hif2a-targeted rnai therapeutic inhibits clear cell renal ... · an rnai therapeutic for hif2a that...

Large Molecule Therapeutics

HIF2a-Targeted RNAi Therapeutic Inhibits ClearCell Renal Cell CarcinomaSo C.Wong,Weijun Cheng, Holly Hamilton, Anthony L. Nicholas,Darren H.Wakefield, Aaron Almeida, Andrei V. Blokhin, Jeffrey Carlson, Zane C. Neal,Vladimir Subbotin, Guofeng Zhang, Julia Hegge, Stephanie Bertin,Vladimir S.Trubetskoy,David B. Rozema, David L. Lewis, and Steven B. Kanner

Abstract

Targeted therapy against VEGF and mTOR pathways has beenestablished as the standard-of-care for metastatic clear cell renalcell carcinoma (ccRCC); however, these treatments frequently failand most patients become refractory requiring subsequent alter-native therapeutic options. Therefore, development of innovativeand effective treatments is imperative. About 80%–90%of ccRCCtumors express an inactivemutant formof the vonHippel-Lindauprotein (pVHL), an E3 ubiquitin ligase that promotes targetprotein degradation. Strong genetic and experimental evidencesupports the correlate that pVHL functional loss leads to the

accumulation of the transcription factor hypoxia-inducible factor2a (HIF2a) and that an overabundance of HIF2a functions as atumorigenic driver of ccRCC. In this report, we describean RNAi therapeutic for HIF2a that utilizes a targeting ligandthat selectively binds to integrins avb3 and avb5 frequentlyoverexpressed in ccRCC. We demonstrate that functional deliv-ery of a HIF2a-specific RNAi trigger resulted in HIF2a genesilencing and subsequent tumor growth inhibition and degen-eration in an established orthotopic ccRCC xenograft model.Mol Cancer Ther; 17(1); 140–9. �2017 AACR.

IntroductionPromising targeted treatment options derived from better

understanding of molecular characteristics of cancer biologyhave resulted in significant improvement in the overall survivalof patients with metastatic clear cell renal cell carcinoma(ccRCC). These treatments include therapies that target mole-cules regulating angiogenesis and proliferation pathways suchas bevacizumab, an antibody against VEGF, and several multi-kinase inhibitors that inhibit multiple receptor tyrosine kinasesincluding VEGFR (1–3). These targeted therapies are distinctfrom small-molecule inhibitors that target the mTOR signalingpathways, such as temsirolimus and everolimus, which disruptpathways that regulate cell growth, metabolism, and survival(4, 5). These therapies exhibited significantly improved treat-ment outcomes over the last decade and have become thestandard-of-care in both first- and second-line settings sincethey were first introduced (1, 6, 7). However, emergence ofresistance to these agents is a frequent limitation for long-termsuccess (8–10). More recently, nivolumab (Opdivo), an mAbimmunotherapeutic targeting the programmed cell death-1(PD-1) immune checkpoint pathway (11) was approved as asecond-line option for ccRCC. The excitement surrounding this

promising new treatment class for oncology in general signalsthe importance of targeting orthogonal pathologic pathways tocounter the frequent genomic shifts of heterogeneous cancercell populations and the complexity of tumor microenviron-ment cellular interactions (12).

About 80%–90% of ccRCC tumors express a mutant form ofpVHL, anE3ubiquitin ligase (13–15). Themutant pVHL is unableto polyubiquitinate the transcription factor hypoxia-induciblefactor (HIF), which regulates genes involved in angiogenesis,proliferation, metabolism, and invasion/metastasis pathways(16–19). Strong genetic and experimental evidence supports theobservation that pVHL functional loss leads to the accumulationof the transcription factor HIF2a, and that an overabundance ofHIF2a functions as a tumorigenic driver of ccRCC (16). Incontrast, HIF1a is frequently not expressed due to chromosome14q deletions commonly occurring in ccRCC. Evidence suggeststhat HIF1a functions as a tumor suppressor in renal cancer (16).Accordingly, potential new therapies targeting HIF2a function,including in this study, are being explored. Recently, preclinicaland limited early clinical results using small-molecule inhibitorstargeting HIF2a were reported (16, 20–22). In these studies, theHIF2a antagonists showedantitumor activity in several tumor cellline xenograft mouse models and patient-derived xenograft(PDX) models. Early clinical data showed a patient treated withthe HIF2a antagonist PT2385 remained free of progression formore than 11 months (20). These encouraging results lendvalidation towards developing novel therapeutics that target thepVHL/HIF2a pathway. However, these studies also uncoveredseveral preclinical mouse models that are not responsive to theseHIF2a antagonists (20, 21). In some models, the lack of antitu-mor activity was attributed tomissensemutations that resulted inamino acid substitutions surrounding the binding pocket ofHIF2a antagonists (20, 21). These observations indicate that an

Arrowhead Pharmaceuticals Inc., Madison, Wisconsin.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: So C. Wong, Arrowhead Pharmaceuticals Inc., 502 S.Rosa Road, Madison, WI 53719. Phone: 608-316-3929; Fax: 608-441-0741;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0471

�2017 American Association for Cancer Research.

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effective alternate therapeutic approach to target HIF2a functionis warranted.

The specificity and potency of gene silencing mediated by RNAinterference (RNAi) is well established (23, 24) and targetedtherapeutics utilizing this mechanism to suppress tumor HIF2aexpression canpotentially overcome conventional drug resistanceand improve responsiveness to currently treatments, whichincludes immune checkpoint inhibitors. To realize the potentialof RNAi based therapeutics, efficient delivery of an RNAi trigger(siRNA) has been a major obstacle (25), particularly to targettissues other than liver (26, 27). Previously, we described anefficient and selective liver targeting siRNAdelivery system termeddynamic polyconjugates (DPC; refs. 28, 29). This deliveryapproach included the use of a membrane active polymer thatis reversibly modified with shielding and targeting moieties toprovide highly selective target tissue delivery of siRNA (28, 29).For liver targeting, DPC uses N-acetylgalactosamine as the ligandto target asialoglycoprotein receptors (ASGPr) that are abundant-ly expressed by hepatocytes (30). The incorporation of a targetingligand and the reversible masking of a membrane active polymerallowed efficient target cell uptake and subsequent RNAi triggerescape from endosomes (28, 29). The modular nature of DPCsallows for exchange of targeting moiety and RNAi trigger for aspecific tissue and therapeutic gene of interest. In this report, wedescribe an RNAi trigger delivery system that preferentially bindsto integrin receptors avb3 and avb5 commonly overexpressed intumor cells, including ccRCC (31–33). We demonstrate thetargeted delivery of RNAi-mediated HIF2a gene silencing andthe resulting proof-of-concept effect on tumor growth inhibitionand degeneration in an established orthotopic ccRCC tumorxenograft model.

Materials and MethodsCell culture

A498 (2012), 786-O (2014), Caki-1 (2014), Caki-2 (2015),and Hep3B (2009) cells were purchased directly from ATCC,the year when the cell line was acquired is provided inparenthesis. Cells were maintained at 37�C in a humidifiedatmosphere containing 5% CO2. Cells were subcultured every3 to 4 days and passaged no more than 20 times before use.Mycoplasma testing was conducted every few months usingMycoProbe Mycoplasma Detection Kit (R&D Systems,CUL001B). The A498 and Hep3B cells were cultured in min-imum essential media (Cellgro) supplemented with 10% FBS(Thermo Fisher Scientific) and 1% nonessential amino acidssolution (Gibco). Caki-1 and Caki-2 cells were cultured inMcCoy 5A medium (Cellgro) supplemented with 10% FBS.786-O cells were cultured in RPMI medium (Cellgro) supple-mented with 10% FBS.

In vitro RNAi trigger setIn silico analysis of potential-specific RNAi triggers cross-

reactive to human EPAS1 (accession no. NM_001430.4) andNHP transcripts was performed by Axolabs. After applyingappropriate specificity filters to the sequences, a screen set of187 RNAi triggers was synthesized using standard phosphor-amidite chemistry. The siRNAs contained specific modificationsat the 30 and 50 ends and 20-O-methyl/20flouro-modifiednucleotides to protect against nucleases and reduce potentialinnate immune responses.

In vitro RNAi trigger screeningFor screening purposes, the human EPAS1 (HIF2a) cDNA

sequence (accession #NM_001430.4) was synthesized andcloned (DNA 2.0) into a commercially available reporter-basedscreening plasmid, psiCHECK2 (Promega, C8021), which gen-erated a Renilla/luciferase/EPAS1 fusion mRNA. Hep3B cells wereplated at approximately 1 � 104 cells per well in 96-well format.Each of the 187 EPAS1 RNAi triggers was cotransfected at twoconcentrations, 1 and 0.1 nmol/L, with 25 ng of EPAS1-psiCHECK2 plasmid and 0.2 mL Lipofectamine 2000 (Life Tech-nologies) per well. Gene knockdown was determined by mea-suring Renilla luciferase activities normalized to the levels ofconstitutively expressed firefly luciferase using theDual LuciferaseReporter Assay (Promega, E1910).

Candidate RNAi triggers for DPC conjugationFor the RNA triggers used for in vivo testing, the sense strand

was synthesized with a 50 primary amine containing phosphor-amidite for attaching click chemistry alkyne functional groupfor polymer conjugation. Following standard cleavage anddeprotection, the sense strand was dissolved in sodium acetatesolution (1.2 mol/L) and precipitated with seven volumeequivalents of ethanol. The precipitate was then dissolved insodium bicarbonate solution (0.1 mol/L). Six equivalents ofthe disulfide containing dibezocyclooctyne-methyl-S-S-N-hydroysuccinimidyl ester (DBCO-Me-S-S-NHS) reagent dis-solved in dimethylformamide (DMF, 10 mg/mL) was added.The reaction progress was monitored by RP-HPLC. Once thereaction was complete, the solution was precipitated withethanol and purified using RP-HPLC.

Small-molecule synthesisProtease masking reagent PEG12-L-analine-L-citrulline-p-ami-

nobenzyl alcohol p-nitrophenyl carbonate (PEG12-ACit-PABC-PNP, PEG12 refers to a discrete 12-mer of PEG) was synthesizedas described previously (28). RGD- or RGE mimetic–conjugated-PEG masking reagents, RGD-PEG20-FCitFP-TFP or RGE-PEG20-FCitFP-TFP (PEG20 is a discrete 20-mer PEG, FCitFP refers to theamino acid sequence phenylalanine-citrulline-phenylalanine-proline, TFP (tetrafluorophenyl ester), were synthesized usingstandard amide coupling reagents as described previously(34, 35). RGD or RGEmimetics are arginine-glycine-aspartic acidor glutamic acid mimics, respectively (34). The copper-free clickchemistry reagent DBCO-Me-S-S NHS was synthesized asdescribed previously (36). See Supplementary Data for structures.

Competitive solid-phase ligand binding assayThe binding constant of the RGDmimetic was evaluated using

an ELISA-based competitive assay using recombinant humanintegrin avb3, avb5, and avb6 (see Supplementary Methods fordetails)

RAFT copolymer of N-Boc-ethoxyethylamine acrylate andpropyl acrylate

Polymer N-Boc-ethoxyethylamine acrylate and propyl acrylate(EAP) synthesis is similar as described for N-Boc-ethoxyethyla-mine acrylate and sec-butyl acrylate (EAB) (28). For polymer EAP,propyl acrylate was used instead of sec-butyl acrylate. Additionaldetails are provided in Supplementary Data.

HIF2a-Targeted RNAi Therapeutic

www.aacrjournals.org Mol Cancer Ther; 17(1) January 2018 141




Assembly and characterization of RGD-DPCRGD-PEGandPEG-onlymasking reagentswere added to azide-

functionalized EAP polymer in 5 mmol/L pH 8.0 HEPES bufferand incubated at room temperature. Masked polymer was thenmixedwith alkyne-functionalized RNAi trigger at a weight ratio of1:0.3, respectively. Trigger-conjugated RGD-DPC was then puri-fied with tangential flow filtration and characterized. Furtherdetails on polymer functionalization, assembly, and analyticmethods used for characterizations are provided in Supplemen-tary Data.

RGD-Cy3-DPC binding and internalization in cultured cellsCy3-NHS ester (GE Healthcare) was coupled to polymer fol-

lowing the manufacturer's recommendations. Cells (1 � 105)plated on cover glass were incubated with 2.5 mg/mL of Cy3-labeled DPC polymer that was modified with PEG12-ACit-PABCand RGD-PEG20-FCitFP-TFP or PEG12-ACit-PABC alone for24 hours in complete media. No RNAi triggers were included inthese imaging DPCs. Cells were fixed in 10% formalin andcounterstained with 20 nmol/L Alexa Fluor 488-phalloidin(A12379; Invitrogen) and 40 nmol/L TO-PRO-3 (T3605; Invitro-gen) in PBS for 30 minutes. Cover glass was then mountedonto slides with Vectashield mounting medium (H-1000; VectorLaboratories) and evaluated by confocal microscopy (LSM710;Carl Zeiss).

Orthotopic ccRCC xenografts with A498 cells stably expressingSEAP

Female athymic nude mice 6–8 weeks old were obtained fromEnvigo Bioproducts Inc. All animal studies followed proceduresapproved by the Institutional Animal Care and Use Committee atArrowhead Pharmaceuticals. The mice were housed in individu-ally ventilated cages (Super Mouse 1800, Lab Products) withaccess to food and water ad libitum. For tumor implantation, a10 mL aliquot of SEAP-expressing A498 cell/Matrigel mixturecontaining about 4� 105 cells [2:1 (v/v) of cell:Matrigel; Corning,catalog no. 354248], using a 27-gauge needle catheter wasinjected into the left kidney subcapsular space using a syringepump. Serumwas collected every 7–14 days after implantation tomonitor tumor growthusing a commercially available SEAPAssayKit (Life Technologies, T1016). For most studies, tumor-bearingmice were used 5–6 weeks after implantation, when tumors weretypically 4–8 mm in length and width.

RGD-DPC binding and internalization in tumor-bearing miceCy3-RGD-DPCpreparedwithCy3-labeled polymer (100mg) as

described above was injected via the tail vein into tumor-bearingmice. Tumor and other organs were harvested 4 hours postinjec-tion and frozen tissue sections were prepared as described previ-ously (29). Tissue sections were counterstained and analyzed asdescribed above for in vitro binding studies.

In vivo efficacy studiesHIF2a-RGD-DPCwas injected via the tail vein into A498-SEAP

tumor-bearing mice. Dosage was calculated on the basis of theamount of polymer before conjugation to masking reagents andsiRNA. In multi-dosing studies, serum was collected weekly forSEAP and clinical chemistry evaluations. Clinical chemistry wasanalyzed using a Cobas Integra 400 Plus clinical analyzer (RocheDiagnostics). Tumors were excised at the end of the study and

total RNA extracted using TriReagent (Molecular Research Cen-ter). Caliper measurements of tumor length and width (shortermeasurement) were recorded, and in some instances tumor grossmorphology was photographed. Tumor volume was calculatedusing the formula for a hemi-ellipsoid (p/6 x L x W x D), wheretumorwidthwas used as an estimate for depth (D). Tumorweightwas determined by subtracting the weight of the contralateralkidney from the kidney weight implanted with tumor.

Tumor gene expression assaysApproximately 500 ng RNA was reverse-transcribed using the

High Capacity cDNA Reverse Transcription Kit (Life Technolo-gies). Quantitative PCR was performed by using a 7500 Fast orStepOnePlus Real-Time PCR system (Life Technologies). TheDDCt method was used to calculate relative gene expression.Primer sets used are described in Supplementary Data.

Statistical analysisSignificance of relative mRNA expression between treatment

and control groups was determined using a Student t test. Signif-icance of changes in tumor volume or weight was determinedusing a Mann–Whitney U test. Data were analyzed using Graph-Pad Prism.

Additional methodsFlow cytometry, IHC, RGD-DPC plasma clearance, and crea-

tionof SEAP-expressingA498 cells are provided in SupplementaryData.

ResultsFunctional components and characteristics of RGD-DPC

The DPC delivery system described in this study utilizedprotease cleavable masking reagents similar to those describedpreviously (28). The protease cleavable masking reagents weredeveloped to provide the DPC with longer systemic circulationtime compared to the pH-labile maleic anhydride (CDM) mask-ing chemistry used in first-generation DPC (29, 37). For targetingtissue outside of the liver, longer circulation times are necessaryfor targeting receptors that are likely less abundant andpotentiallyless efficient than the Ashwell-ASGPr expressed on hepatocytes(30, 38). The exploitation of RGD-binding integrins frequentlyoverexpressed in cancer but more restrictive in normal tissue hasbeen explored by others as a delivery tool, as well as an integrinantagonist in cancer therapy (31, 39, 40). The current construct,RGD-DPC, targets integrin receptors avb3 and avb5 using apeptidomimetic RGD molecule (34). This RGD mimetic boundto bothavb3 andavb5with high affinity and selectivity, with IC50

values of 1.3� 0.4 nmol/L (n¼ 4) and 4.9� 2.3 nmol/L (n¼ 3),respectively, evaluated using an ELISA-based competitive bindingassay (Supplementary Fig. S1). In contrast, binding to the relatedintegrinavb6 had an IC50 value of 116� 4.9 nmol/L (n¼ 3). LiketheNAG-DPCdescribed previously (28), RGD-DPC comprises anethoxyethylamine acrylate polymer modified with a mixture ofprotease cleavable PEG masking reagents with or without theligand conjugated. The optimal number of RGD mimetics perpolymer was determined empirically as 10–20 using in vitrocellular uptake, in vivo tumor uptake, and gene knockdown asreadouts. Upon unmasking, the polymer is expected to perturbendosomal membranes to facilitate cytoplasmic delivery. Thetrigger was attached to the polymer using a hindered-disulfide

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Mol Cancer Ther; 17(1) January 2018 Molecular Cancer Therapeutics142




linker (41) to confer significant serum and extracellular compart-ment stability while enabling efficient dissociation from thedelivery vehicle in the tumor cell cytoplasm. Using Cy5-labeledRNAi trigger tomeasure the intact conjugate, the serumhalf-life ofRGD-DPC was 2 hours, whereas unconjugated RNAi trigger was<5 minutes. The serum half-life for PEGylated DPC (withoutRGD) was 12 hours. The shorter serum half-life for RGD-DPCsupports an RGD-mediated cellular uptake mechanism. Thenanoparticles are approximately 20 nm in size and with a nearneutral zeta potential, features that are essential for tumor extrav-asation and to minimize nonspecific cellular binding (42, 43). Asummary of the physical characteristics of the RGD-DPC issummarized (Supplementary Table S1). A schematic of the DPCnanoparticle is shown in Fig. 1.

Expression of integrins avb3 and avb5 and RGD-DPCinternalization

To evaluate the utility of integrin receptors as an effectivemeansto deliver RNAi therapeutics, the cell-surface expression profile ofintegrins avb3 and avb5 were evaluated on four commerciallyavailable RCC cell lines, A498, 786-O, Caki-1, andCaki-2, by flowcytometry. The expression of avb3 and avb5 tabulated as specificmean fluorescence intensity (sMFI) is summarized in Table 1. Theexpression of avb3 is several-fold higher in A498 and Caki-2 cellscompared to 786-O and Caki-1. In contrast, the expression ofavb5 was similar between the four cell lines. Binding and inter-nalization of Cy3-RGD-DPC was strongest in A498 and Caki-2cells, consistentwith their relatively high expression levels ofavb3(Fig. 2). These results suggest that RGD-DPC exhibits a preference

foravb3 in vitro.However, as theRGDmimetic showshigh affinityto both receptors, this may reflect the combined abundance ofboth receptors rather than preference of avb3 versus avb5 integ-rin. Further demonstration of RGD-dependent binding wasobserved with a DPC without ligand or a DPC with RGE-controlligand showing background fluorescent signals (SupplementaryFig. S2A). Consistent with an endocytic uptake mechanism, afteran overnight incubation, approximately 80% of the internalizedRGD-DPC in A498 cells was localized to lysosomemarker LAMP-1–positive vesicles (Supplementary Fig. S2B).

To evaluate the potential usage of this delivery approach,the expression of integrin avb3 in a commercially availablerenal cancer tissue microarray was profiled (CT565907; Ori-Gene Technologies). Among the samples showing histologicmorphology consistent with ccRCC (25/50), approximately80% (20/25) showed good (medium to strong) b3 membranestaining, and nine of these (36% of all ccRCC samples)showed strong b3 expression, similar in staining intensity asA498 cells derived tumors (Supplementary Fig. S3). Theseobservations are consistent with those reported in the litera-ture (32, 33). The selectivity of the b3 antibody was validatedusing tumor tissue sections derived from cell lines known toexpress or lack b3 as determined by flow cytometry. As integrinb3 is known to pair with only av or aIIb (platelet-specific)subunits (44), this should provide a good representation oftumor avb3 staining.

In vivo DPC uptake in established orthotopic xenograftsAlthough in vitro validation of ligand binding provided spec-

ificity, efficient in vivo delivery to established tumors is an oblig-atory step for a successful model and ultimately an effectivetherapeutic. To evaluate in vivo delivery to established tumors,100 mg of Cy3-RGD-polymer was injected intravenously via thetail vein into tumor bearing mice established with either A498,786-O, Caki-2, or Caki-1 cells in the left kidney subcapsule.Delivery of Cy3-RGD-DPC to tumor was evaluated by confocalmicroscopy examination of frozen tissue sections. The efficiencyof uptake and internalization to tumor parenchymal cells showeda correlation with the cell lines expressing higher levels of avb3integrin, A498 (Fig. 2A and E) and Caki-2 (Fig. 2C and G). A498

Figure 1.

Schematic illustration of RGD-DPC.Polymer EAP (green curve with amineside chains presented as green barsand alkyl groups depicted as purplebars). Theamine side chains of EAParemodified with PEG (gray squiggles) orintegrin-targeting ligand RGD (orangespheres) via protease-labile bonds(blue spheres). RNAi trigger isattached to the polymer with a linkercontaining a hindered-disulfide bond(twomagenta spheres) as depicted onthe end of the polymer. Thisillustration is used with permissionfrom Rozema and colleagues (28).

Table 1. Relative expression of integrins avb3 and avb5 analyzed by flowcytometry

Integrin receptor expression levels (sMFI)Cell lines avb3 avb5

A498 15.13 5.93786-O 7.04 6.11Caki-1 5.14 4.85Caki-2 35.26 6.72

NOTE: Values of sMFI were calculated by dividing MFI values from cells stainedwith antibody by MFI values from unstained cells alone.






tumors exhibited morphology typical of ccRCC whereas Caki-2showedmorphology consistentwithpapillary renal carcinoma, asreported (45). RGD-polymer with no ligand or RGE controlligand showed no tumor uptake in A498 tumor cells (Supple-mentary Fig. S2A, bottom row).

Differences in the tumor architecture of these tumors may alsohave influenced effective delivery to cancer cells. The parenchymalcancer cells in 786-O and Caki-1 tumors were heavily surroundedby a layer of stromal myofibrocytes (strongly stained by AlexaFluor 488-phalloidin) that potentially form a more restrictivemicroenvironment that hinders efficiency of RGD-DPC delivery(Fig. 2F and H).

Selection of HIF2a RNAi triggerIn addition to an efficient delivery vehicle, a potent and

specific RNAi trigger is critical to an effective RNAi therapeutic.To identify a lead therapeutic candidate, 187 candidate RNAitrigger sequences highly specific to human EPAS1 (HIF2a),cross-reacting with rhesus and cynomolgus non-human pri-mate sequences, and exhibiting minimal potential off-targethybridization to nontarget genes, including HIF1a, and seedregions of microRNAs were selected by in silico analysis among5,166 potential human-specific EPAS1 RNAi triggers. Sequencescontaining known SNPs directed against target sites wereexcluded from the screen set. The candidate sequences weresynthesized with 20-O-methyl/20-fluoro-modified nucleotidesto provide nuclease resistance, and to abrogate potentialimmune activation (46). The gene inhibition potency of theRNAi trigger screen set was evaluated by in vitro transfection.The relative potency of the siRNAs was ranked using a waterfall

plot. Additional sequence selection information is available inour published patent application (35). Five of the most potentsequences were then further evaluated in a gene silencing studyin tumor-bearing mice (Supplementary Fig. S4). The mostpotent trigger (sequence ID T4) was selected for further eval-uation and used in subsequent animal studies.

Ligand-dependent dose–response and duration of HIF2a genesilencing in A498 tumor-bearing mice

HIF2a-conjugated DPC modified with or without RGD wasinjected into A498 tumor-bearing mice (n ¼ 3 per group) viathe tail vein at 5, 10, and 15 mg/kg. Animals were enrolled intothe efficacy study 4 to 5 weeks after tumor establishment(confirmed by serum SEAP levels). Serum SEAP levels wereused to randomize animal group assignment. HIF2a mRNAexpression relative to mice receiving delivery buffer alone wasdetermined on study day 5 (study day 1¼ injection day). Dose-dependent HIF2a gene silencing was observed in the micereceiving DPC with the RGD mimetic targeting ligand, whereasDPC without RGD was ineffective (Fig. 3). At the highest doseadministered, an 87% � 2.5% reduction in relative HIF2amRNA expression level was observed.

Todetermine thebest dosing regimen formultidose studies, theduration ofHIF2a gene silencing after a single injection of 15mg/kg (polymer) of RGD-DPC was evaluated on study days 4, 8, 11,and 15 (day 1 ¼ injection day). Each time point represented aseparate group of animals (n¼ 3). Tumor samples were collectedat each timepoint and relative gene expressionwasbatch analyzedat the end of the study. The results showed substantial genesilencing on study day 4 (78% � 7.1%; Fig. 4A), and maximal

Figure 2.

In vitro and in vivo cellular uptake of RGD-DPC. A–D, RGD-DPC internalization in cultured cells. Indicated renal cancer cell lines cultured on a cover glasswere incubated with RGD-DPC formulated using Cy3-labeled polymer (red) in complete media at 2.5 mg/mL for 24 hours. Cell were fixed and counterstained

with Alexa Fluor 488-phalloidin (20 nmol/L) for actin (green) and TO-PRO�-3 (40 nmol/L) for cell nuclei (blue). Cover glass mounted slides were analyzedusing a LSM710 confocal microscope. Confocal micrographs depicting a single 0.5-mm optical section are shown (magnification, 630�). E–H, RGD-DCPinternalization in tumors implanted in mouse kidneys. Indicated cell lines were implanted into kidney subcapsule. RGD-DPC containing 100 mg of Cy3-labeledpolymer (red) was injected via the tail vein. Tumors were excised 4 hours after injection and frozen tissue sections were prepared as described previously (29).Tissue sections were counterstained and analyzed as described above. Scale bars for A–D and E–H are 20 mm.

Wong et al.





gene silencing (89%� 2.1% reduced) on study day 8. Maximumgene silencing was sustained until study day 11. By study day 15,recovery of HIF2a expression began but remained significantlyreduced by 79� 7.7%. As a tool tomonitor tumor growth, serum

SEAP levels were evaluated and were lower in the RGD-DPC–treated group (Supplementary Fig. S5), suggesting an effect ongrowth after a single injection. In parallel to HIF2a mRNAexpression, recovery of SEAP expression was apparent at the end

Figure 3.

Ligand-dependent dose titration inA498-SEAP orthotopic tumor bearingmice. Relative HIF2a mRNAexpression on day 5 (day 1 ¼ injectionday) compared with control (5%dextrose in water, D5W) measured byqRT-PCR in animals receiving 5, 10, or15 mg/kg of DPC (base polymer) withor without ligand, approximately 1.5, 3,or 5 mg/kg of siRNA, respectively,was dosed. Relative expressionshown were normalized geometricmeans � SD.

Figure 4.

Duration of mRNA knockdown andeffects on tumor growth. A, tumor-bearing mice injected with a singledose of 15 mg/kg of RGD-DPC(polymer; 5 mg/kg siRNA equivalent)were evaluated for HIF2a mRNAexpression over time. Relativeexpression normalized to miceinjected with D5W was used ascontrol. Values shown werenormalized geometric means � SD.Separate groups of mice (n ¼ 3) wereused for each time point. D5W,dextrose 5% in water; � , P < 0.05. B,Effects on tumor weight, mean � SD.Day 15 tumor weights were notstatistically significantly different(NS). C, Tumor histology by H&Estaining on day 4, 8, 11, and 15 (day 1¼injection day). Scale bars ¼ 100 mm.






of the duration study (Supplementary Fig. S5). The effect ontumor growth was further substantiated by the overall smaller,although not statistically significant, tumors observed on day 15compared with those of the control-treated group after excisingand weighing (Fig. 4B). Furthermore, histologic examinationshowed evidence of tumor degeneration and the presence ofblood-filled cavities surrounding areas of collapsing tumor integ-rity (Fig. 4C).Maximal antitumor effectswere observed during thetime points corresponding to peak HIF2a gene silencing.

HIF2a-RGD-DPC multidosing study in A498-SEAP tumor-bearing mice

A multidosing study was conducted to further assess the effectof HIF-2a gene silencing on tumor growth. On the basis of theobservation that HIF2a gene silencing after a single dose of RGD-DPC injection was sustained for up to 14 days, a multidosingstudy in which four biweekly doses of 10 mg/kg RGD-DPC wasconducted. A submaximal efficacious dose of 10 mg/kg waschosen to allow for better evaluation of potential additive effectsof repeat dosing. Fourmice per treatment groupwere randomizedon the basis of pretreatment SEAP expression. Serum SEAP levelswere determined predose and throughout the course of treatmentto indirectly evaluate tumor growth. Potential adverse effects ofrepeat dosing were monitored by daily general physical healthcheck, weekly clinical chemistry evaluations, and body weightmeasurements. One week after the fourth and final RGD-DPCinjection, contralateral and tumor-bearing kidneys were excised,photographed, examined for gross morphology, and tumor mea-surements were recorded. Baseline tumor volume was obtainedby collecting tumor measurements from a similar cohort ofanimals on the day treatment was initiated for reference. Tumortissues were then processed for gene expression and histologyevaluations.

Upon excision of kidney tumors on day 49 and study comple-tion, dramatic differences in tumor sizes were apparent by visualexamination of gross tumor morphology (Fig. 5A). Animals thatwere injected with delivery buffer D5W showed significant tumorgrowth. In contrast, calculated tumor volumes from the RGD-DPC–treated group were not significantly different as comparedwith baseline pretreatment tumor volume, suggesting highlyeffective tumor growth inhibition over the course of RGD-DPCtreatment (Fig. 5B). Tumor growth inhibition over the course oftreatmentwas also apparent based on relatively unchanged serumSEAP levels (Fig. 5C). Relative HIF2amRNA expression was 85�7% reduced compared to animals injected with D5W (Fig. 5D).The expression of several known HIF2a-regulated genes, VEGFa,Glut1, and EGLN3 were also reduced by 47% � 10.6%, 42% �18.6%, and 49% � 6.4%, respectively. The expression of thetumor suppressor gene p53 was elevated by 28% � 9%(Fig. 5D). In a separate study, the effects of RGD-DPC containinga control RNAi trigger (luciferase) were evaluated, indicating nosignificant effects on tumor growth or HIF2a mRNA expression.

Histologic examination further revealed widespread tumorstructural degeneration in the HIF2a-RGD-DPC–treated group.Tumor degeneration was associated with blood-filled cavitieswhere CD31-positive tumor vasculature was significantlyreduced, indicative of vascular collapse (Fig. 5E), a phenotypepotentially a result of the reduction in tumor VEGFa expression,secondary to HIF2a gene silencing. However, given the histologicevidence of general destruction of tumor architecture showingsignificant tumor degeneration, the overall effects on tumor

growth is profound and not fully characterized by evaluatingtumor volume alone. The blood-filled cavities are histologicallydistinct from spontaneous tumor necrosis, which is oftenobserved in overgrown untreated tumors. These features likelyresult from downregulation of HIF2a-regulated tumor growth–promoting genes (Fig. 5D), leading to cell death and disruption oftumor integrity. Importantly, the repeat dosing regimen was well-tolerated, as no significant changes were observed in either bodyweight ormajor clinical chemistry parameters including AST, ALT,BUN, creatinine, and total bilirubin (Supplementary Table S2).No significant histologic findings were observed in the contralat-eral kidney, liver, or spleen.

DiscussionDevelopment of drug resistance, particularly to small-molecule

drugs, are frequent challenges in cancer therapy. The currentstandard-of-care treatments for metastatic ccRCC have notescaped from this fate. Thehigh frequency ofVHL loss-of-functionmutation in ccRCC and the subsequent overexpression of HIF2ahas long been an attractive therapeutic target (16). In general,transcription factors such as HIF2a have generally been consid-ered undruggable therapeutic targets (47). Recently, severalreports described the antitumor effects of a small-moleculeHIF2aantagonist that binds to ahydrophobic binding pocket discoveredin HIF2a PAS-B domain (16, 20–22). Although these studiesshowed encouraging results that validated HIF2a as a therapeutictarget, certain limitations are apparent. These included preexistingdrug-resistant mutations, posttreatment mutations discoveredsurrounding the binding pocket of this small molecule, and thedependence of some tumors on cellular pathways other thanVHL-HIF (20–22).

The modular nature of the RGD-DPC delivery system allowseach of the individual components to be independently opti-mized to maximize therapeutic potential. In addition, the recep-tor/ligand pair or the target gene can be substituted when appro-priate. TheRGDmimetic used in the current studywas designed totarget integrins overexpressed in some ccRCC patients (32, 33).We showed that the efficiency of RGD-DPC delivery to varioustumor cells correlated with overall abundance of RGD-bindingintegrin expression, particularly avb3. The RGD mimetic is nec-essary for efficient tumor delivery of the DPC.Our results indicatethat RGD-binding integrin expression in tumor cells facilitatesefficient delivery of RNAi triggers likely through receptor-medi-ated internalization. Furthermore, expression of avb3 wasreported to correlate with tumor progression and metastasis(32, 48), a patient population where alternate treatment overcurrently available therapies is needed. The RNAi trigger thera-peutic payload may be specific to one or more potential targetgenes. Alternate effective RNAi sequences of the same target genesequence can be readily substituted, if necessary.

The Caki-2 cell line is characterized as a papillary renal cancerwith no VHLmutation that expresses bothHIF1a andHIF2a (45,49). Although the contribution of HIF in tumor progression inthis subtype of renal cancer is unclear, 2 weekly doses of 10mg/kgof HIF2a-RGD-DPC induced signs of tumor degeneration andapoptosis (unpublished results). HIF2a expression was inhibitedapproximately 70%, but no effect was observed on HIF1a expres-sion, validating the specificity of the HIF2a trigger.

The RNAi delivery system described in the current reportprovided a proof-of-concept approach to an alternative

Wong et al.





therapeutic modality. Questions such as maximum tolerateddose, biodegradability, and toxicity upon repeat dosing, needto be assessed before this technology advances to the clinic.Despite significant efforts to reduce the immunomodulatorypotential of the HIF2a-RGD-DPC therapeutic, evaluation ofpossible cytokine induction, complement activation and otherimmune reactions warrants attention (46, 50). A successful

first-in-class RNAi therapeutic for ccRCC will provide a much-needed alternative treatment choice, not only as a potentialmonotherapy, but more attractively in combination with cur-rent treatment options. Tumor targeting by HIF2a inhibitionmay augment immune-checkpoint control therapies, metabol-ic regulators, or other therapeutics that elicit orthogonalmechanisms of action.

Figure 5.

Multidose tumor growth inhibitionstudy in orthotopic A498-SEAP tumorbearingmice.Micewere injected every2 weeks with either 10 mg/kg RGD-DPC or D5W. A total of 4 doses wereadministered to 4 mice per group. Acohort of na€�ve tumor bearing micewas terminated on day 1 (start ofstudy) as baseline reference. Studywas terminated onday49, 1week afterfinal injection. A, gross tumormorphologies. From each mouse, thekidney implanted with tumor cells isshown on the left and the contralateralkidney without tumor cellimplantation is shown on the right. B,tumor volumes calculated with calipermeasurements; � , P < 0.05. C, SEAPexpression during treatment. TheSEAP reporter protein expression wasused to monitor tumor growth. Serumwas collected weekly. Fold-changesrelative to predose levels of eachanimal are shown. D, HIF2a andHIF2a-regulated VEGF-A, GLUT-1,EGLN3, and p53 mRNA expression.Normalized geometric mean � SD areshown. � , P < 0.05. E, Tumor histology(H&E) and CD31 staining.Representative images are shown.Scale bar for H&E ¼ 100 mm, CD31staining ¼ 50 mm.






Disclosure of Potential Conflicts of InterestS.B. Kanner has ownership interest (including patents) in Arrowhead Phar-

maceuticals.Nopotential conflictsof interestweredisclosedby theotherauthors.

Authors' ContributionsConception and design: S.C. Wong, W. Cheng, A.L. Nicholas, D.H. Wakefield,A. Almeida, J. Carlson, D.B. Rozema, D.L. Lewis, S.B. KannerDevelopment of methodology: S.C. Wong, W. Cheng, A.L. Nicholas,D.H. Wakefield, A. Almeida, A.V. Blokhin, Z.C. Neal, J. Hegge, D.B. RozemaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):W. Cheng, Z.C. Neal, V. Subbotin, J. Hegge, S. Bertin,V.S. TrubetskoyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.C. Wong, W. Cheng, H. Hamilton, D.H. Wakefield,A. Almeida, Z.C. Neal, V. Subbotin, S. Bertin, V.S. Trubetskoy, D.B. Rozema,D.L. LewisWriting, review, and/or revision of the manuscript: S.C. Wong, W. Cheng,H. Hamilton, A.L. Nicholas, A.V. Blokhin, Z.C. Neal, V. Subbotin, J. Hegge,D.B. Rozema, D.L. Lewis, S.B. KannerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.C. Wong, A.L. Nicholas, A.V. Blokhin,G. Zhang

Study supervision: S.C. Wong, D.H. Wakefield, J. Hegge, D.B. Rozema,D.L. LewisOther (managed efforts of small-molecule chemistry group to developsynthetic pathway that allowed to generate required amounts of targetingligands and masking reagents.): A.V. BlokhinOther (synthetic chemistry): J. Carlson

AcknowledgmentsThe authors would like to thank Lauren Almeida andMeganWaters for RNAi

trigger synthesis. We also thank Aaron Andersen, Tracie Milarch, and RachaelSchraufnagel for technical assistance.

The entire work was supported by Arrowhead Pharmaceuticals Inc.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 22, 2017; revised August 28, 2017; accepted October 10, 2017;published OnlineFirst October 27, 2017.

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2018;17:140-149. Published OnlineFirst October 27, 2017.Mol Cancer Ther So C. Wong, Weijun Cheng, Holly Hamilton, et al. Carcinoma

-Targeted RNAi Therapeutic Inhibits Clear Cell Renal CellαHIF2

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