Intratumor RNA interference of cell cycle genes slows down tumor ...

Gene Therapy (2011) 18, 727–733 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt

ORIGINAL ARTICLE

Intratumor RNA interference of cell cycle genes slows down tumor progression S Dharmapuri1,5,6, D Peruzzi1,5,7, E Marra1,8, F Palombo1,9, AJ Bett2, SR Bartz3, M Yong3, G Ciliberto1,10, N La Monica1,11, CA Buser2, C Toniatti4 and L Aurisicchio1,8 Small interfering RNAs (siRNAs) are emerging as promising therapeutic tools. However, the widespread clinical application of such molecules as modulators of gene expression is still dependent on several aspects that limit their bioavailability. One of the most promising strategies to overcome the barriers faced by gene silencing molecules involves the use of lipid-based nanoparticles (LNPs) and viral vectors, such as adenoviruses (Ads). The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. In this study, we tested the capability of LNPs and Ad to transduce and treat locally tumors in vivo. Efficient knockdown of a surrogate reporter (luciferase) and therapeutic target genes such as the kinesin spindle protein (KIF11) and polo-like kinase 1 were observed. Most importantly, this activity led to a cell cycle block as a consequence and slowed down tumor progression in tumor-bearing animals. Our data indicate that it is possible to achieve tumor transduction with si/short hairpin RNAs and further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer gene silencing. Gene Therapy (2011) 18, 727–733; doi:10.1038/gt.2011.27; published online 10 March 2011 Keywords: siRNA; liponanoparticle; KSP; PLK1

INTRODUCTION The process of post-transcriptional double-stranded RNA-dependent gene silencing is called RNA interference (RNAi).1 RNAi is triggered by the presence of 21–23-nucleotide-long double-stranded RNA in the cell and results in the rapid degradation of the mRNA containing identical or nearly-identical sequence. In nature, double-stranded RNAs are recognized by Toll-like receptor 3. Upon recognition, Tolllike receptor 3 induces the activation of nuclear factor-kB to increase the production of type I interferons, which signal other cells to activate their antiviral defenses.2 RNAi in adult mice is well characterized.3 Major targets for small interfering RNA (siRNA) therapy against cancer include oncogenes and genes that are involved in angiogenesis, metastasis, survival, antiapoptosis and resistance to chemotherapy. Recent published data suggest that siRNAs could be used for induction of cytokines, such as interferon-a, silencing suppressors of cytokine signaling and transducing antigen-presenting cells, with siRNAs targeting apoptotic genes Bak, Bax, Casp8 and Fas to prolong antigen-presenting cell lifespan.4–8 An effective siRNA has to be stable in order to achieve long-term inhibition of target genes. In fact, the half-life of unmodified siRNAs is short, especially in vivo, due to rapid elimination by the kidney and rapid degradation by serum RNases,

with a half-life of o10 min.9 The stability of siRNA has been significantly increased by chemical conjugation or modifications of the sugar backbone and by capping the ends of the siRNA. Chemical modifications at the 2¢ position of the ribose, introduction of 2¢-F pyrimidines, deoxy purines and inverted deoxy abasic end cap in the sense strand as well as 2¢-F pyrimidines and 2¢-OMe purines in the antisense strand and incorporation of the 3¢-S-phosphorothioate modification into the strands of RNA led to half-life in the range of hours.10,11 The in vivo use of siRNAs against cancer hinges on the availability of a delivery vehicle that can be systemically administered to reach both primary and metastatic tumor cells. Several different technologies are being explored to solve this complex issue, among them the use of RNA duplexes conjugated with conventional antisense oligonucleotides (Rxi Pharmaceuticals) or complexed with protein moieties, such as the peptide transduction domains linked to a double-stranded RNA binding domain,12 developed by Traversa Therapeutics. Liposomes or liponanoparticles (LNPs) have been used extensively for drug delivery and gene transfer.13,14 They have been shown to enhance the delivery efficiency and protect the siRNA from degradation.15 In particular, a liposomal RNA delivery vehicle, LNP201, has been recently developed

1Merck Research Laboratories, IRBM ‘P Angeletti’, Pomezia (Rome), Italy; 2Merck Research Laboratories, West Point, PA, USA; 3Merck Research Laboratories, Sirna, San Francisco, CA, USA and 4Merck Research Laboratories, Boston, MA, USA Correspondence: Dr L Aurisicchio, Istituto di Ricerche Genetiche ‘Gaetano Salvatore’, Department of Molecular Oncology, BIOGEM,scarl, via Camporeale 83031, Ariano Irpino (AV) 83031, Italy. E-mail: [email protected] 5These authors contributed equally to this work. 6Current address: PharManagement Ltd, London, UK. 7Current address: Department of Medicina Interna, University of Rome ‘La Sapienza’, Italy. 8Current address: BIOGEM,scarl, via Camporeale, Ariano Irpino (Av), Italy. 9Current address: Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. 10Current address: University of Catanzaro, Department of Experimental and Clinical Medicine ‘G Salvatore’, Campus Germaneto, Catanzaro, Italy. 11Current address: Idera Pharmaceuticals, 167 Sidney Street, Cambridge, MA, USA. Received 29 August 2010; revised 22 November 2010; accepted 22 November 2010; published online 10 March 2011

RNAi of cell cycle genes slows down tumor progression S Dharmapuri et al 728

and shown to cause sustained silencing of target genes in the mouse liver.16 Similarly, viral vectors are particularly attractive because they can induce long-term knockdown of gene transcripts in vivo.17 Therefore, the use of RNAi by a stable viral vector system, such as the adenovirus (Ad), is a possible strategy for stable gene knockdown. In fact, constitutive expression of short hairpin RNA by Ad vectors has been shown for several weeks, thereby consolidating this technology as an alternative to generating liver-specific knockout animals,18 as development of potential agents to treat hepatitis B9,19,20 or to generate antitumor therapies against antiapoptotic genes18,21 or genes involved in the cell cycle.22 The objective of this study was to achieve intratumoral local delivery of siRNA using LNPs or Ad vectors. As proof of concept of LNPs delivery and efficacy, we used the reporter gene luciferase (Luc). Two cell cycle genes were then targeted: the kinesin spindle protein (KSP or KIF11) and polo-like kinase 1 (PLK1). KSP has an essential role in centrosome separation and mitotic spindle formation. PLK1 regulates multiple steps of mitosis and phosphorylates proteins involved in the cell cycle. Both genes have been extensively investigated as targets of anticancer drugs.23,24 However, the downstream molecular pathways through which tumor cells undergo cell death in response to mitotic arrest are not well defined. Evidence from microtubule binding drugs suggests that proliferation of non-transformed cells, including hematopoetic precursor cells, might explain the severe myelosuppression and neutropenia observed in patients during therapy.25 RNAi could overcome these adverse effects and show greater efficacy because of localized inhibition.26 This concept has set up the path for therapeutic applications, both locally27 and systemically.28 Recently, the demonstration of RNAi in humans from systemically administered siRNA by targeted nanoparticles has been achieved29 and Alnylam Pharmaceuticals has started clinical trials for the evaluation of a KSP–RNAi therapeutic in a wide variety of cancers. Here, we show efficient silencing of target genes and the impact of specific RNAi on tumor volumes upon local delivery of LNP201 particles and adenoviral vectors in vivo. Our findings could open new therapeutic avenues to the treatment of accessible solid tumors. RESULTS LNP201 delivery leads to luciferase gene knockdown To validate luciferase as a surrogate marker of siRNA-mediated silencing, in vitro studies were conducted on HeLa-Luc cells plated in 96-well plates and treated with LNP201 delivering Luc, KSP or control siRNA. After 24 h, the expression of luciferase and cell viability was evaluated. Figures 1a (luminescence) and b (viability) show the knockdown efficiency of the LNP201–siRNAs: HeLa-Luc cells were transfected with LNP201–siRNAs ranging from a concentration of 200–0 nM. The maximal knockdown of luciferase expression as reported by photon emission was achieved at B2 nM. At similar concentrations, the LNP201–KSP showed the maximal biological activity by induction of cell death. It is important to note that at higher concentrations (67 and 200 nM) all the LNPs showed a toxic effect on plated cells and that the luciferase expression is also diminished as a consequence of cell death induced by LNP201–KSP. These data show that LNPs are effective in targeting the desired mRNA and achieving a biological effect. To assess the effects of LNP201 in vivo, HeLa-Luc tumors were implanted in nude mice. After 12 days, when the average tumor volume was 80–100 mm3, the tumors were injected with LNP201-Luc or LNP201-Control (LowHex) at the dose of 3 mg kg1 (B60 mg per tumor). Mice were killed after two intratumoral (i.t.) injections of LNPs 3 days apart. Tumors were well vascularized and no differences Gene Therapy

were observed in terms of tumor weight (not shown). Expression of luciferase varied between the groups (Figure 1c). Tumors were normalized to weights and those injected with LNP201-Luc showed a distinct and significant reduction in luminescence as compared with control and LNP201-control treated groups. However, as observed from the in vitro experiment, luciferase expression was also affected in LNP201-control injected tumors as a consequence of potential toxicity and/or immunomodulatory effects exerted by LNP201. LNP201 delivery mediates antitumor effects To perform efficacy studies, we first selected the HEPA1-6 hepatoma cell line, as LNP201 are optimal for liver cell transduction and KSP and PLK-1 are highly expressed at mRNA level (data not shown). HEPA1-6 cells were transfected with LNPs delivering KSP (LNP201KSP), either of two PLK1 siRNA oligos (LNP201-PLK1-1 and LNP201-PLK1-2) or the control LNP201-Control at 30, 6, 1.2 and 0.24 nM concentrations and subjected to cell cycle analysis 48 h later. Cell cycle arrest was evident as a higher percentage of cells were measured in S and G2/M phase after transfection with LNP201–KSP and LNP201–PLK1-1 (Figure 2a). Untreated cells or cells transfected with LNP201-Control showed similar cell cycle profiles. These data indicated that the HEPA1-6 cell line, once grafted in mice to form tumors, could be treated with LNPs to assess the therapeutic impact. Groups of CD1 nude mice were implanted with 1107 HEPA1-6 cells. Tumors measuring at least 80 mm3 in volume were injected i.t. 5 times, once every 3 days with LNPs. A number of tumors were collected 24 h after the second injection. KSP expression was measured by quantitative PCR and distinct knockdown of KSP expression was observed (Figure 2b). LNP201–KSP particles induced highly significant 88% of gene knockdown compared with the untreated control. However, also the negative control LNP201-Control was able to partially inhibit gene expression (67% knock down (KD)), thus indicating a potential toxicity obtained by the injection of the particles themselves. A significant reduction in tumor growth was observed targeting KSP (Figure 2c) following complete treatment. LNP201–KSP-injected tumors were reduced in volume by nearly 40% when compared with phosphorus-buffered saline-injected tumors. The reduction was lesser (20%) when compared with the negative control LNP201-Control (but significant after 4 i.t. injections). LNP–PLK1-1 showed a significant delay in tumor growth with a 30% decrease in volume after 4 i.t. injections. To extend these observations to another model, we injected HCT-116 human colon cancer xenografts. In this model, LNP201– PLK1-1 significantly reduced tumor volume after 5 i.t. injections. LNP201–KSP and LNP201–PLK1-2 showed a trend toward reducing tumor volume (Figure 2d), but were less efficacious. Again, an antitumor effect by LNP201-Control was observed and attributed to toxicity. LNP201 delivery affects head and neck and BCC tumor cell cycle The most straightforward translational application of intratumoral delivery of siRNAs is the treatment of accessible tumors, such as head and neck or basal cell carcinomas (BCC). To further extend the data obtained from HEPA1-6 and HCT-116 cells, we have analyzed the impact of LNP201 particles on the cell cycle of tumor cell lines of different origin, such as CCL138 (tongue carcinoma), HTB43 (pharyngeal squamous cell carcinoma), CRL2095 (pharyngeal carcinoma) and CRL7804 (BCC). The transfection was carried over as described above and results are shown in Figure 3. All lines showed diverse sensitivity to the LNPs. In CRL2095 cells (Figure 3a), cell cycle arrest was evident as a higher percentage of cells were measured in S and G2


Figure 1 LNP201–siRNAs result in biological activity in vitro and in vivo. (a) Quantification of luciferase expression. Photons emitted at the indicated LNP concentrations were measured through Living Image software (Caliper, Hopkinton, MA, USA). (b) LNP201–KSP induce cell death. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide was added to the cells, and following incubation for 2 h at 37 1C, dimethylsulfoxide was added and absorbance at 570 nM was measured using an enzyme-linked immunosorbent assay reader. The knockdown efficiency was 90 and 95% at 2 and 20 nM, respectively. (c) Tumor weights and their associated luminescence were as measured by imaging. Ratio of photons per mg tumor tissue is shown. Empty circles indicate the value for each single mouse. The filled circle represents the group average value. Each group was composed of five mice. Student’s t-test P-values are shown.

phase after treatment with LNP201–KSP and both LNP201–PLK1 formulations. HTB43 (Figure 3b) appeared to be sensitive only to LNP–PLK1-2, whereas another pharyngeal carcinoma line—CCL138 (Figure 3c)—showed a high percentage of cells in S and subG1 phase. CRL7804 (Figure 3d) was sensitive to LNPs targeting both KSP and PLK1, and showed an increased number of cells in the S phase. In vivo tumor transduction and antitumor effects by Ad vectors To confirm intratumor knockdown and growth effects observed in the HCT-116 model with a different delivery system, we used an Ad vector carrying a dicistronic cassette expressing enhanced green fluorescent protein (EGFP) and a short hairpin RNA against PLK1 (Figure 4a). Ad-EGFP was used as a control. To verify that Ad infection could lead to RNAi of the target gene, PLK1 was quantified by quantitative PCR, 48 h after infection at multiplicity of infection¼100 of HCT-116 cells in vitro. An efficient knockdown effect was measured in cells infected with Ad-EGFP–shPLK1 (Figure 4a).

To verify if Ad vectors were capable of transducing tumors in vivo, groups of CD1 nu/nu mice were implanted with 2106 HCT-116 tumor cells. When tumors reached a size of B100 mm3, 1108 EGFPtransducing units of each vector were injected intratumorally. Two days later, tumors were explanted and digested with collagenase upto single cell suspension, and then analyzed using fluorescence-activated cell sorting (FACS). About 10–20% of tumor cells resulted EGFP+ after a single injection, thus showing that Ad can efficiently transduce tumors in vivo (Figure 4b). To assess whether inhibition of PLK-1 by Ad could lead to in vivo therapeutic effects, CD1 nu/nu mice bearing 80–100 mm3 HCT-116 tumors were treated with Ad vectors (109 EGFP-transducing units). A further 2 injections were performed after a week’s interval and growth was followed over time. A significant delay in tumor growth was observed in Ad-EGFP–shPLK1 compared with the other groups (Figure 4c). No effect was observed with Ad-EGFP. These data confirmed that silencing of PLK1 can reduce tumor progression. Gene Therapy


Figure 2 Biological effects and impact of i.t. LNP injections on HEPA1-6 and HCT-116 tumors. (a) Cell cycle analysis of HEPA1-6 cells after transfection in vitro with LNPs. Untreated (UT) cells were used to define the baseline data. (b) HEPA-1–6 tumor-bearing mice were injected twice with 60 mg LNPs and killed 24 h after the second injection. RNA was extracted from the tumor and quantitative PCR analysis was performed with KSP and normalized with glyceraldehyde-3-phosphate dehydrogenase. Percentage of knockdown is indicated. Student’s t-test P-values are shown. (c) HEPA1–6 xenograft tumor volume index measured after five intratumoral LNP injections, indicated by the arrows. Tumor index was calculated as the ratio of tumor volume at the indicated time point and the volume pretreatment for each mouse. P-values (Student’s t-test): Po0.05 (KSP vs phosphorus-buffered saline (PBS); KSP vs Control; PLK1-1 vs untreated). Time points at which P-values were significant are marked with an asterisk. (d) HCT-116 xenograft tumor volumes measured after 5 i.t. LNP injections (see arrows). P-values (Student’s t-test): Po0.05 (PLK1 vs PBS). Time points at which P-values were significant are marked with an asterisk.

DISCUSSION The in vivo use of siRNAs against cancer is limited by the lack of appropriate delivery vehicles that can be systemically administered to reach both primary and metastatic tumor cells. LNPs, with their inherent and tested ability to merge with cell membranes and deliver their payload, offer an opportunity to address the issue of siRNA delivery. Similarly, Ad vectors with their ability to infect replicating and quiescent cells of several tissue origins are valuable tools for longlasting short hairpin RNA expression and target KD effects. Here, we have tested an LNP formulation, LNP201, originally designed for liver transduction16 with the objective of delivering siRNAs targeting the cell cycle genes, KSP and PLK1. KSP is currently the target of intense research for the development of novel anticancer therapeutics. Several inhibitors of KSP have progressed into clinical trials and many others are in preclinical development.30–32 Similarly, PLK1 inhibitors have moved into clinical studies in patients with locally advanced or metastatic cancers,33 and in vitro studies have shown that the combination of PLK1-specific siRNAs with modern breast cancer drugs could represent rational combinations to improve the sensitivity in a synergistic manner.34 Using luciferase as a biomarker, we first showed in vitro and in vivo KD (in tumor tissue) of luminescence (Figure 1a). However, aspecific effects on reporter expression with the negative control LNP201Control were observed, which could be attributed to the potential toxicity of LNP formulation or to the relatively high-dose utilized. Off target effects are also possible. Gene Therapy

Repeated intratumoral injections in HEPA-1-6 and HCT-116 xenografts showed KD effects of target genes by LNP-delivered KSP and PLK1 siRNAs. Tumor volumes were significantly reduced most likely due to blocking of the cell cycle (Figure 2a). Again, we observed aspecific KD and tumor growth effects: it is likely that the 60 mg dose that was tested may have had some toxic effects as evident from the results with LNP201-Control, which nevertheless shows mRNA KD (Figure 1c). In vivo cytokine release and inflammatory gene induction may also contribute to these effects. Tests of lower doses and new LNPs formulations are currently in progress, which comprises different combinations of degradable cationic lipids (ester and carbamate lipids), neutral lipids (DSPC, DOPC, cholesterol, DOPE) and Peglipids (DMG-Peg, DSG-Peg) with enhanced endosomal release properties and compatible with tumor acidic environment. Cell cycle analysis after LNP-mediated siRNA delivery represents a very useful tool for prediction of in vivo efficacy. Head and neck and BCC tumor cell lines have also been treated with the LNPs showing diverse degrees of sensitivity (Figure 3). This suggests that the range of tumors targeted with this therapy can be enlarged and in vitro experiments can allow appropriate selection of treatable tumor types. Importantly, these tumor types are generally accessible and ideal for intratumoral injections with RNAi agents. We also show that Ad vectors are efficacious delivery tool for target gene KD and intratumor treatment. One single injection achieved 10–20% tumor cell transduction and most importantly, repeated treatment specifically inhibited HCT-116 tumor growth over time


Figure 3 Effects of LNP on head and neck and BCC cell lines. A cell cycle analysis of tumor cell lines was performed 48 h after transfection in vitro with LNPs. (a) Tongue carcinoma (CRL2095); (b) Pharyngeal squamous cell carcinoma (HTB43); (c) Pharyngeal carcinoma (CCL138); (d) BCC (CRL7804). Untreated (UT) cells were used to define the baseline data.

(Figure 4c). As control Ad-EGFP was ineffective, the antitumor effect was not mediated by the induction of innate immunity upon vector injection.35 These data further showed that RNAi of cell cycle-related genes (such as PLK-1) represents a viable option in the treatment of cancer. However, as our experiments have been performed in immunodeficient mice, we have not assessed the potential impact of the immune response against Ad vectors. As pre-existing and vectorinduced immunity represents a serious issue for Ad as vaccine carriers, especially for cancer vaccine applications,36 it is likely that neutralizing antibodies and/or T-cell response would affect tumor transduction. In conclusion, our study shows that intratumoral delivery of siRNA/ short hairpin RNA to target cell cycle genes such as KSP and PLK1 in accessible tumors is achievable and has the potential to confer therapeutic effects. To our knowledge, this is the first report describing the application of intratumor injections of LNPs. Other studies are warranted to better define the chemical properties of LNPs in order to decrease their toxicity and enhance the in vivo tumor transduction potency.

GTTTACCGAAGTTT-3¢) and two different sequences targeted human Plk1 (target sites: PLK1-1, 5¢-AGCGTGCAGATCAACTTCTTT-3¢ and PLK1-2, 5¢-TCCGGATCAAGAAGAATGATT-3¢, respectively). A nonspecific siRNA (control), 5¢-UCUUUUAACUCUCUUCAGG-3¢ was always included as a negative control in all experiments. All siRNAs were designed through a specific algorithm.

Ad vectors Ad5-EGFP-shPLK1 and Ad5-EGFP were generated by homologous recombination as described elsewhere.37 H14 is an E1–E3 deleted empty Ad5 vector utilized as negative control. Preadenoviral plasmids were cut with PacI to release the Ad inverted terminal repeats and transfected in PerC-6 cells (Crucell, Leiden, the Netherlands) by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Vectors were amplified through serial passages. Viruses were purified through standard CsCl purification protocol and extensively dialyzed against A105 buffer (5 mM Tris, pH 8.0, 1 mM MgCl2, 75 mM NaCl, 5% sucrose, 0.005% Tween-20). Batches titers ranged from 1 to 61012 vp ml1 and were normalized for EGFP-transducing units.

Cell lines MATERIALS AND METHODS Liponanoparticles LNP201 encapsulating siRNAs were synthesized by Sirna (San Francisco, CA, USA) as described in Abrams et al.16 The LNPs used in this study have a size o150 nm, contain diffusible 1-[8¢-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3¢, 6¢-dioxaoctanyl]carbam-oyl-methyl-poly(ethylene glycol) (PEG-DMG; designed for liver uptake) and the following composition: 50.3% Clin-DMA, 44.3% cholesterol, 5.4% 1-[8¢-(1,2-dimyristoyl-3-propanoxy)-carboxamido3¢,6¢-dioxaoctanyl]carbam-oyl-methyl-poly(ethylene glycol). siRNA targeting firefly luciferase (luc) was used to demonstrate in vitro and in vivo knockdown activity. One siRNA targeted Kif11 or human Ksp (target site: 5¢-GATTGAT

The human endometrial tumor HeLa-Luc (Caliper, Hopkinton, MA, USA), HEPA-1-6 (mouse hepatoma, ATCC, Manassas, VA, USA), HCT-116 (human colon adenocarcinoma, ATCC), CCL138 (tongue carcinoma, ATCC), HTB43 (pharyngeal squamous cell carcinoma, ATCC), CRL2095 (pharyngeal carcinoma, ATCC) and CRL7804 (BCC, ATCC). Cell lines were used and cultured according to providers’ indications.

Mice and intratumoral delivery Six- to 8-week-old CD1nu/nu male mice were purchased from Charles River (Calco, Italy). Tumor cells were implanted on the right flank with or without matrigel. Tumors were injected with LNPs containing to 60 mg siRNA in a Gene Therapy


Figure 4 In vitro and in vivo effects of Ad-EGFP–shPLK1 vector injections on HCT-116 tumors. (a) HCT-116 cells were infected in vitro at multiplicity of infection¼100. After 48 h, the RNA was extracted and PLK1 mRNA was quantified by Taqman quantitative PCR. The experiment has been performed in triplicates. (b) HCT116 tumors implanted in CD1 nu/nu mice were injected with 108 EGFP-transducing units (TU). Collagenase-digested tumors were analyzed using FACS for detection of EGFP-positive cells. Examples of untreated, Ad-EGFP and Ad-EGFP–shPLK1 tumor samples are shown. Percentage of EGFP-positive cells is indicated. (c) HCT-116 tumors in CD1 nu/nu mice were injected three times with 109 EGFP-TU at weekly intervals (see arrows). Tumor growth index in mice treated with the indicated Ad vectors. Time points at which P-values were significant (Po0.05) are marked with an asterisk.

volume of 50 ml at the indicated time points and measured with a caliper twice a week. Mice were killed when tumors exceeded the size of 2000 mm3.

Statistics Student’s t-test was performed where indicated. P-value o0.05 were considered significant.

In vitro assays Cells were plated in 96-well black plates (Costar, Lowell, MA, USA) at 5000– 10 000 cells per well, transfected with the indicated concentrations of LNPs and 24 h later analyzed for emission of photons at an IVIS-200 (Caliper). A volume of 15 ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (10 mg ml1, Sigma, St Louis, MO, USA) were added to 125 ml cell monolayer in each well and incubated for 1 h at 37 1C and formazan crystals solubilized in 100 ml per well dimethylsulfoxide. Plates were read using an enzyme-linked immunosorbent assay reader at a wavelength of 570 nm. Gene expression was measured by Taqman gene expression assay for human KSP, mouse KSP, human PLK1 (all reagents from Applied Biosystems, Foster City, CA, USA). Cell cycle analysis was performed and analyzed with a Guava Easycyte (Guava Technologies, Hayward, CA, USA) according to the manufacturer’s instructions.

FACS analysis Cells were trypsinized, washed with phosphorus-buffered saline and analyzed at a FACScalibur (Becton Dickinson, Franklin Lakes, NJ, USA). Tumors were explanted, digested in RPMI 10% fetal calf serum containing 2 mg ml1 collagenase for 2 h, then filtered, washed in FACS buffer (phosphorus-buffered saline, 1% fetal calf serum) and analyzed at a FACS CantoII (Becton Dickinson). Gene Therapy

CONFLICT OF INTEREST The authors declare no conflict of interest.

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