Therapeutic perspectives of in vivo cell electropermeabilization

Ž .Bioelectrochemistry 53 2000 1–10www.elsevier.comrlocaterbioelechem

Review article

Therapeutic perspectives of in vivo cell electropermeabilizationq

Lluis M. Mir )

Laboratoire de Physicochimie et Pharmacologie des Macromolecules Biologiques, UMR 8532 CNRS, Institut GustaÕe-Roussy, 39 rue C. Desmoulins,´F-94805 Villejuif, Cedex, France´

Received 1 November 1999; received in revised form 6 June 2000; accepted 15 September 2000

Abstract

Ž .Cell electropermeabilization also termed cell electroporation is nowadays a routine technique used in biochemical and pharmacologi-cal studies for the in vitro introduction of nonpermeant molecules into living cells. But electric pulses can be used as well in vivo for thedelivery of drugs or DNA into cells of tissues. This review then gives an updated overview of the therapeutic perspectives of cell

Želectropermeabilization in vivo, in particular of the antitumour electrochemotherapy i.e., the combination of a cytotoxic nonpermeant.drug with permeabilizing electric pulses delivered to the tumours and of in vivo DNA electrotransfer for gene therapy. After a shortŽ .summary of the present knowledge on cell electropermeabilization particularly in vivo , the basis, the present achievements, and the

challenges of electrochemotherapy are described and discussed, which includes an overview of still open questions and an update onrecent clinical trials. DNA electrotransfer for gene therapy is an emerging field in which results are rapidly accumulating. Presentknowledge on DNA electrotransfer mechanisms, as wel as the potentialities of DNA electrotransfer to become an efficient non-viralapproach for gene therapy, are reviewed. q 2000 Elsevier Science S.A. All rights reserved.

Keywords: Electropermeabilization; Electroporation; Electrochemotherapy; DNA electrotransfer; Gene therapy

1. Introduction

Cell electropermeabilization, also termed cell electropo-Ž .ration see Section 2.1 , is nowadays a routine technique

for the introduction of nonpermeant molecules into livingcells in vitro, for biochemical and pharmacological studiesw x w x1 . Since the initial reports by Wong and Neumann 2 and

w xNeumann et al. 3 on the achievement of cell transfectionin vitro by DNA electrotransfer into electropermeabilizedcells, the technique became rapidly one of the most widelyused methods for introducing DNA into bacteria, animal

w xcells, plant cells and yeast 4 . DNA electrotransfection canbe easily done in vitro with simple generators that produceexponentially decaying pulses, even if such type of electricpulses leads to a large cell killing in the optimal conditionsfor gene transfer. Later on, square-wave electric pulsegenerators were developed, that allowed, on the one hand,to specify the effect of the various electric parameters

q Part of this review was presented at the XVth Symposium onŽ .Bioelectrochemistry and Bioenergetics held at Strasbourg France ,

September 1999, under the auspices of the Bioelectrochemical Society.) Tel.: q33-1-42-11-47-92; fax: q33-1-42-11-52-76.

Ž .E-mail address: [email protected] L.M. Mir .

Ž . w xpulse intensity, pulse length, number of pulses 5 , and onthe other hand, to obtain electropermeabilization condi-tions under which a very large proportion of cells was

w xsimultaneously permeabilized and alive 6 . A new cellbiology and pharmacology was then possible using nonper-meant molecules that could enter the transiently permeabi-

w xlized cells and act on intracellular targets 1 . As expected,Žthe entry of oligonucleotides a typical nonpermeant.molecule like all the nucleic acids into cell cytosol was

w xalso achieved 7–9 . But unexpectedly, the largest develop-ment resulted from the observation that bleomycin, a cur-rently used anticancer drug, was also a nonpermeant

w x Žmolecule 10 bleomycin molecules enter intact cells notby diffusion through the plasma membrane but by a mech-

w x.anism of receptor-mediated endocytosis 11 . Because ofŽthe huge intrinsic cytotoxicity of this molecule several

hundreds of molecules of bleomycin inside the cell are.sufficient to kill the cell , the combination of bleomycin

and cell electropermeabilization increased the toxicity ofthis anticancer drug by hundreds of thousands times in

w xvitro 12,13 .A perspective of the potential biomedical applications

w xof the electric pulses was published in 1995 14 . In thispaper, special emphasis was put on the antitumor elec-

0302-4598r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved.Ž .PII: S0302-4598 00 00112-4

( )L.M. MirrBioelectrochemistry 53 2000 1–102

Žtrochemotherapy the combination of bleomycin and of.electric pulses delivered in vivo to the tumours . A very

important progress has been achieved since then. A newtherapeutical use of in vivo electropermeabilization, namelyin vivo DNA electrotransfer, has emerged since that time.The purpose here is to give an updated overview of thetherapeutic perspectives of cell electropermeabilization invivo, in particular on the antitumor electrochemotherapyand on the DNA electrotransfer for gene therapy. Electricpulses delivery is being also considered as a promisingway to allow the transfer of molecules of pharmacologicalinterest across the skin, in particular across the stratum

w xcorneum 15–17 . The bases of effects of electric pulses onskin have been extensively analysed by Pliquett and Weaverw x w x18 and Weaver et al. 19 . However, the potential uses ofstrong electric field pulses in transdermal drug deliverywill not be discussed here for various reasons. First of all,purposes are different: the goal of both electrochemother-apy and DNA electrotransfer is the electroloading of for-eign molecules into living cells of various tissues, whiletransdermal drug delivery aims at the introduction of ex-

w xogenous molecules into the body 14–16 . Second, the«electropermeabilization» achieved in transdermal drug de-livery is very different from that required for efficientelectrochemotherapy or convenient DNA electrotransfer.For transdermal drug delivery the layer of «cells» thatmust be permeabilized is the stratum corneum, an insulat-

Žing layer of dead packed corneocytes that is of cellswithout resting membrane potential, with a membrane andcell inside quite different from those of living cells, in

.particular with a dehydrated cell inside . The stratumcorneum structure is only affected in small areas, either atthe appendages vicinity or at the LTR, the Large Transport

w xRegions generated by the electric pulses 20,21 . Clearly,the generation of aqueous pathways at the substratumcorneum level, a supracellular structure, is an endpointquite different from the achievement of reversible changesat the membrane structure of each individual living cell,like those of muscles, tumours, liver, etc., even if bothsituations are the expression of a «strong electric fieldpulse-induced behaviour».

Before describing the present achievements and chal-Ž .lenges of electrochemotherapy Section 3 and of DNA

Ž .electrotransfer Section 4 , a short presentation of knowl-Ž .edge on cell electropermeabilization particularly in vivo

is given in Section 2.

2. In vivo electropermeabilization: what do we know?

2.1. General background on cells in suspension

2.1.1. Consequences of cell exposure to strong electricfield short pulses

Just for the readers who are not familiar with the effectsof short intense electric pulses on living cells, it must be

recalled that these effects can be easily analysed throughone of their consequences at the cell level, that is the

Želectropermeabilization of the exposed cells i.e. the factthat the cell becomes permeable to otherwise nonpermeant

. w xmolecules 1 .At the level of the entire cell, the consequences of cell

exposure to the electric pulses are understood. In thepresence of the external electric field, a change in the

w xtransmembrane potential difference is generated 22–24 . Itsuperimposes upon the resting transmembrane potentialdifference and it may be calculated from the Maxwell’s

Žequations, providing a few approximations are made veryreduced thickness of the cell membrane, null membrane

.conductivity, etc. . These changes in the transmembranepotential difference have been experimentally observedw x25,26 . Analytically, the effects of the exposure of cells toelectric pulses are well described in the case of isolated

w xcells in suspension 27,28 .At the molecular level of analysis, the explanation of

the phenomena occurring at the cell membrane level is stillhypothetical. It is assumed that above a threshold value of

Žthe net transmembrane potential which could depend on.the particular parameters of the pulse protocol used , the

changes occurring in membrane structure will be enoughas to render that membrane permeable to otherwise non-permeant molecules of given physicochemical character-

Ž .istics molecular mass, radius, etc .

2.1.2. Theories of cell electropermeabilizationOne of the most commonly accepted theories, the cell

w xelectroporation 22–24 , is based on the generation of aŽlarge number of «transient electropores» half-life in the

.order of 1 ms and of a reduced number of «stablelong-lived electropores», responsible for the transport ofmolecules across the membrane. However, theory does notgive the origin of the energy necessary to induce themolecular rearrangements required for the closure of thelong-lived electropores. Moreover, these pores have neverbeen observed in membranes of cells submitted to ‘‘effec-tive’’ electric pulses. Secondary large pores, arising longafter the pulsing and not related to the primary poresŽ .electropores have been detected in cells pulsed in a

w xhypoosmotic medium 29 . In that case, the immediateinflux of water molecules resulted in a rapid swelling and

Žincrease in cell volume at a constant surface of the cell.membrane that caused the formation of secondary «pores»

at any place where structural defaults of the membranewere present.

In fact, many models have been developed to explaineither the irreversible electrical breakdown in lipid mem-

w xbranes 30–32 or the reversible electrical breakdown, asŽ .observed in living cells. These include i the phase transi-

w xtion model 33 based on the statistical mechanical modelw x Ž .of lipid membrane structure 34 , ii the denaturation

w xmodel 35 based on specific effects at the level of theŽ .membrane proteins, and iii the «electroporation» model,

( )L.M. MirrBioelectrochemistry 53 2000 1–10 3

based on the possibility of spontaneous pore formation inw xlipid bilayers 36 and on the hypothesis that some of these

w xpores could reach a stable conformation 37 .The term electroporation is well known among biolo-

gists not specialised in bioelectrochemistry, and there is acommon habit to use the terms «cell electroporation» and«cell electropermeabilization» synonymously. Obviouslythe former is related to one of the theories, while the latteris derived from the most commonly determined and quan-tified effect of cell exposure to electric pulses. Both termsthus highlight a particular aspect and, in view of the stillexisting uncertainties about the precise mechanisms, amore neutral term such as «strong electric field pulse-in-duced behaviour» of the exposed cells would be moreappropriate. However, this would be very clumsy, hencethe term electropermeabilization is retained here for rea-sons of convenience.

2.1.3. Mechanisms of molecular uptake into electroperme-abilized cells

In any case, after exposure of cells to appropriateelectric pulses, small hydrophilic molecules can cross theplasma membrane and enter the still living cell, essentiallyby simply diffusing through the electropermeabilized areasof the cell membrane. While electrophoretic transport of

Žcharged molecules can occur only during the pulses e.g..100 ms , diffusion and possibly convection can take place

Žduring all the lifetime of the permeabilized regions forexample during several minutes after exposures to eightreversibly permeabilizing pulses of 100 ms for cells incu-

.bated in vitro at 258C or for muscle fibres in vivo .Transport thus may occur for time periods which areorders of magnitude greater than pulse durations. More-over, a large molecule such as DNA is not yet inside the

w xcell at the end of the pulse 38 although the pulses usedŽare 200 times longer than those for small molecules see

.Section 4.2 , and an electrophoretic component in theinteraction of cell and DNA was actually demonstratedw x39 .

2.2. In ÕiÕo cell electropermeabilization

2.2.1. Methods of inÕestigationIn all the aspects, the knowledge on cell electroperme-

abilization in vivo is by far much more fragmentary andincomplete than the knowledge accumulated from in vitrostudies. The importance of electric field distribution foreffective in vivo electroporation of tissues has been high-

w xlighted 40,41 but many questions are still open, such asthe influence of the organisation of the cells inside thetissues. The situation could rapidly progress in the nearfuture because several complementary methods for study-ing cell electropermeabilization in vivo have been de-

w xscribed recently 42–45 . These methods could allow acomplete description of the main features of electroperme-

abilization of cells in tissues because of the various waysto approach the phenomenon:

v indirectly, e.g. in tumors, by the determination of thetoxicity of bleomycin or cisplatinum as a function ofexternal electric field strength, pulse length, number

w xof pulses, etc. 42,46 ;v directly, either by noninvasive approaches, e.g. by

118 w x w xmeans of In–bleomycin 44 or DTCA 45 and agamma-camera, or by invasive quantitative appro-

57 w x 51aches, e.g. using Co–bleomycin 42 or Cr–EDTAw x43 ;

v topologically, e.g. using very high doses of bleomycinw x47 .

It is important to note that the assessment of in vivo cellelectropermeabilization using these approaches is essen-tially independent of any theoretical model or mechanistichypothesis.

2.2.2. Electric field threshold for the achieÕement of cellelectropermeabilization in ÕiÕo

One of the parameters that has already been quantifiedin vivo is the electric field threshold value for some casesof cell electropermeabilization in tissues. When eightsquare-wave electric pulses of 100 ms are delivered at a

Ž .frequency of 1 Hz electrochemotherapy conditions , thethreshold value is between 300 and 500 Vrcm in tumorsw x w x42 , is very close to 450 Vrcm in skeletal muscle 43 and

w xis 360"20 Vrcm in liver 47 . It is important to note thatunder optimal conditions for DNA electrotransfer for gene

Ždelivery eight square-wave electric pulses of 20 ms deliv-.ered at 1 or 2 Hz , the threshold for skeletal muscle

permeabilization, using exactly the same set-up than forthreshold determination under electrochemotherapy condi-

w xtions, is only 90 Vrcm 48 , far below the 450 Vrcmw xreported above 43 .

2.2.3. Electric field distribution in ÕiÕoThe electric field distribution in tissues is another point

w xthat it is still necessary to precise 40,41 . A two-dimen-sional model for the electric field distribution in the skele-

w xtal muscle was published in 1999 43 while three-dimen-w xsional models are also becoming available 47 . In fact

these models describe the «initial situation», i.e. the elec-tric field distribution just at the beginning of the pulsewhen the pulse itself has not yet begun to change thebiological and physicochemical situation of the unexposedtissue. With these models, in an ulterior step, it will bepossible to analyse the factors influencing the electric fielddistribution in vivo.

2.2.4. Other in ÕiÕo effects of permeabilizing electricpulses

Almost nothing is yet known about other potentialeffects of the electric pulses delivered in vivo to various


tissues, except that too intense electric pulses can irre-w xversibly damage the tissues 49,50 . Obviously, therapeuti-

cal applications imply that deleterious electric pulses shouldbe avoided. In the future, it will then be necessary todescribe, quantify and evaluate the beneficial or detrimen-tal inputs of other effects of the electric pulses. In particu-lar, it appears more and more clearly that electric pulsestransiently impair the blood flow in the volume of tissue

w xexposed to the electric pulses 51–53 . The causes of thisperturbation, as well as its consequences, are not yetprecisely known.

3. Electrochemotherapy

3.1. Definition

Electrochemotherapy can be defined as the local poten-tiation, by means of permeabilizing electric pulses, of theantitumor activity of a nonpermeant anticancer drug pos-sessing a high intrinsic cytotoxicity.

[ ]3.2. Principal aspects 54

3.2.1. AchieÕement of in ÕiÕo cell electropermeabilizationOf course this is the essential basis of the treatment. As

discussed in the Introduction, knowledge in cell electroper-meabilization in vivo should make important progress in anear future. Electropermeabilization can be achieved using

Ž .either invasive electrodes e.g. needles or surface elec-Ž . Žtrodes metallic plates . In the latter case use of transcuta-

.neous pulses , pulse duration is in fact sufficient to disruptthe complex electrical barrier constituted by the stratumcorneum. Once the skin electrical resistance is reduced to avalue of tens of ohms, the situation is not very differentfrom that with intratissular electrodes, even though thedrop of voltage at the level of the skin requires the

w xapplication of higher voltages 19,42 .

3.2.2. Nonpermeant drugs possessing high intrinsic cyto-toxicity

This point was anticipated because of the biologicalw xproperties of bleomycin described by our team 55 . It is

important to note that, in spite of several attempts to findmore appropriate cytotoxic molecules, bleomycin is stillthe most adequate candidate for the combined use withelectric pulses because of its very high intrinsic cytotoxic-ity.

3.2.3. Participation of the host immune responseVery soon after the initial preclinical trials on elec-

w xtrochemotherapy 56,57 , it was shown that the host im-mune response was participating in the achievement of the

w xcures after electrochemotherapy 58 . The immunothera-py-mediated enhancement of the local effects of the elec-

trochemotherapy was also accompanied by the achieve-Ž .ment of systemic antitumor effects see Section 3.5.3 .

3.2.4. Vascular effects of the electric pulsesSeveral reports mention the fact that blood flow changes

w xoccur after tissue electropulsation in vivo 51–53 . Thesemodifications in blood flow could be advantageous forintratumoral drug injection, as this would decrease drugwashout. The impact on the systemic route for bleomycinadministration has to be analysed, too.

3.3. Recent clinical trials

The initial clinical trials were performed from 1991 onw xat the Institut Gustave-Roussy 59–61 and the technique

Ž . Ž .soon spread to Toulouse France , Ljubljana Slovenia ,Ž . Ž . w xTampa Florida, USA , and Reims France 62 . Results

of recent clinical trials were published since 1998.

( ) [ ]3.3.1. Basal cell carcinomas Tampa, USA 63The results are quite exciting because 99% of the

nodules treated have been in complete regression for morethan three years now. It is therefore possible that cureŽ .complete regression for a period longer than 5 years willbe achieved in most of the nodules treated.

(3.3.2. Oral caÕity squamous cell carcinomas Chicago,) [ ]USA 64

ŽAmong the 10 tumors treated, the five T2 nodules size.less or equal to 4 cm in diameter went into complete

regression, and for the five larger lesions treated, twopartial regressions were also obtained.

3.3.3. Metastases of bladder transitional cell carcinomas( ) [ ]Yamagata, Japan 65

The authors reported the treatment of a patient sufferingof 17 painful metastases on the skull. After the treatment,14 totally disappeared, and three others dried, becamepainless, and ulterior biopsies demonstrated the absence ofliving cells in the remnant of the treated nodules.

(3.3.4. Malignant melanomas, using cisplatinum Ljubljana,) [ ]SloÕenia 66

This work is very interesting because it demonstratesthat electrochemotherapy can also be performed usingcisplatinum instead of bleomycin. Of course, because theincrease in cisplatinum efficacy is much lower than that ofbleomycin, cisplatinum must be given at doses that arealready active and that lead to the usual side effects of thisdrug. Therefore, the operational approach consists in sup-plementing the treatment of the largest nodules with thelocal delivery of the electric pulses during the course of aregular cisplatinum infusion for the treatment, for example,of malignant melanomas. With respect to control nodulesnot exposed to the electric pulses, the electrochemother-apy-treated nodules presented larger index of response tothe antitumor cisplatinum infusion. Cisplatinum can also


be delivered by intratumoral injection: this administrationroute has also been used in electrochemotherapy, withinteresting results. A last improvement in the efficiency ofelectrochemotherapy with cisplatinum has been reportedrecently in a few patients through the combination of this

w xtreatment with radiotherapy 67 .

3.4. Open questions and additional aspects

In spite of the increasing number of preclinical andŽ .clinical trials that have been recently published see above ,

several questions are still open.

3.4.1. Intratumoral Õs. intraÕenous injection of thebleomycin

For example, two different ways to administratebleomycin have been explored in preclinical and clinical

Ž wtrials, namely the systemic essentially intravenous 51,58–x w x61,65,68 , but also intraperitoneal 69 and, at least in two

w x.cases of patients, intra-arterial 61 delivery and the localŽ . w xintratumoral delivery 63,64,70–73 .

The amounts of bleomycin required for efficient antitu-mor results in the presence of the electric pulses deliveryare low enough as to be injected systemically without theneed to take safety procedures in case of eventual majorside effects. The only precaution may be the administrationof an anti-histaminic drug to prevent the light febrileallergic reactions that can occur 2 h after the bolus admin-istration of the bleomycin. The advantage of the systemicinjection lies in the possibility to treat several nodules at atime, or large nodules, within the therapeutical window

Ž w xdetermined in humans from 8 to 28 min 61 after theintravenous bolus injection of 22 500 IUrm2 of body

2 .surface, that is 15 mgrm . It is important to recall thatthe growing parts of the tumors are well vascularised.Therefore, the parts of the tumors that must be treated arethose that will be easiest reached by bleomycin, assuringelectrochemotherapy efficacy. Nevertheless, it is also im-portant to note that bleomycin must be administered in abolus and not in a long perfusion, because it is necessaryto reach a minimal concentration in the tissue.

Obviously, the intratumoral injection of bleomycin re-quires less bleomycin on a per nodule basis. The recom-mended amounts of bleomycin are 1000 IUrcm3 of tumorŽ 3 3.that is 1 Urcm , or 0.66 mgrcm . Therefore, totalinjected amount depends on nodule volume, and on thenumber of nodules to be treated. Thus, it is also obviousthat for the treatment of large tumor masses or of a largenumber of nodules, physicians or surgeons lose the advan-tage of a reduction in the dose with respect to the systemicinjection. On the contrary, the need to inject each noduleseparately makes the procedure longer, and also less securebecause everybody who has tried to inject a product inlarge tumors knows how difficult it is to reliably perfuse

Žthe whole mass personal experience shared with many. Žcolleagues . Moreover, in some cases e.g. pancreatic car-

.cinomas , direct injection into the tumor mass is almostimpossible.

ŽTherefore, in the absence of comparative studies not.yet performed , the present recommendation would be the

use of intratumoral bleomycin delivery in the case of alimited number of small nodules and the use of systemicbleomycin delivery in other situations.

3.4.2. Effects on the marginsMargins of the tumors are the normal tissues located

just around the tumor nodule: they are often alreadyinfiltrated by a few tumor cells that can originate diseaserelapse after an unpredictable period of time. Because ofthe aggressivity of classical tumor treatments, margins areusually not extensively treated. In electrochemotherapyusing low systemic doses of bleomycin, there is an inter-esting difference in the biological response in mice be-

Ž .tween an actively growing tissue tumors and a normalŽ . Ž .tissue liver Mekid and Mir, unpublished results . Per-

meabilizing electric pulses were delivered after the ad-ministration of therapeutic doses of bleomycin using anintravenous injection in order to have a more or lesshomogeneous distribution of low concentrations ofbleomycin in the tissues. This results in a low uptake ofbleomycin molecules per cell. Under these conditions, theDNA double strand breaks generated by the bleomycin canonly kill the cells that will try to divide because theirmitosis will not be properly resolved. The cells that willnot divide will not be affected by the presence of a fewdouble strand breaks in their genome. Due to this, under

Žthese conditions that may or may not apply to the case of.the intratumoral bleomycin delivery , differential effects

are obtained between tumor and normal tissues. Therefore,these observations open the possibility of a safe treatmentof large margins around the treated nodules. This topic stillrequires further investigations.

3.4.3. Actual dosages requiredDose–effect studies in patients with intratumoral or

systemic administration of bleomycin were not yet per-formed. The dosage for the intratumoral injection was

w xextrapolated from animal studies 70,71 . The doses usedin the clinical trials were efficient and devoid of sideeffects because the administered amounts were very re-

w xduced 63,72 except for the treatment of large tumoralw xmasses 64 . Even in this last case no side effect due to

bleomycin was observed.Usually, bleomycin is delivered to patients for 5 con-

secutive days during routine treatments. The dosage for acomplete session of electrochemotherapy with systemicbleomycin administration comprises the amount ofbleomycin corresponding to one daily dose in the commontreatment. Therefore, the total amounts of bleomycin usedin electrochemotherapy already represent a decrease by afactor of five with respect to those used in commonlyaccepted dosages, which are known to cause no side


Žeffects except for the lung fibrosis that may appear after.five consecutive treatments of 5 days with bleomycin .

Again, the dosage used in the first clinical trials wasw xeffective and devoid of side effects 59–61 . Therefore, it

was not necessary to make escalating dose trials.However, it would be important to know if lower

dosages could still be efficient. Such de-escalating trialsŽi.e. trials in which patients receive lower and lower drug

.doses are ethically unacceptable because, if a given doseis known to be safe and effective, patient’s life cannot beendangered by the administration of a lower dose theefficacy of which is not known. Therefore, it will bedifficult to solve this issue.

3.4.4. Efficacy in patients who are not beyond the possibil-ity of treatment by conÕentional therapies

For ethical reasons, all the trials performed until nowhave involved patients that were beyond the possibility oftreatment by conventional therapies. One exception werethe trials dealing with patients affected by basal cellcarcinomas. This is a non-aggressive pathology that is welltreated by surgery. However, patients often suffer from

Žrepeated primary lesions in the skin, in particular in theareas of the body exposed to external aggressions, essen-

.tially the face and the neck . Since surgery is often mutilat-ing and its consequences very visible, a patient may refusethe surgical treatment. Thus, these patients could be en-rolled in the clinical trials of electrochemotherapy. A veryimportant finding was that electrochemotherapy resulted in

Žan efficacy close to that of the surgery 98% of complete.regressions for at least 3 years, study in progress and

Žtotally preserved the tissues concerned ears, lips, neck,. w xetc. 72 .Nowadays, in order to perform demonstrative trials of

the real efficacy of electrochemotherapy in patients whoare not beyond the possibility of treatment by conventionaltherapies, it will be necessary to find other situationsethically acceptable, in which it will be possible to treatmore aggressive pathologies using electrochemotherapy asfirst line treatment.

3.5. Challenges of electrochemotherapy

3.5.1. Comparison with surgery: efficacy, treatment of themargins, organ preserÕation

A comparison of electrochemotherapy with surgery hasbeen presented above, for the treatment of basal cellcarcinomas. Other comparisons are performed at the pre-clinical level. The feasibility of electrochemotherapy as amodality of limb-preserving treatment for patients withsarcoma of the extremities has been recently demonstrated

w xin an animal model 73 . The present studies dealing withthe effects of the treatment on the normal nondividing

Ž .tissues see above are also important to open the possibil-ity of a safe treatment of very large margins that couldresult in a reduction of the recurrences occurring aftersurgery.

( )3.5.2. Comparison with brachytherapy curietherapyIn many cases, the indications of electrochemotherapy

can be the regular indications for brachytherapyŽ .curietherapy . This type of localised radiotherapy isachieved by depositing a radioactive gamma-rays emitter

Ž .source iridium wires close to the tumor mass for a periodof a few days. However, protection of the patients’ envi-ronment is necessary because of the long distance reachedby the emitted gamma-rays, and patients must be isolatedduring the treatment. In terms of accessibility, brachyther-apy and electrochemotherapy are comparable because in-stalling the guides for the iridium wires and inserting theelectrochemotherapy electrodes require similar accessibili-ties. However, inserting the electrodes takes considerablyless time than the long process of installing the guides.Moreover, the level of safety is also tremendously differentsince electrochemotherapy does not require the rules ofstrict isolation of the patient during the time of irradiationgenerated by the iridium wires. Therefore, electro-chemotherapy could probably advantageously replacebrachytherapy in many of its indications. Moreover, itshould be pointed out that eliminating yet another sourceof radioactive material from the health care system wouldhave the benefit of being «environment friendly» and

Žwould also reduce the costs acquisition and disposal of.radioactive materials .

3.5.3. Possibility of enhancement of the local effects, andachieÕement of systemic effects, by the combination ofelectrochemotherapy with immunotherapy

Immunotherapy is essentially based on the administra-Žtion of biological response modifiers BRM like cytokines,

.lymphokines, etc, in particular interleukin-2 in order toenhance the host’s immune responses. Several preclinicaltrials performed at the Institut Gustave-Roussy with a

w xmetastazing murine tumor model 74 , a non-metastazingw xmurine tumor model 75 , and a carcinoma model trans-

w xplanted in the liver of rabbits 51 have shown that thecombination of electrochemotherapy with immunotherapynot only results in an enhancement of the local effects butalso gives rise to systemic antitumor effects. Therefore,this combination is an attractive possibility which, how-ever, requires further experimental developments.

3.5.4. Transfer of progress in knowledge on cell electrop-ermeabilization into clinical use

Since knowledge about cell electropermeabilization isŽ .rapidly growing see before , it will be important to ascer-

tain a proper and rapid transfer of this progress to thephysicians and surgeons that will treat cancer patients. Inthis respect, new clinical trials have been recently acti-vated. One of them, at Herlev Hospital, Copenhagen,concerns the use of the combination of electrochemother-

Ž .apy with immunotherapy interleukin-2 in malignantmelanomas. This study has already shown an immediate

w xand lasting interruption of tumor bleeding 53 , an observa-


tion that was already described in patients by Kubota et al.w x65 .

4. In vivo DNA electrotransfer for gene therapy

As mentioned in the Introduction, electroporation isvery often used to transfect mammalian cells in vitro.Therefore, it was tempting to try the electrotransfection invivo.

4.1. Preclinical trials on mammalian tissues

Three steps can be distinguished. In 1991, Titomirov etw xal. 76 reported positive results of the transfer of plasmids

to skin cells in mice using exponentially decaying pulses,but transfer efficacy was difficult to quantify.

In 1996, a report was published which corresponds tothe second step in the development of this application of

w x Želectropermeabilization. Heller et al. 77 , using short 100.ms square-wave electric pulses, obtained good transfec-

tion levels in the liver cells treated in situ.The third step can be defined by the use of square-wave

electric pulses of a much longer duration. Several papersw xhave been published since 1998 78–85 . Experiments

w xwere performed on liver tissue again 78 , skeletal musclew x w x79–81,85 , and tumors 82–84 .

It is important to note that long trains of bipolar pulseshave also been used recently for the successful transfer of

w x w xreporter genes 86 or of therapeutic genes 87 . Shortsquare-wave pulses have also proved to be efficient for

w xtherapeutic gene transfer in tumors 88 .

4.2. DNA electrotransfer to the skeletal muscle

Because of the accessibility of skeletal muscles andtheir ability to secrete foreign gene products, and in viewof all the experiments with injections of naked DNA or ofcombinations of naked DNA with many kinds of chemicalvectors, skeletal muscles must be considered as one of themost interesting potential targets for DNA electrotransferin vivo.

ŽResults have shown that using long pulses e.g. 20 msw x Ž . w x.80,81 or more 50 ms 79 , high levels of DNA electro-transfer can be achieved using electric fields of moderate

Ž w x w x.intensity 200 80,81 or 250 Vrcm 79 . In comparison tothe expression of genes transferred by the simple injectionof the naked DNA into skeletal muscles, the main resultsobtained by the delivery of these electric pulses after DNAinjection in the muscle are an increase in the expression of

Ž .the electrotransferred gene over 200 times and a largedecrease in the variability of the electrotransferred gene.Moreover, a sustained expression for more than 9 monthshas been obtained with the gene coding for the firefly

w x w xluciferase 80 . The analysis of the mechanisms 38,81shows that DNA must be injected prior to the delivery of

the electric pulses. This strongly suggests that a mecha-nism of electrophoretically induced increase of contact ofthe DNA with the membrane andror transport across it ismost likely responsible for the high efficacy of DNA

w xuptake by muscle cells in vivo 38,81,82 . This is inw xagreement with previous results in vitro 89 .

4.3. Potentialities of DNA electrotransfer for gene therapy

The results obtained, whatever the type of tissue inves-Ž .tigated skeletal muscle, liver, tumors , show that DNA

electrotransfer possesses great potentialities for use in genetherapy. One of its main advantages, besides its efficacy

Žand its reproducibility as detailed in the case of the.skeletal muscle , is that DNA electrotransfer is an easy

procedure which uses naked DNA easy to prepare. Forexample, this means that, for research purposes, the influ-ence of mutations in gene expression in vivo can beanalysed very rapidly after the modification of the DNAsequences because these sequences do not need to beinserted into particular vectors. The obstacles that remain

Žto be solved are the accessibility of the electrodes as for.the electrochemotherapy , the still insufficient level of

Žknowledge on in situ electropermeabilization see previous.discussion , and the missing detailed evaluation of theŽ . Ž .sensations pain in humans when long pulses 20 ms will

be used. This is reflected by the present situation of theclinical trials.

4.4. Clinical trials

Direct delivery of electric pulses in situ for DNAelectrotransfer for gene therapy has not yet been performedbecause of the novelty of the approach and because of thepresent lack of studies dealing with the safety of theprocedure and the pain associated with it. Among 252clinical trials of gene therapy for cancer treatment, elec-troporation was used in only two cases, and in these twotrials electroporation was used ex vivo, i.e. on cellsremoved from the patients and exposed to the electric

Žpulses outside the body for further details consult thewebsite http:rrwww.wiley.co.ukrgenetherapyrclinicalr

.DATABASE . As of June 2, 2000, no trial using DNAelectrotransfer for gene therapy of monogenic diseases,infectious diseases, on healthy volunteers, for gene mark-ing or other purposes was reported among the 144 clinicaltrials listed in the same specialised databank. However,with the recent results on preclinical trials, it is possiblethat these numbers will change in a near future.

5. Conclusions: therapeutic perspectives of the use of invivo electropermeabilization

For the electrochemotherapy, it can be concluded thatthe new series of clinical trials that have been recently


activated will allow to show the large potentialities of themethod, or its limitations. In the latter case, there isnevertheless a large place for the electrochemotherapy in

Ž .the treatment of small lesions e.g. basal cell carcinomaand as a palliative treatment that will not impair patient’sstatus. In the former case, it is still too early to describe thefull range of potential applications of the method.

Moreover, electrochemotherapy with bleomycin mayfind in the future an extension through the use of veryactive oligonucleotides directed to specific DNA se-quences responsible for specific diseases because they aremutated or because they have lost their regulated expres-sion.

For DNA electrotransfer, one should not forget that alarge range of other vectors for DNA introduction into thecells have been prepared and are still under investigation.This includes viral vectors, like adenoviruses, retroviruses,the adenovirus associated virus, etc. It also includes non-viral vectors, essentially chemical vectors like liposomes,cationic lipids, etc. or physical vectors like the ballisticŽ .gene gun , the stress waves, the heat pulses or the ultra-sounds. However, because the electric pulses act simulta-neously on the targeted cell and on the molecule to bevectorised, it is possible to expect that in the future thebioelectrochemical vectorisation resulting from the use ofappropriate electric pulses will have an important placeamong the methods of DNA delivery for gene therapy.

Acknowledgements

The author thanks Tadej Kotnik for reading themanuscript and his criticism.

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Therapeutic perspectives of in vivo cell electropermeabilization