1521-0103/349/1/107–117$25.00 http://dx.doi.org/10.1124/jpet.113.209346THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 349:107–117, April 2014Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics

The Antiangiogenic Insulin Receptor Substrate-1 AntisenseOligonucleotide Aganirsen Impairs AU-Rich mRNA Stability byReducing 14-3-3b–Tristetraprolin Protein Complex, ReducingInflammation and Psoriatic Lesion Size in Patients s

Sylvie Colin, Bernadette Darné, Amin Kadi, Antoine Ferry, Maryline Favier, Corinne Lesaffre,Jean-Pascal Conduzorgues, Salman Al-Mahmood, and Nejib DossGene Signal SAS, Evry, France (S.C., A.F., S.A.-M.); Monitoring Force Group, Maisons-Laffitte, France (B.D., A.K.); InstitutCochin, Institut National de la Santé et de la Recherche Médicale U1016, CNRS UMR8104, Morphology and Histology Platformand Université Paris Descartes, Paris, France (M.F., C.L.); AMATSI, St. Gely du Fesc, France (J.-P.C.); and Université de Tunis ElManar, Faculté de Médecine de Tunis, Military Hospital of Tunis, Department of Dermatology, Tunis, Tunisia (N.D.)

Received September 5, 2013; accepted February 6, 2014

ABSTRACTIncreased inflammation and aberrant angiogenesis underliepsoriasis. Here, we report that the inhibition of insulin receptorsubstrate-1 (IRS-1) expression with aganirsen resulted in adose-dependent reduction (P , 0.0001) in IRS-1 protein in thecytoplasm, while IRS-1 protein remained quantitatively un-changed in the perinuclear environment. Aganirsen induceda dose-dependent increase in serine-phosphorylated IRS-1 inthe soluble perinuclear-nuclear fraction, inducing IRS-1–14-3-3bprotein association (P , 0.001), thereby impairing 14-3-3b–tristetraprolin protein complex and AU-rich mRNA’s stability(P , 0.001). Accordingly, aganirsen inhibited (P , 0.001) invitro the expression of interleukin-8 (IL-8), IL-12, IL-22, andtumor necrosis factor alpha (TNFa), four inflammatory media-tors containing mRNA with AU-rich regions. To demonstratethe clinical relevance of this pathway, we tested the efficacy ofaganirsen by topical application in a pilot, double-blind, randomized,

dose-ranging study in 12 psoriatic human patients. After 6 weeks oftreatment, least square mean differences with placebo were238.9% (95% confidence interval,275.8 to22.0%) and237.4%(274.3 to20.5%) at the doses of 0.86 and 1.72mg/g, respectively.Lesion size reduction was associated with reduced expressionof IRS-1 (P, 0.01), TNFa (P, 0.0001), and vascular endothelialgrowth factor (P , 0.01); reduced keratinocyte proliferation(P , 0.01); and the restoration (P , 0.02) of normal levels ofinfiltrating CD41 and CD31 lymphocytes in psoriatic skinlesions. These results suggest that aganirsen is a first-in-class ofa new generation of antiangiogenic medicines combining anti-inflammatory activities. Aganirsen-induced downregulation ofinflammatory mediators characterized by AU-rich mRNA likelyunderlies its beneficial clinical outcome in psoriasis. These resultsjustify further large-scale clinical studies to establish the dose ofaganirsen and its long-term efficacy in psoriasis.

IntroductionPsoriasis is a common chronic disease occurring in ∼2% of

the population inWestern countries (Sabat et al., 2007; Nestleet al., 2009). Extensive work has established that increasedinflammation, aberrant angiogenesis, and vascular remodel-ing as well as keratinocyte hyperproliferation are involved inthe pathogenesis of psoriasis (Heidenreich et al., 2009).

T cell–mediated immune response to an as-yet-unknownautoantigen seems to play a key role in the pathogenesis ofpsoriasis. This deregulated immune response is primarilydriven by CD41 T cells with a T(h)1 and/or T(h)17 phenotype(Nograles et al., 2008; Nestle et al., 2009; Coimbra et al., 2012)and by the production of tumor necrosis factor alpha (TNFa)(Orlinick and Chao, 1998; Caldarola et al., 2009). A significantincrease in the development of the microvasculature (angio-genesis) has been observed in psoriatic lesions compared withhealthy skin (Heidenreich et al., 2009), especially in the earlyphase of the development of the lesions (Henno et al., 2010).Vascular endothelial growth factor (VEGF), which has a centralrole in angiogenesis, is upregulated in psoriasis (Detmar et al.,1994), and its levels of expression represent a good indicator ofactive psoriatic arthritis (Heidenreich et al., 2009).

This study was supported by Gene Signal SAS.J.-P.C. is a consultant in product formulation for Gene Signal. S.C. and S.A.-M.

are cofounders of Gene Signal, have stock ownership, and are employees of GeneSignal. A.F. possesses stock ownership of Gene Signal. B.D., A.K., M.F., C.L.,and N.D. declare no conflicts of interest.

S.C., B.D., S.A.-M., and N.D. contributed equally to this work.dx.doi.org/10.1124/jpet.113.209346.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: CK-16, cytokeratin 16; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;hEC, human endothelial cell; HRP, horseradish peroxidase; IL, interleukin; IRS-1, insulin receptor substrate-1; mAb, monoclonal antibody; PBS,phosphate-buffered saline; PCR, polymerase chain reaction; PGA, Physician’s Global Assessment; pIRS-1, phosphorylated insulin receptorsubstrate-1; PVDF, polyvinylidene difluoride; RT-PCR, reverse-transcription polymerase chain reaction; SPN, soluble perinuclear-nuclear; TNFa,tumor necrosis factor alpha; TSS, Total Sign Score; TTP, tristetraprolin protein; VEGF, vascular endothelial growth factor.

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Conventional systemic therapies for psoriasis, such asmethotrexate, cyclosporin A, retinoids, or psoralen and UV Atherapies, can result in long-term toxicity and may not beeffective. The development of novel therapies targeting in-flammatory factors including TNFa, interleukin-12 (IL-12),and IL-22 now provide new and efficient treatment options(Weger, 2010). Nonetheless, because both angiogenesis andinflammation are involved in the development of psoriasis,we speculated that aganirsen (GS-101) could be efficient inpsoriatic patients. Aganirsen is an antisense oligonucleotidethat inhibits the expression of insulin receptor substrate-1(IRS-1) and has antiangiogenic activities, including inhibi-tion of both VEGF and IL-1b expression (Andrieu-Soleret al., 2005; Al-Mahmood et al., 2009; Cloutier et al., 2012).In this study, we show that, by inhibiting IRS-1 expression inthe cytoplasmic compartment, aganirsen impaired 14-3-3b–tristetraprolin protein (TTP) complex formation, leadingto the inhibition of the expression of several cytokines withAU-rich mRNA, including IL-8, IL-12, IL-22, and TNFa. Wetested the efficacy of aganirsen by a 6-week topicalapplication in patients with chronic mild-to-moderate pla-que psoriasis and showed that aganirsen reduced plaquearea. This was associated with a reduction in the expressionof VEGF and TNFa protein, as well as a normalization of thelevels of CD41 and CD31 lymphocytes and reduced kerati-nocyte proliferation in psoriatic skin lesion biopsies. Thesedata strongly support that the combined antiangiogenic andanti-inflammatory properties of aganirsen synergized toimprove the clinical status of the patients.

Materials and MethodsReal-Time Reverse-Transcription Polymerase Chain Re-

action Assay. The human microvascular endothelial cell line(hEC) was provided by Dr. E. W. Ades (Centers for Disease Controland Prevention, Atlanta, GA), who established this line by trans-fecting human dermal endothelial cells with SV40A gene product andlarge T antigen (Ades et al., 1992). Cells were cultured in EGM-2-MVmedium (Lonza, Levallois, France). After exposure to variousconcentrations of aganirsen (0–10 mM) or vehicle for 24 hours, hEC(5 � 105 cells/ml) total mRNAs were isolated using the NucleoSpinRNA II kit (Macherey-Nagel, Hoerd, France). RNA yields and puritywere assessed by spectrophotometric analysis. The real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed asdescribed previously (Al-Mahmood et al., 2009). In brief, 0.5mg of totalRNA was reverse-transcribed with random hexamer primers andMoloney murine leukemia virus (200 U; Invitrogen, Carlsbad, CA),and the synthesized cDNA was used immediately for real-time PCR am-plification using the DNA-binding dye SYBR Green I for the detection ofPCR products and the following primers: TNFa (sense, 59-GCTGCAGCA-CATTATAATACAGAGA-39; antisense, 59-GGTGTTTGTCGCGACTCC-39); IL-8 (sense, 59-AGTGGACCACACTGCGCCAAC-39; antisense,59-CCACAACCCTCTGCACCCAGT-39); IL-12 (sense, 59-GAATGCAAAGCTTCTGATGGA-39; antisense, 59-GTGGCACAGTCTCACTGTTGA-39); IL-22 (sense, 59-CCCTCAATCTGATAGGTTCCAG-39; antisense,59-GCAGGTCATCACCTTCAATATG-39); and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (sense, 59-TGAAGGTCGGAGTCAACGGA-39; antisense, 59-CATTGATGACAAGCTTCCCG-39). The real-time PCRreactions were carried out with the DNA Light Cycler 480 (RocheDiagnostics, Meylan, France). The results were quantified using theequation: CopyTF/CopyGAPDH5 2C(t)GAPDH2C(t)TF, in which CopyTF isthe number of copy of the targeted fragment gene andCopyGAPDH is thenumber of copy of GAPDH gene fragment used as internal control. AllPCR products were analyzed by electrophoresis on 1.5% agarose gel,visualized with ethidium bromide, and analyzed using the Genesnap

6.00.26 software (Syngene, Frederick, MD). Densitometric analysis wasperformed using GeneTools Analysis Software version 3.02.00 (Syngene).

Subcellular Protein Fractioning and Protein Quantifica-tion. Subcellular protein fractioning was realized using the ThermoScientific subcellular protein fractionation kit for cultured cells(Product No. 78840; Pierce Biotechnology, Rockford, IL) according tothe manufacturer’s instructions. The fractionation kit permitted theseparation and preparation of five fractions: membrane, cytoplasmic,soluble perinuclear-nuclear (SPN), chromatin-bound, and cytoskele-tal protein fractions from hECs. IRS-1 protein was only detected inthe cytoplasmic and the SPN fractions. The protein content of thedifferent fractions was measured by Bradford’s method and adjusted.For purity control within the subfractions, equivalent amounts ofproteins were resolved by SDS-PAGE, followed by the transfer ofproteins to a polyvinylidene difluoride (PVDF) membrane. Themembrane was incubated with 5% defatted milk solution inphosphate-buffered saline (PBS)–0.5% Tween for 1 hour, followedby two washes and immunoblotting with an anti–epidermal growthfactor receptor monoclonal antibody (mAb) (clone D38B1, rabbitmAb; Cell Signaling Technology Inc., Danvers, MA) as plasmamembrane marker; an anti–heat shock protein p90 (cytoplasmicfraction marker) (clone C45G5, rabbit mAb; Cell Signaling Technol-ogy, Inc.), an anti–histone deacetylase 2 mAb (rabbit mAb; CellSignaling Technology, Inc.), and an anti–transcription factor Sp1mAb as markers of the SPN fraction; an anti-vimentin mAb (cloneD21H3, rabbit mAb; Cell Signaling Technology, Inc.) as a marker ofthe cytoskeletal fraction; and an anti–histone 3 mAb as a marker ofthe chromatin-bound insoluble nuclear fraction. The results of thepurity controls of the obtained fractions are shown in SupplementalFig. 1.

Serum-deprived hECs were incubated with different concentra-tions of aganirsen or scramble oligonucleotide at 37°C under 5% CO2

for 6 hours. After three washes with ice-cold PBS, cells were sus-pended with the protein extraction buffer [20 mM Tris-HCl (pH 7.5),150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mMsodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4,1 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride]. Theprotein content wasmeasured by Bradford. IRS-1 concentration in theextracts was determined by the Path-Scan Total IRS-1 Sandwichenzyme-linked immunosorbent assay (ELISA) kit (Cell SignalingTechnology, Inc.) according to the manufacturer’s instructions. The

TABLE 1Main characteristics of the patients included in the pilot clinical study

Number of Patients 12 (2 Women and 10 Men)

Patients characteristicsAverage age (yr) 48 6 14Mean body weight at baseline (kg) 75.8 6 11.9Number of nonsmokers 6 (50%)Number of smokers 4 (33.3%)Number of former smokers 2 (16.7%)Mean heart rate (beats/min) 77.0 6 5.4Mean blood pressure (mm Hg)

Systolic 133 6 11Diastolic 71 6 8

Psoriasis historic and severityPsoriasis diagnosis (yr) .2Psoriasis Area and Severity Index score 4–7; median, 6Body surface area (%) 3–6; median, 5 (scale n°1)Number of refractory lesions

Placebo group 11/12 (91.7%)0.86 mg/g group 10/12 (83.3%)1.72 mg/g group 11/12 (91.7%)

Lesions at the upper limbPlacebo group 7 (58.3%)0.86 mg/g group 6 (50%)1.72 mg/g group 8 (66.7%)

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data were collected from four separate experiments performed induplicate and expressed relative to control cells (vehicle).

To confirm the results obtained with the Path-Scan Total IRS-1Sandwich ELISA kit, equal volumes of the adjusted cell extracts werealso resolved by SDS-PAGE, followed by the transfer of proteins toa PVDF membrane. The membrane was incubated with 5% defattedmilk solution in PBS–0.5% Tween for 1 hour, followed by two washesand immunoblotting with either an anti–IRS-1–horseradish peroxi-dase (HRP) conjugate (1:300 dilution; Santa Cruz Biotechnology, Inc.,Santa Cruz, CA), anti–GAPDH-HRP conjugate (Santa Cruz Bio-technology, Inc.), goat anti-TNFa (1:200 dilution; reference sc52746;Santa Cruz Biotechnology, Inc.), and anti–goat-HRP conjugate (1:20,000 dilution; reference sc2302; Santa Cruz Biotechnology, Inc.).Proteins were then monitored by ECLplus (GE Healthcare, ChalfontSt. Giles, UK).

For the quantification of IL-8, IL-12, IL-22, and TNFa in theculture medium, hECs in culture medium without serum wereincubated with different concentrations of aganirsen or scrambleoligonucleotide at 37°C under 5% CO2 for 24 hours. Culture mediumwas recovered and used to quantify TNFa, IL-8, IL-12, and IL-22using human TNF sandwich ELISA kit (reference 900-k25; Pepro-tech, Neuilly-sur-Seine, France) and human IL8, IL-12, and IL-22sandwich ELISA kits (reference 900-k18, 900-K96, and 900-K246;Peprotech), respectively, according to the manufacturer’sinstructions.

Immunoprecipitation. hECs in culture medium without serumwere incubated with different concentrations of aganirsen or scramble

oligonucleotide at 37°C under 5% CO2 for 6 hours. The cells werewashed twice with cold PBS and lysed with 1% Triton X-100 buffer for1 hour at 4°C. The protein 14-3-3 was immunoprecipitated by adding4 mg of anti–14-3-3bmAb (reference sc628; Santa Cruz Biotechnology,Inc.), and the immune complexes were harvested with protein G-Sepharose beads (Santa Cruz Biotechnology, Inc.), washed with lysisbuffer, and resolved in SDS-PAGE; and proteins were transferred toPVDF membrane and revealed with the indicated antibody.

Pilot Clinical Study. The clinical study was approved by theinstitutional ethics committee and was conducted in accordance withGood Clinical Practices including International Conference of Harmo-nization Guidelines and all applicable local laws and regulatoryrequirements. It was designed as a double-blind, randomized, placebo-controlled, dose-ranging, Latin-square-design, single-center studyfrom the Department of Dermatology of the Military Hospital of Tunis(see study protocol and results in Supplemental Data). It was set up toevaluate the safety and the efficacy of two doses of aganirsen versusplacebo in 12 patients with chronic mild-to-moderate plaque psoriasis(Feldman and Krueger, 2005). Each patient had three psoriasisplaques (including two refractory lesions; refractory areas were elbowand knee). Each lesion was treated either by a low dose (0.86 mg/g) ora high dose (1.72 mg/g) of aganirsen or the ointment alone [placebo;60% paraffin oil and 40% white Vaseline (v/v)]. At the inclusion andrandomization visit (visit 1), the investigator selected for treatment atleast two discoids or plaques from two refractory sites (elbow and knee)representative of the disease and a third plaque in a refractory or notrefractory site. At visit 1 (inclusion in the study) and at visit 2 (3 weeks

Fig. 1. Aganirsen reduced IRS-1 protein expression in the cytoplasmic compartment but not in the SPN. (A) Equivalent amounts of protein extractsfrom hECs incubated with increasing concentrations of aganirsen were used to quantify IRS-1 protein by Western blot (upper). Membranes werestripped and reprobed with anti-GAPDHmAb to control for equal loading (lower). (B) The same hEC protein extracts as in A were used to quantify IRS-1protein by ELISA sandwich assay. (C) Equivalent amounts of protein extracts from human keratinocytes incubated with increasing concentrations ofaganirsen were used to quantify IRS-1 protein by Western blot (upper). Membranes were stripped and reprobed with anti-GAPDH mAb to control forequal loading (lower). (D) The same human keratinocyte protein extracts as in C were used to quantify IRS-1 protein by ELISA sandwich assay. (E)Equivalent amounts of the cytoplasmic fractions from hECs incubated with increasing concentrations of aganirsen were resolved by SDS-PAGE,transferred to PVDF membranes, and probed with anti–IRS-1 mAb (upper). The membranes were stripped and reprobed with anti-GAPDH mAb tocontrol for equal loading (lower). (F) Equivalent amounts of SPN fractions from hECs incubated with increasing concentrations of aganirsen wereresolved by SDS-PAGE, transferred to PVDF membrane, and probed with anti–pIRS-1 mAb (upper). The same membranes were stripped and reprobedwith anti-actin mAb to control for equal loading (lower). (G) Equivalent amounts of the cytoplasmic fraction from hECs incubated with increasingconcentrations of aganirsen were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with anti–pIRS-1 mAb. (H) Equivalent amountsof SPN fractions from hECs incubated with increasing concentrations of aganirsen were resolved by SDS-PAGE, transferred to PVDF membrane, andprobed with anti–pIRS-1 mAb. **P , 0.001; ***P , 0.0001 versus vehicle-treated group (0.9% NaCl). Scramble oligonucleotide (10 mM).

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postinclusion), 75 tubes were delivered, i.e., 25 tubes of each type (A, B,and C). The investigator completed a form that was given to the patienttogether with the ointment tubes explaining what type (A, B, and C) ofointment tube must be used for each plaque. The order of plaquescorresponded to the order recorded in the case report form. “A” and “B”types were assigned to refractory sites only. “A” ointment tubes wereused for the first plaque, “B” ointment tubes were used for the secondplaque, and “C” ointment tubes were used for the third plaque. Thesame one type of ointment tube was always used to treat a givenpsoriasis plaque throughout the study for a given patient.

Each index psoriasis plaque was photographed at inclusion beforestarting the treatment and every 3 weeks. A study nurse was trainedto take these photos according to standards specifically developed forthis study.

Population of the Pilot Clinical Study. Before starting thepilot clinical study, the percutaneous absorption of aganirsen wasstudied quantitatively ex vivo on human dermatome skin biopsiesmounted in Franz diffusion cells. Results showed that single topicalapplication of aganirsen ointment resulted in a therapeutic concentrationin both the epidermis and the dermis (Supplemental Fig. 2, A and B). Nochange in protocol of the pilot clinical study occurred. The maincharacteristics of the included patients were summarized in Table 1.

Immunohistochemistry Analysis. We collected three types ofbiopsies from three patients randomly selected at the end of the trial:

healthy skin; placebo-treated psoriatic skin; and aganirsen (0.86 mg/g)-treated psoriatic skin. Tissue sections were stored at 280°C. Forimmunolabeling, they were thawed at room temperature, followed byincubation with a 3% bovine serum albumin solution in PBS for 1 hourat room temperature. Then they were labeled with the indicatedmAb diluted to 1/100 in PBS–3%bovine serumalbumin. The antibodieswere incubated for 1 to 2 hours for the anti-TNFa mAb (mouse anti-human TNFa; clone 52B83; reference sc-52746; CliniSciences,Montrouge, France) and anti-VEGF mAb (clone C-1; Santa CruzBiotechnology, Inc.), overnight at 4°C for anti-human CD3 polyclonalantibody (reference 14-0038-82; Affymetrix eBioscience, Santa Clara,CA) and anti-human CD4 polyclonal antibody (reference 14-0048-82;Affymetrix eBioscience). Tissue sections were then washed with PBSat room temperature and incubated with the horse anti-mouse IgG(H1L)-Fluorescein conjugate (reference FI-2000; CliniSciences) di-luted to 1/100 in PBS for 1 hour. Following washes with PBS, tissuesections were mounted in aqueous medium (Vectashield; reference H-1500; Vector Laboratories, Burlingame, CA). The labeled tissuesections were examined with a Leica DMR fluorescence microscopeequipped with a DC300F camera for image acquisition. Thequantification of the labeling was performed using ImageJ softwarefromNIH Image, and staining data were normalized and presented as% labeling area per lesion area. For each labeling, the n correspondsto the number of tissue sections analyzed. Immunohistochemistry

Fig. 2. Aganirsen impairs 14-3-3b–TTP complex leading to the inhibition of both IL-8 and TNFa expression. (A) hECs incubated with increasingconcentrations of aganirsen were lysed and proteins were immunoprecipitated with anti–14-3-3b mAb. Equivalent amounts of the immunoprecipitateswere resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti–IRS-1 mAb. (B) The same equivalent amounts of im-munoprecipitates as in A were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-TTP mAb. (C) The membranes used in Awere stripped and reprobed with anti–14-3-3b mAb to control for equal loading in A and B. (D) hECs incubated with increasing concentrations ofaganirsen were lysed and equivalent amounts of proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-TNFamAb to reveal the membrane-bound TNFa. (E) The PVDF membranes used in (D) were stripped and reprobed with anti-actin mAb to control for equalloading in D. (F) hECs were incubated with increasing concentrations of aganirsen, and TNFa mRNA was measured by quantitative RT-PCR andexpressed as mean6 S.E.M. (n = 4). (G) hECs in serum-deprived culture medium were incubated overnight with increasing concentrations of aganirsen,and the culture supernatants were collected to quantify secreted TNFa proteins by ELISA sandwich assay. Results are expressed as picograms of TNFaper milliliter of culture supernatant and presented as mean6 S.E.M. (n = 4). (H) hECs were incubated with increasing concentrations of aganirsen, andIL-8 mRNAwasmeasured by quantitative RT-PCR and expressed as mean6 S.E.M. (n = 4). (I) hECs in serum-deprived culture mediumwere incubatedovernight with increasing concentrations of aganirsen, and the culture supernatants were collected to quantify secreted IL-8 protein by ELISA sandwichassay. Results were expressed as picograms of IL-8 per milliliter of culture supernatant and presented as mean 6 S.E.M. (n = 4). **P , 0.001; ***P ,0.0001 versus vehicle-treated group (0.9% NaCl). Scr, scramble oligonucleotide (10 mM).

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procedure and analysis were repeated three times in independentseries of experiments using the same tissue sections. This measurewas made blinded to treatment.

Study Endpoints. The primary efficacy endpoint was the percent-age evolution of area of plaque size between baseline and week 6 asmeasured on photographs. Thismeasurewasmade blinded to treatment.This efficacy assessment relied on a millimetric surface scale measure-ment of target lesions (pictures). All lesions within the scale square weremeasured. The sum of all areas of all lesions within the square was used.Therefore, the primary efficacy parameter was defined as:

area  at  visit  32 area  at  visit  1area  at  visit  1

� 100

Major secondary efficacy endpoints were the percentage change inarea after 3 weeks from baseline and change after 3 and 6 weeks frombaseline in Total Sign Score (TSS) on physical examination and inPhysician’s Global Assessment (PGA). Safety assessments were madeat all visits by investigating adverse events, serious adverse events,and routine hematologic and laboratory values.

Statistical Analysis. Primary and secondary efficacy analyseswere based on the intent-to-treat population. The intent-to-treat pop-ulation included all randomized patients. Safety analyses were basedon the dataset comprising all randomized patients who received atleast one dose of study treatment. All statistical tests were two-sidedat the 5% significance level. The power level of the study was notconsidered given the small sample size. Normality of areas of targetlesions, percentage changes in area of target lesions, and lesiondiameter variables were checked visually and with the Kolmogorov-Smirnov test and Anderson-Darling test (Anderson and Darling,1954). All parameters related to psoriatic lesion data were summa-rized by treatment group (placebo, 0.86 mg/g, and 1.72 mg/g) andtarget lesions. Continuous parameters, such as primary efficacy, werecompared using analysis of variance. All data were analyzed using theSAS software version 9.2.

ResultsAganirsen Reduced IRS-1 Protein Expression in the

Cytoplasmic Fraction, But Not in the SPN Fraction.Aganirsen dose-dependently inhibited IRS-1 expression inhECs (Fig. 1, A and B) and human keratinocytes (Fig. 1, C andD). Subcellular fractionation of hECs permitted the separa-tion of five fractions: membrane, cytoplasmic, SPN, chromatin-bound, and cytoskeletal protein. IRS-1 protein was onlydetected in the cytoplasmic and the SPN fractions. Closerexamination of IRS-1 protein expression revealed thataganirsen inhibited IRS-1 protein expression in the cytoplas-mic fraction only (Fig. 1E) and not in the SPN fraction (Fig.1F). Analysis of the serine phosphorylation state of IRS-1protein (pIRS-1Ser) revealed that pIRS-1Ser was undetectablein the cytoplasmic compartment (Fig. 1G); in contrast, therewas a dose-dependent increase in pIRS-1Ser in the SPNfraction (Fig. 1H).Aganirsen Impairs 14-3-3b–TTP Complex, Leading to

the Inhibition of TNFa Expression. In the SPN fraction, theincreased serine phosphorylation of IRS-1 (Fig. 1H) paralleledthe increased association of IRS-1 with the protein 14-3-3b.Increasing amounts of IRS-1 coimmunoprecipitated with theprotein 14-3-3b (Fig. 2A); this was accompanied with decreasingquantities of the zinc finger protein TTP coimmunoprecipitatedwith 14-3-3b (Fig. 2B). These results suggest that the asso-ciation 14-3-3b–IRS-1 impaired the formation of the complex14-3-3b–TTP. TTP is a hyperphosphorylated protein thatdestabilizes mRNAs by binding to their AU-rich elements(Blackshear et al., 2003; Cao et al., 2003). The mRNA ofmany cytokines contains AU-rich elements, including those ofTNFa, IL-8, IL-12, and IL-22 (Cao et al., 2003). The scramble

Fig. 3. Aganirsen inhibits both IL-12and IL-22 expression. (A) hECs wereincubated with increasing concentrationsof aganirsen, and IL-12 mRNA was mea-sured by quantitative RT-PCR andexpressed as mean 6 S.E.M. (n = 4). (B)hECs in serum-deprived culture mediumwere incubated overnight with increasingconcentrations of aganirsen, and the cul-ture supernatant was collected to quantifysecreted IL-12 protein by ELISA sandwichassay. Results were expressed as pico-grams of IL-12 per milliliter of culturesupernatant and presented as mean 6S.E.M. (n = 4). (C) hECs were incubatedwith increasing concentrations of aganirsen,and IL-22 mRNA was measured by quan-titative RT-PCR and expressed as mean6S.E.M. (n = 4). (D) hECs in serum-deprivedculture medium were incubated overnightwith increasing concentrations of aga-nirsen, and the culture supernatant wascollected to quantify secreted IL-22 proteinby ELISA sandwich assay. Results wereexpressed as picograms of IL-22 per milli-liter of culture supernatant and presentedas mean 6 S.E.M. (n = 4). **P , 0.001;***P , 0.0001 versus vehicle-treatedgroup (0.9% NaCl). Scr, scramble oligonu-cleotide (10 mM).

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oligonucleotide had no effect on the membrane-bound TNFa inhECs (Fig. 2D). In contrast, there was a dose-dependentdecrease in membrane-bound TNFa in hECs incubated with

aganirsen (Fig. 2D). This dose-dependent inhibition of TNFaexpression by aganirsen was confirmed by the measurement ofthe secreted TNFa protein in the culture medium (Fig. 2F) and

TABLE 2Study clinical endpoints

Variables Placebo Aganirsen (0.86 mg/g) Aganirsen (1.72 mg/g)

% Evolution in lesion area (visit 3)a 24.5 6 41.7 214.4 6 38.6* 212.9 6 52.4*% Evolution in lesion area (visit 2) 18.5 6 30.9 218.8 6 28.4** 219.8 6 31.4**TSS scoreb

Baseline 5.5 6 1.0 5.7 6 1.5 5.5 6 1.26 weeks 3.4 6 1.2 3.6 6 1.9 3.2 6 1.3

PGAc

Baseline 2.5 6 0.7 2.5 6 0.8 2.5 6 0.76 weeks 3.7 6 1.1 3.7 6 1.2 3.9 6 1.0

Toleranced

Pruritus baseline 0.3 6 0.6 0.3 6 0.6 0.3 6 0.66 weeks 0.2 6 0.6 0.2 6 0.6 0.2 6 0.6Change in irritation 0 6 0 0 6 0 0 6 0Change in burning 0 6 0 0 6 0 0 6 0Change in exudation 0 6 0 0 6 0 0 6 0Change in itching after 6 weeks

21 1 (8.3%) 1 (8.3%) 1 (8.3%)0 10 (83.3%) 10 (83.3%) 10 (83.3%)2 1 (8.3%) 1 (8.3%) 1 (8.3%)

aPercentage evolution (mean 6 S.D.) between week 6 (visit 3) and week 3 (visit 2) and baseline (visit 1) in area oftreated psoriasis plaque asmeasured by photography; two types of diameters weremeasured, both at baseline, 3 weeks, and 6weeks after treatment: sum of diameters (mm) from the Case Report Form and diameters (cm) as measured on pictures.

bTSS: sum of signs (redness/erythema, scale/crusting, thickening/elevation) and symptoms (pruritus) using 4-pointscales (e.g., none/clear = 0, mild = 1, moderate = 2, extensive = 3). Score varies from 0 to 12 for each lesion.

cFor PGA, three items were noted: erythema, induration, and scale, according to Feldman and Krueger (2005).dLocal tolerance of treatment: patient’s assessment on a 4-point scale of irritation, itching, pruritus, sensation of

burning, and exudation.*P , 0.05 vs. placebo; **P , 0.01 vs. placebo.

Fig. 4. Topical application of aganirsen reduced psoriaticlesion area compared with placebo. (A) Treatment of 12patients with topical application of 0.86 mg/g or 1.72 mg/gaganirsen led to a significant reduction at 3 (†P , 0.01) and6 weeks (‡P , 0.05) in the treated lesions compared withplacebo. Data are mean 6 S.D. (B) Representative images ofthe evolution of psoriatic lesions of patients treated withaganirsen at 1.72mg/g (upper), patients treatedwith aganirsenat 0.86 mg/g (middle), and patients treated with placebo(lower). Visit 1, visit 2, and visit 3 correspond to baseline,after 3 weeks, and after 6 weeks of treatment, respectively.

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by the measurement of TNFa transcripts by quantitative RT-PCR (Fig. 2G). In addition, aganirsen led to a dose-dependentinhibition of IL-8 expression at both the translational (Fig. 2H)and the transcriptional levels (Fig. 2I), of IL-12 expression atboth the transcriptional (Fig. 3A) and the translational levels(Fig. 3B), and of IL-22 expression at both the transcriptional(Fig. 3C) and the translational levels (Fig. 3D).Efficacy of Aganirsen in the Treatment of Psoriatic

Lesions. Treatment of 12 patients with topical application of0.86 mg/g or 1.72 mg/g aganirsen for 6 weeks led to a sig-nificant reduction (P , 0.05) in the area of the treated lesionscompared with placebo (Fig. 4, A and B; Table 2). By contrast,a slight increase in the lesion area was observed (median,22%) in the placebo-treated group. Least square mean dif-ferences with placebo were 238.9% (95% confidence interval,275.8 to22.0%) and237.4% (274.3 to20.5%) for the 0.86mg/gand 1.72 mg/g groups, respectively. A significant decrease inaganirsen-treated lesion size was observed at as early as3 weeks (Fig. 4, A and B; Table 2) of treatment compared withplacebo (P , 0.01). Least square mean differences withplacebo were237.3% (262.7 to211.9%) and238.3% (263.8 to212.9%) for the 0.86 mg/g and 1.72 mg/g groups, respectively.

We found no difference in the evolution of the TSS score(P5 0.63 and P5 0.82 at weeks 3 and 6, respectively; Table 2)between the three groups. At baseline, TSS was a median of 6;at week 3, a median of 4; and at week 6, a median of 3 in theplacebo group. In the 0.86 mg/g treated group, the medianTSS score was 5 at both baseline and week 3 and 4 at week 6,whereas in the 1.72 mg/g treated group, the median TSS scorewas 6 at baseline, 4 at week 3, and 3 at week 6. Likewise, thePGA score remained stable over the 6-week period and wassimilar in the three groups (Table 2).Safety of Aganirsen. Safety analysis was carried out on

the 12 patients. Aganirsen’s safety was good, without anyreported adverse effects. Patients reported no irritation,burning, or exudation. In each treatment group, mild itchingwas reported for one lesion at baseline, no itching was reportedat 3 weeks, and a moderate itching for one lesion at 6 weeks.Aganirsen Inhibits IRS-1 and VEGF Expression in

Psoriatic Lesions of Patients. Treatment with aganirsen(0.86 mg/g) reduced IRS-1 expression by 81% 6 12% (P 50.0132; n 5 22) in human psoriatic skin lesions relative toplacebo-treated psoriatic skin lesions (Fig. 5A; SupplementalFig. 3). No difference in IRS-1 expression was detected

Fig. 5. Topical application of aganirsen for6 weeks decreased both IRS-1 and VEGFexpression in psoriatic lesion biopsies. (A)Inhibition of IRS-1 expression (measured byimmunofluorescence using an IRS-1 anti-body) after daily topical treatment of 6 weekswith aganirsen (0.86 mg/g ointment) inpsoriatic skin lesion biopsies from patients.Data are mean 6 S.E.M. *P = 0.0008 versusplacebo-treated psoriatic lesions. (B) Inhibi-tion of VEGF expression after daily topicaltreatment of 6 weeks with aganirsen (0.86mg/g ointment). Data aremean6 S.E.M. *P =0.0074 versus placebo-treated psoriaticlesions. (C) Representative images of VEGFlabeling in human skin biopsies using anti-VEGF mAb in healthy untreated skin (left),placebo-treated psoriatic lesions (middle),and aganirsen-treated psoriatic lesions (right).Negative controls (secondary antibody only)are presented in the top row of images, cellnuclei are stained with 49,6-diamidino-2-phe-nylindole (DAPI) in blue (second row ofimages), immunostaining of VEGF is in green(third row of images), and the merge imagesincorporate both nuclei and VEGF immunos-taining-derived fluorescence (fourth row ofimages).

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between biopsies of healthy skin and placebo-treated lesions(Fig. 5A). In psoriatic lesion biopsies, we found that VEGFlabeling was lowered by 42% 6 8% (P 5 0.0074) in 0.86 mg/gtreated psoriatic lesion biopsies compared with placebo-treated pathologic skin lesions (Fig. 5B).Effects of Aganirsen on TNFa Expression, Inflamma-

tory Cell Recruitment, and Keratinocyte Proliferationin Psoriatic Lesions of Patients. Treatment with aganirsen(0.86 mg/g) was also associated with a lower TNFa labeling by77%6 11% (P5 0.0047; n5 14) (Fig. 6A; Supplemental Fig. 4)in skin biopsies of psoriatic patients compared with placebo-treated psoriatic skin samples. Measurement of CD41 labelingindicated that psoriatic skin contained 65% 6 21% more cells(P5 0.013; n5 12) than healthy skin. In contrast, CD41 labelingin aganirsen (0.86 mg/g)–treated pathologic tissue samples waslower by 43% 6 6% (P 5 0.035; n 5 12) compared with placebo-treated pathologic tissues (Fig. 6B; Supplemental Fig. 5). Similarresults were obtained with CD31 labeling. The psoriatic skincontained 115%6 40%more (P5 0.0089; n5 14) CD31 labelingthan healthy skin, confirming our precedent results with CD41

cells and further supporting the role of inflammation inpsoriasis. CD31 labeling in aganirsen-treated pathologic skinbiopsies was reduced (47% 6 6%; P 5 0.0355) compared withplacebo-treated pathologic tissues (Fig. 6C; Supplemental Fig.6); we found CD31 labeling to be similar between healthy skintissues and aganirsen-treated skin tissues. Finally, after 6weeks of treatment with aganirsen, expression of proliferationmarker Ki-67 and cytokeratin 16 were lower by 59%6 3% (P50.0132; n 5 10) and by 46% 6 8% (P 5 0.0265; n 5 5),respectively, compared with placebo-treated lesions (Figs. 7, A

and B, and 8, A and B). Taken together, these findingsdemonstrate that aganirsen has potent anti-inflammatoryeffects and inhibits both the infiltration of lymphocytes andthe proliferation of keratinocytes in the psoriatic skin.

DiscussionThis study demonstrates that aganirsen is the first in its

class of a new generation of antiangiogenic medicines thatcombines both antiangiogenic and anti-inflammatory activi-ties and could be an effective treatment of patients withpsoriasis. This statement is supported by several pieces ofdirect evidence, including the following: 1) in vitro, aganirseninduced IRS-1–14-3-3b association, which impaired 14-3-3b–TTP complex, leading to the inhibition of the expressionof cytokines with AU-rich mRNA, including IL-8, IL-12, IL-22,and TNFa; 2) in psoriatic patients, aganirsen inhibited theexpression of both VEGF and TNFa, thereby reducing theinfiltration of inflammatory CD41 and CD31 lymphocytes; andaccordingly 3) aganirsen inhibited keratinocyte proliferationand consequently reduced psoriatic lesion area in patients.Psoriasis is a chronic inflammatory disease in which many

inflammatory cytokines, including IL-12, Il-17, IL-22, andTNFa, play important pathologic roles. Thus, the most de-sired therapy is one that targets all these inflammatoryfactors. The mRNAs of IL-12, IL-17, IL-22, and TNFa shareone characteristic, containing AU-rich elements at their39-untranslated regions (Bak and Mikkelsen 2010; Khabar,2010). TTP is an mRNA-binding protein with high bindingspecificity for the so-called class II AU-rich elements within

Fig. 6. Topical application of aganirsen inhibitsTNFa, CD4+, and CD3+ lymphocyte infiltrationin psoriatic lesions. Twice-daily topical treat-ment of 6 weeks with aganirsen at 0.86 mg/gointment inhibited TNFa expression (A) inpsoriatic skin lesions and reduced the levels ofinfiltrated CD4+ (B) and CD3+ (C) lymphocytescompared with placebo-treated psoriatic lesions.Data are mean 6 S.E.M. (A) *P = 0.047 versuspathologic placebo-treated lesions; ‡P = 0.007versus healthy skin. (B) *P = 0.002 versuspathologic placebo-treated lesions; †P , 0.0001versus healthy skin. (C) *P = 0.0355 versuspathologic placebo-treated lesions; †P = 0.0089versus healthy skin.

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the 39-untranslated mRNAs (Blackshear et al., 2003; Caoet al., 2003); specific binding of TTP to the AU-rich elementsresults in the destabilization and the decay of the mRNAs(Carballo et al., 1998; Lai et al., 1999). TTP occurs in acomplex with 14-3-3, preventing TTP from binding to and/ordirecting mRNA to the degradation machinery (Johnsonet al., 2002; Zhao et al., 2011). Our results show thataganirsen-induced association of IRS-1 with 14-3-3b proteinimpairs 14-3-3b–TTP complex, revealing a novel regulatoryrole for IRS-1. Indeed, IRS-1 associates with many of theseven isoforms of 14-3-3 proteins (Ogihara et al., 1997; Kosakiet al., 1998; Xiang et al., 2002; Oriente et al., 2005); yet thefunctional consequence for these associations was unknown.Our results are therefore the first to demonstrate that bypromoting the association of IRS-1 to 14-3-3b protein andimpairing 14-3-3b–TTP complex, aganirsen inhibits theexpression of IL-8, IL-12, IL-22, and TNFa at both thetranscriptional and the translational levels. This is in linewith the importance of the liberation of TTP from the 14-3-3–TTP complex, and with the role of TTP in the regulation ofexpression of AU-rich elements in the destabilization and thedecay of mRNAs (Carballo et al., 1998; Lai et al., 1999). Theseresults, combined with those showing that aganirsen inhibited

VEGF and IL-1b expression (Andrieu-Soler et al., 2005; Al-Mahmood et al., 2009), identify aganirsen as a dual-targettherapeutic agent that is highly desirable for the treatment ofpsoriasis.Topical applications of aganirsen led to therapeutic con-

centrations in both epidermis and dermis of human skin(Supplemental Fig. 2, A and B). It has been suggested that theimpaired barrier function of psoriatic lesions (Schittek, 2011)and the amphipathic nature of aganirsen (Cloutier et al.,2012) could facilitate uptake of antisense drugs in general andaganirsen in particular. In agreement, topical applications ofaganirsen decreased psoriatic plaque area in both groups ofpatients (0.86 and 1.72 mg/g) compared with placebo. Fur-thermore, aganirsen had a rapid onset of action as illustratedby the significant decrease in lesion area after 3 weeks oftreatment of 214 and 213% in the 0.86 mg/g and 1.72 mg/gtreated groups, respectively. By contrast, the plaque areaincreased by ∼15% in the placebo-treated group over thisperiod. We did not find any significant differences in theevolution of TSS and PGA scores after either 3 or 6 weeks oftreatment. This is most likely related to the short term of thetrial; at least 12–16 weeks of treatment is required toconfirm drug efficacy against psoriatic lesions (Papp et al.,

Fig. 7. Topical application of aganirsen inhibitskeratinocyte proliferation in psoriatic lesions. (A)Twice-daily topical treatment of 6 weeks withaganirsen at 0.86 mg/g ointment inhibited kerati-nocyte proliferation as evidenced by the reducedexpression of the keratinocyte proliferation markerKi-67 in psoriatic lesions. Data are mean 6 S.E.M.*P = 0.0022 versus pathologic placebo-treated lesions;†P = 0.021 versus healthy skin. (B) Representativeimages of Ki-67 labeling in human skin samples usinganti–Ki-67 mAb in healthy nontreated skin (left) andpsoriatic lesions either placebo-treated (middle) oraganirsen-treated (right). Negative controls (second-ary antibody only) are presented in the top row ofimages, cell nuclei are stained with 49,6-diamidino-2-phenylindole (DAPI) in blue (second row of images),immunostaining of Ki-67 is in green (third row ofimages), and themerge images incorporate both nucleiand Ki-67 immunostaining-derived fluorescence (fourthrow of images).

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2012a,b). In addition, our study did not reveal a dose-dependent effect of aganirsen; both 0.86 and 1.72 mg/g led toa similar decrease in psoriatic lesion area. These resultssuggest that the two doses tested in this pilot study induceda maximal effect, at least within the time frame of the currentprotocol. Lower daily doses of aganirsen combined with a longerperiod of treatment will therefore have to be tested in futureclinical studies. Importantly, aganirsen was well toleratedlocally, and no serious adverse effects were observed in anygroup. Nonetheless, our study was neither large enough nor ofsufficiently long duration to ascertain uncommon adverse effects.Psoriasis is characterized by chronic inflammation and

enhanced lymphocyte infiltration (Nestle et al., 2009; Coimbraet al., 2012). Consistently, psoriatic lesions contained moreCD41 and CD31 labeling compared with biopsies of healthyskin. Lymphocyte recruitment requires blood vessels, andaberrant angiogenesis has been linked to psoriasis (Detmaret al., 1998; Sabat et al., 2007; Heidenreich et al., 2009). Inagreement with the antiangiogenic property of aganirsen, ourresults showed that VEGF expression was significantly lowerin psoriatic lesions treated with aganirsen compared withplacebo-treated psoriatic lesions, although VEGF expressionwas not elevated in the latter compared with that in thehealthy skin. In contrast, CD311 and CD341 labeling wasincreased in psoriatic lesions compared with the healthy skin,and this increase was normalized following the treatment with

aganirsen. In aggregate, these results suggest that the anti-inflammatory activity of aganirsen rather than its antiangio-genic activity underlies the observed beneficial clinical outcome.VEGF, nonetheless, is chemoattractant (Sawano et al., 2001;Ferrara et al., 2003) and promotes lymphocyte rolling andadhesion in skin microvessels (Detmar et al., 1998). Conse-quently, we would like to suggest that the inhibition of VEGFexpression by aganirsen contributes to limit the recruitment ofimmune cells within the psoriatic lesions. This would explainthe important decrease in TNFa protein in aganirsen-treatedpsoriatic lesions, which is mostly produced by activated leu-kocytes (Orlinick and Chao, 1998).Psoriasis is characterized by epidermal thickening and

keratinocyte hyperproliferation, which is directly linked tothe inflammation within the psoriatic lesion (Sabat et al.,2007; Nestle et al., 2009). The expression of VEGF and TNFawas reduced by aganirsen in psoriatic lesions compared withplacebo-treated lesions and was associated with .50% in-hibition of keratinocyte proliferation as measured by Ki-67and cytokeratin 16 (CK-16) labeling. The loss of CK-16–associatedfluorescence following a 6-week therapy is a crucial finding sinceCK-16 is an important marker predicting therapeutic outcome(Krueger et al., 1995). The potent inhibitory action of aganirsen onkeratinocyte proliferation is most probably linked to its anti-inflammatory action through inhibition of the expression ofVEGF, TNFa, IL-12, and IL-22.

Fig. 8. Topical application of aganirsen inhibits cytoker-atin 16 (CK-16) expression in psoriatic lesions. (A) Twice-daily topical treatment of 6 weeks with aganirsen at 0.86mg/g ointment inhibited keratinocyte proliferation asevidenced by the reduced expression of CK-16 in psoriaticlesions. Data are mean 6 S.E.M. *P = 0.0265 versus path-ologic placebo-treated lesions. (B) Representative images ofCK-16 labeling in human skin samples using anti–CK-16mAb in healthy nontreated skin (left) and psoriatic lesionseither placebo-treated (middle) or aganirsen-treated (right).Negative controls (secondary antibody only) are presentedin the top row of images, cell nuclei are stained with 49,6-diamidino-2-phenylindole (DAPI) in blue (second row ofimages), immunostaining of CK-16 is in green (third row ofimages), and the merge images incorporate both nuclei andCK-16 immunostaining-derived fluorescence (fourth row ofimages).

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Finally, the levels of VEGF and TNFa were elevated in thebiopsies of healthy skin, confirming the global proinflamma-tory status of these patients (Sabat et al., 2007; Nestle et al.,2009; Weger, 2010). Many anti-inflammatory drugs targetingTNFa, IL-12, IL-17, and IL-23 have been approved or are inadvanced development stage for the treatment of psoriasis(Coimbra et al., 2012; Papp et al., 2012b). However, unlikeaganirsen, which is active topically, these orally adminis-trated new drugs have been associated with numerous sideeffects. Nonetheless, these new biologics have been used inpatients with moderate-to-severe forms of psoriasis, whileaganirsen was used in mild-to-moderate forms of the disease(Psoriasis Area and Severity Index score ranged from 4–7)and therefore cannot be compared.In conclusion, our results demonstrate that aganirsen

inhibits the expression of cytokines whose mRNA containsAU-rich elements at their 39-untranslated regions, includingTNFa, IL21b, IL-8, IL-12, and IL-22, known targets of bothinflammation and angiogenesis. On the other hand, we con-firmed that aganirsen is a potent inhibitor of VEGF expression,the only known growth factor that induces inflammation andpromotes angiogenesis and lymphangiogenesis (Al-Mahmoodet al., 2009; Koch et al., 2011; Ji, 2012). Despite the limitations ofour trial (the small sample size and the short-term duration ofthe treatment), these results suggest that aganirsen is a dualantiangiogenic and anti-inflammatory agent that could repre-sent an innovative safe and effective alternative for the treatmentof psoriasis, a disease in need of newand safe therapeutic options.

Acknowledgments

The authors thank Dr. Ekat Kritikou, Dr. Eric Thorin, and EricViaud for help in the writing and editing of the manuscript, andCéline Steverlynck (research engineer, Gene Signal SAS) and MaudBongaerts (technician, Gene Signal SAS) for expert technicalassistance.

Authorship Contributions

Participated in research design: Ferry, Doss, Al-Mahmood.Conducted experiments: Colin, Darné, Favier, Lesaffre, Kadi.Contributed new reagents or analytic tools: Conduzorgues,

Al-Mahmood.Performed data analysis: Al-Mahmood.Wrote or contributed to the writing of the manuscript: Al-Mahmood.

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