Toxicogenomics of Non-viral Vectors for Gene Therapy: AMicroarray Study of Lipofectin- and Oligofectamine-induced

Gene Expression Changes in Human Epithelial Cells

YADOLLAH OMIDIa, ANDREW J. HOLLINSa, MUSTAPHA BENBOUBETRAa, ROSS DRAYTONa, IBRAHIM F. BENTERb andSAGHIR AKHTARa,*

aCentre for Genome-based Therapeutics, The Welsh School of Pharmacy, Cardiff University, Redwood Building,King Edward VII Avenue, Cardiff CF10 3XF, UK

bDepartment of Pharmacology, Kuwait University, Kuwait

(Received 3 October 2003)

Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery ofgenes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs.However, little is known about the impact of these delivery systems on global gene expression in targetcells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have usedmicroarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles ofhuman A431 epithelial cells. Using the manufacturer’s recommended CL concentrations routinely usedfor gene delivery, cDNA microarray expression profiling revealed marked changes in the expression ofseveral genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrayshousing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cellsresulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstreamfunctional consequences of CL-induced gene expression alterations led to an increased tendency ofcells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect wasgreater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient toinduce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay.These data highlight the fact that inadvertent gene expression changes can be induced by the deliveryformulation alone and that these may, ultimately, have important safety implications for the use of thesenon-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be takeninto consideration in gene therapy or gene silencing experiments using CL formulations where theymay potentially mask or interfere with the desired genotype and/or phenotype end-points.

Keywords: Cationic lipids; Gene delivery; Gene silencing; Gene expression profiling; siRNA;Antisense

INTRODUCTION

Gene-based therapies such as gene therapy, antisense

oligonucleotides, ribozymes, DNAzymes and RNA-

interference all represent promising therapeutic para-

digms for future healthcare in the post-genomic era (for

reviews see Akhtar et al., 2000; Somia and Verma,

2000; Zallen, 2000; Hughes et al., 2001; McManus and

Sharp, 2002; Dykxhoorn et al., 2003; Kerr, 2003).

Gene-based therapies require acceptable and efficient

delivery along with minimal toxicity and maximum

patient safety. These issues are important for both ex

vivo, where cells undergo gene therapy in culture prior

to implantation into the patient, or in vivo gene therapy

where nucleic acids are administered directly to

the patient to effect the desired gene change. In either

approach, ideally only the therapy-intended gene

expression changes should occur. However, this is not

always the case. For example, viral vectors are known

to be efficient delivery systems for nucleic acids but

can also induce toxic responses including inadvertent

gene expression changes following random integration

into the host genome (Tarahovsky and Ivanitsky, 1998;

Pleyer et al., 2001; Audouy et al., 2002; Whitehouse,

2003). Hence, several non-viral vector systems have

been developed for gene and oligonucleotide delivery.

Of these the cationic lipid (CL) formulations that can

readily condense DNA into complexes termed

ISSN 1061-186X print/ISSN 1029-2330 online q 2003 Taylor & Francis Ltd

DOI: 10.1080/10611860310001636908

*Corresponding author. Tel.: þ44-29-2087-6309. Fax: þ44-29-2087-4149. E-mail: saghirakhtar@cf.ac.uk

Journal of Drug Targeting, 2003 Vol. 11 (6), pp. 311–323

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lipoplexes have been the most widely studied for ex vivo

and in vivo gene therapy approaches (for reviews see

Juliano and Akhtar, 1992; Akhtar et al., 2000; Aissaoui

et al., 2002; Hirko et al., 2003).

CL can be classified into several categories based on

their structure, size and charge (Pedroso de Lima et al.,

2001). Due to their high delivery efficiency, particular

interest has been given to the positively charged CL or

liposome formulations for DNA transfer (Pedroso de Lima

et al., 2001). These reagents form unilamellar liposomes

bearing positive charges which interact spontaneously

with polyanionic DNA to form lipoplexes (Duzgunes

et al., 2003). The lipid-DNA lipoplex is thought to enter

cells via adsorptive endocytosis and, by mechanisms not

fully understood as yet, release nucleic acids out of the

endosomal/lysosomal compartments with the net effect of

yielding high uptake and intracellular delivery of genes

and oligonucleotides (Zelphati and Szoka, 1996; Pedroso

de Lima et al., 2001).

Despite the enhanced delivery and the resultant

improved biological activity of nucleic acids delivered

with CLs, they can often, but not always (e.g. Canonico

et al., 1994), result in cellular toxicity (Campbell, 1983;

Filion and Phillips, 1997; Scheule et al., 1997; Nagahiro

et al., 2000; Pedroso de Lima et al., 2001). However, little

is known about the impact of CL delivery systems on global

gene expression in target cells. In a previous study aimed at

screening the downstream changes in gene expression

following treatment of cells with antisense oligonucleo-

tides targeting the epidermal growth factor receptor, we

became aware of non-target gene expression changes that

may have been due to the Lipofectin-delivery system used

(Petch et al., 2003). Here, in an attempt to study the geno-

compatibility and toxicogenomics of CL formulations in

target cells, we have used gene expression microarrays to

compare the effect of two commercially available CL

formulations, Lipofectin and Oligofectamine, on gene

expression in human A431 epithelial cells.

MATERIALS AND METHODS

Materials

Total RNA isolation kit Trireagente, Ethidum Bromide,

isopropanol, chloroform, formaldehyde and low melting

point agarose (LMPA) were from Sigma (Poole, UK).

CL (Lipofectin and Oligogectamne), Dulbecco’s modi-

fied Eagle’s medium (DMEM) containing 25 mM

HEPES, fetal bovine serum (FBS), penicillin G,

streptomycin, L-glutamine 200 mM ( £ 100), first strand

PCR buffer ( £ 5), Moloney Murine Leukaemia Virus

reverse transcription (MMLV-rt), dithiotheritol (DTT)

and RNase/DNase free ddH2O were purchased from

Invitrogen, (Paisley, UK). The deoxynucleotide triphos-

phate monomers (dNTPs) and random hexamer

primers (pdN6) were from Amersham Biosciences

(Amersham, Bucks, UK). The cyanine fluorescent dyes

(Cy3 and Cy5) were obtained from Amersham Life

Science (Little Chalfont, UK). Tissue culture treated

multi-well plates and flasks were obtained from Corning

Costar (High Wycombe, UK). RNasine was from

Promega (Southampton, UK) and Taq polymerase from

Qiagen Ltd (Crawley, UK). The dual-window Comet

slide was obtained from Trevigen (Gaithersburg, MD).

Annexin V-FITC apoptosis detection kit was obtained

from Oncogene Research Products, (San Diego, CA,

USA). Human epidermoid carcinoma A431 cell line was

purchase from ECACC, (Salisbury, UK).

Cell Culture

A431 cells were seeded at 5 £ 104 cells/cm2 into either 6- or

96-well plates and were subjected to a standard transfection

protocol. Briefly, following the manufacturer protocol, CL

(Lipofectin or Oligofectamine) was prepared initially at a

concentration of 0.2mg/ml in serum-free medium (SFM)

and then diluted, after 30–45 min incubation at room

temperature, to 0.045mg/ml in SFM. Cells, at the 40–50%

confluency, were washed twice with SFM, exposed to CL

mixture (0.8 ml/well for 6 well plates) and incubated at

378C for 4 h. Cells were replenished with normal growth

medium, incubated at 378C for 24 h, and were then used for

gene expression or other assays.

Total RNA Isolation

Total RNA was isolated, from CL treated and untreated

A431 cells, using TRIREAGENTe according to the

manufacturer’s (Sigma) protocol. Briefly, cells were lysed

directly on the culture dish where the medium was

discarded and 1 ml of the TRIREAGENTe per 10 cm2 of

culture area was added. The homogenous lysate was then

transferred to a RNase/DNase free 1.5 ml micro-

centrifuge tube and 0.2 ml of chloroform per 1 ml of

TRIREAGENTe was added, mixed and kept for 5 min at

room temperature. After centrifugation, 12,000g for

15 min at 48C, using Eppendorf Centrifuge 5417R,

(Eppendorf, Hamburg, Germany), the upper aqueous

phase of three separated phases was mixed with 0.5 ml of

isopropanol, kept for 5 min at room temperature, and then

centrifuged at 12,000g for 10 min at 48C and the RNA was

recovered as a precipitate. RNA pellet was washed twice

with 1 ml of 75% ethanol, spun at 10,000g for 5 min, then

dried and dissolved in 50ml DNase/RNase free water.

Quantification and purity of extracted total RNA were

assessed by UV spectrophotometry scans (Ultraspec 3100

pro, Amersham, Bucks, UK) and by electrophoresis of a

sample on 1% denaturing agarose gel followed by UV

visualisation on the Gel Doc 1000 gel documentation

system (BioRad, Hertfordshire, UK).

The cDNA Microarray Labelling Procedure

The aminoallyl (aa)-cDNA labelling was undertaken

using a modified version of the TIGR group protocol

(Hegde et al., 2000). Briefly, a 100 mM aa-dUTP was

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prepared by dissolving 1 mg in 19.1ml 0.1 M KPO4

buffer, pH 7.5. The (50 £ ) labelling mix of aminoallyl-

dNTP (aa-dNTP) containing 2:3 ratio of aa-dUTP to dTTP

with a final concentration of 25 mM of each dNTPs,

(i.e. dATP, 25 mM dCTP, 25 mM dGTP, 15 mM dTTP,

10 mM aa-dUTP). Ten micrograms of total RNA was

mixed with 6mg of Random Hexamer primers in a final

volume of 25ml. The mixture was incubated at 708C for

10 min, then placed on ice for 3 min. In the RT reaction

6ml of 5 £ first strand buffer, 3ml of 0.1 M DTT, 0.6ml

50 £ aa-dNTP mix and 2ml SuperScript II RT (200 U/ml)

were added to RNA/pdN6 mix and incubated at 428C for

6 h in a PCR thermal cycler (Techne Flexigene,

Cambridge, UK). The remaining total RNA was

hydrolysed in 10ml of 1 M NaOH and 10ml of 0.5 M

EDTA at 658C for 15 min with subsequent neutralisation

with 10ml of 1 M HCl.

To purify the aa-cDNA from unincorporated aa-dUTP

and free amines prior to conjugation with NHS-ester Cy

dyes, a Qiagen PCR purification kit was used. This

purification step prevented Cy dyes coupling to free amine

groups in the reaction. The aa-cDNA product was mixed

with 350ml (5 £ reaction volume) binding buffer PB,

transferred to QIAquick column and centrifuged for 1 min

at 12,000g for 1 min. To maximize aa-cDNA binding,

eluate was re-spun using same column. Washing steps

(twice) were carried out with 500ml Tris-free phosphate

wash buffer (5 mM KPO4, pH 8.0 and 80% EtOH)

spinning at 12,000g for 45 s. Column was then transferred

to a fresh tube and 30ml 4 mM KPO4, pH 8.5 was added.

Column was incubated at room temperature for 1 min and

centrifuged at 12,000g for 1 min to elute the aa-cDNA. To

maximize the aa-cDNA recovery an additional 30ml

4 mM KPO4, pH 8.5 was introduced to column, incubated

for 1 min, and centrifuged to elute with to a total volume

of 60ml. Finally, the labelled sample was dried using a

speed vacuum lyophiliser (DNA120 SpeedVac, Thermo-

Savant, Thermo Life Science, NY, USA). The aa-cDNA

was quantified using UV spectrophotometry scans

(Ultraspec 3100 pro, Amersham, Bucks, UK) prior to

conjugation with NHS-ester Cy dyes. Normally 10mg

total RNA in such reactions should provide 4–6mg

aa-cDNA after QIAquick purification.

The aminoallyl-labelled cDNA was resuspended in 5ml

of 0.1 M carbonate buffer, pH 9.0. For covalent coupling

of the dye-ester to the aminoallyl-labelled cDNA, 5ml of

3 mM stock solution of fluorescent cyanine dye was added

and incubated in the dark at room temperature for 1 h.

Thirty five microlitres of 100 mM NaOAc pH 5.2 was then

added to reaction. Uncoupled dye was removed prior to

hybridisation using QIAquick PCR purification kit.

Elution was performed twice in 30ml EB for a total of

60ml as described above. Cy3 or Cy5 coupled cDNA

was quantified using UV spectrophotometry (Ultraspec

3100 pro, Amersham, Bucks, UK). Coupled aa-cDNA

with Cy-dye was finally dried using a speed vacuum.

Approximately 150–200 pmol of dye incorporation per

sample (aa-cDNA product of 10mg total RNA) and a ratio

of 40–50 nucleotides/dye molecules yielded optimal

hybridisation results.

Hybridisation of Cy-dye Coupled cDNA

MWG-Biotech human starter arrays (housing 200 gene

spots) were prepared with Gene Framesw 1 day prior to

hybridisation following MWG protocol (Milton Keynes,

UK). The Gene Frames were used because gas-tight

sealing system can withstand temperatures of up to 978C

and prevents reagent loss due to evaporation, thus

improving the hybridisation reliability. Hybridisation

was performed following MWG-Biotech protocol with

slight modifications. Briefly, dried Cy-dye coupled cDNA

was dissolved in 60ml of MWG hybridisation solution.

A measured quantity of 1.5–2mg of each probe was

mixed in one tube in a final volume of 120ml salt-based

hybridisation buffer and incubated at 958C for 3 min. The

combined probes were incubated on ice for 3 min, then

introduced to one end of the Gene frame after removing

the thin polyester backing sheet from the frame. The

polyester cover slip was then carefully placed over the

Gene Frame. The arrays were incubated at 428C for

18–24 h in a shaking water bath. Following hybridisation,

the array slides were carefully removed from the

hybridisation chamber and washed with wash buffer A

(2 £ SSC, 0.1% SDS), wash buffer B (1 £ SSC), and

wash buffer C (0.5 £ SSC), 4 times in each wash, 2 min

each time. The array slide was briefly rinsed with

distilled water, centrifuged at 1600g for 5 min using

Avanti J-25 cetrifuge, Beckman Coulter (California,

USA). Arrays were scanned for Cy3 (green laser

532 nm; filter FM570-10) and Cy5 (red laser 635 nm;

filter FM665-12) at gain 35, resolution normal (10mm)

and line average 1 using Affymetrix 428 Array Scanner

(California, USA). In some experiments the actual Cy-dye

used for labelling treated versus control cDNA samples

was interchanged (referred to as “Dye-flipping” exper-

iments).

Analysis of cDNA Microarrays

The microarray data analysis packages, ImaGene and

GeneSight, from BioDiscovery (California, USA) were

used for gene expression profiling analyses. In this study

data for each gene were typically reported as an

“expression ratio” or as the base 2 logarithm of the

expression ratio of experiment to control. Normalisation

was fulfilled based on the total intensity by dividing mean

value of each spot by mean values of entire slide. Genes

were assumed to be up- or down-regulated if they revealed

an expression ratio of .2 and ,0.5 (or .1 and ,21 for

log2 transformed data), respectively.

One way analysis of variance (ANOVA) followed by

multiple comparison test (post-hoc) and/or unpaired two-

tailed t-test were used with an assumption of p value less

than 0.05 for significant differences using GraphPad prism

software.

TOXICOGENOMICS OF NON-VIRAL VECTORS 313

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Semi-quantitative RT-PCR

Standard RT-PCR reactions were performed using the

following primers: b-actin (forward: 50-GGC ATG GGT

CAG AAG GAT T-30 and reverse: 50-GGG GTG TTG

AAG GTC TCA AA-30, 254 bp); proteasomeb4 (forward:

50-CCA GGA CAG TTT TAC CGC AT-30 and reverse:

50-CAA GCA TGT CCA CAT AAC CG-30, 469 bp);

annexin 2 (forward: 50-CTC TAC ACC CCC AAG TGC

AT-30 and reverse: 50-TGA GAG AGT CCT CGT CGG

TT-30, 335 bp). The primers were designed using internet-

based primer design program Primer3 (http://

www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/).

The PCR thermocycling program was as follows:

denaturation at 948C for 30 s, annealing at 57–648C for

45 s and extension at 728C for 45 s through a total of 30

cycles. The PCR recipe was (for 25ml reaction): 8.5ml

RNase/DNase free ddH2O, 2.5ml Taq 10 £ buffer

containing MgCl2 (15 mM), 1ml MgCl2 (25 mM), 2ml

dNTPs (10mM), 2ml cDNA (100 ng/1ml), 0.02–0.5ml

forward and reverse primers (10 pmol/1 ml), and

0.1ml Qiagen Taq polymerase enzyme (5 Units/1ml).

The PCR products were electrophoresed through a 1.2%

agarose gel in the presence of 1mg ethidium bromide/1 ml

gel and visualised under UV light. As positive controls,

mouse brain and kidney total RNA were used whereas for

negative controls, RT-PCR reactions were performed

without the desired cDNA and/or primer. The density of

expressed bands was measured using the GS-700

densitometer (Bio-Rad Molecular analysis software,

Hemel, Hempstead, UK). The density of each spot was

subjected to local background density subtraction and

normalised to the density of the house keeping gene,

b-actin. The ratio of treated over untreated were

performed for two independent replicates.

Annexin V-FITC Flow Cytometric Assay for Apoptosis

Flow cytometry (fluorescence-activated cell sorting or

FACS) analysis was performed using annexin V-FITC to

detect the induction of early and/or late apoptosis in

CL-treated A431 cells as described (Zhang et al., 1997;

Peng et al., 2002). Briefly, CL-treated cells, untreated

control cells or, as a positive control for apoptosis, cells

treated with 5% DMSO were stored on ice following

removal of culture media and gently washed once with

PBS. Cells were then detached by gentle trypsinisation

using 0.5 ml 0.5 £ trypsin, and resuspended in the original

media to include detached cells in the suspension for

analysis. Approximately, 1 £ 106 cells were exposed to

10ml media binding reagent and 1.25ml Annexin V-FITC

and incubated in the dark for 15 min at room temperature.

Cell suspension was centrifuged at 1000g for 5 min, the

supernatant was discarded and the cells resuspended in

0.5 ml ice-cold 1 £ binding buffer. Finally, 10ml

propidium iodide was added and the cells were transferred

to FACS tubes (Fahrenheit, UK) and analysed immedi-

ately. Cell associated fluorescence distributions were

obtained from 20,000 events per cell sample through

a FL1 band-pass filter (FITC-Annexin V binding) and FL2

band-pass filter (Propidium iodide) using a FACScalibar

flow cytometer (BD Biosciences, Oxford, UK). The

fluorescence of cell populations was analysed using

validated analysis software, WinMDI 2.8 (http://facs.

scripps.edu/).

Assessment of DNA Damage by COMET Assay

COMETassays on Lipofectin-treated cells were performed

as described previously (Singh et al., 1988), with slight

modifications. In brief, a suspension of cells (5 £

105 cells/ml) in LMPA was prepared. A desired small

amount of the suspension was added into a dual-window

COMET slide (Trevigen, Gaithersburg, MD), such that

each window contained approximately 5–10 £ 105 cells

suspended in 75ml LMPA. Slides were then immersed

overnight in lysis solution (2.5 M NaCl, 100 mM

Na2EDTA, 10 mM Tris, 10% (v/v) DMSO, 1% Triton X-

100—pH 10). Slides were then exposed in an alkaline

solution (300 mM NaOH, 1 mM EDTA—pH . 13) for

30 min. The slides were placed for electrophoresis, using

alkaline solution, at 2 V/cm for 20 min. In order to

minimise the risk of LMPA lifting from the slide, all the

solutions were kept at 48C during the assay. Following

electrophoresis, the slides were neutralised using the

supplied neutralising buffer (Ikzus, Genoa), before being

fixed in ethanol and air dried. Slides were stained with

FLUO-Plus DNA stain (Ikzus, Genoa) and visualised on a

fluorescent microscope. Image data were analysed using

CASP software (Konca, 2003). To quantify the DNA

damage, olive tail moment (OTM, i.e. %DNA £ distance

of centre of gravity of DNA) and/or tail moment (TM, i.e.

%DNA £ tail length) of 30–50 cells from each window of

each slide were analysed. Replicates, 3–4, were performed

for all experiments including Lipofectin treated, SFM as

negative control and H2O2 treated (100mM) cells, the latter

serving as a positive control for induced DNA damage.

RESULTS

Lipofectin and Oligofectamine Induce Gene

Expression in A431 Cells as Shown by Microarray-based Gene Expression Profiling

In our previous studies with microarray-based gene

expression profiling (e.g. Petch et al., 2003), we learned

that the data handling of the large amount of information

generated was more manageable with low-density arrays.

Thus, in this study we used low-density cDNA

microarrays housing 200 gene spots containing 160

different genes that were replicated twice on the same

glass slide to yield a total of 400 gene spots per slide.

Human A431 epithelial cells treated with either Lipofectin

or Oligofectamine (at the recommended concentrations

routinely used for nucleic acid delivery) for 4 h were then

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grown in serum-containing medium for a total period of

24 h. Gene expression profiles of CL-treated cells were

compared to those of control cells that had been handled in

exactly the same manner except that no lipids were added

during the 4 h treatment period.

Figure 1 shows typical results from microarrays

hybridised with cy3- or cy-5 labelled cDNA from either

Lipofectin (left hand panels A to D) or Oligofectamine

(right hand panels E to H) treated A431 cells. Images

from their respective experiments for the untreated

control cell samples are also shown (panels B and G).

The top panels A and E show hybridisation intensity of

gene spots from a section of the microarray housing 4

grids of 50 gene spots each. A magnified image of the

selected grid is given in the middle panels B–C and F–G

for treated and untreated samples. Finally, in the lower

panels (D and H), gene expression images from treated

and untreated cells are superimposed. The high

reproducibility of the control spots can be observed by

noting the equal expression intensity of the control gene,

FIGURE 1 Representative cDNA microarrays showing the intensity of hybridisation for Lipofectin (LF)—or Oligofectamine (OF)-treated and untreatedsamples. Panels A and E show 200 gene spots (i.e. 4 £ 50 grids) for LF- and OF-treated cells, respectively. Panels B–C and F–G is the selected 50-genegrid for Cy3 or Cy5 labelled cDNA of treated or untreated samples. Panels D and H show superimposed Cy-dyes for treated and untreated samples.The white arrow heads represent the internal control spots of ubiquitin showing the equal intensities of this control protein within arrays.

TOXICOGENOMICS OF NON-VIRAL VECTORS 315

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ubiquitin, as marked by white arrowheads in all the

arrays shown. The expression of ubiquitin and several

other control gene spots were used as quality assurance

markers. Furthermore, in panels B, F, C and G, the

treated and controls for the two CL formulations are

shown with the Cy-dyes “flipped” to highlight the

reproducibility of our hybridisations as the ubiquitin

control highlighted gave equal intensities of spots

irrespective of the dye used in these experiments.

To achieve this the number of dye molecules per

nucleotide were carefully controlled during the labelling

procedure as described in the “Methods” section.

Arrays showing non-equal intensity or variable intensity

of control gene spots in replicates on the same slide or

between slides in dye-flipping experiments were nor-

mally rejected.

Figure 2 shows a scatter plot of all the genes represented

on the array for either Lipofectin or Oligofectamine-

treated cells. The gene spots highlighted in solid circles

represent those that change their expression by 2-fold or

more in the treated versus untreated controls. It can be

seen that a greater number of genes are highlighted for

Oligofecatmine compared to Lipofectin treatment with

a greater number of these genes falling into the range of

4-fold or greater change in expression. The exact identity

of these genes are given in Tables I and II.

Genes from different functional categories showed

expression changes in A431 epithelial cells upon treatment

of CL formulations. Of the genes represented on our array,

Lipofectin initiated up-regulation of the replication protein

a1 (rpa1) as shown in Table I, whereas Oligofectamine

induced the over-expression of 24 genes (Table II),

including genes involved in apoptosis such as bcl2-related

protein a1 (bcl2a1), Caspase 8 isoform c (casp8), heat

shock protein 70 (hsp70) and heat shock 60 kDa protein 1,

chaperonin (hspd1). Lipofectin triggered suppression of

some genes (Table I), e.g. endothelin receptor type b

isoform 2 (ednrb), Ribosomal protein 16 (rp16) and

endothelin receptor type b, isoform 1 (ednrb), where

Oligofectamine inhibited the expression of the genes

annexin a2 (anxa2), retinoid x receptor alpha (rxra) and

s100 calcium-binding protein a8 (s100a8).

Figure 3 represents gene expression data for

Oligofectamine as a single linkage Hierarchical Cluster-

ing Plot as performed on the GeneSight software. The

algorithm used subjects the expression intensity ratio of

treated versus untreated samples to single-linkage

Hierarchical clustering using Euclidean distance metric

analyses in order to arrange each gene with its related

group members exhibiting a similar ratio of change in

expression (Panel A). Gene-expression families were

colour coded for comparison, i.e. the over-expressed and

FIGURE 2 Scatter plots of gene expression changes induced by CL in A431 cells. Data represent Log2 transformed gene expression valuesfor Lipofectin (panel A) and Oligofectamine (panel B). The reference lines shown indicate the intensity ratios of genes exhibiting no changes (0),under-expression by 2-fold (21) or 4-fold (22) or over-expression by 2-fold (1) or 4-fold (2).

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TABLE I Lipofectin-induced gene expression changes in A431 epithelial cells

Accession No Gene function and annotation Ratio (T:UT) Changes

NM_002945 Replication protein a1 (70 kd); rpa1 2.02 +NM_007209 Ribosomal protein l35; rpl35 1.65 NCNM_005319 H1 histone family, member 2; h1f2 1.64 NCNM_033158 Hyaluronoglucosaminidase 2; hyal2 1.64 NCNM_021103 Thymosin beta tmsb10. 1.60 NCNM_021065 H2a histone family, member g; h2afg 1.56 NCNM_006086 Tubulin, beta, 4; tubb4 1.39 NCNM_004537 Nucleosome assembly protein 1-like 1; nap1l1 1.10 NCNM_001943 Desmoglein 2 preproprotein; dsg2 1.07 NCNM_002690 Polymerase (dna directed), beta; polb 1.04 NCNM_006716 Activator of s phase kinase; ask 1.02 NCNM_006013 Ribosomal protein l10; rpl10 1.02 NC– b-actin 1.00 NCNM_002623 Prefoldin 4; pfdn4 1.00 NCNM_006087 Tubulin, beta, 5; tubb5 0.96 NCNM_002796 Proteasome (prosome, macropain) subunit, beta type, 4; psmb4 0.96 NCNM_004039 Annexin a2; anxa2 0.94 NCNM_004383 C-src tyrosine kinase; csk 0.67 NCXM_016685 Similar to activator of s phase kinase (h. sapiens); loc143173 0.45 –NM_002416 Monokine induced by gamma interferon; mig 0.44 –NM_002064 Glutaredoxin (thioltransferase); glrx 0.36 –NM_004417 Dual specificity phosphatase 1; dusp1 0.31 –NM_001870 Mast cell carboxypeptidase a3 precursor; cpa3 0.23 –NM_006890 Carcinoembryonic antigen-related cell adhesion molecule 7; ceacam7 0.23 –NM_003991 Endothelin receptor type b isoform 2; ednrb 0.23 –NM_000970 Ribosomal protein l6; rpl6 0.21 –NM_000115 Endothelin receptor type b, isoform 1; ednrb 0.17 –

Ratio of treated: untreated cells is given and genes that are overexpressed (þ), underexpressed (2 ) or unchanged (NC) are indicated.

TABLE II Oligofectamine-induced gene expression changes in A431 epithelial cells

Accession No Gene function and annotation Ratio (T:UT) Changes

NM_004417 Dual specificity phosphatase 1; dusp1 3.69 +NM_001801 Cysteine dioxygenase, type i; cdo1 3.64 +XM_016685 Similar to activator of s phase kinase (h. sapiens); loc143173 3.11 +NM_002467 v-myc Myelocytomatosis viral oncogene homolog (avian); myc 2.97 +NM_004049 bcl2-Related protein a1; bcl2a1 2.93 +NM_002424 Matrix metalloproteinase 8 preproprotein; mmp8 2.90 +NM_033356 Caspase 8, isoform c; casp8 2.89 +NM_003991 Endothelin receptor type b isoform 2; ednrb 2.63 +NM_003875 Guanine monophosphate synthetase; gmps 2.52 +NM_003195 Transcription elongation factor a (sii), 2; tcea2 2.46 +NM_002698 Pou domain, class 2, transcription factor 2; pou2f2 2.46 +NM_002623 Prefoldin 4; pfdn4 2.39 +NM_033158 Hyaluronoglucosaminidase 2; hyal2 2.28 +L12723 Heat shock protein 70; hsp70 2.22 +NM_000245 Met proto-oncogene precursor; met 2.21 +NM_006716 Activator of s phase kinase; ask 2.20 +NM_006235 Pou domain, class 2, associating factor 1; pou2af1 2.18 +NM_001983 Excision repair cross-complementing rodent repair deficiency, ercc1 2.16 +NM_000994 Ribosomal protein l32; rpl32 2.11 +NM_004094 Eukaryotic translation initiation factor 2, subunit 1 (alpha, 35 kd); eif2s1 2.10 +NM_000698 Arachidonate 5-lipoxygenase; alox5 2.09 +NM_002849 Protein tyrosine phosphatase, receptor type, r; ptprr 2.03 +NM_002156 Heat shock 60 kd protein 1 (chaperonin); hspd1 2.02 +NM_021067 Kiaa0186 gene product; kiaa0186 2.01 +NM_004383 c-src tyrosine kinase; csk 1.94 NC– b-actin 1.08 NCNM_001943 Desmoglein 2 preproprotein; dsg2 1.04 NCNM_001469 Thyroid autoantigen 70 kd (ku antigen); g22p1 1.03 NCNM_001274 chk1 checkpoint homolog (s. pombe); chek1 1.00 NCNM_002796 Proteasome (prosome, macropain) subunit, beta type, 4; psmb4 0.70 NCNM_006087 Tubulin, beta, 5; tubb5 0.57 NCNM_005566 Ldha 0.54 –NM_004039 Annexin a2; anxa2 0.50 –NM_002957 Retinoid £ receptor, alpha; rxra 0.49 –

Ratio of treated: untreated cells is given and genes that are overexpressed (þ), underexpressed (2 ) or unchanged (NC) are indicated.

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under-expressed genes appear in red and green,

respectively, with the relative ratio reflected by the

intensity of the colour. Example genes from these

different expression families are magnified into groups

and single genes of interest highlighted (Panel B). The

purpose of such clustering is that this methodology

allows classification of genes by their relative expression

ratios and thus allows any patterns to be easily

visualised. For instance, in the examples given in

Panel B it can be seen that Oligofectamine treatment of

epithelial cells resulted in overexpression (red bars) of

several genes linked with apoptosis including casp8,

bcl2a1 and hsp70.

Semi-quantitative Reverse Transcriptase Polymerase

Chain Reaction (RT-PCR)

The gene expression data obtained with cDNA micro-

arrays was validated by semi-quantitative RT-PCR.

Figure 4 shows the expression of b-actin, proteasome

beta 4 and annexin 2 as example genes. The mRNA

expression of these genes for untreated controls and CL

treated samples of A431 cells (panel A), and their

normalised ratio represented as an histogram (panel B).

The expression ratio of Proteasome beta 4 and annexin 2

relative to actin as obtained by RT-PCR correlated well

and exhibited similar trends to those obtained by cDNA

microarray analysis (see Tables I and II).

Impact of CL-induced Gene Changes on Apoptosis and

DNA Damage

In an attempt to understand the functional consequences

of the gene changes induced by Oligofectamine and

Lipofectin in A431 cells we investigated their effect on the

apoptosis and DNA damage status of these cells especially

as some of the genes altered were known to be associated

with programmed cell death. Figure 5 shows that

CL-treatment of A431 cells significantly altered the

FIGURE 3 Single-linkage hierarchical clustering of gene expression induced by Oligofectamine in A431 cells. Gene-expression families were colourcoded for comparison, i.e. the over-expressed or under-expressed genes appear in red or green, respectively, with the relative ratio reflected by theintensity of the colour (see Panel A inset). Panel A is the clustered representation for entire array. Panel B is the selected examples of genes in the clustersof over-expressed, no changes and under-expressed gene families.

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apoptosis status of these cells as assessed by annexin

V-FITC flow cytometry analyses. Treatment of cells with

5% DMSO, as a positive inducer of apoptosis, led to a

marked increase in the proportion of cells entering early

(e.g. indicative of membrane damage) and late (e.g.

indicative of membrane and DNA damage) apoptosis can

be observed in Panels B and E. Lipofectin treatment

induced a significant increase in the number of late

apoptotic cells and a non-significant increase in early

apoptotic cells (( p . 0.05); Panels C and E) whereas

Oligofectamine induced a significant increase in the

number of cells undergoing early apoptosis and a non-

significant increase in cells in late apoptosis (( p . 0.05);

Panels D and E). These results confirm that CL treatment

leads to an altered apoptosis status of cells.

Focusing on Lipofectin, the most widely used of the CL

lipids, we next examined its effect on inducing DNA

damage. Using the same concentration of Lipofectin that

induced the above mentioned gene expression changes and

the enhanced entry of cells to late apoptosis (shown above)

we used the single cell electrophoresis (the so called

COMET) assay to assess the degree of DNA damage. This

assay is widely used for the study of DNA damage in cells

(Singh et al., 1988; Schindewolf et al., 2000).

Figure 6 shows the typical results obtained with the

COMET assay of A431 cells treated with Lipofectin or

hydrogen peroxide, H2O2, a positive control that is known

to induce DNA damage. Panel C shows the typical diffuse

migration (in the shape of an astronomical comet) of

damaged DNA around each of the single cells during

electrophoresis of samples treated with the positive control.

In contrast, statistical analysis showed no significant

( p . 0.05) difference between untreated and Lipofectin-

treated A431 cells (Fig. 6) implying that the observed gene

expression changes induced by this CL were not associated

with a marked increase in DNA damage.

DISCUSSION

The use of viral vectors for ex vivo and in vivo gene

therapy typically results in high transfection efficiencies

but this advantage is often offset by important safety

concerns relating to immunological and toxicological

responses in cells, animals and human patients (for

reviews see Somia and Verma, 2000; Zallen, 2000).

Indeed, recent gene therapy trials were halted due to

unexplained toxicity surrounding the death of a patient

FIGURE 4 Semi-quantitative RT-PCR for b-actin, annexin2 and Proteasome beta 4. Panel A shows the expression of the amplified fragments in 1%agarose gel in the presence of ethidium bromide. Panel B presents histograms of the relative expressions of these genes normalised to b-actin andrepresents a ratio of treated over untreated. Asterisk represents a significant ( p , 0.05) gene expression change.

TOXICOGENOMICS OF NON-VIRAL VECTORS 319

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undergoing ornithine transcarbamylase gene therapy in a

Phase 1 clinical trial using second generation adenoviral

vectors (Somia and Verma, 2000). Non-viral vectors,

though often less efficient as gene transfection agents,

have been investigated as potentially safer alternatives

for the delivery of genes. Of these the CL lipid

formulations are the widely used. By virtue of their net

positive charge, which is important for binding to the

nucleic acid and association with the negatively charged

cell membrane to achieve high uptake and transfection

efficiency, these CL formulations can also be cytotoxic

(Filion and Phillips, 1997; Pedrosa de Lima et al., 2001).

However, little is known about the extent or nature of the

gene changes induced by CL in target cells. Here, we

report on the toxicogenomics and geno-compatibility of

these lipids in human epithelial cells by studying

FIGURE 5 The influence of Lipofectin- or Oligofectamine-induced gene expression changes on apoptosis in A431 cells using flow cytometric AnnexinV-FITC apoptosis assay. Panels A–D show A431 cell population for untreated (control), DMSO (5%) Lipofectin-treated (LF) and Oligofectamine-treated (OF) cells, respectively. In each panel, C, N, EA and LA represent abbreviations for control, necrotic, early apoptotic and late apoptotic,respectively. Panel E represents a histogram of the relative proportion of the cell population undergoing EA or LA. Significant differences ( p , 0.05)are as follow: ‡LA vs EA in LF-treated cells; † EA vs LA in OF-treated cells; *EA of OF-treated vs EA of untreated cells; **EA of vs LA inOF-treated cells.

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the CL-induced gene expression changes using cDNA

microarrays.

At concentrations routinely used to obtain

efficient delivery of genes, oligonucleotides and gene

silencing siRNAs, we found that both Lipofectin and

Oligofectamine-induced marked gene changes in human

epithelial cells (Figs. 1–3; Tables I and II). In the case

of Oligofectamine, this CL elicited the expression of up to

27 genes representing 16% of the genes on the microarray.

The affected genes were functionally known to be

involved in various cellular processes, including cell

proliferation, differentiation and programmed cell death

(apoptosis). Of the genes involved in apoptosis, these

lipids triggered the over- or under-expression of some

important genes such as bcl2-related protein a1 (bcl2a1),

caspase 8, isoform c (casp8), heat shock protein 70

(hsp70) and 60 (hspd1), annexin a2 (anxa2) and tubulin

beta 5 (tubb5). For example, the Oligofectamine-induced

up-regulation of caspase-8 suggests that this CL initiates

the activation of procaspases and caspases, a process that

would normally result in activation of a series of apoptotic

signalling cascades involving, amongst others, the

electron carrier protein cytochrome C, adaptor protein

Apaf-1, Bcl-2 family, p53 and various transcription factors

(Sedlakova et al., 1999; Kanduc et al., 2002). However, as

we only used low-density microarrays (for ease of data

handling) in the present study, many of the downstream

genes were not represented on our array. Thus, the use of

higher density or more focused custom arrays housing all

apoptotic genes may be more useful to explore the exact

nature of these changes. Further studies using such arrays

are planned.

The gene expression changes observed for CL treated

cells ranged in magnitude from more than 5-fold under-

expressed to almost 4-fold over-expressed (see Tables I

and II). This degree of change was similar to that observed

for downstream signalling molecules when A431 cells

were treated with an inhibitor of epidermal growth factor

receptor expression (Petch et al., 2003). Thus, these

changes are reflective of what might happen during

normal signalling processes such as those following

switching on or off of receptor signalling cascades as

exemplified by EGFR. Furthermore, the observed gene

changes were reproducibly seen on replicate arrays even

during dye-flipping experiments. Thus, these findings

coupled with independent validation of the array data with

RT-PCR analyses further confirmed the observed gene

changes upon CL treatment of A431 cells were real

changes that may have important functional consequences

in cells.

In an attempt to study the functional consequences of

the altered gene expression arising from CL treatment,

we examined the effects of Lipofectin or Oligofectamine

on apoptosis and DNA damage in epithelial cells.

FIGURE 6 The single cell gel (SCG) electrophoresis (COMETassay) for DNA damage detection induced by CL in A431 cells. Lipofectin-treated cells(2mg/cm2 of cultured cells) showed no significant difference compare to untreated, whereas the positive control hydrogen peroxide-treated cells(100mM) revealed the characteristic comet around cells indicative of DNA damage (Panel A–C). Panels E shows the expressed DNA damagerepresented in the olive tail moment (OTM, i.e. %DNA £ distance of centre of gravity of DNA).

TOXICOGENOMICS OF NON-VIRAL VECTORS 321

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Consistent with the nature of the gene changes described

above, increased apoptosis was observed in cells treated

with the lipids compared to untreated cells (Fig. 5). In the

case of Oligofectamine, this involved an increased number

of cells in early apoptosis whereas for Lipofectin this was

associated with an increased population of cells in late

apoptosis. However, the latter did not appear to involve

extensive DNA damage (Fig. 6) further implying that

apoptosis in Lipofectin-treated cells was associated with

mostly membrane damage and not with extensive DNA

scission.

Of interest, our data suggested that gene changes, and

thus, geno-toxicity, elicited by the CL formulations were

lipid-dependent as no extensive overlap was observed in

those genes exhibiting expression changes upon either

Lipofectin or Oligofectamine treatment. However, this

may simply be reflective of the low number of genes

present on the arrays used; the likelihood of finding

common gene changes would be greater for high density

arrays. Alternatively, it may be a function of the

responsiveness of these cells to the lipids used. Indeed,

it is well known that the transfection capability of these

CL formulations is both cell type and lipid dependent

(for reviews see Akhtar et al., 2000; Hirko et al., 2003),

thus it is not surprising they have markedly different

effects on the gene expression profile of a given cell type.

Also for example, Filion and Phillips (1997) reported high

toxicity of some CL in phagocytic cells (macrophages and

U937 cells), but not in non-phagocytic T lymphocytes.

Thus, for a detailed characterisation of the toxicogenomics

of these lipid delivery systems, it is, therefore, likely that

gene expression patterns/profiles will need to be

determined in many cell types of interest.

In addition, CL formulations may also affect the

outcome of gene therapy experiments. In studies with

antisense nucleic acids, siRNA or plasmid DNA where

often only a single desired genetic change is sought, it is

possible that gene changes induced by the lipid delivery

system itself could exacerbate, attenuate or even mask the

desired effects of the nucleic acid. The net effect is likely

to be determined by many factors but will include the

relative magnitude of the over- or under-expression of the

genes of interest. Potentially, this could be of particular

concern where the gene targeted by the nucleic acid is the

same as the one directly affected by the CL formulation

used. Furthermore, the presence of nucleic acid in the CL

formulation may modulate the induced gene expression

changes and thus needs to be determined in such studies.

In conclusion, our studies highlight the fact that

inadvertent gene expression changes can be induced by

the CL delivery formulation itself and that these

alterations in gene expression can lead to downstream

functional consequences such as increased apoptosis.

Thus, gene expression changes that, ultimately, lead to

changes in cell phenotype will have important safety

implications for the use of these non-viral vectors in gene-

based therapies. Also, the induced or inadvertent gene

expression changes should be taken into consideration

in gene therapy or gene silencing experiments using

CL formulations where they may potentially mask

or interfere with the desired genotype and/or phenotype

end-points.

References

Aissaoui, A., Oudrhiri, N., Petit, L., Hauchecorne, M., Kan, E., Sainlos,M., Julia, S., Navarro, J., Vigneron, J.P., Lehn, J.M. and Lehn, P.(2002) “Progress in gene delivery by cationic lipids: guanidinium-cholesterol-based systems as an example”, Curr. Drug Target. 3,1–16.

Akhtar, S., Hughes, M.D., Khan, A., Bibby, M., Hussain, M., Nawaz, Q.,Double, J. and Sayyed, P. (2000) “The delivery of antisensetherapeutics”, Adv. Drug Deliv. Rev. 44, 3–21.

Audouy, S.A., de Leij, L.F., Hoekstra, D. and Molema, G. (2002) “In vivocharacteristics of cationic liposomes as delivery vectors for genetherapy”, Pharm. Res. 19, 1599–1605.

Campbell, P.I. (1983) “Toxicity of some charged lipids used in liposomepreparations”, Cytobios 37, 21–26.

Canonico, A.E., Plitman, J.D., Conary, J.T., Meyrick, B.O. and Brigham,K.L. (1994) “No lung toxicity after repeated aerosol or intravenousdelivery of plasmid-cationic liposome complexes”, J. Appl. Physiol.77, 415–419.

Duzgunes, N., De Ilarduya, C.T., Simoes, S., Zhdanov, R.I., Konopka, K.and Pedroso de Lima, M.C. (2003) “Cationic liposomes for genedelivery: novel cationic lipids and enhancement by proteins andpeptides”, Curr. Med. Chem. 10, 1213–1220.

Dykxhoorn, D.M., Novian, C.D. and Sharp, P.A. (2003) “Killing themessenger: short RNAs that silence expression”, Nat. Rev. 4,257–467.

Filion, M.C. and Phillips, N.C. (1997) “Toxicity and immunomodulatoryactivity of liposomal vectors formulated with cationic lipids towardimmune effector cells”, Biochim. Biophys. Acta 1329, 345–356.

Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R.,Hughes, J.E., Snesrud, E., Lee, N. and Quackenbush, J. (2000)“A concise guide to cDNA microarray analysis”, Biotechniques 29,548–554, See also page 556.

Hirko, A., Tang, F. and Hughes, J.A. (2003) “Cationic lipid vectors forplasmid DNA delivery”, Curr. Med. Chem. 10, 1185–1193.

Hughes, M.D., Hussain, M., Nawaz, Q., Petch, A.K., Sayyed, P. andAkhtar, S. (2001) “Cellular delivery of antisense oligonucleotides andribozymes”, Drug Discov. Today 6(6), 303–315.

Juliano, R.L. and Akhtar, S. (1992) “Liposomes as a drug deliverysystem for antisense oligonucleotides”, Antisense Res. Dev. 2,165–176.

Kanduc, D., Mittelman, A., Serpico, R., Sinigaglia, E., Sinha, A.A.,Natale, C., Santacroce, R., Di Corcia, M.G., Lucchese, A., Dini, L.,Pani, P., Santacroce, S., Simone, S., Bucci, R. and Farber, E. (2002)“Cell death: apoptosis versus necrosis (review)”, Int. J. Oncol. 21,165–170.

Kerr, D. (2003) “Nature reviews cancer, clinical development of genetherapy for colorectal cancer”, Nat. Rev. Cancer 3(8), 615–622.

Konca, K., Lankoff, A., Banasik, A., Lisowska, H., Kuszewski, T.,Gozdz, S., Koza, Z. and Wojcik, A. (2003) “A cross-platform publicdomain PC image-analysis program for the comet assay”, Mutat Res534, 15–20.

McManus, M.T. and Sharp, P.A. (2002) “Gene silencing in mammals bysmall interfering RNAs”, Nat. Rev. 3, 737–747.

Nagahiro, I., Mora, B.N., Boasquevisque, C.H., Scheule, R.K. andPatterson, G.A. (2000) “Toxicity of cationic liposome-DNA complexin lung isografts”, Transplantation 69, 1802–1805.

Pedroso de Lima, M.C., Simoes, S., Pires, P., Faneca, H. andDuzgunes, N. (2001) “Cationic lipid-DNA complexes in genedelivery: from biophysics to biological applications”, Adv. DrugDeliv. Rev. 47, 277–294.

Peng, L., Jiang, H. and Bradely, C. (2002) “Detection of B lymphomacells undergoing apoptosis by Annexin-V assay”, Chin. Med. Sci. J.17, 17–21.

Petch, A.K., Sohail, M., Hughes, M.D., Benter, I., Darling, J.,Southern, E.M. and Akhtar, S. (2003) “Messenger RNA expressionprofiling of genes involved in epidermal growth factor receptorsignalling in human cancer cells treated with scanning array-designed antisense oligonucleotides”, Biochem. Pharmacol. 66,819–830.

Y. OMIDI et al.322

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

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rsita

et Z

ueri

ch o

n 07

/12/

14Fo

r pe

rson

al u

se o

nly.

Pleyer, U., Groth, D., Hinz, B., Keil, O., Bertelmann, E., Rieck, P. andReszka, R. (2001) “Efficiency and toxicity of liposome-mediated gene transfer to corneal endothelial cells”, Exp. Eye Res.73, 1–7.

Scheule, R.K., St George, J.A., Bagley, R.G., Marshall, J., Kaplan, J.M.,Akita, G.Y., Wang, K.X., Lee, E.R., Harris, D.J., Jiang, C., Yew, N.S.,Smith, A.E. and Cheng, S.H. (1997) “Basis of pulmonary toxicityassociated with cationic lipid-mediated gene transfer to themammalian lung”, Hum. Gene. Ther. 8, 689–707.

Schindewolf, C., Lobenwein, K., Trinczek, K., Gomolka, M.,Soewarto, D., Fella, C., Pargent, W., Singh, N., Jung, T. and Hrabede Angelis, M. (2000) “Comet assay as a tool to screen for mousemodels with inherited radiation sensitivity”, Mamm. Genome 11,552–554.

Sedlakova, A., Kohut, A. and Kalina, I. (1999) “Biochemical changes inapoptosis and methods for their determination (review)”, Cesk.Fysiol. 48, 107–118.

Singh, N.P., McCoy, M.T., Tice, R.R. and Schneider, E.L. (1988) “A simpletechnique for quantitation of low levels of DNA damage in individualcells”, Exp. Cell Res. 175, 184–191.

Somia, N. and Verma, I.M. (2000) “Gene therapy: trials and tribulations”,Nat. Rev. Genet. 1, 91–99.

Tarahovsky, Y.S. and Ivanitsky, G.R. (1998) “Liposomes in gene therapy.Structural polymorphism of lipids and effectiveness of genedelivery”, Biochemistry (Mosc. 63, 607–618.

Whitehouse, A. (2003) “Herpesvirus saimiri: a potential gene deliveryvector (Review)”, Int. J. Mol. Med. 11, 139–148.

Zallen, D.T. (2000) “US gene therapy in crisis”, Trend Genet. 16, 272–275.Zelphati, O. and Szoka, F.C. Jr. (1996) “Mechanism of oligonucleotide

release from cationic liposomes”, Proc. Natl Acad. Sci. USA 93(21),11493–11498.

Zhang, G., Gurtu, V., Kain, S.R. and Yan, G. (1997) “Early detection ofapoptosis using a fluorescent conjugate of annexin V”, Biotechniques23, 525–531.

TOXICOGENOMICS OF NON-VIRAL VECTORS 323

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