receptor-mediated delivery of an antisense gene to human brain cancer cells

RESEARCH ARTICLE

Receptor-mediated delivery of an antisense geneto human brain cancer cells

Yun Zhang

Hwa Jeong Lee

Ruben J. Boado

William M. Pardridge*

Department of Medicine, UCLASchool of Medicine, Los Angeles,CA 90024, USA

*Correspondence to: William M.Pardridge, MD, Department ofMedicine, UCLA Warren Hall,Room 13-164, 900 VeteranAvenue, Los Angeles, CA 90024,USA.E-mail:[email protected]

Received: 8 October 2001

Revised: 23 November 2001

Accepted: 27 November 2001

Abstract

Background The goal of this work was the development of a gene targetingtechnology that will enable the delivery of therapeutic genes to brain cancercells in vivo following intravenous administration. High-grade brain gliomasoverexpress the epidermal growth factor receptor (EGFR) and EGFR anti-sense gene therapy could reduce the growth of EGFR-dependent gliomas.

Methods A human EGFR antisense gene driven by the SV40 promoter ina non-viral plasmid carrying elements that facilitate extra-chromosomalreplication was packaged in the interior of 85 nm pegylated immuno-liposomes (PILs). The PILs were targeted to U87 human glioma cells withthe 83-14 murine monoclonal antibody (MAb) to the human insulin receptor(HIR).

Results Confocal fluorescent microscopy demonstrated that the unconju-gated HIR MAb is rapidly internalized by the glioma cells. Endocytosisfollowed by entry into the nucleus was also demonstrated for the HIR MAbconjugated PILs carrying fluorescein-labeled plasmid DNA. The PILs deliveredexogenous genes to virtually all cells in culture, based on b-galactosidasehistochemistry. The targeting of a luciferase gene to the U87 cells with thePILs resulted in luciferase levels in excess of 150 pg/mg protein after 72 h ofincubation. The level of luciferase gene expression in the U87 cells achievedwith the PIL gene targeting system was comparable to that with lipo-fectamine. Targeting the EGFR antisense gene to U87 glioma cells with thePILs resulted in more than 70% reduction in [3H]thymidine incorporationinto the cells; this was paralleled by a 79% reduction in the level ofimmunoreactive EGFR.

Conclusion The present work describes the targeting of an EGFR antisensegene to human brain cancer cells, which results in a 70–80% inhibition incancer cell growth. PILs provide a new approach to gene targeting that iseffective in vivo following intravenous administration without viral vectors.Copyright # 2002 John Wiley & Sons, Ltd.

Keywords blood–brain barrier; epidermal growth factor receptor; genetargeting; insulin receptor; gene therapy

Introduction

High-grade human brain gliomas overexpress the receptor for epidermalgrowth factor (EGF) [1–4]. U87 human brain glioma cells have beentransfected in cell culture with a gene encoding for antisense mRNAcomplementary to EGF receptor (EGFR) mRNA; this results in a loss intumorigenicity of the cell line [5,6]. Similarly, EGFR is overexpressed in

THE JOURNAL OF GENE MEDICINEJ Gene Med 2002; 4: 183–194.Published online 14 February 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ jgm.255

Copyright # 2002 John Wiley & Sons, Ltd.

cancers of peripheral origin and the proliferative capacityof these cell lines is decreased by transfection with EGFRantisense genes [7,8].

Gene transfection in tissue culture is possible withcationic lipids that form micro-aggregates with theplasmid DNA, and the cationic liposomes transfect cellsbased on the phagocytosis, not the endocytosis, of thecomplex [9]. However, with respect to gene therapy ofbrain tumors in vivo, it is not possible to target exogenousgenes to the brain in vivo with cationic lipid/DNAcomplexes. Following intravenous injection of cationiclipid/DNA complexes, there is rapid sequestration of thecomplex by lung, with no uptake by brain [10]. Ifantisense gene therapy is to be used for brain cancer, thenalternative forms of gene targeting technology need tobe developed. Viral vectors are limited by the lack oftransport of the virus across the blood–brain barrier(BBB) and by the inflammation in brain followingintracerebral administration of a single dose of eitheradenovirus or herpes simplex virus [11,12]. Genetargeting technology should enable the delivery to braincancer of a non-viral formulation of the therapeutic genevia a simple intravenous injection. Such a gene targetingtechnology should have the following characteristics:(i) stability in blood with a prolonged residence time;(ii) transport across the brain capillary endothelial wall,which forms the BBB in vivo; (iii) endocytosis across thetumor cell membrane; and (iv) gene expression in tumorcells with the desired biologic end-point [13]. Non-invasive, non-viral gene targeting to the brain is possibleusing pegylated immunoliposomes (PILs) wherein theplasmid DNA is encapsulated within the interior of an85 nm liposome [14,15]. The surface of the liposome isdecorated with several thousand strands of polyethyleneglycol (PEG) to promote stabilization in the bloodstreamand the tips of 1–2% of the PEG strands are conjugatedwith a targeting monoclonal antibody (MAb). Thispeptidomimetic MAb triggers receptor-mediated trans-cytosis across the BBB and receptor-mediated endocytosisinto brain or tumor cells. The liposome consists of fuso-genic lipids [14,15], which release the plasmid DNA tothe cytosol of the target cell prior to gene expression. Thepattern of gene expression in vivo is determined by thereceptor specificity of the targeting MAb and no geneexpression is observed in vivo if the targeting MAb isreplaced by an isotype control antibody that does nottarget the receptor [15].

The present studies were designed to develop a genetherapy formulation suitable for use in human braincancer following intravenous administration of the gene.A gene encoding antisense mRNA directed to the humanEGFR was packaged in a non-viral plasmid under theinfluence of the SV40 promoter and the Epstein–Barrnuclear antigen (EBNA)-1 gene to promote persistence ofthe transgene and extra-chromosomal replication. Thehuman EGFR antisense gene is packaged in the interior ofthe PIL that is targeted to human glioma cells with the83-14 murine MAb to the human insulin receptor (HIR),designated HIR MAb. The HIR is expressed at both the

BBB perfusing human brain gliomas and at the plasmamembrane of human glioma cells [13]. Therefore, theHIR MAb could target the gene formulation to braintumors in vivo similarly to that demonstrated previouslyfor gene delivery to brain cells in vivo in the rat usinga targeting MAb to the rat transferrin receptor (TfR)[14,15]. For gene targeting in humans, the HIR MAb ispreferred over the TfR MAb, because the MAb directed atthe human insulin receptor is transported across theprimate BBB nearly ten-fold faster than the MAb directedat the human TfR [13].

Materials and methods

Materials

POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine)and DDAB (didodecyldimethylammonium bromide)were purchased from Avanti-Polar Lipids Inc. (Alabaster,AL, USA). Distearoylphosphatidylethanolamine (DSPE)-PEG2000 (PEG2000 is PEG of 2000 daltons) was obtainedfrom Shearwater Polymers (Huntsville, AL, USA). DSPE-PEG 2000-maleimide was custom-synthesized by Shear-water Polymers. [a-32P]dCTP (3000 Ci/mmol) was fromNEN Life Science Products Inc. (Boston, MA, USA). N-succinimidyl[2,3-3H]propionate (3H-NSP, 101 Ci/mmol),[methyl-3H]thymidine (86 Ci/mmol), enhanced chemi-luminescence (ECL) western blotting detection reagents,and protein G Sepharose CL-4B were purchased fromAmersham-Pharmacia Biotech (Arlington Heights, IL,USA). The lipofectamine was from Life TechnologiesInc. (Rockville, MD, USA). Pancreatic DNase I, with aspecific activity of 2000 Kunitz units/mg, 5-bromo-4-chloro-3-indoyl-b-galactopyranoside (X-gal), and othermolecular biology grade reagents were purchasedfrom Sigma Chemical Co. (St. Louis, MO, USA). Luci-ferase reagents, recombinant luciferase, exonuclease III,Klenow fragment, and restriction endonucleases wereobtained from Promega (Madison, WI, USA). N-hydroxysuccinimide (NHS)-fluorescein, 2-iminothiolane(Traut’s reagent), and bicinchoninic acid (BCA) proteinassay reagents were obtained from Pierce Chemical Co.(Rockford, IL, USA). Mouse IgG2a myeloma ascites wasfrom Cappel Division of ICN Pharmaceuticals (Aurora,OH, USA). Centriprep-30 (molecular weight cut-off:30 000) micro-concentrators were obtained from AmiconCo. (Beverly, MA, USA). Clone 753 plasmid DNA isderived from the pGL2 luciferase expression plasmid(Promega) and was prepared in the laboratory aspreviously described [16,17]. The pCEP4 luciferaseexpression vector was obtained from Invitrogen (SanDiego, CA, USA). The mouse hybridoma line secreting the528 MAb, the pE7 plasmid containing the human EGFRcDNA (ATCC 57346), and the U87 human brain gliomacells (ATCC HTB 14) were obtained from the AmericanType Culture Collection (ATCC) (Rockville, MD, USA).Anti-EGFR MAb, A431+EGF cell lysate (positive control),and EGFR blocking peptide were purchased from

184 Y. Zhang et al.

Copyright # 2002 John Wiley & Sons, Ltd. J Gene Med 2002; 4: 183–194.

BD Transduction Laboratories (Lexington, NY, USA).Immobilon-P membranes were from Millipore Corp-oration (Bedford, MA, USA). Fluorescein-12-2k-deoxy-uridine-5k-triphosphate (fluorescein-12-dUTP) was fromBoehringer Mannheim (Indianapolis, IN, USA). Thenick translation kit was purchased from Roche (Basel,Switzerland).

Fluorescein labeling of MAb and DNA

The 528 MAb, a murine monoclonal antibody against thehuman EGF receptor, and the 83-14 MAb, a murinemonoclonal antibody to the HIR, were purified by proteinG affinity chromatography from serum-free hybridoma-conditioned media as described previously [18]. Both ofthese MAbs are mouse IgG2a; the mouse isotype controlantibody was also purified by protein G Sepharose affinitychromatography from commercially obtained mouseIgG2a ascites. The 528 MAb, the 83-14 MAb, and themouse IgG2a were individually fluoresceinated with NHS-fluorescein as described previously [19].

For production of fluorescein-conjugated DNA, 15 mg ofclone 882 plasmid DNA (described below) was labeledwith fluorescein-12-dUTP by nick translation and purifiedby LiCl/ethanol precipitation according to the manu-facturer’s instructions. The HIR MAb PILs carrying thefluorescein-882 DNA were prepared with 10 mmol oflipid, 15 mg of fluorescein-882 DNA, and 1.5 mg of HIRMAb, as described below.

Confocal microscopy

Approximately 200 000 U87 cells were plated per well ina six-well cluster dish that contained a Corning glasscoverslip at the bottom of the dish. The cells werecultured in MEM medium with 10% fetal bovine serum(FBS) and the coverslips containing the cultured cellswere transferred to individual 35-mm Petri dishes. Toeach dish was added 1 ml of fresh medium containingno serum. After cooling each dish on ice, 50 ml/well of1 mg/ml fluoresceinated antibody or mIgG2a was addedto a final concentration of 50 mg/ml, followed by incuba-tion for 60 min at 4uC. The medium was removed byaspiration; 1 ml of fresh medium without serum that wasprewarmed at 37uC was added to each well; and indi-vidual wells were incubated at 37uC for 3 or 30 min. Themedium was removed by aspiration and the wells werewashed with cold phosphate buffered saline (PBS) andfixed with 1 ml/well of 10% formalin in PBS at 4uC for20 min. The formalin was aspirated; the cells werewashed with PBS; and the coverslips were mounted onglass slides with 5% n-propyl gallate in 100% glycerol.Confocal microscopy was performed using a Leica TCSSP confocal microscope with a Leica DM-IRBE invertedmicroscope stand and an argon laser for blue excitation(488 nm for fluorescein).

For confocal microscopy of the fluorescein-conjugatedclone 882 plasmid DNA, the U87 cells were incubated foreither 3 or 24 h with 1 mg of fluo-882 DNA encapsulated

within HIR MAb PILs in MEMmedium with 10% FBS. Theclone 882 plasmid expresses EGFR antisense RNA, asdescribed below. Confocal microscopy was performedwith a Zeiss LSM 5 PASCAL microscope with an argonlaser.

Construction of an hEGFR antisensemRNA expression vector (clone 882)

The y0.7 kb EcoRI–HindIII hEGFR cDNA fragment wasobtained by double digestion of the pE7 plasmid [20] andpurified by gel electrophoresis, followed by centrifuga-tion with a Spin-X filter unit. This region of the hEGFRcorresponds to nucleotides (nts) 2317–3006 of the openreading frame (orf) and contains the tyrosine kinasedomain [20]. The hEGFR fragment was subcloned intothe eukaryotic expression vector clone 753 at HindIII–EcoNI, using cloning techniques previously described[17]. Clone 753 was derived from the pGL2 luciferaseexpression plasmid (Promega, Madison, WI, USA), aspreviously described [16]. The 3k-end of the EcoRI andEcoNI fragments was filled with deoxynucleotide tripho-sphates (dNTPs) and the Klenow fragment of DNApolymerase I to generate clone 879. This clone is drivenby the SV40 promoter, followed by antisense hEGFRnts 2317–3006, and the SV40 T antigen intron andpolyadenylation signals. Clone 879 also contains nts2100–2300 of the bovine GLUT1 glucose transporter 3k-untranslated region (UTR), which introduces a cis-actingstabilizing element in the 3k-UTR of the expressed mRNA[17]. Positive clones were confirmed by restrictionendonuclease mapping (ClaI) and by DNA sequencingusing the pGL2-1 sequencing primer (Promega) [21]. TheSV40-antisense hEGFR expression cassette from clone879 was further subcloned in pCEP4 (Invitrogen, SanDiego, CA, USA) to introduce the Epstein–Barr nuclearantigen (EBNA)-1 and replication origin (oriP), whichenable extra-chromosomal replication in human cells[22]. Clone 879 was double-digested with XhoI and SalIto release the y2.2 kb SV40-antisense hEGFR fragment.In parallel, the pCEP4 vector was digested with SalI/XhoIto release the CMV cassette and purified as previouslydescribed [17]. The SV40-antisense hEGFR expressioncassette was ligated into the pCEP4 to generate clone882, in reverse orientation, and clone 884 in forwardorientation. The insert orientation of the clones wasidentified by restriction endonuclease mapping with NruIand HindIII, and confirmed by DNA sequencing. Clone882 is under the influence of the SV40 promoter; itexpresses the EGFR antisense mRNA containing theGLUT1 glucose transporter 3k-UTR for mRNA stabilization[17] and contains the EBNA-1 and oriP elements derivedfrom pCEP4 to enable persistence of gene expression viaextra-chromosomal replication in human cells.

DNA radiolabeling

Supercoiled DNA was labeled with [a-32P]dCTP by thenick translation system as described previously [14],

Antisense Gene Targeting 185


using DNA polymerase I and DNase I. Unincorporatednucleotide was removed by a G25 Sephadex column(Roche Diagnostics Co., Indianapolis, IN, USA). Thespecific activity of the labeled probe was 5 mCi/mg; thepercentage of incorporation and purity were 77% and96%, respectively.

Pegylated liposome synthesis andplasmid DNA encapsulation

POPC (18.8 mmol), DDAB (0.4 mmol), DSPE-PEG2000

(0.6 mmol), and DSPE-PEG2000-maleimide (0.2 mmol)were dissolved in chloroform, followed by evaporation.Owing to the 2 : 1 molar ratio of anionic lipid (DSPE-PEG)relative to the cationic lipid (DDAB), the net charge of theliposome is anionic. The lipids were dispersed in 0.2 ml of0.05 M Tris–HCl buffer (pH 7.0) and vortexed for 1 min,followed by 2 min of bath sonication. Supercoiled DNA(150 mg) and 2 mCi of [32P]DNA were added to the lipids.The dispersion was frozen in ethanol/dry ice for 5 min,thawed at 40uC for 1–2 min, and incubated at room tem-perature for 5 min; this freeze–thaw cycle was repeatedten times. The liposome dispersion was diluted to a lipidconcentration of 40 mM, followed by extrusion four toeight times through two stacks each of 400-nm, 200-nm,and 100-nm pore size polycarbonate membranes, with ahand-held LipoFast2-Basic extruder (Avestin, Ottawa,Canada), as described previously [14]. The mean vesiclediameters were determined by quasi-elastic light scat-tering using a Microtrac Ultrafine Particle Analyzer(Leeds-Northrup, St. Petersburg, FL, USA), as describedpreviously [23].

The plasmid absorbed to the exterior of the liposomeswas removed by nuclease digestion, and 5 units of pan-creatic endonuclease I and 5 units of exonuclease III wereadded in 5 mM MgCl2 to the liposome/DNA mixtureafter extrusion [24]. After incubation at 37uC for 1 h, thereaction was stopped by adding 20 mM EDTA. Thenuclease digestion removed any exteriorized plasmidDNA, as demonstrated by agarose gel electrophoresis andethidium bromide staining of aliquots taken before andafter nuclease treatment, as described previously [14].The formulation prior to antibody conjugation is desig-nated a pegylated liposome (PL) and the formulationafter antibody conjugation is called a pegylated immu-noliposome (PIL).

Conjugation of the HIR MAb to thepegylated liposome/DNA complex

The HIR MAb was radiolabeled with [3H]NSP, as descri-bed previously [25]. The [3H]MAb had a specific activityof 0.11 mCi/mg and a trichloroacetic acid (TCA) precipit-ability of 97%. The HIR MAb (3.0 mg, 20 nmol) wasthiolated with a 40 : 1 molar excess of 2-iminothiolane(Traut’s reagent), as described previously [23]. The thio-lated HIR MAb, which contained a trace amount of3H-labeled HIR MAb, was conjugated to the pegylatedliposome overnight and unconjugated MAb was separated

by Sepharose CL-4B column chromatography, asdescribed previously [14]. The number of HIR MAbmolecules conjugated per liposome was calculated fromthe total [3H]HIR MAb cpm in the liposome pool and thespecific activity of the labeled HIR MAb, assuming100 000 lipid molecules per liposome, as describedpreviously [23]. The average number of MAb moleculesconjugated per liposome was 39t4 (meantSE, n=5syntheses). The final percentage entrapment of 150 mg ofplasmid DNA in the liposome preparation was computedfrom the 32P radioactivity and was 16t2% (meantSE,n=5 syntheses) or 24 mg of plasmid DNA.The immunoliposome solution was sterilized for use

in tissue culture with a 0.22 mm filter (Millipore Co.,Bedford, MA) and the recovery for 3H or 32P was >95%after filtration sterilization. An aliquot of the filtered HIRMAb-PIL was re-applied to the CL-4B column to examinethe extent to which the sterilization procedure disruptedthe PIL carrying the encapsulated DNA.

Luciferase gene expression withlipofectamine – comparison of SV40and cytomegalovirus (CMV) promoters

The effect of either CMV or SV40 promoters on expres-sion of the luciferase gene was investigated in U87 cells.These studies were performed with clones 781 and 790,which are both pCEP4-derived luciferase expression plas-mids. In the pCEP-derived clone 790, the luciferase geneis driven by the SV40 promoter, as described previously[17]. To investigate the effect of the CMV promoter, theluciferase gene was also subcloned at the HindIII andBamHI sites of the pCEP4 multiple cloning region to gene-rate clone 781, which is driven by the CMV promoter.U87 glioma cells were grown in 35 mm dishes with

Eagle’s minimal essential medium (MEM) containing 10%FBS. Transfection of cells was performed with lipofecta-mine as previously described [17]; 2 mg of plasmid DNAin 20 ml of lipofectamine was individually mixed with100 ml of MEM without serum, combined, and incubatedat 22uC for 10 min. Cells were rinsed twice with 3 ml ofMEM without serum and incubated with the transfectionmixture for 16 h in a humidified incubator at 37uC in anatmosphere of 95% air–5% CO2. The transfection mediumwas discarded and the cells were incubated in completeMEM–10% FBS for 24–48 h. After incubation, cells werelysed with 200 ml of Reporter Lysis Buffer (Promega). Celllysates were transferred to a microcentrifuge tube, vor-texed, and centrifuged at 12 000 g for 2 min at 4uC topellet the cell debris. The supernatant (cell extract) wastransferred to a new tube. Luciferase activity was deter-mined using 20 ml of cell extract and 100 ml of luciferinsubstrate (Promega) in a Berthold luminometer (LumatLB 9507, BadWildbad, Germany), as previously described[17]. Luciferase activity was recorded in relative lightunits (RLU) and converted to pg of luciferase using astandard curve determined in parallel. The backgroundlevel was determined by measuring a sample containing

186 Y. Zhang et al.


only lysis buffer. The protein concentration in the cellextract was determined with the BCA protein assayreagent and the results were expressed as pg luciferaseper mg cell protein.

Luciferase gene expression withpegylated immunoliposomes (PILs)

U87 cells were grown on 35-mm collagen-treated dishes.When the cells reached 80% confluence, the medium wasremoved by aspiration and 3 ml of fresh MEM mediumcontaining 10% FBS was added to the cells. Milliporefilter-sterilized aliquots (0.24 ml) of PILs conjugated withHIR MAb, and containing 2 mg of clone 790 plasmid DNA,were added and the cells were incubated for 24, 48 or72 h. At the end of the incubation, the medium wasdiscarded and cells were washed twice with 0.01 M PBSprior to extraction for luciferase activity as describedabove. Serum was added to the medium containing theHIR MAb PILs because it does not inhibit the activity ofthe PIL gene targeting system. In contrast, seruminhibited the activity of lipofectamine; it was omittedfrom the medium during the initial incubation withlipofectamine/DNA, as described above.

b-galactosidase gene expression

The pSV-b-galactosidase expression plasmid, designatedclone 756, was obtained from Promega. This plasmid

DNA was encapsulated in either PLs without MAb or inPILs conjugated with the HIR MAb. U87 cells were grownto 80% confluence on 35-mm collagen-treated dishes. Themedium was aspirated and 4 ml of MEM medium wasadded per dish; 1 ml of either PLs or HIR MAb PILscontaining 0.3–1.5 mg of clone 756 DNA was the added tothe cells and the cells were incubated for 24 h at 37uC. Atthe end of the incubation, the medium was aspirated andwashed gently with 0.01 M PBS/pH7.0. The cells werefixed with 0.5% glutaraldehyde in 0.1 M NaH2PO4/pH7.0at room temperature for 5 min. The fixative was aspiratedand the plate was washed with 0.1 M NaH2PO4/pH7.0once; 25 ml of 40 mg/ml X-gal in 100% DMSO and 1 ml ofdeveloping solution [0.01 M NaH2PO4 (pH 7.0), 0.15 MNaCl, 35 mM K3Fe(CN)6, 35 mM K4Fe(CN)6, 0.01%sodium deoxycholate, 0.02% Np-40] were added to thefixed cells and the cells were incubated overnight at 37uCin a humidified chamber without added CO2. The disheswere scanned with a 1200 dpi UMAX flatbed scanner witha trans-illuminator and the images were cropped withAdobe Photoshop 5.5 on a G4 Power Macintosh.

[3H]thymidine incorporation in U87cells

U87 cells were grown on 35-mm collagen-treated dishes.The medium was aspirated and 1.7 ml of fresh MEMmedium was added. Clone 882 plasmid DNA, whichencodes the EGFR antisense RNA, was encapsulated in

Figure 1. Confocal fluorescent microscopy of U87 glioma cells in tissue culture incubated with either the HIR MAb (A, D), theEGFR MAb (B, E), or the mouse IgG2a isotype control (C, F). All three antibodies were directly conjugated with fluorescein.Cells were incubated with the antibody for either 3 or 30 min at 37uC prior to fixation and confocal microscopy. The magnifi-cation bar in A is 8 mm. The fluorescein-conjugated HIR MAb is detected within intra-nuclear vesicles after 3 or 30 min ofincubation, which is indicative of endocytosis into the cell (A and D)



either PLs without HIR MAb or in PILs conjugated withHIR MAb. In addition, clone 790 DNA, which encodesfor luciferase RNA, was encapsulated in PILs conjugatedwith HIR MAb. The Millipore filter-sterilized PLs or PILscontaining 1.5 mg of either clone 882 DNA or clone 790DNA, 4 mCi of [3H]thymidine, and a final concentration of10 mM unlabeled thymidine were added to the cells andthe cells were incubated at 37uC for 6, 24 or 48 h in MEMwith 10% FBS. The medium was aspirated and the cellswere washed three times with cold 0.01 M PBS; 1 ml ofcold 10% TCA was added to each dish and the cells wereincubated on ice for 10 min. The cells were collected witha rubber policeman, transferred to a microfuge tube, andcentrifuged for 60 s at 4uC. The TCA supernatant wasdiscarded by aspiration; 0.5 ml of 1 N NaOH–0.25% SDSwas added to the pellet, vortexed, and heated for 30 minat 60uC to solubilize the cells; 250 ml of solubilized cellsolution was removed and 3H radioactivity was countedby adding 10 ml of Ultima GOLD scintillation cocktail(Packard Instrument Co., Downers Grove, IL, USA). Theremainder of the solubilized cell solution was used forBCA protein assay and the data were expressed as nmolthymidine incorporated per mg protein.

Western blot analysis

U87 cells were grown on 35-mm collagen-treated dishes.When the cells reached 80% confluence, the medium wasremoved by aspiration and 3 ml of fresh MEM mediumcontaining 10% FBS was added to the cells. Followingproduction of the HIR MAb PILs carrying the plasmidDNA, the preparation was sterilized by passage througha 0.22 mm Millipore filter; aliquots (0.3 ml) containing1.5 mg of clone 882 or clone 790 plasmid DNA were addedand the cells were incubated for 72 h. At the endof the incubation, the washed cells were suspended inreducing SDS sample buffer, electrophoresed througha 7.5% SDS-PAGE gel, and the proteins blotted to anImmobilon-P membrane. The membrane was blockedwith 5% non-fat dried milk overnight and then incubatedfor 1 h at room temperature with 5 mg/ml anti-EGFRMAb or 5 mg/ml anti-EGFR MAb plus 33.3 mg/ml EGFRblocking peptide as a control. After three washes for10 min each with 0.05% Tween-20 in PBS, the membranewas incubated for 20 min at room temperature with1:2000 horseradish peroxidase-conjugated anti-mouseIgG. After the membrane was washed, peroxidase wasdetected with the ECL system. The membrane wasexposed to an Amersham Hyperfilm for 2 min and thefilm was quantitated by scanning densitometry.

Statistical analysis

Statistically significant differences in the incorporation ofthymidine were determined by an analysis of variance(ANOVA) with Bonferroni correction using program 7D ofthe BMDP Statistical Software package developed by theUCLA Biomedical Computing Series. A p value less than0.05 was considered significant.

Results

The targeting MAb must not only bind to the tumor cell

plasma membrane, but also trigger active endocytosis in

the tumor cell. The endocytosis in U87 cells of fluor-

esceinated 528 MAb to the human EGFR, of fluorescei-

nated 8314 MAb to the HIR, and of fluoresceinated mouse

IgG2a isotype control was investigated by confocal

fluorescent microscopy (Figure 1). The HIR MAb targets

the insulin receptor on the plasma membrane and this

antibody is rapidly endocytosed into the U87 human

glioma cells, as demonstrated by the accumulation of the

antibody within intra-nuclear vesicles (Figures 1A and

1D). The HIR MAb is detected in vesicular structures as

early as 3 min of incubation (Figure 1A). In contrast, no

intracellular vesicles were detected either with the 528

MAb to the human EGFR (Figures 1B and 1E), or with the

mouse IgG2a isotype control (Figures 1C and 1F). On the

basis of these studies showing prominent intracellular

distribution of the HIR MAb, the PILs were subsequently

formulated with the HIR MAb.Following encapsulation of the plasmid DNA within the

pegylated liposomes, the thiolated and 3H-radiolabeled

HIR MAb was conjugated to the tips of the PEG strands.

Figure 2. (Top) Elution of pegylated immunoliposomes (PILs)through a Sepharose CL-4B gel filtration column allows forseparation of the PILs from smaller size unconjugated MAband exteriorized nuclease-digested DNA. The [32P]-plasmidDNA is encapsulated in the interior of the PIL, which is con-jugated with [3H]HIR MAb. (Bottom) The purified PIL wassterilized for use in tissue culture by filtration through a0.22 mm Millipore filter and an aliquot of the sterilized PILwas re-applied to the CL-4B column. The PIL was intactfollowing passage through the 0.22 mm filter, as both the[3H]-targeting antibody and the [32P]-plasmid DNA co-elutedin the first peak of the column

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The PIL was separated from unconjugated HIR MAb and

from digested exteriorized [32P]DNA by CL-4B gel

filtration chromatography as shown in Figure 2 (top

panel). The final PIL peak was sterilized by passage

through a 0.22 mm Millipore filter to enable use in tissue

culture and an aliquot of the sterilized PIL preparation

was re-analyzed on the CL-4B column (Figure 2, bottom

panel). These data show exact co-migration of the

[32P]plasmid DNA and the [3H]HIR MAb and no elution

from the column of low-molecular-weight impurities

(Figure 2, bottom panel), which indicates that the

integrity of the PIL is not altered by filtration through a

0.22 mm filter.The SV40-b-galactosidase plasmid was packaged in

the interior of either pegylated liposomes (PLs) carrying

no MAb, or inside HIR MAb-conjugated PILs, and the

preparation was added to cultured U87 glioma cells. A

diagram of the PL is shown in Figure 3A and the structure

of the PIL conjugated with the HIR MAb is shown in

Figure 3B. Following 24, 48, and 72 h of culture with

either the PL or the PIL, the cells were fixed for b-

galactosidase histochemistry. When the SV40-b-galacto-

sidase plasmid was packaged inside non-targeting PLs,

there was no specific gene expression for b-galactosidase

in the cultured cells, although occasional cells expressing

endogenous enzyme activity were visible (Figure 3C).

However, when the SV40-b-galactosidase plasmid was

packaged in the interior of HIR MAb PILs, there was a

linear increase with time in the level of b-galactosidase

histochemical product; the histochemistry at 24 h is

shown in Figure 3D. These studies showed that more

than 90% of the cells in tissue culture are transfected with

the exogenous gene using the HIR MAb PILs.The delivery of the luciferase reporter gene to the U87

cells was examined with either lipofectamine or the HIR

MAb-PIL delivery systems (Figure 4). In initial studies

with lipofectamine only, luciferase gene expression in the

U87 cells was measured for either clone 781, which is

driven by the CMV promoter, or for clone 790, which is

under the influence of the SV40 promoter. The data in

Figure 4A demonstrate a large increase in expression of

the luciferase reporter gene driven by the SV40 promoter,

compared with the CMV promoter. Therefore, subsequent

luciferase reporter constructs utilized the SV40 promoter.

The SV40-luciferase plasmid was delivered to U87 cells

with either lipofectamine or HIR MAb PIL; comparable

levels of gene expression were observed with either

system (Figure 4B). The luciferase gene expression with

lipofectamine at 48 h exceeds the luciferase gene expres-

sion at 48 h with the HIR MAb PIL (Figure 4B). However,

Figure 3. (A) Structure of a pegylated liposome (PL) carrying plasmid DNA. (B) Structure of a pegylated immunoliposome(PIL) carrying DNA. The targeting MAb was the HIR MAb. b-galactosidase histochemistry of U87 cells grown in tissue culturewas performed at 24 h after incubation with either the PL carrying the b-galactosidase plasmid (C) or the HIR MAb PIL carry-ing the b-galactosidase plasmid (D). Bar=29 mm. The cells were not counter-stained



following a single addition of HIR MAb PIL at time 0, thelevel of luciferase gene expression versus time of incu-

bation increased linearly over a 72-h period (Figure 4C).

The level of luciferase enzyme activity continued to

increase over time and peaked at 419t31 pg luciferase

per mg protein (meantSD) at 7 days of incubation

following the single addition to the cells at time 0 of the

luciferase plasmid encapsulated in the HIR MAb PIL.The third gene that was targeted to the U87 cells with

the HIR MAb-PIL gene delivery system was a plasmidencoding for EGFR antisense mRNA (clone 882). An

approximate 700 base pair fragment of the human EGFR

cDNA was released by double restriction endonuclease

digestion of the pE7 plasmid (Figure 5A). This 0.7 kb

fragment included nucleotides 2317–3006 of the human

EGFR open reading frame (Figure 5C) and was subcloned

in reverse orientation in the pCEP4 plasmid, as outlined in

Figure 5B. The orientation of the EGFR antisense gene

within the plasmid was confirmed by restriction endonu-

clease digestion (Figure 5D). The final plasmid was

double-digested with NruI and HindIII, resulting in the

release of 2.0 and 9.0 kb fragments for the reverse

orientation (lane 1, Figure 5D) and 3.7 and 7.5 kb

fragments for the forward orientation (lane 2, Figure 5D).The biologic activity of the EGFR antisense gene,

designated clone 882 (Figure 5), was examined by

treating the U87 cells with one of three different

formulations over a 6–48 h period: (a) clone 882 DNA

was encapsulated in PLs conjugated with no MAb and

designated PL/882; (b) clone 790 DNA was encapsulated

in HIR MAb PILs and designated HIR MAb PIL/790; and

(c) clone 882 DNA was encapsulated in HIR MAb PILs and

designated HIR MAb PIL/882. The rate of thymidine

incorporation into the cells was linear if the cells were

exposed with PL/882, which encodes for EGFR antisense

mRNA, which is packaged in non-targeted PL (Figure 6).

As shown by the histochemistry (Figure 3C), the PL

formulation (Figure 3A) does not lead to gene expression

and a high rate of thymidine incorporation was observed

(Figure 6). A similarly high rate of thymidine incorpora-

tion was observed when the clone 790 plasmid DNA,

which encodes for luciferase, was delivered to the U87

cells with the HIR MAb PILs (Figure 6). However, there

was more than a 70% reduction in the rate of thymidine

incorporation into the U87 cells exposed to the clone 882

EGFR antisense gene packaged in the interior of the HIR

MAb targeted PILs (Figure 6). The amount of clone 882

EGFR antisense gene that was packaged in the HIR

MAb PILs was varied from 3 ng to 3000 ng, and a dose

response was observed (data not shown).The pharmacologic effect of the HIR MAb PIL/882 was

confirmed by EGFR western blot analysis. As shown in

Figure 7A (left panel), incubation of the U87 cells with

the HIR MAb-PIL carrying the clone 882 DNA resulted

in a decrease in the amount of immunoreactive EGFR,

compared with the receptor level in the cells exposed to

an equal amount of HIR MAb PILs carrying a control DNA

comprised of clone 790. Co-incubation of the primary

anti-EGFR MAb with the EGFR blocking peptide elimi-

nated the EGFR signal (Figure 7A, right panel). The film

was quantitated with NIH Image software and incuba-

tion of the cells with the HIR MAb PIL/882 resulted in a

79% decrease in the level of the immunoreactive EGFR,

compared with incubation of the cells with the HIR MAb

PIL/790 formulation (Figure 7B).The intracellular delivery of clone 882 DNA into the

U87 cells by the HIR MAb PILs was demonstrated by

confocal microscopy. Clone 882 DNA was fluorescein-

conjugated prior to encapsulation in the HIR MAb PILs

and the U87 cells were incubated for either 3 or 24 h

with HIR Mab PIL/fluoro-882. By 3 h, the fluorescein-

conjugated DNA was distributed throughout the U87

cytoplasm and was also observed within intra-nuclear

vesicles (Figure 8A). Conversely, after 24 h of incubation,

the fluorescein-conjugated DNA was primarily seques-

tered within the nucleus (Figure 8B).

Figure 4. (A) Luciferase activity expressed as pg luciferaseequivalent per 35 mm dish is shown for U87 cells transfectedwith the pCEP4 expression plasmid driven by either the CMV(clone 781) or the SV40 promoter (clone 790). Data aremeantSE (n=3). (B) Luciferase activity is expressed as pgper dish for U87 cells transfected with the SV40-luciferaseexpression plasmid (clone 790) with either lipofectamine(open bars) or the HIR MAb-conjugated pegylated immunoli-posome (PIL, closed bars). (C) Luciferase activity in the U87cells transfected with the SV40-luciferase plasmid (clone 790)packaged in the interior of the HIR MAb PIL is plottedagainst the time of incubation. Data are meantSE (n=3dishes per point)

190 Y. Zhang et al.


Discussion

The results of these studies are consistent with thefollowing conclusions. First, exogenous genes may betargeted to glioma cells with a non-viral gene targetingtechnology using antibody-directed pegylated immuno-liposomes (PILs). Second, the PIL technology gives levelsof gene expression comparable to that observed with acationic lipid, lipofectamine (Figure 4). Third, virtuallyall cells in tissue culture are targeted by the HIR MAbPILs based on b-galactosidase histochemistry (Figure 3).Fourth, the desired biologic end-point, inhibition of EGFRexpression (Figure 7) and inhibition of cell proliferation(Figure 6), is achieved with an EGFR antisense gene,provided the gene is packaged in the interior of thetargeted PIL. Fifth, the HIR-PIL delivery system enablesrapid endocytosis of the therapeutic gene, followed bysequestration within the nucleus (Figure 8).

The 83-14 HIR MAb is an endocytosing antibody thatenters the cytosol after binding to the insulin receptor onthe plasma membrane of the cell [26]. Confocal micro-scopy shows selective endocytosis of the 83-14 HIR MAb,

compared with the 528 EGFR MAb (Figure 1). Radi-

oligand studies show that the 528 MAb avidly binds to

U87 cells in culture (unpublished observations); however,

the confocal microscopy of the U87 cells with fluorescei-

nated 528 MAb shows that this antibody is internalized at

a much slower rate than is the HIR MAb (Figure 1). The

HIR MAb is found in intracellular vesicles after only 3 min

of incubation, indicating active endocytosis into the

intracellular system. Prior work has shown that the 83-

14 HIR MAb is an active in vivo BBB drug delivery vector

in Old World primates such as the rhesus monkey [27]. In

addition, immunocytochemistry of human glioblastoma

multiforme shows persistent expression of the HIR on the

capillary endothelium perfusing the tumor and HIR on

the plasma membrane of tumor cells [13]. Therefore, the

HIR MAb could be used as a vector for targeting to human

brain cancer PILs carrying tumor-directed antisense

genes. The HIR MAb can cause both the receptor-

mediated transcytosis across the BBB at the tumor

microvasculature and the receptor-mediated endocytosis

into the tumor cell.In addition to transport across cellular barriers, an

Figure 5. (A) pE7 plasmid was digested with EcoRI and HindIII to release a 0.7 kb fragment near the 3k end of the codingsequence of the human EGFR cDNA. (B) The 0.7 kb fragment was subcloned in reverse orientation in the pCEP4 expressionplasmid to generate the pCEP4-882 plasmid encoding mRNA antisense to the human EGFR. (C) The EGFR antisense mRNA corre-sponds to nucleotides 2317–3006 of the human EGFR open reading frame. (D) Ethidium bromide-stained agarose gel shows restric-tion endonuclease-digested plasmid (lanes 1 and 2) and DNA molecular weight standards (lanes 3 and 4). Restriction endonucleasedigestion indicates that clone 882 (lane 1) is in the reverse orientation, whereas clone 884 is in the forward direction (lane 2).Double digestion with NruI and HindIII results in the release of 2.0 and 9.0 kb fragments when the insert is in the reverse orientation(lane 1) and in the release of 7.5 and 3.7 kb fragments when the insert is in the forward orientation (lane 2)



in vivo gene targeting technology must protect the

therapeutic gene from the ubiquitous endonucleases in

the body [13]. This is accomplished with the use of PILs,

which carry the gene in the interior of the liposome

[14,15]. The liposome structure provides a formulation

that allows for conjugation of a targeting MAb to a

plasmid gene that is encapsulated within the interior of

the liposome and this is demonstrated by gel filtration

chromatography (Figure 2). Liposomes, per se, are not

stable in the blood and have very short plasma residence

times. The surface of a naked liposome is immediately

coated with serum proteins in vivo [28] and this triggers

uptake of the liposome by cells lining the reticulo-

endothelial system (RES). However, tethering approxi-

mately 2000 strands of PEG polymers to the surface of the

liposome inhibits the binding of plasma proteins to the

liposome surface and reduces uptake by the RES [29].

This ‘pegylation’ of the liposome results in a stabilized

formulation in blood and a prolonged plasma residence

time [23]. In contrast, cationic lipid/DNA complexes are

rapidly removed from blood by the lung [10]; in tissue

culture, cationic lipid/DNA complexes are taken up by

phagocytosis, not endocytosis [9]. Conversely, the PIL

delivers the gene to the target cell via receptor-mediated

endocytosis and virtually all cells in culture are trans-

fected with this delivery system, as shown by the b-

galactosidase histochemistry (Figure 3).Targeting the EGFR antisense gene (clone 882) to U87

cells with the HIR MAb-PIL gene delivery system results in

a reduction in cell proliferation that is more than 70%based on [3H]thymidine incorporation (Figure 6) and areduction of 79% in the level of immunoreactive EGFR(Figure 7). The EGFR antisense construct targets nucleo-tides 2317–3006 of the human EGFR mRNA and thisoverlaps with the tyrosine kinase domain (Figure 5C).The orientation in the reverse direction was confirmed byrestriction endonuclease mapping (Figure 5D). The EGFRantisense expression plasmid is 11.0 kb and has thefollowing characteristics: (i) the antisense gene is drivenby the SV40 promoter, which gives higher levels of geneexpression than the CMV promoter (Figure 4A); (ii) the3k-UTR of the antisense mRNA contains a cis element fromthe GLUT1 glucose transporter mRNA, which stabilizesthe transcript in cells [16,17]; and (iii) the constructcontains EBNA-1 and oriP gene elements to promoteextra-chromosomal replication and persistence of geneexpression in cells [17].The HIR MAb-PIL gene delivery system enables the

nuclear accumulation of the therapeutic gene within thetarget cell, as shown by the confocal microscopy study inFigure 8. There is rapid uptake of either the uncon-jugated HIR MAb (Figures 1A and 1D) or the HIR MAbPIL/fluoro-DNA (Figure 8A) into intra-nuclear vesiclesof the U87 cells. Certain endocytosing receptors enablethe movement of the ligand from the extracellular space

Figure 6. Nanomoles of [3H]thymidine incorporated per mgof U87 cell protein at 6, 24, and 48 h of incubation of thecells with (i) pegylated liposomes (PLs) carrying the clone882 plasmid DNA (1.5 mg/dish), (ii) HIR MAb pegylatedimmunoliposomes (PILs) carrying the clone 790 plasmid DNA(1.5 mg/dish), or (iii) HIR MAb PILs carrying the clone 882plasmid DNA (1.5 mg/dish). Clone 882 plasmid DNA encodesfor the EGFR antisense mRNA (Figure 5) and clone 790 plas-mid DNA encodes for luciferase mRNA. Data are meantSE(n=3). The decrease in thymidine incorporation at both 24and 48 h in the cells incubated with the clone 882 DNA pack-aged in the HIR MAb PILs (PIL/882) was significant at thep<0.01 level. There is no inhibition of thymidine incorpora-tion into the cells following 6 h incubation with the HIR MAbPIL/882 formulation

Figure 7. (A) EGFR western blot of U87 cells incubated for72 h with either the HIR MAb PIL/882 or the HIR MAb PIL/790 formulations. The western blot with the primary anti-EGFR MAb alone is shown in the left panel and the westernblot with the primary anti-EGFR MAb plus EGFR blockingpeptide is shown in the right panel. The incubations ofthe cells were performed in triplicate. (B) The ECL film fromthe western blot was scanned and the integrated density forthe EGFR signal was computed with NIH Image software andnormalized per mg protein of U87 cells in each dish. Thedata are meantSE (n=3). Incubation of the cells with theclone 882 DNA, encapsulated within the HIR MAb PILs,resulted in a 79% decrease in the level of immunoreactiveEGFR, compared with the cells incubated with clone 790DNA, encapsulated within the HIR MAb PILs

192 Y. Zhang et al.


into the nuclear compartment [30] and these pathways

may facilitate the nuclear delivery of DNA conjugated to

the receptor ligands [31]. The intra-nuclear delivery of

therapeutic DNA by the HIR MAb-PIL system may allow

for intra-nuclear production of the EGFR antisense RNA.

The intra-nuclear EGFR antisense RNA may then bind the

endogenous EGFR sense mRNA to promote the pharma-

cologic effect of the antisense gene therapy.In conclusion, a gene targeting technology has been

developed that may enable the delivery to brain cancer

cells in vivo of antisense genes, which are intended to

suppress the activity of tumor-specific transcripts. The

targeting technology results in selective delivery of drugs

to brain tumors in vivo relative to normal brain, and

uniform targeting to the tumor center and periphery [32].

The PIL gene targeting technology allows for widespread

gene expression in the brain in vivo following the intra-

venous injection of non-viral gene formulations [14,15].

Gene expression is restricted to the brain followingintravenous administration if the exogenous gene that isencapsulated within the PIL is under the influence of abrain-specific gene promoter [33]. The expression oftherapeutic genes may be restricted to brain cancer in vivo

following intravenous administration of the exogenousgene with the combined use of tumor-specific genepromoters and gene targeting technology using PILs.

Acknowledgements

Daniel Jeong skilfully prepared the manuscript. Drs Matt

Schibler and Chunni Zhu assisted with the confocal microscopy.

This work was supported by a grant from Accelerate Brain

Cancer Cure, Inc. (ABC2).

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receptor-mediated delivery of an antisense gene to human brain cancer cells

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