optimization of retroviral vector-mediated gene transfer into

1508

Optimization of Retroviral Vector-MediatedGene Transfer Into Endothelial Cells In Vitro

Mark L. Kahn, Sung W. Lee, and David A. Dichek

Retroviral vector-mediated gene transfer into endothelial cells is relatively inefficient with transductionrates as low as 1-2% in vitro and even lower in vivo. To increase the efficiency of gene transfer intoendothelial cells, we used retroviral vectors expressing f-galactosidase and urokinase and measuredendothelial cell transduction efficiencies with quantitative assays for /-galactosidase and urokinaseprotein. We evaluated several techniques reported to improve the efficiency of retroviral transduction invitro, including 1) extended periods of exposure to vector, 2) repeated exposures to vector, 3) maximizationof the ratio of vector particles to endothelial cells by increasing the volume and concentration of vectorparticles or by decreasing the number of endothelial cells exposed, 4) cocultivation of endothelial cells withvector-producing cells, and 5) variation of the type and concentration of polycation used with theretroviral vector. Only the use of more concentrated (higher titer) vector-containing supernatant and theuse of the polycation DEAE-dextran improved the efficiency of gene transfer into endothelial cells in vitro.In an optimized transduction protocol, a 60-second exposure to 1 mg/ml DEAE-dextran followed by a

single 6-hour exposure to supernatant of a titer of 105_106 colony-forming units/ml resulted intransduction efficiencies of 50-90%o with both vectors. Decreasing the time of the supernatant exposure to15 minutes permitted transduction efficiencies of 15-20%Yo while significantly minimizing the duration ofthe transduction. Therefore, the optimized protocol allows high efficiency in vitro gene transfer intoendothelial cells within several hours. The briefer protocol may prove useful for in vivo gene transfer inwhich the time of exposure to the supernatant is limited. (Circulation Research 1992;71:1508-1517)KEY WoRDs * gene transfer * endothelial cell * retrovirus * DEAE-dextran

T he endothelium has received much attention as atarget for gene transfer because of its potentialability to deliver functional gene products in

vivo at sites of vascular disease.1-4 The location ofendothelial cells (ECs) along the blood vessel lumenmay be ideal for the expression of proteins that controlfocal vascular disease processes, such as thrombosis andintimal proliferation.56 Although both liposome-medi-ated gene transfer and calcium phosphate transfectionof ECs have been reported,78 retroviral vector-medi-ated gene transfer remains the most versatile and widelyused method of gene transfer into ECs.19 Retroviralvectors possess the advantages of stable integration andresultant long-term expression of transferred geneticmaterial10 as well as a proven safety record in humanclinical protocols.1'

Realization of the potential of retroviral vector-mediated gene transfer to deliver significant levels ofrecombinant gene products from the endothelium hasbeen limited by low transduction efficiency (TE). Zwie-bel et al'2 reported a TE of 0.5-1%, as determined byneomycin selection of rabbit aortic ECs after a singleexposure to a retroviral vector-containing supernatantwith a titer of 5 x 105 colony-forming units (CFU)/ml.Wilson et a14 reported variable TE with canine ECs

From the Molecular Hematology Branch, National Heart, Lung,and Blood Institute, Bethesda, Md.Address for correspondence: Dr. David A. Dichek, Molecular

Hematology Branch, National Institutes of Health, Building 10,Room 7D-18, 9000 Rockville Pike, Bethesda, MD 20892.

Received June 24, 1992; accepted September 8, 1992.

(5-60%), as determined by visual analysis of histochem-ically stained plates after successive exposures to f8-ga-lactosidase-containing retroviral vector with titers of0.5-5x 105 CFU/ml. In our laboratory, the efficiency ofretroviral vector-mediated gene transfer into bovineand rabbit aortic ECs, sheep venous and arterial ECs,human umbilical vein ECs, and baboon external jugularvein ECs has been between 1% and 15% (Reference 13;and unpublished observations, M. Kahn and D.Dichek). Production of a purified population of genet-ically engineered ECs has therefore required the use ofvectors containing the selectable neomycin phospho-transferase gene (which occupies precious space withinthe vector) followed by 2 weeks of antibiotic selectionand expansion of the surviving cells in culture.'4 Directin vivo gene transfer into vascular cells with retroviralvectors has also been attempted with transduction effi-ciencies even lower than those found in vitro."",16

For purely in vitro experiments, a delay of 2-3 weeksto obtain a purified population of transduced cells is notclearly problematic. However, for protocols in whicheither transduced cells are reimplanted in vivo or trans-duction itself is performed in vivo, low TE is a majorobstacle. Antibiotic selection is time consuming andexpensive. Prolonged in vitro culture may alter the ECphenotype17"18 and expose the cells to a continual dan-ger of microbial contamination, with consequent failureof a reimplantation protocol. Low efficiency of genetransfer renders an in vivo gene transfer protocol un-likely to produce a biological effect. Therefore, high TEis desirable for ex vivo protocols and essential for in vivo

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Kahn et al Optimization of Gene Transfer 1509

protocols. Despite the importance of increasing theefficiency of retroviral vector-mediated gene transferinto ECs, no study has systematically investigated TEand attempted to optimize it. Although one finds tech-niques in use that purportedly enhance the rate oftransduction, such as repetitive exposure to retroviralvector-containing supernatants,4 optimization of thecalculated multiplicity of infection (MOI),'9 and addi-tion of particular concentrations of polycation such asPolybrene or Protamine to vector-containing superna-tant,20 there are little or no published data to supportthese practices. In the present study, we used bovineaortic ECs (BAECs) and vectors encoding both intra-cellular and secreted gene products to evaluate the roleof many of the factors commonly believed to affect TEboth in ECs as well as in other cell types. Specifically,we determined 1) the effects of varying either length ofexposure or frequency of exposure to retroviral vectors,2) the importance of the titer of the retroviral vector-containing supernatant and the effect of concentratingsupernatant by centrifugation to increase titer, 3) thecontribution of the type and concentration of polycationused to enhance TE, and 4) the effect on TE ofcocultivating BAECs with the cells that produce theretroviral vector. Two optimized transduction schemesare proposed for either 6-hour or 15-minute exposuresto retroviral vector-containing supernatants. The for-mer protocol is useful for optimizing in vitro genetransfer; the latter may eventually be useful for in vivogene transfer protocols, in which time of exposure ofcells to the vector must be kept to a minimum.

Materials and MethodsCell Harvest and CultureBAECs were isolated from a fresh aorta using the

method of Jaffe et al.21 Endothelial cell identity wasconfirmed using immunoperoxidase staining with anti-body to human von Willebrand's factor (Atlantic Anti-bodies, Scarborough, Me.) and by fluorescent stainingfor the presence of acetylated low density lipoproteinreceptors22 (Biomedical Technologies Inc., Stoughton,Mass.). BAECs were maintained in Dulbecco's modi-fied Eagle's medium (DMEM, Biofluids, Inc., Rock-ville, Md.) containing 10% heat-inactivated fetal calfserum (Hyclone Laboratories Inc., Logan, Utah) at37°C and 5% CO2. This medium formulation is hereaf-ter referred to as D-10. Cells were passaged usingtrypsin-EDTA digestion, and all experiments were per-formed with cells passaged fewer than 12 times.

Retroviral Vectors and Vector SupernatantLBgSN23 contains the Escherichia coli gene for a-ga-

lactosidase ligated into the Xho I site of LXSN.10G1BgSVNa,24 a kind gift of Genetic Therapy, Inc.,Gaithersburg, Md., is identical to LBgSN except for anexpanded multiple cloning site downstream of the 5'long terminal repeat (LTR). The G1BgSVNa vector wasused, as it became available, in addition to LBgSN,because of its more consistently high titer. LUKSNcontains a 1.5-kb human urokinase cDNA cloned intothe Hpa I site of LXSN.25 A schematic diagram is shownin Figure 1. In all vectors, the reporter gene (13-galactosidase or urokinase) is expressed from a tran-script initiated in the Moloney murine leukemia virus

LBgSN/G 1 BgSVNa

LAC-Z

6'LTR SV-40 NEO 3UTR

LUKSN

'~~~U5'UR SV-40 NE 3'LUR

1.0 kb

FIGURE 1. Schematic diagram of retroviral vectors. Tworetroviral vectors allowed measurement of rate of endothelialcell transduction using either an intracellular protein, 13-ga-lactosidase (termed LAC-Z), or a secreted protein, humanurokinase (UK). LBgSN contains the Escherichia coli genefor f3-galactosidase and the gene for neomycin phosphotrans-ferase (NEO). LUKSN contains a cDNA for human uroki-nase. Both reporter genes are under the transcriptional controlofthe viral long terminal repeat (LTR). The earlypromoter ofsimian virus-40 (SV-40) and the 5' and 3' viral LTRs ofMoloney murine leukemia virus are shown. A size markerindicates approximate length in kilobases.

LTR, and neomycin phosphotransferase is expressedfrom the simian virus 40 early promoter. Multiplevectors were used to ensure that results were generallyapplicable and not vector or protein specific.High titer amphotropic retroviral vector-containing

supernatant was generated in PA-31726 packaging cellsby transinfection, as described previously.'3 We definedtiter, as have others,'9 as the number of infectious vectorparticles per milliliter of supernatant, which was mea-sured by the ability of a sample of the supernatant totransfer antibiotic resistance to NIH 3T3 cells. Titer wasmeasured by plating 2.5 x 104 3T3 cells per 35-mm well24 hours before exposure to 1.5 ml serially dilutedsupernatant containing 8 gg/ml Polybrene (AldrichChemical Co., Milwaukee, Wis.) for 2 hours. After 24hours, the medium was changed to D-10 containing 0.8mg/ml G418 (a neomycin analogue that kills untrans-duced 3T3 cells in 5-7 days), and resistant colonies werecounted 12-14 days later. All viral supernatants werefree of helper virus, as assessed by the S+L- assay.27 Thetiter of supernatant after one freeze-thaw cycle was5-10x 1iO for LBgSN and 1-l0x 105 for G1BgSVNa andLUKSN. Supernatant that had been frozen once andthawed for use was used for all experiments, exceptthose in which fresh supernatant was concentrated andused without freezing. We defined MOI as the calcu-lated ratio of the number of infectious vector particles(determined by multiplying titer by supernatant vol-ume) to the total number of target cells at the time ofexposure to supernatant: MOI (CFU/cell)=titer of su-pernatant (CFU/ml)xvolume of supernatant (ml)/tar-get cell number. Target cell number at the time ofexposure to the supernatant was determined bytrypsinization and counting of cells in wells maintainedin parallel to those being transduced.



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1510 Circulation Research Vol 71, No 6 December 1992

Measurement of TETE was measured by quantitation of reporter gene

protein production. The advantages of measuring TE byprotein production as opposed to Southern analysis orpolymerase chain reaction for inserted vector DNA areas follows: 1) Recombinant protein production is themost important end point of gene transfer, whereasmeasurement of DNA gives only an indirect indicationof the ultimate success of gene transfer. Because ofpotential inefficiencies of transcription and translation,quantitation of DNA may not correlate with proteinproduction.28 2) The linearity and reproducibility of theprotein assays permit quantification of TE with refer-ence to a standard curve within the typical range of TEmeasurements (5-80%) and allow confident discrimi-nation of TEs within this range (e.g., 15% versus 30%transduction). 3) The sensitivity of the protein assaysallows the measurement of TE even in the setting ofvery low levels of gene transfer (<5%). Southern anal-ysis is far less sensitive than the protein assays. Poly-merase chain reaction is extremely sensitive but must bevery carefully controlled to permit even relative quan-tification.29 A caveat in this analysis is that a higher TE,as measured by protein production, may be due toeither a greater percentage of transduced cells or ahigher number of vector copies per cell in the samenumber of transduced cells. However, these possibilitiescan be differentiated experimentally (see below).We used f3-galactosidase and urokinase as reporter

genes. Cells transduced by LBgSN or G1BgSVNa ex-pressed intracellular ,B-galactosidase, which was mea-sured by a standard enzymatic assay of cell extracts30,31adapted to a 150-,ul volume reaction in a 96-wellmicrotiter plate. Briefly, the cells of a 35-mm well weretrypsinized, pelleted, and resuspended in 40 gl of 0.1%Nonidet-40 (Pierce Chemical Co., Rockford, Ill.). Ly-sate (15 gl) was used to measure 83-galactosidase activ-ity by using o-nitrophenyl f3-D-galactopyranoside(ONPG, Sigma Chemical Co., St. Louis, Mo.) as asubstrate and reading absorbance at 405 nm. Extracts ofuntransduced cells contained only very low backgroundlevels of /8-galactosidase activity. Total protein in thecell lysate was measured by the Bradford protein assay(Bio-Rad Laboratories, Richmond, Calif.) with bovineimmunoglobulin G as a standard. ,3-Galactosidase ac-tivity was also detected by histochemical staining, aspreviously described.13 Cells transduced by the LUKSNvector secreted human urokinase into the conditionedmedium. Secreted urokinase antigen was collected in 1ml conditioned medium over defined time periods rang-ing in individual experiments from 16 to 24 hours andmeasured using an enzyme-linked immunosorbent assayin which bovine urokinase is unreactive (AmericanDiagnostica Inc., Greenwich, Conn.). Total cellularprotein was measured with the BCA protein assay(Pierce) after addition of 0.25 ml of 0.5% Triton X-100to each 35-mm well. Urokinase secretion was calculatedwith reference to both time of collection and totalcellular protein for each well.By using the measured amount of f3-galactosidase

activity or urokinase antigen, TE was calculated as thepercentage of transduced (i.e., 83-galactosidase-contain-ing or urokinase-secreting) cells in each sample. To

we constructed standard curves of f3-galactosidase ac-tivity per transduced cell and urokinase secretion pertransduced cell using mixed populations of untrans-duced cells and transduced G418-selected cells. Briefly,a pure population of transduced cells was created byselection over 14 days in 0.4 mg/ml G418, a concentra-tion that kills 100% of untransduced cells in 7 days.Transduced selected cells and untransduced cells werethen counted and plated together in triplicate wells atconstant total cell number in proportions of 80%, 40%,20%, 10%, 5%, 2.5%, 1.25%, and 0% transduced cells.,8-Galactosidase activity or urokinase antigen secretionwas determined for each of the mixtures as describedabove, and linear regression equations were derivedfrom plots of activity or antigen secretion versus thepercentage of transduced cells. The initial equation thatwas derived for 83-galactosidase-expressing cells andapplied for calculation of TE was as follows: transducedcells (%)=(microunits ,3-galactosidase activity per mil-ligram cellular protein) x 1.16+0.84. In later experi-ments, a second equation was generated in which a newmixture of f-galactosidase-expressing cells and a newenzyme standard were used to express the units of,8-galactosidase activity according to the strict definitionof 1 unit of enzyme as the ability to convert 1 molsubstrate ONPG.31 This second equation was as follows:transduced cells (%)=(microunits f3-galactosidase ac-tivity per milligram cellular protein) x0.29-5.9. As thecalculated enzyme activity drops out of the final TEcalculation after reference to the standard curve, thedata generated with the two equations may be com-bined. The equation derived for the urokinase-secretingcells was as follows: transduced cells (%)= (nanogramsurokinase secreted per hour per milligram cellularprotein)x2.24+4.5. These equations were applied tothe individual experiments to determine the percentageof transduced cells after exposure to a vector-containingsupernatant.

Transduction ProtocolsStandard transduction conditions. The following pro-

tocol, used regularly in our laboratory before the pre-sent study, was defined as a standard transduction.BAECs were plated at 5 x 104 cells per well onto 35-mmwells on day 1. On day 2 (20 hours after plating), themedium was replaced with 1.5 ml viral supernatantcontaining 8 ,ug/ml Polybrene (Aldrich) for 6 hours,after which the medium was replaced with fresh D-10medium. The medium was again changed on day 4. Onday 5 (72 hours after exposure to the supernatant), cellsand conditioned medium were collected as describedabove for measurement of either ,B-galactosidase orurokinase. Standard transduction, as defined in thisarticle, refers specifically to this protocol.Determination of the effect of the duration of exposure

to viral supernatant on TE. To determine if the practiceof exposing target cells to supernatant for extendedperiods of time improves TE in BAECs, we varied theduration of a single supernatant exposure. BAECs were

exposed to supernatant for 2, 6, 12, or 24 hours, withother conditions unchanged from those of the standardtransduction.

Determination of the effect offrequency of exposure toviral supernatant on TE. To determine if the practice of

allow the calculation of percentage of transduced cells, exposing target cells to retroviral vector repetitively



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improves TE, BAECs were exposed to the supernatanton multiple occasions. As an additional variable, thetime intervals between the repeated exposures werealso changed. In the first experimental design, BAECsunderwent one, two, or three standard transductionsseparated by time intervals of 24 hours (time betweenthe start of one supernatant exposure and the start ofany subsequent exposures). In the second experimentaldesign, BAECs underwent three standard transductionsat 48-hour intervals or two standard transductions at72-hour intervals. In this manner, BAECs were exposedmultiple times to new solutions of retroviral vector with1) adequate time between exposures to regeneratesurface receptor proteins potentially necessary for ret-roviral vector binding and cell entry and 2) minimalchance that, over the course of the experiment, theBAEC cell cycle would precisely coincide with the timeinterval between supernatant exposures and therebyrepetitively expose all cells at the same point in their cellcycle. In all cases, TE was determined by harvestingcells and assaying for recombinant protein expression 72hours after the final supernatant exposure.

Determination of the effect ofMOI on TE by alterationof either 1) supernatant volume, 2) cell number, or 3)supernatant concentration. 1) To alter MOI by varyingthe volume of supernatant, standard transductions wereperformed with 0.5, 1, 2, 4, or 6 ml supernatant. Thevolume range was limited by the need to cover the cellswith a minimal amount of medium and by the maximumvolume of the wells. 2) To alter MOI by varying thenumber of target cells, standard transductions wereperformed with 103, 5 X 103, 104, and 5x 104 BAECs. 3)To alter MOI by varying the vector concentration in thesupernatant, standard transductions were performedwith either thawed viral supernatant or maximally con-centrated fresh viral supernatant as well as serial dilu-tions of both in D-10 medium containing 8 gg/mlPolybrene. To achieve maximal vector concentration,the supernatant was concentrated using a sterile tech-nique by centrifuging 36 ml fresh supernatant at 15,000rpm in a Beckman SW28 rotor for 2 hours at 4°C with arecovery of 1-3 ml from the bottom of the tube andrepetitive trituration around the bottom of the tube.32 Incontrast to experiments done with unconcentrated su-pernatants, the concentrated supernatants were notfrozen but were used immediately both for experimentsand for simultaneous titering on 3T3 cells.

Determination of the contributions of the polycationsPolybrene and DEAE-dextran to TE. Standard transduc-tions were performed with Polybrene and DEAE-dex-tran (Pharmacia, Uppsala, Sweden) using concentra-tions of either of these two polycations varying from 4 to80 ,ug/ml. In addition, in a separate experiment de-signed to minimize the time of exposure to the polycat-ions with their associated toxicity, BAECs were incu-bated in DMEM containing high concentrations (0.1 or1 mg/ml) of either Polybrene or DEAE-dextran for only60 seconds before exposure to the supernatant for 6hours. In a third experimental design based on favorableresults obtained with a 60-second DEAE-dextran expo-sure, BAECs were exposed to DEAE-dextran for 60seconds and then to the supernatant for periods rangingfrom 15 to 360 minutes. In all of these experiments, cells

sures. TE was measured 72 hours after the end of thesupernatant exposure.

Cocultivation of BAECs with viral producer cells. Acocultivation apparatus was used in which BAECs were

grown on tissue culture inserts of 25-mm diameter and0.45-,um pore size (Falcon, Lincoln Park, N.J.) abovevector producer cells. This apparatus prevented mixingof the cells but theoretically allowed vector particles(approximately 0.08 ,um in diameter33) to come intocontact with the basolateral membrane of platedBAECs through the pores of the tissue culture insert.Unlike the cocultivation applied to transduction ofhematopoietic cells in which the target cells are mixedwith producer cells that have been lethally irradiated,34this design was chosen to allow continuous access tofreshly produced vector but to prevent the potentialmixing of cell types. BAECs (2.5 x 104) were plated ontothe tissue culture inserts located in 35-mm wells. Twen-ty-four hours after plating, this tissue culture insert wastransferred to a 35-mm well above a 70% confluentlayer of producer cells. Medium was replaced every 24hours with fresh D-10 containing 4 ,ug/ml Polybrene. TEwas determined as described above 72 hours after thetermination of exposure to producer cells.

ResultsDuration and Frequency of Exposure toViral Supernatant

In this series of experiments as well as in othersdescribed below, we report results from individual ex-

periments in which the same batch of supernatant was

used for each experimental group rather than pooledresults from several experiments in which differentbatches of supernatant were used, a practice that wouldhave allowed small differences in the titer of the super-natant used to become an uncontrolled variable. Indi-vidual experiments include data points obtained withtriplicate wells and were virtually always repeated bothwith the same vector and with at least one differentvector, as detailed below.A single 2-hour exposure to the LBgSN vector trans-

duced 2-4% of BAECs; under the same conditions, theLUKSN vector transduced 10-25% of the target cells.Increasing the duration of supernatant exposure to 6hours resulted in a small increase in TE over a 2-hourexposure. However, this difference did not consistentlyachieve statistical significance. Incubations of 12 or 24hours did not further improve TE over a 6-hour incu-bation (Figure 2A and data not shown).

Multiple supernatant exposures were attempted toincrease TE on the theoretical basis that 1) a greatertotal percentage of cells might be exposed to vectorduring active cell replication and thus be susceptible toretroviral vector-mediated transduction35 and 2) cellsmight receive multiple copies of vector DNA withconsequently increased recombinant protein productionif successfully infected by retroviral vectors on multipleoccasions. In these experiments, TE after a single6-hour exposure to supernatant was approximately 6%for LBgSN and 10% for LUKSN. Use of either two or

three separate 6-hour exposures at 24-hour intervalsfailed to improve TE significantly with either of these

were washed three times with phosphate-buffered saline(Biofluids) after both polycation and supernatant expo-

vectors (Figure 2B and data not shown). Lengthening ofthe time interval between exposures from 24 to either



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A

2 6 12 24Time of Exposure (hours)

'-c 40

3030

V 2000V 10

b 0

LB9SN

LUKSN

1 2 3Number of Exposures

FIGURE 2. Bar graphs showing the effect of duration and frequency of supernatant exposure on transduction efficiency. Todetermine whether transduction efficiency is improved by exposing target cells to supernatant for extended periods or by repetitiveexposures to fresh supernatant, we tested both methods using LBgSN (open bars) and LUKSN (hatched bars). Panel A: Bovineaortic endothelial cells were exposed to LBgSN or LUKSN supernatant with 8 ,g/ml Polybrene (Aldrich Chemical Co.,Milwaukee, Wis.) for 2, 6, 12, or 24 hours. Data are mean ±SD of triplicate wells from individual experiments. This experimentwasperformed twice with each of the vectors; results were similar to those illustrated. Panel B: Bovine aortic endothelial cells wereexposed to LBgSN- or LUKSN-containing supernatant with 8 pg/ml Polybrene for 6 hours at 24-hour intervals one to three times.Data are mean±SD oftriplicate wells. As discussed in the text and illustrated herein, interexperimental transduction efficiency (thedifference in percent transduction by LUKSN in panelsA and B) was sufficiently variable that the data are more clearly presentedas individual experiments. This experiment was performed twice with LBgSN and once with LUKSN.

48 or 72 hours, which would be expected to reducefurther any synchronization between cell cycle andvirion exposure, also failed to augment TE over thatobtained with a single 6-hour exposure to aliquots of thesame batch of vector-containing supernatant (data notshown).

Variation of Multiplicity of InfectionWe investigated the relation between MOI and TE by

varying each of the three elements that determine MOI:supernatant volume, target cell number, and vectorconcentration. In the first set of experiments, MOI wasvaried over a 12-fold range for LBgSN and LUKSN byincreasing the volume of virion-containing supernatantwith a constant target cell population (Table 1). In thesecond set of experiments, MOI for G1BgSVNa andLUKSN was varied over a 50-fold range by reducing thenumber of BAECs without a change in supernatantvolume (Table 1). When the lowest MOI was used ineach of the experiments, TE was approximately 4% withLBgSN, 20% with G1BgSVNa, and 12-22% withLUKSN. Substantial increases in MOI in each of thetwo experimental designs and with each of the threevectors revealed no significant increases in TE overthese baseline values. Thus, neither an increase inabsolute virion number nor a decrease in plated targetcell number resulted in higher TE. There was actually adecrease in TE of approximately 50% at target cellplating densities below 5 X 104 cells per 35-mm well.We next attempted to increase MOI by concentrating

the virion-containing supernatant. The ability of theconcentration protocol to increase supernatant titer wastested by comparing the titer of aliquots of supernatant,which were either concentrated then titered or titeredwithout prior concentration. A 20-fold concentration ofsupernatant (by volume) raised titer approximately 1 log(n=2). However, despite the ability of the concentra-tion protocol to increase supernatant titer substantially,use of concentrated supernatant to transduce BAECsresulted in only a marginal increase in TE (20-30%

TABLE 1. Transduction Efficiency With the Multiplicity ofInfection Varied by Manipulation of Supernatant Volume orTarget Cell Number

MOI Transduced cells (%)Supernatant volume (Exp A)LBgSN

0.5 ml 0.6 3.7+1.61.0 ml 1.2 4.0+0.82.0 ml 2.4 4.7±0.84.0 ml 4.8 4.2+0.56.0 ml 7.2 4.1+0.3

LUKSN0.5 ml 5 12.2±1.41.0 ml 10 11.4±0.22.0 ml 20 9.1±0.54.0 ml 40 9.4±1.16.0 ml 60 10.3±0.6

No. of target cells (Exp B)GlBgSVNa

50,000 2 20.2± 1.710,000 10 10.2±1.85,000 20 10.7+2.41,000 100 15.4±2.5

LUKSN50,000 2 22.0±2.110,000 10 12.6±2.75,000 20 10.7+ 1.41,000 100 12.0±1.1

MOI, multiplicity of infection; Exp, experiment. Values aremean±SD of triplicate wells.

Experiment A was performed twice with LBgSN and once withLUKSN, using a constant target cell number of 5 x 104. ExperimentB was performed once each with LBgSN, LUKSN, andG1BgSVNa, using a constant supernatant volume of 1.5 ml.Representative experiments are shown. MOI is calculated bymultiplying titer (colony-forming units per milliliter)xvolume ofsupernatant (milliliters) and dividing by the cell number present atthe time of transduction.

B

40

30

20

10

0

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(D

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Multiplicity of Infection (cfu/cell)FIGURE 3. Graph showing the effect ofsupernatant dilutionand concentration on transduction efficiency. The relation ofthe concentration of infectious vector particles (as defined bytiter on 3T3 cells) to transduction efficiency was investigatedusing supernatant that was first concentrated then seriallydiluted with D-10 medium containing 8 1ug/ml Polybrene(Aldrich Chemical Co., Milwaukee, Wis.). Fresh LBgSN-containing supernatant was harvested and concentrated bycentrifugation. An aliquot of concentrated supernatant was

usedfor titering, and the remainder was usedfor transductionof endothelial cells either undiluted or serially diluted. Trans-duction efficiency was measured as described in "Materialsand Methods. "After completion of the experiment, superna-

tant volume (1.5 ml) and the measured titer (106 colony-forming units [cfulIml in this case) of the concentratedsupernatant were used along with target cell number tocalculate multiplicity of infection (MOI) at the time oftransduction. MOI for transductions done with aliquots ofdiluted supernatant was calculated by taking into account theextent ofsupernatant dilution. With one exception, data pointsare mean +SD of triplicate wells, each of which represents a

separate transduction (transduction efficiency at MOI=5 isrepresented by a single well). By using the data points obtainedwith higher supernatant dilutions (lower MOI), a predictedlinear relation of MOI to transduction efficiency for more

concentrated supernatant is indicated (dashed line). Theobserved relation is also indicated (solid line). Similar resultswere obtained in two other experiments using concentratedLBgSN supernatant and in two experiments using concen-

trated LUKSN supernatant.

increase over the maximum TE attainable with uncon-centrated supernatant) with either LBgSN (n =3) or

LUKSN (n =2) (Figure 3 and data not shown).To further discern the relation between vector con-

centration and TE, transductions were performed withserial dilutions of either unconcentrated or concen-trated supernatant. A striking relation was found be-tween MOI and TE: after an initial plateau, TE de-creased with a linear relation to MOI (Figure 3 and datanot shown). Therefore, TE is related to MOI in a linearfashion if one dilutes a given aliquot of supernatantbeyond twofold to fourfold. However, if one uses thislinear relation to predict the effect of supernatantconcentration on TE, the actual TE obtained usingconcentrated supernatant consistently falls below thepredicted value (Figure 3 and data not shown). Thisobservation correlates with the failure of supernatantconcentration to increase TE proportionately.

TABLE 2. Transduction Efficiency With Polybrene andDEAE-Dextran

Polycation Tranduced cells (%)concentration (,ug/ml) G1BgSVNa LUKSN

None <1 1.2+0.2Polybrene

4 15.2+0.7 16.0+2.08 20.0+1.5 14.1+1.8

16 19.9+0.6 15.9+0.732 20.7+2.0 13.2+0.6

DEAE-dextran5 29.8±0.9 35.4±3.310 52.5±12.0 38.7±11.120 * 44.0±11.940 * *

Values are mean±SD of transduction efficiencies determinedfrom triplicate wells.

Endothelial cells were exposed to supernatant for 6 hours in thepresence of the polycations Polybrene (Aldrich Chemical Co.,Milwaukee, Wis.) or DEAE-dextran at the indicated concentra-tions. Data are from a single experiment with each of the vectors.

*Cell death of >50%.

Polycation EffectThe enhancement of TE with the polycations Poly-

brene and DEAE-dextran was first investigated byadding increasing concentrations of polycation to thevector-containing supernatant and incubating the su-pernatant with the target cells for 6 hours. Omission ofpolycation resulted in virtually undetectable TE (Table2). Addition of Polybrene at a concentration of 4 ,ug/mlresulted in TE of approximately 15% for bothG1BgSVNa and LUKSN (Table 2 and data not shown).Increasing the concentration of Polybrene over therange of 4-32 ,ug/ml did not consistently result insignificant increases in TE using either of the vectors.Addition of DEAE-dextran at a concentration of 5,ug/ml resulted in increased TE over that obtained withany of the concentrations of Polybrene. In the experi-ment described in Table 2, TE was 30% for G1BgSVNaand 35% for LUKSN, approximately twice that ob-tained using the same supernatant with added Poly-brene at any concentration. As with Polybrene, increas-ing DEAE-dextran concentration above 5 ,ug/ml did notresult in consistent dose-dependent increases in TEwith either vector (Table 2 and data not shown). In-creasing DEAE-dextran above 10 ,ug/ml was limited byassociated cellular toxicity. With both vectors, DEAE-dextran concentrations of 20, 40, and 80 ug/ml resultedin >50% cell death, as observed directly with phase-contrast microscopy and confirmed by comparison oftotal cellular protein with that of cells in parallel wellsreceiving lower concentrations of DEAE-dextran orPolybrene (data not shown). Because of the toxicity ofDEAE-dextran, TE could be measured reliably onlyoccasionally at 20 gg/ml DEAE-dextran and never at 40or 80 ,ug/ml. When either LUKSN or G1BgSVNa wasused, TE with 20 ,ug/ml DEAE-dextran (when it couldbe measured) was not significantly different from thatobtained at 10 ,ug/ml (Table 2 and data not shown,n=3). The entire experiment illustrated in Table 2 wasrepeated with similar results.



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Cd)-C,

-o

(L)

m

(D

I-

100

80

60

40

20

0.008 mg/mI 0.1 mg/mI 1.0 mg/mI

Polybrene DEAE-Dextran

FIGURE 4. Bar graph comparing transduction by using60-second exposure to DEAE-dextran to standard Polybrene(Aldrich Chemical Co., Milwaukee, Wis.) transduction. Tominimize cellular toxicity and improve transduction efficiency,endothelial cells were exposed briefly to high concentrations ofDEAE-dextran before supernatant exposure. Cells weretransduced under three conditions: 1) a 6-hour exposure tosupernatant containing 8 uglml Polybrene, 2) a 60-secondexposure to 0.1 mglml DE,AE-dextran in Dulbecco's modifiedEagle's medium (DMEM) followed by 6 hours ofsupernatantexposure without polycation, and 3) a 60-second exposure to1.0 mglml DEAE-dextran in DMEM followed by 6 hours ofsupernatant exposure without polycation. Data presented aremean±SD of triplicate wells from individual experimentsperformed with either GJBgSVNa- or LUKSN-containingsupernatant. This experiment was performed a total of threetimes with each vector, always with similar results.

To increase TE while minimizing associated cellulartoxicity, we attempted brief high-concentration DEAE-dextran exposure before the addition of supernatant. A60-second exposure to DEAE-dextran at a concentra-tion of either 0.1 or 1 mg/ml before supernatant expo-sure resulted in a TE of 50-90% for both G1BgSVNaand LUKSN compared with 9-17% for standard trans-ductions done in parallel with either of the two vectors(Figure 4 and data not shown, n=6). Thus, brief expo-sure to high-concentration DEAE-dextran resulted in a2.5-fold to eightfold increase in TE over that obtainedwith a standard transduction protocol. Cellular toxicitywas 20-30% with both 0.1 and 1 mg/ml DEAE-dextran,as observed directly by phase-contrast microscopy andquantified by comparison of total cellular protein withthat of cells grown in parallel wells and transduced usinga standard Polybrene transduction (data not shown). Incontrast to the results obtained with DEAE-dextran,60-second exposures to Polybrene at concentrations of0.1 and 1 mg/ml did not improve TE over that obtainedwith a standard transduction (data not shown). Giventhat a 60-second exposure to DEAE-dextran was effec-tive at enhancing TE, the time of subsequent superna-tant exposure was varied to determine whether it couldalso be reduced. Again, with either G1BgSVNa orLUKSN, 6 hours of supernatant exposure resulted inTE of 60-70% (Figure 5 and data not shown). Decreas-ing the time of supernatant exposure resulted in a dropin TE. However, with as little as 15 minutes of super-natant exposure after a 60-second exposure to 1 mg/mlDEAE-dextran, TE remained at 15-20%, equivalent tothe efficiency of a standard 6-hour Polybrene transduc-tion done in parallel. Cellular toxicity with 1 mg/mlDEAE-dextran was again 20-30%.

100

19~ ~80

O 60~0 ~ 9

40

~0

Time (mi) 360

Polycation Polybrene

0.008 mg/ml

] G1BgSVNa

LUKSN

ALrS.F

15 30 60 360

DEAE-Dextran1.0 mg/mI

FIGURE 5. Bar graph showing relation of transductionefficiency to time of supernatant exposure after 60-secondexposure to high concentration DEAE-dextran. To determinethe minimum time necessary for transduction after exposureto high concentration DEAE-dextran, the time ofexposure tosupernatant was varied. Endothelial cells were exposed to 1.0mglml DEAE-dextran in Dulbecco's modified Eagle's me-dium for 60 seconds and washed three times with phosphate-buffered saline. Bovine aortic endothelial cells were thenexposed to either G1BgSVNa- (open bars) or LUKSN-con-taining supernatant (hatched bars) for 15, 30, 60, or 360minutes. Transduction efficiency is presented as mean±SD oftriplicate wells from each of the two individual experiments.This experiment was repeated once with GJBgSVNa withsimilar results (not shown).

Higher TE, as Measured by Recombinant ProteinProduction, Is Due to a Greater Percentage ofTransduced CellsBecause we used the level of recombinant protein to

quantitate TE, it is possible that an increase in reportergene expression is due to a higher level of expressionper cell rather than a greater percentage of transducedcells. To discriminate these two possibilities, two ap-proaches were used. In the first approach, curves of,B-galactosidase activity and urokinase secretion wereconstructed (as described in "Materials and Methods"for use in the determination of percentage positivecells) with G418-selected cell populations obtained withthree different methods of transduction: 1) a standard(Polybrene) transduction, 2) a 60-second exposure to 1mg/ml DEAE-dextran followed by 6 hours of superna-tant exposure, and 3) a 60-second exposure to 1 mg/mlDEAE-dextran followed by 15 minutes of supernatantexposure. The curves for each group obtained by trans-duction with the LBgSN vector are shown in Figure 6.For each group of cells, the normalized activity (inmicrounits per milligram cellular protein) per trans-duced cell was virtually identical. Similar standardcurves resulted from a repetition of this experimentusing the LUKSN vector (data not shown). Therefore,the measured increases in reporter gene expressionusing the DEAE-dextran transduction protocols resultfrom a higher percentage of transduced cells rather thanan increase in reporter gene expression per cell. In asecond, confirmatory approach, BAECs were trans-duced with the G1BgSVNa vector by either the "stan-dard" or "optimized" protocols. Histochemical stainingfor 13-galactosidase expression revealed that a twofold tothreefold increase in the number of transduced cells was

3



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Polycation

Exposure Time (I

FIGURE 6. (sidase after i

curves of f-g

generated by tiEndothelial cdthree ways: 1dextran (DE1supernatant (mg/ml DEAEtant (solid trig6-hour exposu(Aldrich CheTransducedp(standard curvg

cells with untrMethods." Vamicrounits pertions from tril

achieved by(data not shi

CocultivatiocWhen the

was used, nm

after 72 houLBgSN procknown to prmThis experinLUKSN.

Retroviraldescribed as

gene transfeiison with elliposome-mecells are st(<10%) hasclinical genetion, W. FreBlood Institipresent studused by indciency. We dcess obtainecells to virioor by increa


supernatants.19 The experiments reported herein de-scribe our attempt to quantitate the contribution (if

-/~any) of these described protocols to the improvement of/A the efficiency of retroviral vector-mediated gene trans-

fer into endothelial cells.The use of extended periods of exposure to retroviral

vector and/or multiple exposures to vector have beenamong the most commonly used techniques to improvetransduction in hematopoietic cells40 and fibroblasts.4'

0 20 40 60 80 A rationale for these methods is that retroviral vector-mediated gene transfer occurs only in actively replicat-

Transduced Cells (%) ing cells35'42; therefore, the longer and more frequentlythat target cells are exposed to vector, the greater the

-O-Polybrene -v- DEAE-Dex -A- DEAE-Dex probability that any given cell will be cycling at the timemin) 360 360 15 of exposure. Serial exposures could conceivably result inraph showingcellular expression of ,B-galacto- the transfer of more than one vector copy per cell.different methods of transduction. Standard Despite these theoretical arguments, we were unable toalactosidase expression in G418-selected cells improve TB in BABCs with >6 hours of exposure orhree different transduction protocols are shown. with up to three different exposures at either constantells were transduced with the LBgSN vector in or variable time intervals. The failure to improve TE') a 60-second exposure to 1 mglml DFAE- with supernatant exposures longer than 6 hours may be

a460-sec)follonedbexposureitot1mgpmluDEAE- due to the instability of retroviral vector at 37°C. In thisIE-Dex) followed by a 15-minute exposure toy'p tige, 6sore t

context, a loss of greater than 1 log in titer has beenopen trlangles), 2) a 60-second exposure to 1 observed when the supernatant is kept at 37°C for 24- oo- hours (personal communication, Ofer Nussbaum, Na-

angles), and 3) a standard transduction with a tional Heart, Lung, and Blood Institute, Bethesda,ire to supernatant containing 8 ug/ml Polybrene Md.). The lack of change in TE with repetitive expo-mical Co., Milwaukee, Wis.) (open circles). sures suggests both that the cell cycle does not signifi-opulations were selected in 0.4 mg/ml G418, and cantly limit TE in cultured BAECs (perhaps because ates were generated by mixing transduced selected the plating densities used the cells are continuouslyransduced cells, as described in "Materials and replicating) and that repeated transduction of the samelues of normalized f3-galactosidase activity (in cells may be an infrequent event.r milligram) represent mean ±SD of determina- Experiments reported herein in which the relation ofplicate wells. MOI to TE was investigated by changing the superna-

tant volume or target cell number indicate that withinthe range of MOI tested (0.6-100) TE is not a simple

using teotmzdtasutoprtcl function of the vector particle/target cell ratio andown). cannot be improved merely by increasing the volume of

n With Viral Producer Cells the supernatant or decreasing the number of targetcells. In fact, as cell density was lowered, TE decreased

cocultivation apparatus described above modestly, possibly because of a detrimental effect ofo 3-galactosidase was detected in BAECs lower plating densities on the health of these nonim-irs. The quantity of amphotropic PA-317 mortalized cells.ducer cells used in this experiment was In contrast to the results obtained by varying volumeoduce > 104 CFU infectious virion per day. or target cell number, TE of endothelial cells was clearlynent was not repeated with G1BgSVNa or dependent on MOI when MOI was altered by variations

in the concentration of vector to which the cells were

Discussion exposed (with concentration determined by titering the

vector-mdiated gene transfer has beenvector-containing supernatant on 3T3 cells). The rela-

vector-mediated gene transfer has been tion of TE to vector concentration was, however, not aan "efficient" means of achieving stable simple one; whereas dilution of the supernatant could

r.12,36 Although this may be true in compar- reliably decrease TE over a wide range, concentrationlectroporation and calcium phosphate or only marginally increased TE. For example, even withdiated transfection in which extremely few concentrated supernatant titered at 107 CFU/ml pre-[ably transduced,37 fairly low efficiency sented to endothelial cells at an MOI of 10, a maximumin fact plagued both experimental38'39 and TE of only 30% was achieved. This apparent inconsis-transfer protocols (personal communica- tency results from the fact that supernatant titer is

nch Anderson, National Heart, Lung, and measured with limiting dilutions of the supernatant, inute, Bethesda, Md.). Before beginning the which the titer is determined from wells that containly, we surveyed the literature for methods only a few transduced cells whose progeny can beividual investigators to increase this effi- accurately counted as individual colonies. Transduc-liscovered mostly anecdotal reports of suc- tions of target cells, however, are performed with eitherd by extending the period of exposure of undiluted or concentrated supernatant, in which theins,40,41 by concentrating the supernatant,32 goal is to transduce every cell. The present data suggestLsing MOI through the use of higher titer that one cannot extrapolate in a linear fashion from a

CDE

0

400

300

200

100

0



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titer done on a diluted sample of supernatant to deter-mine the effective vector concentration in more concen-

trated samples. Although the biological basis of thisobservation is not addressed in the present study (thedisproportionate presence in concentrated supernatantof damaged virions or other inhibitors of transductioncould conceivably play a role43), the practical conse-quence is that the concentration of supernatant raisesthe titer to a far greater extent than it raises TE.Concentration is therefore not a practical means bywhich to increase TE of endothelial cells.

Given that the majority of BAECs could be exposedto transducing virions at an MOI of up to 100 withoutbeing transduced (Table 1), we reasoned that a morefruitful approach might consist of efforts to improvevirion-target cell interaction. More than 20 years ago,polycations were shown to enhance the infection effi-ciencies of a number of viruses, including the murineleukemia virus, from which retroviral vectors are de-rived.4445 Certain of these previous experiments44 re-

vealed that the infectivity of murine leukemia virus in3T3 cells was enhanced 20-fold by DEAE-dextran (atan optimal concentration of 20 ,ug/ml) and 10-fold byPolybrene (at concentrations between 2 and 8 ,ug/ml).The proposed mechanism of action of polycations is thecreation of a cell/polycation/virus particle sandwich inwhich the polycation mediates apposition of the nega-tively charged cell and viral surface membranes,444647resulting in a higher effective local concentration ofvector particles and in an improvement in the efficiencyof uptake of virions. This hypothesis is supported byprevious observations47 and by our own data (notshown) demonstrating that the beneficial effects ofDEAE-dextran preincubation on TE can be reversed bythe addition of a polyanion, such as heparin, to theDEAE-dextran solution. In sum, our results confirm thedependence of TE on the presence of polycations andindicate that DEAE-dextran is the polycation of choicefor transduction of BAECs.To decrease the cellular toxicity associated with pro-

longed exposure to DEAE-dextran, we used brief expo-

sures to higher DEAE-dextran concentrations. This ap-

proach has been reported with transfection of DNA4849into eukaryotic cells but has not been applied previouslyto retroviral vector-mediated gene transfer. Our datademonstrate that this technique is effective not only inlimiting cellular toxicity but results in reliably high (50-90%) TE. These high TE values are achievable withsupernatant of only moderately high titer (105_106 CFU/ml) and are the highest yet reported in endothelial cells,therefore suggesting that we have developed a protocolfor reliable high efficiency gene transfer into endothelialcells in vitro. It is significant that this protocol is success-ful when using supernatant of a titer that, based on our

experience, is easily attainable with most retroviral vec-

tors, for this obviates the need for extensive manipulationof producer cells that may lead to contamination withpathogenic helper virus.5051

Despite the success of the in vitro protocol, there are

several issues that will need to be examined as theprotocol is extended to optimize direct in vivo endothe-lial transduction. These issues include reduction of thetotal duration of the protocol (due to the requirementfor short periods of vessel occlusion), probable de-

creased efficiency of retroviral vector-mediated gene

transfer into the normally nondividing ECs of the vesselwall, and utilization of endogenous cellular promoterelements that may be less active (though more stablyexpressed in vivo) than the viral LTR.52 We have madesome progress toward resolution of the first of theseissues by reduction of the total duration of the trans-duction protocol to under 20 minutes (Figure 5), al-though this is achieved at a significant cost in TE. It willbe of interest to compare this protocol and improve-ments thereupon to thce e used by ourselves and byothers to achieve in vivo gene transfer into the vesselwall.2'3'16 We anticipate that the conceptual frameworkprovided by the present in vitro study will be useful inthe design of protocols to optimize in vivo transductionas well.

AcknowledgmentsWe thank Genetic Therapy, Inc., Gaithersburg, Md., for the

kind gift of the LBgSN and G1BgSVNa producer cell lines andDr. W. French Anderson for his ongoing support.

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M L Kahn, S W Lee and D A DichekOptimization of retroviral vector-mediated gene transfer into endothelial cells in vitro.

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