.142T or with ICLV.ET.InsB9–23.142T (n =4, n = 21 islets; n = 6, n = 22 islets; ***P < 0.001, ANOVA) or at 43 weeksof age (n = 2, n = 16 islets; n = 6, n = 26 islets; ***P < 0.001, ANOVA). Thenumber of FoxP3+ cells was determined and data are reported as meanof FoxP3+ cell sorting for grade of infiltration ± SEM. (C) Representa-tive images are reported for 16-week-old untreated and 43-week-oldICLV.ET.InsB9–23.142T–treated NODmice (scale bar, 50 mm). Splenocytes, liver-infiltrating lymphocytes, and PLN cells were isolated from ICLV.ET.InsB9–23.142T–treated (n = 2, 16-week-old) NOD mice and pooled to magnetically sortCD4+CD25+ T cells, which are highly enriched in FoxP3+ Tregs. CD4

+CD25−

responder T cells were isolated and pooled from diabetic NOD mice(TT1D) (n = 2). (D and E) The capacity of enriched FoxP3+ Tregs to suppressT cell responses was tested after polyclonal stimulation (anti-CD3 mAb platebound, 10 mg/ml) (D) or after Ag-specific stimuli (InsB9–23 or IGRP195–214 pep-tides, 10 mg/ml) (E). Proliferation of TT1D cells was quantified by [3H]thymidineincorporation. Percentage of suppression is shown. Mean CPM × 10−3 ± SEMfor each experimental condition is reported.

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to generate transgene-specific Tregs (28), indicates that InsB9–23–specificTregs are induced in the liver and accumulate in the peripheral ana-tomical sites where the cognate Ag is presented to T cells.

Active protection from T1D induced by ICLV.ET.InsB9–23.142Ttreatment is Treg-dependentTo determine whether protection from T1D is due to an activemechanism of Ag-specific immune regulation mediated by FoxP3+

Tregs, splenocytes isolated from 16-week-old ICLV.ET.InsB9–23.142T–or ICLV.ET.OVA.142T–treated NOD mice were cotransferred withsplenocytes isolated from diabetic NOD mice in NOD.scidmice. Signif-icant protection from T1D was observed in the group cotransferredwith splenocytes from ICLV.ET.InsB9–23.142T–treated mice (Fig. 4A).On the other hand, T1D onset occurred at a similar rate in NOD.scidmice repopulated with splenocytes from diabetic mice alone or cotrans-ferred with splenocytes from ICLV.ET.OVA.142T-treated NODmice. Theprotection mediated by splenocytes isolated from ICLV.ET.InsB9–23.142T–treated NOD mice was completely abrogated when CD4+CD25+ cellswere depleted from the splenocytes before the cotransfer (Fig. 4B).The depletion procedure removed 75% of CD4+CD25+FoxP3+ spleno-cytes (fig. S5).

To define whether Ag-specific Tregs induced by ICLV.ET.InsB9–23.142Ttreatment also exert bystander suppression toward other b cell–specificAgs, we evaluated the IGRP206–214–specific CD8+ T cell response indiabetic and age-matched ICLV.ET.InsB9–23.142T–treated NOD mice.Results indicate that ICLV.ET.InsB9–23.142T treatment also reducedIGRP206–214–specific CD8+ T cell response (fig. S6). Overall, theseresults demonstrate that the suppression of diabetogenic responsesby ICLV.ET.InsB9–23.142T treatment is mediated by Ag-specificCD25+FoxP3+ Tregs, which also exert bystander suppression.

Integrase-defective LV InsB9–23 gene transfer protectsfrom T1DThe use of LVs as a vaccine-based platform to generate immune tol-erance may raise concerns due to the risks of insertional mutagenesis,although extensive preclinical and clinical studies support the safety ofLV-mediated gene transfer (34–37). Moreover, long-term transgeneexpression may not be required to maintain immune tolerance, in par-ticular to auto-Ags, which are present in the organism. We previouslydemonstrated that the intravenous administration of an integrase-defective LVs (IDLVs) encoding for green fluorescent protein (GFP),IDLV.ET.GFP.142T, led to transient expression of GFP in hepatocyteswhile retaining the ability to induce a robust state of Ag-specifictolerance (29).

To determine whether liver-directed, IDLV-mediated InsB9–23 genetransfer arrests T1D, 10-week-old NOD mice were treated with IDLV.ET.InsB9–23.142T. Blood glucose levels were monitored for 15 weeksafter IDLV.ET.InsB9–23.142T treatment, revealing that transient expres-sion of InsB9–23 by hepatocytes suppresses T1D development in 80% oftreatedNODmice (mean blood glucose: 127 ± 49mg/dl) (Fig. 5A). His-tological analysis of the pancreata revealed that IDLV.ET.InsB9–23.142Ttreatment inhibited the progressive infiltration of the pancreatic islets(Fig. 5B).On the contrary, in IDLV.ET.OVA.142T-treatedmice, diabetesoccurred as in untreated control mice. VCN analysis confirmed absentto very low IDLV genomes in the liver of treated mice at the end of ex-periment (fig. S7C). Similar to what was observed after ICLV treatment,transgene-specific effector cells were detected in the spleen and liverafter IDLV.ET.142T gene transfer (fig. S7, A and B), and the frequency

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of FoxP3+Tregs was increased in the PLN and in pancreatic islets only inmice treated with IDLV.ET.InsB9–23.142T (Fig. 6A and figs. S8 and S9).Cotransfer of splenocytes isolated from IDLV.ET.InsB9–23.142T–treatedmice with splenocytes from diabetic NOD mice demonstrated thatIDLV.ET.InsB9–23.142T treatment induces Ag-specific active immuno-regulation of diabetogenic T cells (Fig. 6B). Moreover, depletion ofCD4+CD25+ splenocytes before cotransfer demonstrated that Ag-specific active immunoregulation was dependent on the activity ofCD4+CD25+FoxP3+ Tregs (Fig. 6C), as shown for ICLV treatment.

Fig. 4. T1D protection in InsB9–23 mice is Treg-dependent. (A) Spleno-cytes isolated from diabetic NOD mice were cotransferred into NOD.scid

mice at 1:1 ratio with splenocytes isolated from ICLV.ET.InsB9–23.142T–treated (n = 6, 16-week-old; *P < 0.05, **P < 0.01, Kaplan-Meier log-ranktest) and ICLV.ET.OVA.142T-treated (n = 5, 16-week-old) NOD mice ortransferred alone. (B) To evaluate the role of FoxP3+ Tregs, which are mainly in-cluded in the CD4+CD25+ T lymphocytes, splenocytes isolated from diabeticNOD mice were cotransferred into NOD.scidmice at 1:1 ratio with CD4+CD25+-depleted splenocytes isolated from ICLV.ET.InsB9–23.142T–treated (n = 6,16-week-old) or ICLV.ET.OVA.142T-treated (n = 5, 16-week-old) NOD mice.Blood glucose levels were measured to monitor T1D transfer in NOD.scidmice. Diabetes incidence is shown.

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Hepatocyte-targeted InsB9–23 expression combined withsuboptimal dose of anti-CD3 reverses T1DThe efficacy of hepatocyte-directed InsB9–23 gene transfer in control-ling T1D was also tested at later stages of the disease progression.ICLV.ET.InsB9–23.142T treatment administered in NOD mice at theend of the presymptomatic phase when glycemic levels range from200 to 250 mg/dl protected 27% of the mice (fig. S10A). We also testedICLV.ET.InsB9–23.142T treatment in diabetic NOD mice with bloodglucose levels ranging from 250 to 300 mg/dl; none of the treated micewere cured with reversal to normoglycemic levels (fig. S10B). We next

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combined InsB9–23 gene transfer with anti-CD3 mAb treatment. Treat-ment with optimal doses of anti-CD3 mAb alone can reverse T1D inNOD mice (31, 32). Therefore, to identify the suboptimal dose of anti-CD3 mAb with no protective effect, decreasing doses of anti-CD3 mAbwere tested in diabetic NOD mice with blood glucose levels rangingfrom 250 to 300 mg/dl. We observed that a single administration ofanti-CD3 mAb at 5 mg per mouse instead of 10 mg per mouse was noteffective in protecting from T1D (fig. S11).

On the basis of these results, we treated NOD mice with bloodglucose level ranging from 250 to 300 mg/dl with this suboptimal doseof anti-CD3 mAb (1 × 5 mg) together with ICLV.ET.InsB9–23.142T.This treatment achieved 75% T1D reversal (9 of 12 mice tested; Fig. 7).In 4 of the reverted mice, we observed temporary spikes of blood glu-cose levels, which returned below the 250 mg/dl threshold, indicatingthat a significant portion of b cell mass was still present and functional.

These data indicate that treatment with a suboptimal dose of anti-CD3 mAb is sufficient to reestablish a permissive condition for toler-ance induction mediated by hepatocyte-targeted gene transfer.

Overall, these results demonstrate that the ectopic expression ofauto-Ags in hepatocytes generates Ag-specific CTL but also Tregs,which actively suppress the already ongoing diabetogenic responsesin presymptomatic phase and reverse T1D, in combination with a sub-optimal dose of anti-CD3 mAb. The comparable efficacy of the ICLVand IDLV platforms demonstrates that the stable persistence of trans-gene expression is not required to achieve long-lasting Ag-specific im-mune tolerance.

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DISCUSSION

A growing body of evidence indicates that the autoimmune attackunderlying b cell destruction does not lead to clinical symptoms ofT1D for many years in most patients. At clinical onset, T1D patientshave already lost more than 80% of b cell mass, and therefore, an ef-fective therapy should specifically suppress ongoing diabetogenic re-sponses while preserving the remaining insulin-producing b cells.

Here, we show that LV-based gene transfer targeting auto-Ag ex-pression to hepatocytes protects NOD mice from T1D. Systemic ad-ministration of a single dose of ICLV.ET.InsB9–23.142T arrested b celldestruction in NOD at advanced prediabetic stage, “freezing” islet in-filtration at the stage observed at the time of treatment, and maintain-ing insulin independence in 90% of LV-treated NOD mice. TargetingInsB9–23 expression to hepatocytes was required to generate InsB9–23–specific FoxP3+ Tregs, which allowed the maintenance of stable normo-glycemia, suppressing diabetogenic T cell responses at the site ofcognate Ag presentation.

We previously showed that hepatocyte-directed gene therapy byICLVs encoding for factor IX (FIX) eradicated preexisting anti-FIX–neutralizing response, and provided therapeutic levels of the missingclotting factor in a mouse model of hemophilia B (FIX-deficient), re-solving the disease (30). Here, we developed a hepatocyte-directed LVgene transfer strategy, which exploits naturally occurring hepatic tol-erogenic pathways to suppress immune responses to auto-Ags. Wedemonstrate that hepatocyte-directed ICLV-mediated auto-Ag (InsB9–23)gene transfer efficiently suppresses ongoing autoimmune responsesagainst a variety of self-Ags expressed by pancreatic b cells, arrestingT1D development. Gene transfer using IDLVs led to comparable re-sults, indicating that persistent high levels of InsB9–23 expression in

Fig. 5. Integrase-defective InsB9–23 LV treatment protects from T1D.(A) Ten-week-old NOD females at late prediabetic stage were treated with

single dose of IDLVs by systemic injection of IDLV.ET.InsB9–23.142T (n = 6; *P <0.05, Kaplan-Meier log-rank test) and IDLV.ET.OVA.142T (n = 4) or left un-treated as control (n = 15). Blood glucose levels were measured to monitorT1D progression. Diabetes incidence is shown. (B) Immunohistochemicalanalysis of pancreatic islets was performed on sections obtained from nor-moglycemic NOD untreated control mice (n = 4, 10-week-old mice, n = 63islets; n = 4 16-week-old mice, n = 62 islets) and from NOD mice injected at10 weeks of age with IDLV.ET.OVA.142T (n = 4, 16-week-old mice, bloodglucose 202 ± 18 mg/dl, n = 49 islets) or with IDLV.ET.InsB9–23.142T (n =5, 16 weeks of age, glucose 144 ± 19 mg/dl, n = 85 islets; n = 4, 35 weeks ofage, blood glucose 138 ± 23 mg/dl, n = 119 islets) to determine the level oflymphocytic infiltration. Data are shown as percentage of islets with a givenlevel of infiltration (no infiltration; peri-infiltration, infiltration less than 50%,and infiltration more than 50% of the islet area).

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hepatocytes are not required for induction of tolerance to self-Ags andlong-term maintenance of insulin production. Moreover, hepatocyte-directed gene transfer resulted in a significant reversal of T1D whencombined with a suboptimal, per se ineffective, dose of anti-CD3 mAb.

Several gene transfer–based approaches to prevent/treat T1D havebeen investigated (38–44). Administration of AAV (39, 40) or mRNA(41) encoding for cytokines resulted in prevention of T1D. In addition,antisense oligonucleotides have been used ex vivo to down-regulateexpression of costimulatory molecules in DC, which control autoimmu-nity once reinfused in vivo (45). All these approaches are non–Ag-specific and therefore may lead to general immune suppression.

Ag-specific immunoregulatory strategies based on ectopic expres-sion of an auto-Ag–derived fragment in certain tissue (that is, muscle,liver) have been explored (38, 46). Han et al. (38) showed that AAV-mediated GAD65500–585 gene transfer to the muscle of NOD mice re-sulted in prevention of T1D in 80% of mice treated at 7 weeks of agewhen the autoimmune disease is at the very early stage. Lüth et al. (46)showed that myelin basic protein expression in the liver protects fromautoimmune neuroinflammation in a mouse model of multiple sclerosis.

Ag-specific prevention of diabetogenic responses has been shownusing plasmid DNA encoding for auto-Ags (41–43). Recently, pro-insulingene transfer by plasmid DNA administration has been evaluated in pa-tients, showing safety and an increase in residual C-peptide associatedwith a decline of pro-insulin–reactive CD8+ T cells (44). However, inthis study, an active state of tolerance, which could ensure stable pro-tection from b cell destruction, was not demonstrated.

Multiple routes of administration of insulin or its InsB9–23 immu-nodominant epitope have been used to prevent T1D (47–49). Howev-er, insulin-based gene delivery in NOD mice has shown controversialresults with variable levels of protection (41, 42, 50, 51). A commonfeature among the gene delivery systems showing at least partial pro-tection from T1D is the ectopic expression of auto-Ag, leading to therelease of immunoregulatory cytokines by T cells and/or the inductionof Tregs specific for the encoded Ag (52).

Expression of an auto-Ag in cells of NOD mice is a particular con-cern because activation of InsB9–23–specific effector T cells could ex-acerbate b cell autoimmunity, in addition to promoting clearance oftransgene-expressing cells. Treatment of mice with LV carrying onlythe ubiquitous PGK promoter, which permits the expression of theInsB9–23 transgene in all cell types including Ag-presenting cells (APCs),led to induction of effector CTLs, clearance of transduced cells, and

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development of T1D. Although the ICLV.PGK.InsB9–23 induced a po-tent immune response, it did not accelerate disease, suggesting that theboost of effector T cell response to insulin induced by this LV con-struct did not have an additive effect on the already ongoing robust

Fig. 6. IDLVs induce Ag-specific Tregs capable of controlling T1D. (A)The frequency of FoxP3+ Tregs was determined by FACS in spleen, liver, and

PLN 15 weeks after treatment of IDLV.ET.InsB9–23.142T (n = 5; **P < 0.01,***P < 0.001, ANOVA) and IDLV.ET.OVA.142T (n = 3; *P < 0.05, **P < 0.01,ANOVA). Untreated 10-week-old NOD mice (n = 3) were used as controlgroup. Data are presented as means ± SEM. (B) Splenocytes isolated fromdiabetic NODmicewere cotransferred into NOD.scidmice at 1:1 ratio withsplenocytes isolated from IDLV.ET.InsB9–23.142T–treated (n = 4, 16-week-old, **P < 0.01, Kaplan-Meier log-rank test) and IDLV.ET.OVA.142T-treated(n = 4, 16-week-old) mice or transferred alone (n = 4). (C) To evaluate therole of FoxP3+ Tregs, which are mainly included in the CD4+CD25+ T lympho-cytes, splenocytes isolated from diabetic NOD mice were cotransferred intoNOD.scid mice at 1:1 ratio with CD4+CD25+-depleted splenocytes isolatedfrom IDLV.ET.InsB9–23.142T–treated (n = 6, 16-week-old) or IDLV.ET.OVA.142T–treated (n= 4, 16-week-old) NODmice. Blood glucose levels weremeasuredto monitor T1D transfer in NOD.scid mice. Diabetes incidence is shown.

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autoimmune responses. The insertion of microRNA-142 target sequencesinto the vector, to abrogate transgene expression in professional APCsand other hematopoietic lineage cells (27, 28), was not sufficient toconfer immunomodulatory properties to the LV construct, which failedto prevent T1D, and it induced transgene-specific CTLs, albeit at areduced level compared to ICLV.PGK.InsB9–23. The immune responseobserved after ICLV.PGK.InsB9–23.142T administration is likely the re-sult of the PGK promoter, which expresses the transgene at higher levelsthan ET promoter in APCs, thus giving rise to some residual trans-gene expression even if subjected to regulation by microRNA-142 andtriggering an immune response to the encoded Ag (26). Transgene ex-pression in APCs, even at very low levels, can inhibit tolerance induc-tion in NOD mice, in which APCs have enhanced immune functionsdue to hyperactivation of the nuclear factor kB pathway (53).

Replacement of PGK with the hepatocyte-specific ET promoterselectively targeted transgene expression to hepatocytes and dimin-ished the immunogenicity of the LV construct (54, 55). In LV.ET.142T-treated mice, the expression of the transgene is strictly limited to liverparenchyma, and therefore, the priming of effector CTLs is likelymediated directly by the transduced hepatocytes, which can provideonly partial costimulation to major histocompatibility complex class IAg presentation, resulting in suboptimal priming of T cells (56). Indeed,a significant reduction in the absolute number of Ag-specific effectorT cells was observed after ICLV.ET.InsB9–23.142T treatment, comparedto those observed in ICLV.PGK.InsB9–23 and ICLV.PGK.InsB9–23.142T–treated mice.

FoxP3+ Tregs play a key role in protection from T1D development,because they suppress pathogenic effector T cells (9, 57). Indeed, theinduction or adoptive transfer of Tregs has been shown efficacious inthe control of ongoing b cell autoimmunity in NOD mice (58–60).Whether Ag specificity is required for Tregs to prevent T1D remains

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an open debate. Here, we show that expression of an early auto-Ag–derived epitope (InsB9–23) in hepatocytes induces Ag-specific Tregs,which mediate robust long-lasting tolerance. Moreover, our study doesnot suggest any regulatory role of InsB-specific CD8+ T cells, as shownby Santamaria’s group (61). LV.ET.InsB9–23.142T gene transfer led toa preferential expansion of Tregs in the liver, and accumulation in thePLN and endocrine pancreas, where the relevant Ag is present. On thecontrary, LV.ET.OVA.142T-treated mice showed no significant varia-tion in the percentage of Tregs in the pancreas and in the PLN, whereasTregs were expanded in the liver. These data indicate that InsB9–23–specific Tregs accumulated at the site of cognate Ag presentation, whereasOVA-specific Tregs were not attracted to the PLN and pancreas ofNOD mice because of lack of the cognate Ag at these sites.

Once Tregs, induced in the liver by LV.ET.InsB9–23.142T therapy,are present at the site(s) where the auto-Ag is presented, they can con-trol autoreactive responses mediated by the same as well as other auto-Ags. Autoimmunity is well established in NOD mice at 10 weeks ofage (62), and epitope spreading is already under way at this time (63).Therefore, we hypothesize that the InsB9–23–specific Tregs, which hometo the site of Ag expression, regulate autoimmune responses directedtoward the same Ag but also different auto-Ags by bystander non–Ag-specific suppression or by inducing in situ and de novo generationof Tregs.

The mechanism underlying the induction of Tregs in the liver is stillunder investigation. Effector T cells themselves may play a crucial rolein the expansion of Tregs. Grinberg-Bleyer et al. (64) showed that ef-fector T cells can boost the number of Tregs, enhancing their regulatoryactivity in the PLN and pancreas. The induction of transgene-specificeffector T cells in the liver after LV.ET.142T administration may there-fore determine the frequency of Tregs. It is possible that this regulatoryfeedback mechanism aimed at protecting InsB9–23–expressing hepato-cytes from a massive immune-mediated clearance may also lead tothe protection of b cells from diabetogenic responses. On the other hand,the expression of the transgene product in hepatocytes may lead tosuboptimal T cell priming, which favors tolerogenic presentation toCD4+ T cells and Treg induction.

The clinical application of ICLVs for hepatocyte-targeted Ag expres-sion to induce immune modulation may be limited by the concernsassociated with integration of the vector into the hosts’ cell genome.IDLVs are emerging as an attractive platform for transgene expression[reviewed in (65)]. IDLVs offer the possibility to express transgenes fora window of time in hepatocytes while sharply reducing the integra-tion into the genome. The nonintegrating feature of the IDLV plat-form provides important safety advantages due to the negligiblegenotoxic risk and the reversibility of transgene expression. Althoughthe levels of expression achieved with IDLVs are lower compared tothose generated by ICLVs, we showed that tolerance to FIX persistedeven when the transgene expression became barely detectable (29).Here, the IDLV platform was efficient as its integrating counterpartin controlling T1D, generating Ag-specific tolerance through the in-duction of InsB9–23-specific Tregs, and driving migration of Tregs to dis-tant anatomical locations to control immune responses.

In conclusion, our study demonstrated that a single LV treatment(LV.ET.InsB9–23.142T) in NOD mice in advanced prediabetes statesuppresses immune responses to b cell auto-Ags, saves islet b cell mass,and induces long-lasting immune tolerance. Moreover, the IDLV-based therapy showed the same tolerogenic properties as the ICLVplatform, giving the advantage of avoiding any permanent genetic

Fig. 7. ICLV.ET.InsB9–23.142T combined with a suboptimal dose of anti-CD3 reverts T1D. Diabetic NOD mice with blood glucose levels ranging

from 250 to 300 mg/dl (gray area) were treated with ICLV.ET.InsB9–23.142Tcombined with anti-CD3 mAb (1 × 5 mg) (n = 12). Blood glucose levelsof each single mouse are reported starting from the time of treatment(color lines). Mice were considered as reverted when the glucose levelsreturned below 250 mg/dl. Red dashed lines identify NOD mice with bloodglucose levels >600 mg/dl, which is consistent with the absence of insulin-secreting b cells.

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changes of target cells. Large-scale Good Manufacturing Practices (GMP)manufacturing of LVs is now well established, and their clinical testingfor the gene therapy of other diseases has been proven safe and success-ful until now, thus paving the way to broader applications (36, 37).

Multiple genetic and immunological factors, which have been iden-tified as predictive biomarkers for T1D (66–70), allow the selection ofhigh-risk patients, in whom intervention when autologous b cell massis still sufficient can result in maintenance of insulin independence.For these subjects, hepatocyte-targeted gene transfer may representan effective personalized therapeutic approach, although not withoutethical considerations. The efficacy of LV gene transfer, combined witha suboptimal dose of anti-CD3 mAb, to revert T1D and induce Ag-specific tolerance significantly increases the feasibility of its clinicalapplication.

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MATERIALS AND METHODS

Study designTo study the efficacy of hepatocyte-targeted InsB9–23 expression toarrest and cure T1D, NODmice were injected intravenously with ICLVor IDLV encoding for InsB9–23. Mice were treated either at 10 weeks ofage to arrest T1D progression or when blood glucose levels ranged from250 to 300 mg/dl to revert hyperglycemia and cure the disease. Exper-imental groups were dimensioned to allow statistical analysis. Mice wererandomly assigned to each group, but the experimenter was not blindedto group identity.

MiceFemale NOD (NOD/LtJ) and severe combined immunodeficient NOD(NOD.scid) mice were purchased (Charles River Laboratories) andhoused in specific pathogen–free conditions. Mice were considered dia-betic when blood glucose measurements were≥250 mg/dl on two succes-sive days as determined by a Bayer BREEZE Blood Glucose MonitoringSystem (Bayer). All procedures were reviewed and approved by theInstitutional Animal Care and Use Committee (IACUC) at San RaffaeleInstitute, Milan (IUCAC 416 and 604). All treated mice were admin-istered LV by intravenous tail vein injection (TU per mouse ranging from5 × 108 to 10 × 108). Anti-CD3e [2-C11 F(ab′)2] from BioExpress wasadministrated intravenously at the indicated doses.

Adoptive cotransfersSplenocytes prepared from diabetic NOD donors (2.5 × 106 or 5 ×106) were injected intraperitoneally into 8-week-old NOD.scid micewith the indicated population at 1:1 ratio. CD4+CD25+ depletionwas performed by negative selection of CD4, followed by positive se-lection of CD25+ cells. CD4− splenocytes were then combined withthe CD4+CD25− fraction to finally reinfuse splenocytes CD4+CD25+

depleted.

Statistical analysesStatistical analyses were performed using GraphPad Prism software.The incidence of diabetes was compared by Kaplan-Meier log-ranktest. c2 test was used to compare levels of infiltration between exper-imental groups. Two-way ANOVA followed with a Bonferroni post-test was used to determine statistical differences between multipleexperimental groups. Findings were considered significant with valuesfor P ≤ 0.05.

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/289/289ra81/DC1Materials and MethodsFig. S1. InsB9–23–specific CTL response is induced after ICLV.PGK and ICLV.PGK.142T treatment.Fig. S2. InsB15–23–specific CD8+ T cells are functional in ICLV.ET.InsB9–23.142T–treated NODmice.Fig. S3. Genes related to immune regulation and FoxP3+ Tregs are up-regulated after ICLV treatment.Fig. S4. TH1, TH2, and TH17 T cell subsets are not expanded by ICLV.ET.InsB9–23.142T treatment.Fig. S5. Depletion of FoxP3+ Tregs from total splenocytes.Fig. S6. CD8+ T cell response to IGRP is reduced in ICLV.ET.InsB9–23.142T–treated NOD mice.Fig. S7. Transgene-specific CTL response is induced after IDLV treatment.Fig. S8. Frequency of FoxP3+ Tregs is increased in pancreatic islet infiltration after IDLV treatment.Fig. S9. IDLV treatment induces expression of FoxP3+ Treg–related genes.Fig. S10. Efficacy of ICLV.ET.InsB9–23.142T treatment is reduced in hyperglycemic NOD mice,and it is abrogated in overt disease.Fig. S11. Definition of the suboptimal and noneffective dose of anti-CD3 mAb.Supporting material to Fig. 3AData tablesReference (71)

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Acknowledgments: We thank S. Gregori [San Raffaele Telethon Institute for Gene Therapy(HSR-Tiget)] and A. Valle and M. Battaglia (San Raffaele Diabetes Research Institute) for helpfulscientific discussions, A. Innocenzi and A. Vino (Pathology Unit, Department of Oncology,IRCCS San Raffaele Scientific Institute) for technical assistance, and A. Nonis for statisticalconsulting (CUSSB, University Center for Statistics in the Biomedical Sciences). Funding:Supported by Fondazione Telethon (TIGET E1 to M.G.R. and D3 to L.N.), Italian Ministry ofHealth (RF-2009-1501881 to M.G.R.), and JDRF innovative grant (JDRF 47-2013-575 to M.G.R.and A.A.). Author contributions: M.A. and K.S.G. designed and performed experiments, ana-lyzed data, and contributed to the writing of the first version of the paper. A.C. performedexperiments and analyzed data. F.R. performed experiments. F.S. supervised histopathologicalstudies. L.N. coordinated the work and revised the paper. A.A. designed experiments, analyzeddata, supervised the work, and wrote the paper. M.G.R. analyzed data, supervised the work,and wrote the paper. Competing interests: The authors declare that they have no competingfinancial interests. A.A., A.C., L.N., and M.G.R. are inventors on a patent application owned byFondazione Telethon and San Raffaele Hospital describing the tolerogenic IDLV.ET.142T.

Submitted 14 November 2014Accepted 6 April 2015Published 27 May 201510.1126/scitranslmed.aaa3032

Citation: M. Akbarpour, K. S. Goudy, A. Cantore, F. Russo, F. Sanvito, L. Naldini, A. Annoni,M. G. Roncarolo, Insulin B chain 9–23 gene transfer to hepatocytes protects from type 1diabetes by inducing Ag-specific FoxP3+ Tregs. Sci. Transl. Med. 7, 289ra81 (2015).

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nceTranslationalMedicine.org 27 May 2015 Vol 7 Issue 289 289ra81 11

on February 18, 2016em

ag.org/

10.1126/scitranslmed.aaa3032] (289), 289ra81. [doi:7Science Translational Medicine 

Roncarolo (May 27, 2015) Francesca Sanvito, Luigi Naldini, Andrea Annoni and Maria Grazia Mahzad Akbarpour, Kevin S. Goudy, Alessio Cantore, Fabio Russo,

regs T+type 1 diabetes by inducing Ag-specific FoxP323 gene transfer to hepatocytes protects from−Insulin B chain 9

 Editor's Summary

   

antigen-specific tolerance in autoimmune disease.progression in these mice. These data suggest that expressing autoantigen in liver cells may induce therapy was combined with a single dose of anti-CD3 monoclonal antibody, it stopped diseasea control antigen, and halted immune cell infiltration into the pancreatic islet. Moreover, when this in a mouse model of type 1 diabetes. This therapy induced regulatory T cell specific for insulin, but nothelp protect against type 1 diabetes.The authors used a lentiviral vector to express insulin in liver cells

suggest that gene transfer mayet al.ranging from monogenetic diseases to cancer. Now, Akbarpour Gene therapy is being used with increasing success to treat a rapidly growing group of diseases

Gene therapy for diabetes

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