intravesicaldeliveryofsmallactivatingrnaformulatedinto ... · t24-p cells are a highly tumorigenic...

Therapeutics, Targets, and Chemical Biology

Intravesical Delivery of Small Activating RNA Formulated intoLipid Nanoparticles Inhibits Orthotopic Bladder TumorGrowth

Moo Rim Kang1, Glen Yang1, Robert F. Place2, Klaus Charisse3, Hila Epstein-Barash3, Muthiah Manoharan3,and Long-Cheng Li1

AbstractPractical methods for enhancing protein production in vivo remain a challenge. RNA activation (RNAa) is

emerging as one potential solution by using double-stranded RNA (dsRNA) to increase endogenous geneexpression. This approach, although related to RNA interference (RNAi), facilitates a response opposite to genesilencing. Duplex dsP21-322 and its chemically modified variants are examples of RNAa-based drugs that inhibitcancer cell growthby inducing expression of tumor suppressor p21WAF1/CIP1 (p21). In this study, we investigate thetherapeutic potential of dsP21-322 in an orthotopic model of bladder cancer by formulating a 20-fluoro-modifiedderivative (dsP21-322-20F) into lipid nanoparticles (LNP) for intravesical delivery. LNP composition is based uponclinically relevant formulations used in RNAi-based therapies consisting of PEG-stabilized unilamellar liposomesbuilt with lipid DLin-KC2-DMA. We confirm p21 induction, cell-cycle arrest, and apoptosis in vitro followingtreatmentwith LNP-formulated dsP21-322-20F (LNP-dsP21-322-20F) or one of its nonformulated variants. Both 20-fluoro modification and LNP formulation also improve duplex stability in urine. Intravesical delivery of LNP-dsP21-322-20F into mouse bladder results in urothelium uptake and extends survival of mice with establishedorthotopic human bladder cancer. LNP-dsP21-322-20F treatment also facilitates p21 activation in vivo leading toregression/disappearance of tumors in 40% of the treated mice. Our results provide preclinical proof-of-conceptfor a novel method to treat bladder cancer by intravesical administration of LNP-formulated RNA duplexes.Cancer Res; 72(19); 5069–79. �2012 AACR.

IntroductionBladder cancer is thefifthmost commonhumanmalignancy

(1). Approximately 70% of bladder cancer incidences arediagnosed at a superficial stage in which about half will recurfollowing transurethral resection (TUR) despite post-op ther-apy. Intravesical administration of chemical and/or immuno-logic agents (e.g., mitomycin, Bacillus Calmette-Gu�erin, etc.) isoften used to eradicate residual tumor cells; however, suchtreatments can have limited efficacy and adverse side effects(2). Asmany as 10% to 30% of recurrent tumors will progress toa higher grade and stage inevitably forming locally invasivecancer (3, 4). Radical cystectomy remains the standard treat-

ment ofmuscle-invasive bladder tumors, which often results insignificant deterioration of urinary and sexual function. Assuch, new therapeutic options are needed for treating residualtumor after TUR, as well as advanced bladder cancer.

RNA interference (RNAi) is currently one of the leadingplatform technologies being developed in the clinic as anoligonucleotide-based approach to gene therapy. It is anevolutionarily conserved mechanism of gene regulation bywhich small double-stranded RNA (dsRNA) molecules—termed small interfering RNA (siRNA)—degrade complemen-tary messenger RNA to silence gene expression (5). By usingsiRNAs as therapeutic compounds, it is possible to block theproduction of disease-causing proteins. However, effectiveuptake of siRNAs by cells requires support of a delivery vehicleto penetrate the cell membrane. Lipid-based vectors remainthe preferred approach for siRNA delivery both in vitro and invivo. Precise formulation with lipids and other molecularcomponents (i.e., polyethylene glycol-lipid, cholesterol, etc.)allows encapsulation of siRNAs into stable liposomal nano-particles (LNP) for improved bioavailability. Currently, ioniz-able lipid DLin-KC2-DMA is one of the leading materials forLNP formulation to safely deliver siRNA molecules in vivo (6).

Safe strategies to selectively enhance gene and/or proteinproduction remain a challenge in gene therapy. Viral-basedsystems are effective at delivering exogenous constructs tofacilitate gene overexpression, but have serious drawbacks (i.e.,

Authors' Affiliations: 1Department of Urology and Helen Diller Compre-hensive Cancer Center, University of California; 2RNA Therapeutics Inc.,San Francisco, California; and 3AlnylamPharmaceuticals, Inc., Cambridge,Massachusetts

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M.R. Kang and G. Yang authors contributed equally to this work.

Corresponding Author: L-C. Li, Department of Urology and Helen DillerComprehensiveCancerCenter, University ofCalifornia, SanFrancisco,CA.Phone: 415-476-3802; Fax: 415-514-4987; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-1871

�2012 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 5069

on March 16, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst August 6, 2012; DOI: 10.1158/0008-5472.CAN-12-1871

http://cancerres.aacrjournals.org/

adverse effects on host genome integrity, immunologic con-sequences, etc.) that hinder progression in the clinic. Recently,small dsRNAs—termed small activating RNA (saRNA)—havealso been shown to induce gene expression in a phenomenonreferred to as RNA activation (RNAa; refs. 7, 8). Several modelsof RNAa have been described including transcriptional acti-vation by targeting promoter sequences and/or overlappingnoncoding regulatory transcripts using saRNA (7, 9–11). Thistechnique offers a similar approach as RNAi, while represent-ing a new strategy to stimulate endogenous gene expression.

The tumor suppressor protein p53 controls cell-cycle pro-gression, senescence, and apoptosis in response to DNA dam-age through transactivation of numerous growth inhibitorygenes (12). One such downstream target is the CDK inhibitorp21WAF1/CIP1 (p21). By suppressing CDK activity, p21 promoteshypophosphorylation of the retinoblastoma (Rb) protein lead-ing to cell-cycle arrest (13). In addition, p21 interacts withproliferating cell nuclear antigen (PCNA) interfering with DNAreplication and cell division (14). In fact, ectopic expression ofp21 has been shown to inhibit tumor growth and induceapoptosis both in vitro and in vivo (15–17). As such, p21 is amajor downstream effector of p53 signaling and is itselfconsidered a potent tumor suppressor.

Alterations to the p53 pathway represent one of the majorcontributors to bladder tumorigenesis (18, 19). Despite fre-quent disruption of the p53 protein, loss-of-functionmutationsleading to p21 inactivation are a rare event (20, 21). Rather,bladder cancer progression has been associated with generaldecreases in p21 expression (20, 21). In this regard, p21 may bean ideal target for RNAa to inhibit bladder cancer cell growth.Although the therapeutic potential of RNAa has been shown inxenograft tumors following intratumoral injection of LNP-formulated saRNA molecules (22), it has not been evaluatedin a clinically relevantmodel of cancer using a practical route ofdrug administration. In the present study, we assess theantitumor activity of RNAa in an orthotopic model of bladdercancer following intravesical delivery of LNP-formulatedsaRNA to facilitate p21 induction. LNP composition consistedof lipid DLin-KC2-DMA and resembled RNAi-based formula-tions. We confirm that LNP-formulated saRNA facilitates p21induction and possesses antiproliferative activity in bladdercancer cells. In vivo analyses reveal that intravesical treatmentofmicewith orthotopic bladder cancer extends animal survivaland inhibits tumor growth. Our results provide proof-of-prin-ciple that DLin-KC2-DMA-based nanoparticles have applica-tion in delivering RNA duplexes to facilitate RNAa in vivo, aswell as define a candidate RNAa-based drug for the treatmentof localized bladder cancer by intravesical instillation.

Materials and MethodsCell culture and transfection

Human bladder cancer cell line KU-7 engineered to stablyexpress firefly luciferase and green fluorescent protein (GFP)was purchased from Caliper Life Sciences. T24-P cells are ahighly tumorigenic bladder cancer subline derived from T24cells (23) acquired fromDr. Jer-TsongHsieh at theUniversity ofTexas Southwestern Medical Center, Dallas, Texas. No furtherauthentication of cell lines was done by the authors. KU-7 cells

were cultured in RPMI 1640 medium supplemented with 10%fetal bovine serum (FBS) and 50 mg/mL gentamycin sulfate in ahumidified atmosphere of 5% CO2 at 37�C. T24-P cells weremaintained in T-Medium (Invitrogen) also supplemented with10% FBS and 50 mg/mL gentamycin sulfate. The day beforetransfection, cells were plated in growth medium withoutantibiotics at a density of �50% to 60%. Transfection ofnonformulated saRNA was carried out using LipofectamineRNAiMAX (Invitrogen) according to manufacturer's instruc-tions. Treatment of LNP-formulated duplexes was done bysimply adding LNP solution directly to cell culture medium.The following day, transfection media was replaced with freshmedia containing antibiotics. All saRNA sequences includingchemical modifications are listed in Table S1.

Lipid nanoparticle compositionNanoparticles were prepared with the ionizable lipid

1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), disteroylphosphatidylcholine (DSPC), cholesterol,and 1-(monomethoxy polyethyleneglycol)-2,3-dimyristoylgly-cerol (PEG–DMG) using a spontaneous vesicle formationformulation procedure as previously described (24). Detailedprotocol is available in Supplementary Methods.

Urine stability assaySolutions containing 1 mL of 20 mmol/L saRNA and 10 mL

undiluted mouse or human urine were incubated at 37�C. Themixtures were flash frozen in liquid nitrogen at desired timepoints to halt nuclease activity until the time course wascomplete. Samples were subsequently mixed with loadingbuffer and analyzed on an agarose gel to visualize duplex levels.

saRNA uptake analysisT24-P cells (1.0 � 105) were treated with LNP-formulated

FITC-labeled dsP21-322-0F (dsP21-322-20F-FITC) for 24 hours.Female C57BL/6 mice were anaesthetized with isoflurane andbladders rinsed twice with 0.2 mL PBS through a 24-guagecatheter. LNP-formulated dsP21-322-20F-FITC (1.68 mg/kg,�33.5 mg) was administered intravesically and retained in thebladder for 2 hours by tying off the orifice of the urethra.Mice were sacrificed at 2 or 9 hours (including indwelling time)after saRNA delivery. Animals treated for 9 hours were deniedwater to limit excretion of formulated duplex. Whole bladderswere removed, placed in cryomolds containing optimal cuttingtemperature compound, and frozen on dry ice. Cryostat sec-tions were cut to 8 mmandmounted onto glass slides. Both celland tissue slides were fixed in 10% neutral buffered formalinand washed with PBS containing sucrose. The fixed sectionswere counterstained with 40,6-diamidino-2-phenylindole(DAPI) using VECTASHIELD Mounting Medium (Vector Lab-oratories) and saRNAuptakewas visualized using fluorescencemicroscopy.

Orthotopic model of bladder cancer and intravesicaltreatment

The animal study was carried out according to a protocolapproved by the Institutional Animal Care andUseCommittee.The orthotopic murine model of bladder cancer was a

Kang et al.

Cancer Res; 72(19) October 1, 2012 Cancer Research5070




modification of a previously described technique (25, 26).In brief, female nude (nu/nu) mice at 8 to 10 weeks ofage (Simonsen Laboratory) were anaesthetized with isoflur-ane. Chemical lesions to the bladder urothelium were carriedout by injecting 10 mL of 0.5 M silver nitrate into the bladder ofeach animal through a 24-gauge catheter. Bladders werewashed with PBS and subsequently instilled with KU-7 cells(2.0 � 106) suspended in 50 mL PBS via catheter. At this point,animals were dosed with ketamine (100 mg/kg) and xylazine(10 mg/kg) by intraperitoneal injection. Cells were retained inthe bladder for 2 hours by tying off the orifice of the urethra.One day following tumor cell implantation, in vivo biolumi-nescence imaging (BLI) of KU-7 cells was evaluated on aIVIS Spectrum Imaging System (Caliper Life Sciences) follow-ing intraperitoneal administration of 150 mg/kg D-luciferin(Gold Biotechnology). Bioluminescence signal (BLS) wasacquired and analyzed using Living Image software version4.10 (Xenogen). Regions of interest (ROI) were defined man-ually to encompass the bladder and quantify signal intensity.Tumor-bearing mice were randomly divided into 3 treatmentgroups (PBS, LNP-dsCon-20F, and LNP-dsP21-322-20F) eachcontaining 9 to 10 animals. Mice received 3 mg/kg of LNP-formulated duplex in a volume of 50 mL by intravesical admin-istration into the bladder via catheter. Each treatment wasretained in the bladder for 2 hours by tying off the orifice of theurethra while the animal was anaesthetized. PBS treatmentswere done at volumes equivalent to LNP-dsP21-322-20F doses.Animals were treated 14 total times every 3 days beginning atday 4 postimplantation. BLS and body weight were monitoredweekly. A death was recorded if animals met AVMA guidelinesfor euthanasia as a result of tumor burden including bodycondition score (BCS)�2 and�20%weight loss. The studywasterminated 8weeks following tumor installation. Any survivingmice were euthanized and all major organs including bladderswere inspected for cancer. Bladder tumor tissue was divided inhalf and placed in either RIPA buffer containing proteaseinhibitors or 10% neutral buffered formalin for immunoblotanalysis or immunohistochemistry (IHC), respectively.

Statistical analysesThe differences in continuous variables between treatments

were assessed by Student t test (for 2 treatments) or ANOVA(for 3 or more treatments). Log rank tests determined statis-tical significance of Kaplan–Meier survival curves. Significancewas defined as P < 0.05. Additional experimental proceduresare described in Supplementary Methods.

ResultsdsP21-322 induces p21 expression in human bladdercancer cellsBy screening several duplexes targeting the p21 promoter,

dsP21-322 has been identified as a lead saRNA molecule withantitumor activity in prostate cancer (22). Earlier in vitrostudies have showed that dsP21-322 also possesses growthinhibitory function in T24 and J82 bladder cancer cell lines (27).To further evaluate dsP21-322 activity in bladder cancer, wetransfected KU-7 and T24-P cells with dsP21-322 for 72 hours.

Analysis of mRNA expression revealed that dsP21-322 pro-foundly induced p21 levels compared with mock and nonspe-cific control (dsCon) treatments (Fig. 1A). Induction of p21wasfurther confirmedby immunoblot analysis. As shown in Fig. 1B,elevated levels of p21 protein correlate to the increase in p21mRNA expression. Morphologically, dsP21-322 treatmentcaused cells to appear less dense and acquire larger, moreflattened shapes; phenotypes indicative of impeded cell growth(Fig. S1). These results imply dsP21-322 retains RNAa activityand growth inhibitory function in KU-7 and T24-P cells.

Activity of LNP-formulated dsP21-322-20F and itsnonformulated variants

Chemical modification (e.g., 20-fluoro) is often required toimprove the medicinal properties of duplex RNAs for in vivoapplication (28). We previously defined a chemically modifiedvariant of dsP21-322 (dsP21-322-20F) with improved nucleaseresistance in serum and reduced immunostimulatory activityby introducing 20-fluoro modifications to all pyrimidinenucleotides in its antisense strand (22). To further improveits in vivo applicability, we formulated dsP21-322-20F into lipidnanoparticles (LNP-dsP21-322-20F) composed of ionizablelipid DLin-KC2-DMA creating PEG-stabilized unilamellar lipo-somal vesicles. Nonspecific lipid-based nanoparticles (LNP-dsCon-20F) were also formulated as a control. Comparativeanalysis of in vitro potency between nonformulated dsP21-322and dsP21-322-20F in KU-7 cells revealed similar EC50

(half maximal effective concentration) values of 1.42 and1.70 nmol/L at 72 hours, respectively (Fig. 1C). In addition,dsP21-322-20F retained strong RNAa activity as compared withdsP21-322. As shown in Fig. 1C, dsP21-322-20F consistentlyincreased p21 levels by �9-fold at maximal activity, whereasdsP21-322 increased p21 levels by �5-fold. Dose-responseexperiments with LNP-dsP21-322-20F indicated an EC50 of0.24 mg/mL with maximal activity reaching�10-fold increasesin p21 levels (Fig. 1C). Immunoblot analyses also revealed dose-dependent induction of p21 protein by dsP21-322, dsP21-322-20F, and LNP-dsP21-322-20F (Fig. 1D). We conducted dose–response experiments evaluating cell viability by MTS assayand calculated IC50 (half maximal inhibitory concentration)values to measure the antiproliferative potency of LNP-dsP21-322-20F and its nonformulated variants. Nonformulated dsP21-322 and dsP21-322-20F had similar IC50 values of 0.72 and 0.74nmol/L, respectively; whereas LNP-dsP21-322-20F had an IC50

of 0.19mg/mLat 72 hours (Fig. S2A). Phenotypically, dsP21-322-20F and LNP-dsP21-322-20F treatments caused dose-depen-dent changes in cell morphology and density similar todsP21-322 (Fig S2B). Taken together, these results indicatethat neither chemical modification nor LNP formulation neg-atively impacted RNAa activity or the growth inhibitory func-tion of dsP21-322.

LNP-formulated dsP21-322-20F and its nonormulatedvariants cause cell-cycle arrest and apoptosis in bladdercancer cells

To investigate cell-cycle distribution, DNA content wasanalyzed by flow cytometry in cells stained with propidiumiodide (PI) following treatment with LNP-dsP21-322-20F or one

Intravesical Delivery of Small Activating RNA

www.aacrjournals.org Cancer Res; 72(19) October 1, 2012 5071




of its variants. As shown in Fig. 2A and B, transfection of dsP21-322 or dsP21-322-20F caused G0–G1 arrest in KU-7 cells asindicated by the increase in G0–G1 cell number and concurrentdeclines in S and G2–M populations. LNP-dsP21-322-20Ftreatment also caused changes in cell-cycle distribution similarto dsP21-322 and dsP21-322-20F corroborating that LNP for-mulation did not interfere with the growth inhibitory functionof dsP21-322-20F (Fig. 2A and B).

Cell-cycle arrest by p21 is primarily mediated althoughinhibition of CDK activity leading to Rb hypophosphorylation(22, 29). As such, we evaluated phosphorylated Rb (p-Rb)levels in KU-7 cells following treatment with dsP21-322,dsP21-322-20F or LNP-dsP21-322-20F. As shown in Fig. 2C,transfection with nonformulated dsP21-322 and dsP21-322-20F reduced p-Rb levels in a dose-dependent manner, whereastotal Rb protein remained generally unchanged. Similarly,LNP-dsP21-322-20F treatment also reduced p-Rb levels atelevated doses (Fig. 2C). These data suggest that p21 inductionresulted in a functional protein capable of manipulating Rbphosphorylation.

Overexpression of p21 has been reported to have proapop-totic function in several cancer cell types contributing to itstumor suppressor activity (12, 27, 30). To quantify the apopto-tic cell fraction following duplex treatments, KU-7 cells wereanalyzed by flow cytometry following staining with 7-AAD andAnnexin V-PE. As shown in Fig. 3A and B, mock and dsContreatments defined baseline apoptosis in KU-7 cells, whereastransfection with dsP21-322 or dsP21-322-20F caused similardose-dependent increases in early and late stage apoptosis.LNP-dsCon-20F mirrored nonformulated control treatments;however, LNP-dsP21-322-20F caused robust detection of earlyand late stage apoptotic cells in a dose-dependent mannersimilar to dsP21-322 and dsP21-322-20F transfections (Fig. 3Aand B). We also measured caspase-3/7 activity (2 key enzymesof the apoptotic signaling cascade) in culture media followingduplex treatments. Transfection of dsP21-322 or dsP21-322-20Fpossessed significantly higher caspase-3/7 activity than controltreatments (Fig. 3C). Likewise, LNP-dsP21-322-20F caused sim-ilar increases in caspase 3/7 signaling, whereas LNP-dsCon-20Ftreatment had nominal effects on caspase-3/7 activity (Fig. 3C).

Figure 1. Activity of LNP-dsP21-322-20F and its nonformulated derivatives in bladder cancer cells. A, human KU-7 and T24-P cells were transfected with 50nmol/L of the indicated duplexes for 72 hours. Mock samples were transfected in the absence of dsRNA. Semiquantitative RT-PCR was used to assess p21and GAPDHmRNA levels. Amplification of GAPDH served as a loading control. B, protein levels of p21 and GAPDH were evaluated by immunoblot analysisusing protein-specific antibodies. C, KU-7 cells were treated with accumulating concentrations of dsP21-322, dsP21-322-20F, and LNP-dsP21-322-20Ffor 72 hours. Expression of p21 was quantified by real-time PCR (mean � SD from 2 independent experiments). Maximum and minimum levels of inductioncorrelate to 100% and 0% response, respectively. Duplex (nmol/L) and nanoparticle (mg/mL) concentration are shown in log scale. Dotted lines illustrateapproximate EC50 values. D, KU-7 cells were transfected at the indicated concentrations of formulated (LNP-dsCon-20F and LNP-dsP21-322-20F) ornonformulated (dsCon, dsP21-322, and dsP21-322-20F) duplex for 72 hours. Protein levels of p21 andGAPDHwere evaluated by immunoblot analysis usingprotein-specific antibodies. Concentrations of LNP-dsP21-322-20F (mg/mL) correspond to total nanoparticle, whereas dsP21-322 and dsP21-322-20Ftreatments represent concentrations (nmol/L) of only the individual duplexes. Data are representative of n ¼ 3.

Kang et al.





Immunoblot analysis was also carried out to monitor reduc-tions in procaspase-3 levels (a marker for caspase-3 activation)and cleavage of PARP (a downstream substrate of caspasesignaling). Transfection with dsP21-322 or dsP21-322-20F ledto reductions in procaspase-3 (caspase-3) with concurrentdetection of PARP cleavage (Fig. 3D). Similarly, LNP-dsP21-322-20F mirrored nonformulated transfection results (Fig. 3D).Taken together, these results indicate that treatmentwithLNP-dsP21-322-20F or its nonformulated variants also promoteapoptosis contributing to the antitumor activity in bladdercancer cells.

Pharmacokinetic properties of LNP-formulated dsP21-322-20FUrine is a liquid waste product excreted by the kidney and

stored in the bladder that is rich in nucleases includingribonucleases (31). To test the stability of LNP-dsP21-322-20Fand its nonformulated derivatives in urine, we incubated equalquantities of duplex with fresh human ormouse urine for up to6 days. Unmodified dsP21-322was generally unstable in humanurine having completed degraded within 3 hours; however, 20-fluoro modification of dsP21-322 (dsP21-322-20F) significantlyincreased duplex stability extending its life to �24 hours (Fig.4A). Packaging RNA into nanoparticles has also been shown toprotect encapsulated cargo from degradation by nucleases(32). As such, LNP formulation further improved its nuclease

resistance increasing its stability to �6 days in human urine.Surprisingly, duplex stability behaved very different in mouseurine. Almost no apparent degradation of unmodified, mod-ified, or LNP-formulated saRNA was observed (Fig. 4A). Sta-bility studies in sterile water resembled the time course inmouse urine suggesting nominal ribonuclease activity and/orinherent duplex stability in mouse urine (Fig. S3A). In support,analysis of untreated mouse urine yielded stable backgrounddetection of low molecular weight nucleic acids, whereashuman urine was devoid of any inherent nucleic acid signal(Fig. S3B). These data indicate that both 20fluoro modificationand LNP formulation improve duplex stability in human urine;however, inherent differences in nuclease activity/composi-tion between human and mouse urine may influence in vivoproperties in either species.

To assess in vitro and in vivo uptake of LNP-formulatedsaRNA, FITC was conjugated to the 30 terminus of dsP21-322-20F (dsP21-322-20F-FITC) and formulated into unilamellar LNPvesicles with DLin-KC2-DMA. When LNP-formulated dsP21-322-20F-FITC was directly added to cultured cells, uptake wasevident in nearly all cells with fluorescence mainly localized tothe cytoplasm and a few punctate nuclear loci at 24 hours (Fig.4B). In vivo uptake was evaluated by directly treating thebladders of normal mice with LNP-formulated dsP21-322-20F-FITC via intravesical administration. After 2 hours oftreatment, dsP21-322-20F-FITC uptake was observed in cells

Figure 2. LNP-dsP21-322-20F andits nonformulated derivativespromote cell cycle arrest and Rbhypophosphorylation. A, KU-7 cellswere treated at the indicatedconcentrations of formulated (LNP-dsCon-20F and LNP-dsP21-322-20F)or nonformulated (dsCon, dsP21-322, and dsP21-322-20F) duplex for72 hours and analyzed by flowcytometry after PI staining. Shownare examples of resulting FL2Ahistograms. B, flow cytometry datawere analyzed to determine cell cycledistribution (mean � SD from 2independent experiments).Percentages correspond to theamount of cells present in thetreatment populations at theindicatedphasesof cell cycle (G0–G1,S, or G2–M). �, P < 0.05; ��, P < 0.01;��, P < 0.001. C, protein levels ofphosphorylated Rb (p-Rb), total Rb(Rb), and GAPDH were determinedby immunoblot analysis. GAPDHserved as a loading control.






lining the inner bladder wall, as well as the interstitial spaces ofthe urothelium (Fig. 4C). Fluorescence remained readilydetectable in bladder cells by 9 hours (Fig. 4D). Images takenat higher magnification clearly show intracellular distributionof dsP21-322-20F-FITC at 2 and 9 hours (Fig. S4). These dataindicate that LNP formulation effectively delivers saRNAduplex into the bladder epithelium.

Intravesical treatment of LNP-dsP21-322-20F inhibitsgrowth of orthotopic bladder tumors

To test the antitumor activity of LNP-dsP21-322-20F in vivo,we established orthotopic bladder cancer in 40 immunocom-promised (nu/nu) mice by intravesical instillation of KU-7cells. BLS from KU-7 cells was detected by measuringphotons/second following intraperitoneal administration ofluciferase substrate (D-luciferin). The day following tumor cellinstillation, 29 mice (72.5%) produced BLS and were randomlydivided into 3 treatment groups including PBS (n ¼ 9), LNP-

dsCon-20F (n ¼ 10), and LNP-dsP21-322-20F (n ¼ 10). Nostatistical difference in BLS intensity or body weight wasdetected between groups at time of randomization. Intrave-sical treatment was initiated on day 4 postimplantation andrepeated every 3 days for 14 total doses (Fig. 5A). BLI was doneweekly to monitor tumor burden. Animal survival was theprimary endpoint.

At the end of the 8 week study, the median survival timefor PBS, LNP-dsCon-20F, and LNP-dsP21-322-20F treatmentgroups was 9, 13, and 45 days, respectively. Five mice (50%)remained alive in the LNP-dsP21-322-20F population, whereas1 mouse (11%) survived in the PBS group and none (0%)represented LNP-dsCon-20F-treated animals. Kaplan–Meiercumulative survival analysis revealed LNP-dsP21-322-20F treat-ment significantly extended animal survival compared withPBS and LNP-dsCon-20F, whereas no significant difference wasdetected between the 2 control groups (Fig. 5B). Four of thesurvivingmice in the LNP-dsP21-322-20F treatment population

Figure 3. LNP-dsP21-322-20F andits nonformulated derivativespromote apoptosis. A, KU-7 cellswere treated at the indicatedconcentrations of formulated(LNP-dsCon-20F and LNP-dsP21-322-20F) or nonformulated (dsCon,dsP21-322, and dsP21-322-20F)duplex for 72 hours and analyzedby flow cytometry after stainingwith 7-AAD and Annexin V-PE.Shown are examples of resultingscatters plots with indicatedquadrant percentagescorresponding to early and latestage apoptosis. B, flow cytometrydata were plotted to compareapoptotic cell populations in eachtreatment (mean � SD from 3independent experiments).Percentages correspond to theamount of cells in early or late stageapoptosis. C, caspase-3/7 activitywas determined by measuringluminescence of a cleavablereporter substrate. Data are plottedas the mean � SD from 3independent experiments.�, P < 0.05; ��, P < 0.01;��, P < 0.001. D, levels ofprocaspase-3 (caspase-3), PARP,and GAPDH were evaluated byimmunoblot analysis. Detection ofa lower molecular product signifiesPARP cleavage.

Kang et al.





showed gradual reductions in tumor burden in which 3 even-tually became tumor-free as evidenced by the disappearance inBLS (Fig. 6A and B). Removal and dissection of tumor-freebladders were devoid of GFP signal upon inspection by fluo-rescence microscopy to further suggest tumor disappearance(Fig. S5). In one of the tumor-free animals, a white area ofscarred tissue with negative GFP signal was found in thebladder wall suggesting regression of an established tumor(Fig. S5). Similar observations have been noted followingregression of prostate xenograft tumors treated with dsP21-322-20F formulated into lipidoid-based nanoparticles (22).Histologic evaluation revealed in some instances that ortho-topic tumor from control groups invaded into the bladderdetrosalmuscle layer (Fig. S6A). In addition, kidneymetastaseswere found in 2 of 9, 1 of 10, and 3 of 10 mice within the PBS,

LNP-dsCon-20F and LNP-dsP21-322-20F treatment groups,respectively (Fig. S6B). No metastases in other organs werenoted.

IHC analysis of harvested mouse bladders revealed strongnuclear staining for p21 in LNP-dsP21-322-20F-treated tumorsas compared with PBS and LNP-dsCon-20F control tissue (Fig.7A). Detection of cleaved caspase 3 also revealed increasedactivation in LNP-dsP21-322-20F-treated tumors indicative ofapoptosis (Fig. 7A). Hematoxylin and eosin (H&E) staining andKi67 detection were used to identify orthotopic tumor in thetissue slides (Fig. 7A). Increased levels of p21 protein were alsoevaluated by immunoblot analysis in protein extracts preparedfrom total homogenized tissue (Fig. 7B). Densitometry analysismeasured �3.3- to 5.2-fold increase in p21 levels within LNP-dsP21-322-20F-treated tissue (Fig. 7C). These results confirm in

Figure 4. Urine stability andintracellular uptake of LNP-dsP21-322-20F. A, equal quantities ofdsP21-322, dsP21-322-20F, andLNP-dsP21-322-20F were incubatedin mouse or human urine for theindicated lengths of time. Duplexstability was visualized on anagarose gel. B, T24-P cells weretreated with LNP-formulated dsP21-322-20F-FITC (0.3 mg/mL) for 24hours. Cells were subsequentlyfixed, counterstained with DAPI(blue), and analyzed by fluorescentmicroscopy to visualize intracellularuptake of dsP21-322-20F-FITC invitro (green). C and D, one dose(�33.5 mg) of LNP-formulateddsP21-322-20F-FITC was instilled byintravesical administration into thebladder of normal mice and keptindwelling for 2 hours. Bladders weresurgically removed at 2 hours (C) or 9hours (D) following instillation forcryosectioning. Tissue sectionswerecounterstained with DAPI andvisualized by fluorescentmicroscopy. All imageswere taken atequal exposure times.Representative images of in vivodsP21-322-20F-FITC uptake (green)and nuclear staining (DAPI, blue) inbladder tissue are shown at 2 hours(C) and 9 hours (D). Insets containenlarged images of fluorescentstaining.






Figure 6. Intravesical delivery ofLNP-dsP21-322-20F inhibitsorthotopic bladder tumor growth.A, cancer growthwasmonitoredbyevaluating bladder tumorbioluminescence over the lifespanof each animal. Shown are BLIimages of a representative mousefrom each treatment group (PBS,LNP-dsCon20F, or LNP-dsP21-322-20F) at the indicated timepoints (days). Note normalization ofBLS intensities at days 1 and 7differed from days 14 to 49 in orderfor tumor signals to fall within thelimits of scale. B, tumorbioluminescence within eachmouse bladder is plotted overanimal lifespan for all treatmentgroups. Note the general trend oftumor bioluminescence in PBS andLNP-dsCon-20F treatment groupsincreases with time, whereasbioluminescence in animals treatedwith LNP-dsP21-322-20Fcollectively remains flat.Background signal generated byROIs in tumor-free animals of theLNP-dsP21-322-20F group werestill plotted on the Y-axis(Bioluminescence) to prevent linetermination and improperinterpretation as animal death.

Figure 5. Intravesical delivery ofLNP-dsP21-322-20F extendssurvival of mice with orthotopichuman bladder cancer. A,schematic diagram of study designand dosing schedule. Treatmentsbegan 4 days postimplantation for14 total doses every 3 days (solidarrowheads). BLI of mousebladders was done weekly (openarrowheads). B, mouse survival inthe days following tumorimplantation was monitored inPBS, LNP-dsCon-20F, and LNP-dsP21-322-20F treatment groups.Data are plotted as Kaplan–Meiercumulative survival curves.

Kang et al.





vivo activation of p21 expression in orthotopic tumors byintravesical administration of LNP-dsP21-322-20F.

DiscussionWe previously showed that dsP21-322 possesses in vitro

antigrowth activity in T24 and J82 bladder cancer cell lines(27). In the present study, we implement a chemically-modified

variant of dsP21-322 (dsP21-322-20F) with improved nucleaseresistance and reduced immunostimulatory activity toimprove its pharmaceutical properties for in vivo application(22). In addition, we use a clinically relevant LNP deliverysystem currently being implemented for RNAi-based therapiesto achieve intracellular uptake of dsP21-322-20F in targettissue. Intravesical administration of LNP-formulated dsP21-322-20F (LNP-dsP21-322-20F) into murine bladders with

Figure 7. LNP-dsP21-322-20Finduces p21 in orthotopic bladdertumors. A, representative H&Eimmunohistochemical staining ofKi67, p21, and cleaved caspase-3 intissue slides prepared from PBS,LNP-dsCon-20F, and LNP-dsP21-322-20F-treated murine bladders.H&E staining and Ki67 detectionwere carriedout to identify orthotopictumors. Insets are enlarged imagesof tumor cells. Note nuclear stainingfor p21 and Ki67. B, whole bladdertissue was homogenized and p21induction was confirmed byimmunoblot analysis. Detection ofGFP served as a loading control.Samples of LNP-dsP21-322-20Ftreatments are from 3 separatebladders.C, relative expression ofp21 was quantified by densitometryanalysis of the immunoblot in B. Theamounts of p21 protein werenormalized to GFP levels.






orthotopic cancer inhibited tumor growth and extended ani-mal survival. Our results provide proof-of-principle that tar-geted gene induction via RNAa in conjunction with currentdelivery platforms may represent a novel approach to treatbladder cancer.

Two previous studies in mice have reported the antitumoreffects of siRNA delivered intravesically by targeting PLK1 andsurvivin (33, 34). Because these studies used only a limitednumber of animals, tumor burden served as the primaryend point. To assess treatment efficacy, we opted to use animalsurvival as the primary end point, whereas monitoring tumorresponse by BLS throughout the study. To achieve sufficientstatistical power, we used a large population containing 29animals with established orthotopic bladder tumors (9 to 10mice/group). In fact, this study currently represents the largestcohort of animals ever used to evaluate small RNA-basedtherapeutics in an orthotopic bladder cancer model. Weobserved that intravesical delivery of LNP-dsP21-322-20Fcaused regression/disappearance of tumors in 40% of thetreated mice and significantly prolonged animal survival com-pared with control groups. As such, LNP-dsP21-322-20F mayrepresent a putative intravesical drug for treating bladdercancer and controlling recurrence.

KU-7 cells have been used to establish orthotopic bladdertumors in immunocompromised mice (26, 35). A previousstudy indicated that KU-7 tumors are confined to the laminapropria within 4 weeks (35); however, we observed muscleinvasion at longer periods of observation (�4 weeks). In someinstances, metastases were found in the kidney, which mostlikely resulted from retrogrademigration of the instilled tumorcells. Such metastases also seemed to evade treatment deliv-ered locally to the bladder. In our hands, KU-7 cells not onlymodeled superficial bladder cancer, but also recapitulatedmuscle-invasive and metastatic disease.

The target site of dsP21-322 is not shared between mouseand human due to poor conservation of p21 promotersequence (36). As such, it is currently unknown whether p21induction by dsP21-322-20F poses adverse effects to the normalbladder epithelium in humans. RNAa is a bias mechanism ofgene overexpression in which some cell lines/types can beresistant to saRNA treatment. In some cases, highly down-regulated genes are more sensitive to gene activation. Becausep21 is associated with reduced expression in bladder cancer(20, 21), it may possess an innate susceptibility to RNAa;whereas normal bladder cells may be more resilient to p21induction by dsP21-322-20F. In this regard, LNP-dsP21-322-20Fmay possess an inherent bias toward disease tissue. Nonethe-less, further evaluation of dsP21-322-20F needs to be done toidentify its effects on normal tissue health.

Intravesical administration of small RNA drugs has theadvantage of bringing high concentrations of drug into directcontact with diseased tissuewithout the side effects associatedwith systemic drug administration. The dosing schedule usedin our study mirrors current treatment regimens for intrave-sical immunotherapy Bacillus Calmette-Guerin (BCG), whichis used to treat superficial and recurrent bladder cancer inhumans. Furthermore, we used a clinically relevant unilamellarLNP to improve the bioavailability of dsP21-322-20F. LNP-for-mulation drastically enhanced dsP21-322-20F stability in urineand capacity to penetrate the protective glycosaminoglycan(GAG) layer of the bladder delivering duplex to the underlyingurothelial cells. In summary, we show that LNP-dsP21-322-20Fhas antitumor activity in an orthotopicmodel of bladder cancerby elevating p21 levels in vivo. Our results provide preclinicalproof-of-concept that DLin-KC2-DMA-based nanoparticleshave application in delivering RNA duplexes to facilitate RNAain vivo, as well as define a candidate RNAa-based drug for thetreatment of regional bladder cancer that may have clinicalrelevance as an adjuvant therapy after TUR.

Disclosure of Potential Conflicts of InterestR.F. Place is employed at RNA Therapeutics. K. Charisse, H. Epstein-Barash,

M. Manoharan are employees at Alnylam Pharmaceuticals. L-C. Li and R.F. Placeare named inventors onpending patent applications related to RNAa, which havebeen filed by the University of California San Francisco and licensed to AlnylamPharmaceuticals. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: M.R. Kang, M. Manoharan, L-C. LiDevelopment of methodology: M.R. Kang, R.F. Place, H. Epstein-Barash,M. Manoharan, L-C. LiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.R. Kang, K. CharisseAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.R. Kang, R.F. Place, L-C. LiWriting, review, and/or revision of the manuscript: M.R. Kang, R.F. Place,K. Charisse, M. Manoharan, L-C. LiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R.F. Place, L-C. LiStudy supervision: R.F. Place, M. Manoharan, L-C. LiDeveloped the formulation used for the study: H. Epstein-Barash

AcknowledgmentsWe would like to thank Dr. Jer-Tsong Hsieh at the Department of Urology,

University of Texas Southwestern Medical Center for his generous support withthe bladder cancer model. This work is supported by the AACR Henry ShepardBladder Cancer Research Grants (09-60-30-LI).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 21, 2012; revised July 26, 2012; accepted July 28, 2012;published OnlineFirst August 6, 2012.

References1. American Cancer Society. Cancer Facts & Figures 2012. Atlanta:

American Cancer Society; 2012.2. Shen Z, Shen T, Wientjes MG, O'Donnell MA, Au JL. Intravesical

treatments of bladder cancer: review. Pharm Res 2008;25:1500–10.3. Gee J, Sabichi AL, Grossman HB. Chemoprevention of superficial

bladder cancer. Crit Rev Oncol Hematol 2002;43:277–86.

4. CooksonMS,HerrHW,ZhangZF, SolowayS,Sogani PC, FairWR. Thetreated natural history of high risk superficial bladder cancer: 15-yearoutcome. J Urol 1997;158:62–7.

5. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potentand specific genetic interference by double-stranded RNA in Caenor-habditis elegans. Nature 1998;391:806–11.

Kang et al.





6. Huang L, Liu Y. In vivo delivery of RNAi with lipid-based nanoparticles.Annu Rev Biomed Eng 2011;13:507–30.

7. Li LC, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, et al. SmalldsRNAs induce transcriptional activation in human cells. Proc NatlAcad Sci U S A 2006;103:17337–42.

8. Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, Corey DR.Activating gene expression in mammalian cells with promoter-tar-geted duplex RNAs. Nat Chem Biol 2007;3:166–73.

9. Place RF, Noonan EJ, Foldes-Papp Z, Li LC. Defining features andexploring chemical modifications to manipulate RNAa activity. CurrPharm Biotechnol 2010;11:518–26.

10. Schwartz JC, Younger ST, Nguyen NB, Hardy DB, Monia BP, CoreyDR, et al. Antisense transcripts are targets for activating small RNAs.Nat Struct Mol Biol 2008;15:842–8.

11. Modarresi F, Faghihi MA, Lopez-ToledanoMA, Fatemi RP, Magistri M,Brothers SP, et al. Inhibition of natural antisense transcripts in vivoresults in gene-specific transcriptional upregulation. Nat Biotechnol2012;30:453–9.

12. el-Deiry WS, Harper JW, O'Connor PM, Velculescu VE, Canman CE,Jackman J, et al.WAF1/CIP1 is induced inp53-mediatedG1arrest andapoptosis. Cancer Res 1994;54:1169–74.

13. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependentkinases. Cell 1993;75:805–16.

14. Waga S, Hannon GJ, Beach D, Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interactionwith PCNA.Nature 1994;369:574–8.

15. Teraishi F, Kadowaki Y, Tango Y, Kawashima T, Umeoka T, Kagawa S,et al. Ectopic p21sdi1 gene transfer induces retinoic acid receptor betaexpression and sensitizes human cancer cells to retinoid treatment. IntJ Cancer 2003;103:833–9.

16. Wu M, Bellas RE, Shen J, Sonenshein GE. Roles of the tumor sup-pressor p53 and the cyclin-dependent kinase inhibitor p21WAF1/CIP1in receptor-mediated apoptosis of WEHI 231 B lymphoma cells. J ExpMed 1998;187:1671–9.

17. Eastham JA, Hall SJ, Sehgal I, Wang J, Timme TL, YangG, et al. In vivogene therapy with p53 or p21 adenovirus for prostate cancer. CancerRes 1995;55:5151–5.

18. Gao J, Huang HY, Pak J, Cheng J, Zhang ZT, Shapiro E, et al. p53deficiency provokes urothelial proliferation and synergizes with acti-vated Ha-ras in promoting urothelial tumorigenesis. Oncogene2004;23:687–96.

19. Mitra AP, Birkhahn M, Cote RJ. p53 and retinoblastoma pathways inbladder cancer. World J Urol 2007;25:563–71.

20. Stein JP, Ginsberg DA, Grossfeld GD, Chatterjee SJ, Esrig D, Dick-inson MG, et al. Effect of p21WAF1/CIP1 expression on tumor pro-gression in bladder cancer. J Natl Cancer Inst 1998;90:1072–9.

21. Clasen S, Schulz WA, Gerharz CD, Grimm MO, Christoph F, Schmitz-Drager BJ. Frequent and heterogeneous expression of cyclin-depen-

dent kinase inhibitor WAF1/p21 protein and mRNA in urothelial car-cinoma. Br J Cancer 1998;77:515–21.

22. Place RF, Wang J, Noonan EJ, Meyers R, Manoharan M, Charisse K,et al. Formulation of small activating RNA into lipidoid nanoparticlesinhibits xenograft prostate tumor growth by inducing p21 expression.Mol Ther Nucleic Acids 2012;1:e15.

23. Karam JA, Huang S, Fan J, Stanfield J, Schultz RA, Pong RC, et al.Upregulation of TRAG3 gene in urothelial carcinoma of the bladder. IntJ Cancer 2011;128:2823–32.

24. Semple SC, Akinc A, Chen J, Sandhu AP, Mui BL, Cho CK, et al.Rational design of cationic lipids for siRNA delivery. Nat Biotechnol2010;28:172–6.

25. Chade DC, Andrade PM, Borra RC, Leite KR, Andrade E, Villanova FE,et al. Histopathological characterization of a syngeneic orthotopicmurine bladder cancer model. Int Braz J Urol 2008;34:220–6.

26. Watanabe T, Shinohara N, Sazawa A, Harabayashi T, Ogiso Y, Koya-nagi T, et al. An improved intravesical model using human bladdercancer cell lines to optimize gene and other therapies. Cancer GeneTher 2000;7:1575–80.

27. ChenZ, PlaceRF, Jia ZJ, Pookot D, DahiyaR, Li LC. Antitumor effect ofdsRNA-induced p21(WAF1/CIP1) gene activation in human bladdercancer cells. Mol Cancer Ther 2008;7:698–703.

28. Vaishnaw AK, Gollob J, Gamba-Vitalo C, Hutabarat R, Sah D, MeyersR, et al. A status report on RNAi therapeutics. Silence 2010;1:14.

29. Abbas T, Dutta A. p21 in cancer: intricate networks and multipleactivities. Nat Rev Cancer 2009;9:400–14.

30. Gartel AL, Tyner AL. The role of the cyclin-dependent kinase inhibitorp21 in apoptosis. Mol Cancer Ther 2002;1:639–49.

31. Hanke M, Hoefig K, Merz H, Feller AC, Kausch I, Jocham D, et al. Arobust methodology to study urinemicroRNA as tumor marker: micro-RNA-126 and microRNA-182 are related to urinary bladder cancer.Urol Oncol 2010;28:655–61.

32. Bartlett DW, Su H, Hildebrandt IJ, Weber WA, Davis ME. Impact oftumor-specific targeting on the biodistribution and efficacy of siRNAnanoparticles measured by multimodality in vivo imaging. Proc NatlAcad Sci U S A 2007;104:15549–54.

33. Nogawa M, Yuasa T, Kimura S, Tanaka M, Kuroda J, Sato K, et al.Intravesical administration of small interfering RNA targeting PLK-1successfully prevents the growth of bladder cancer. J Clin Invest2005;115:978–85.

34. Seth S, Matsui Y, Fosnaugh K, Liu Y, Vaish N, Adami R, et al. RNAi-based therapeutics targeting survivin and PLK1 for treatment ofbladder cancer. Mol Ther 2011;19:928–35.

35. HadaschikBA,BlackPC,Sea JC,Metwalli AR, Fazli L, DinneyCP, et al.A validated mouse model for orthotopic bladder cancer using trans-urethral tumour inoculation and bioluminescence imaging. BJU Int2007;100:1377–84.

36. Huang V, Qin Y, Wang J, Wang X, Place RF, Lin G, et al. RNAa isconserved in mammalian cells. PLoS One 2010;5:e8848.






2012;72:5069-5079. Published OnlineFirst August 6, 2012.Cancer Res Moo Rim Kang, Glen Yang, Robert F. Place, et al. Nanoparticles Inhibits Orthotopic Bladder Tumor GrowthIntravesical Delivery of Small Activating RNA Formulated into Lipid

Updated version

10.1158/0008-5472.CAN-12-1871doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2012/08/06/0008-5472.CAN-12-1871.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/72/19/5069.full#ref-list-1

This article cites 35 articles, 7 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/72/19/5069.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/72/19/5069To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-12-1871

http://cancerres.aacrjournals.org/content/suppl/2012/08/06/0008-5472.CAN-12-1871.DC1

http://cancerres.aacrjournals.org/content/72/19/5069.full#ref-list-1

http://cancerres.aacrjournals.org/content/72/19/5069.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/72/19/5069


intravesicaldeliveryofsmallactivatingrnaformulatedinto ... · t24-p cells are a highly tumorigenic...

Documents