©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Human Vaccines & Immunotherapeutics 1
Human Vaccines & Immunotherapeutics 8:11, 1–9; November 2012; © 2012 Landes Bioscience
For nearly 20 years there has been a focus on using DNA as a tool
for creating new efﬁcient vaccines.1 The idea of a DNA vaccine is
in fact very simple: inject a plasmid encoding a relevant antigen
into e.g., a muscle, ensure that the plasmid is transcribed and the
protein/antigen is produced, with the initiation of an immune
response. DNA vaccines could be, if working as effectively in the
real world as in theory and preclinical studies, an efﬁcient and
novel opportunity to prevent infectious diseases.
The ease by with it is possible to produce GMP-DNA, the
possibility of multi-potent vaccines, the high level of safety due to
absence of viral elements in the manufacturing and the stability
of the DNA molecule making a ‘cold-chain’ superﬂuous, makes
it well worth exploring the effect of DNA vaccines.
On a global economy perspective DNA technology may enable
cheaper vaccines, and thus greater availability and higher likeli-
hood for successful vaccination programs. Different approaches
for optimizing the immunological response after a DNA vaccina-
tion are under investigation, covering aspects such as conjugates
or molecular adjuvants, often in combination with exploitation of
physical methods to improve the cellular uptake of the plasmid.2
A well-known physical technique is electroporation, which is a
*Correspondence to: Julie Gehl; Email: Julie.firstname.lastname@example.org
Submitted: 06/12/12; Revised: 08/24/12; Accepted: 09/02/12
Vaccinations are increasingly used to ght infectious disease,
and DNA vaccines oer considerable advantages, including
broader possibilities for vaccination and lack of need for cold
storage. It has been amply demonstrated, that electroporation
augments uptake of DNA in both skin and muscle, and it is
foreseen that future DNA vaccination may to a large extent
be coupled with and dependent upon electroporation based
delivery. Understanding the basic science of electroporation
and exploiting knowledge obtained on optimization of DNA
electrotransfe r to muscle and skin, may greatly augment eorts
on vaccine development. The purpose of this review is to give
a succinct but comprehensive overview of electroporation as a
delivery modality including electrotransfer to skin and muscle.
As well, this review will speculate and discuss future uses for
this powerful electrotransfer technology.
What you always needed to know about
electroporation based DNA vaccines
Anita Gothelf and Julie Gehl*
Center for Experimental Drug and Gene Electrotransfer (C*EDGE); Department of Oncology; Copenhagen University Hospital Herlev; Herlev, Denmark
Keywords: electroporation, gene transfection, gene electrotransfer, DNA vaccine, gene therapy, skin, muscle
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
non-viral means for transferring genes and other non-permeant
molecules across the cell membrane.
Studies with electroporation assisted DNA vaccinations have
shown that it is possible to obtain both a cellular and a humoral
immunological response, and in the preclinical setting the tech-
nique has shown promising results.3 However, the effect in large
animals and humans may still be improved.4 The aim of this
paper is to look at electroporation based DNA vaccines from a
practical point of view. We have considerable experience in using
electroporation, both in preclinical studies5-7 and in the clinical
setting8,9 (gene transfection to various tissues and electrochemo-
therapy for treatment of cutaneous metastases) and hope that the
knowledge we have achieved can be of use to researchers working
with electroporation for DNA vaccinations.
Electroporation: Basic Concepts
The principle behind electroporation is strikingly simple; by
applying an electric ﬁeld that surpasses the electrical capacitance
of the cell membrane, cells may be rendered transiently permea-
bilized due to entrance of water into the membrane and forma-
tion of hydrophilic permeation structures.10,11
Figure 1 shows how the cell membrane responds to an exter-
nal electric ﬁeld, and also how electrotransfer of drugs and genes
work differently.12 For drugs, and other small molecules, simple
diffusion happens through the cell membrane after permeabili-
zation. Diffusion will take place as long as the cell membrane is
permeabilized, i.e., also after the pulses have been given. On the
contrary, DNA is too large to enter through the hydrophilic pores
by simple diffusion. DNA (and other nucleotides) are polyan-
ions, with a plethora of negative charges, enabling the molecule
to move in an electric ﬁeld.13 However, moving DNA is not sufﬁ-
cient; the cell membrane also needs to be in a permeabilized state
in order to allow passage of the DNA molecule.
Pulses may be optimized to achieve either a greater degree
of permeability of the cell membrane (for passive diffusion of
drugs), or a greater degree of electrophoretic effect. As mentioned
below, there are various ways to go about this, but generally a
series of short high voltage pulses (e.g., 8 pulses of 0.1 ms at 1,000
V/cm voltage to electrode distance) is used for drug delivery,14
and a combination involving long low voltage pulses is used for
DNA transfer (e.g., 1 pulse of 0.1 ms, 800 V/cm and 1 long pulse
of 400 ms 80 V/cm).15
©2012 Landes Bioscience. Do not distribute.
2 Human Vaccines & Immunotherapeutics Volume 8 Issue 11
numerous ways; the frequency, the amplitude, the duration—
and then different pulse forms may be combined. Below some
examples are mentioned for gene electrotransfer to respectively
skin and muscle—the primary target tissues for DNA vaccina-
tions. Furthermore, electrode geometry greatly inﬂuences the
actual electric ﬁeld and local factors in the tissue may alter ﬁeld
distribution, e.g., the presence of stratum corneum in the skin,
muscle ﬁber direction.13,25
Electroporation nomenclature. As in all scientiﬁc ﬁelds,
nomenclature evolves with the development of the ﬁeld. Here are
some deﬁnitions commonly used.
Electroporation or electropermeabilization; these terms are
used interchangeably. From a scientiﬁc viewpoint electroperme-
abilization may be more correct, since what has been scientiﬁ-
cally documented is the permeabilized state, whereas the term
pore may lead the mind into thinking of more formal pore-like
Electrotransfer: Describes the movement of molecules into
cells, by either passive diffusion or electrophoresis, made possible
by the use of electric pulses. From this term we derive DNA elec-
trotransfer, RNA electrotransfer, PNA electrotransfer, etc.
Electrochemotherapy (ECT): The use of electroporation to
enhance the uptake of chemotherapy in a tumor.
Gene electrotransfer in general. Gene electrotransfer or electro-
poration assisted gene transfection is the combination of electric
pulses with injection of a gene, often naked plasmid DNA. The
response of the gene transfection is dependent on the plasmid
injected and the purpose of the treatment; hence two main pur-
poses for gene electrotransfer exist namely gene therapy and
In gene therapy the aim of gene transfection is to render a tis-
sue, e.g., muscle, skin, or tumor, capable of producing a speciﬁc
The permeabilization structures will start to form in a mat-
ter of microseconds during the ﬁrst pulse, and will reseal in the
order of minutes after the pulses have ended.16-20 Drugs may dif-
fuse into cells as long as the permeabilized state exists, and may
therefore also be added just after pulsation. However, DNA must
be added prior to the electric pulse, in order to be subjected to
the electrophoretic effect necessary to transfer the DNA across
the cell membrane; indeed it has been shown that when DNA
is added after the pulses, but while the cell is still permeabilized,
there is no transfection.21
Other nucleotides, which are similar to DNA (e.g., RNA),
can be optimally transferred using similar pulsing sequences. For
different nucleotides, e.g., oligonucleotides such as PNA (peptide
nucleic acid) or LNA (locked nucleic acid), actually the ‘drug
electrotransfer parameters’ may be more appropriate.22
Pulses in every form and shape. The classical laboratory elec-
troporator operates with exponentially decaying pulses.23 This
is the technologically least complicated, and allows for use as a
reasonable priced and reasonable sized laboratory electroporator.
In the exponentially decaying pulse, pulse duration will increase
when amplitude is increased. This offers fewer possibilities for
optimization – on the other hand for transfection of E-coli, it
is the one transfected clone that matters, not the survival of the
For use in mammalian cell cultures, and in vivo work, it is a
different story. Here, high cell viability together with good trans-
fection rates are in demand. In square wave pulse generators, it is
possible to independently control pulse amplitude and duration,
enabling much better optimization. Finally, for electroporation
in the clinical setting, a number of regulatory standards need to
be met for the equipment.23
The electric pulses may be voltage clamped,23 where the cur-
rent will vary, or current clamped with varying pulse amplitude
or duration.24 The number of possibilities for pulse combinations
is really inﬁnitely large. The number of pulses may be varied in
Figure 1. The gure in the left panel depicts a cell, upon which an electric eld is exerted. As the cell interior is negative, the electric force will be
largest upon the pole of the cell facing the positive electrode, and, therefore, dielec tric breakdown will rst, and foremostly, occur here. A smaller
window, but with more extensive permeabilization will form on the pole facing the negative electrode. In the center panel, a cell permeabilised in the
presence of a small uorescent molecule is shown (from B. Gabriel, CNRS, Toulose, as also described in ref. 12). DNA transfer appears to work dierently
from transfer of small molecules. Thus, the DNA is too large to penetrate through small permeabilisations. Instead, the DNA may be ‘pushed’ through
the membrane already rendered permeable, by electrophoretic forces exerted by the eld. As DNA (and most other nucleotides) is a polyanion, with a
massive presence of negative charges, the electrical forces relative to the molecule size means that DNA may be moved in the eld. In the right panel,
it is seen how uorescently labeled DNA enters the cell on the pole of the cell facing the negative electrode (being attracted toward the positive elec-
trode, and repulsed from the negative electrode) from M. Golzio at CNRS Toulouse, also described in ref. 50.
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Human Vaccines & Immunotherapeutics 3
duration. That makes sense since the purpose for using electro-
poration for transfection with DNA, both for vaccination and for
production of therapeutic proteins, is that the transfected cells
must survive the procedure and restore their equilibrium after the
delivery of the electrical pulses. Crudely, the pulse parameters are
divided in high voltage pulses (HV) exceeding the 400 V/cm and
with a duration in the μs range and the low voltage pulse (LV)
below 400 V/cm with a duration in the millisecond range. The
distinction between the HV and LV is not exact, but arbitrary,
and deﬁnitions may vary.
Figure 2 shows that many pulse combinations have been used
for gene transfection. Some authors have succeeded in achiev-
ing gene expression after delivery of HV pulses only,29 however
there is a general acceptance that there has to be an element of
LV pulses in order to attract the DNA to and through the cell
A convenient aspect of electroporation is that the technique
itself is not immunogenic so it is thus possible to treat the same
animal or patient several times.31 Furthermore each spot of gene
transfection is small and conﬁned to the size of the electrodes and
the procedure can if necessary be repeated on several locations at
the same session.
Gene electrotransfer is a well-tolerated treatment. It is per-
formed very quickly, and the amount of discomfort is tolerable.
Minor adverse effects are mostly related to injection of the plas-
mid and insertion of the electrodes, which can be performed
in combination with local anesthesia if necessary as well as the
protein, which is encoded by the plas-
mid. This can either be a relevant
protein the body needs, e.g., due to a
protein deﬁciency disorder or it can
be a therapeutic compound with e.g.,
antineoplastic effect on cancer cells.26
Normal tissues such as muscle and skin
are frequently investigated for produc-
tion of therapeutic proteins with local or
systemic effect after gene electrotransfer,
but direct transfection of tumors with
plasmids encoding antineopolastic mol-
ecules is a possibility as well.
Development of DNA vaccines
represents the other therapeutic pos-
sibility of gene electrotransfer, where
electroporation acts as an effective tool
for transfecting plasmids encoding
antigens against speciﬁc epitopes and
thus enhancing an immune response.3
DNA vaccines can furthermore be
divided into two groups: (1) prophy-
lactic vaccines, which serves at creating
an immune response against a known
infectious agent, and (2) therapeutic
vaccines, which aims at using the body’s
immune system to react adequately to a
tumor antigen e.g., in order to achieve
an anticancer effect.
In preclinical studies, rodents are the animals most often used
for gene transfection.26 The reasons are naturally the availability
and the price opposed to the cost of larger animals. A number of
studies have been performed with gene electrotransfer to various
tissues, but mainly to muscle, skin and tumor.27 There are how-
ever some issues, which must be taken into account concerning
animal model when it comes to muscle, skin or tumor transfec-
tion. These will be discussed in the following sections.
There exist many types of electrodes for gene transfection,
both to muscle and skin, but they can generally be divided in
two groups: the non-invasive, which consist of plates, patches
and wires that are placed on the skin or around the injected vol-
ume and the invasive electrodes, which consist of different needle
arrays that are inserted into the tissue. The plate electrodes are
primarily used in rodents and small animals, whereas the needle
electrodes often are used in larger animals, such as the pig, and
in clinical studies. There is, however constant research ongoing
in order to minimize the discomfort connected with both injec-
tion of plasmid and penetration of tissue with the needles. To
this end different types of patches and superﬁcial electrodes are
The electric pulses chosen for gene transfection are numerous
and different research groups have found their preferable electri-
cal parameters and electrode conﬁguration suitable for achieving
a desired response.26 As seen in Figure 2, the electrical parameters
look like a scatter plot when depicted on a graph, although there
is a tendency toward the higher ﬁeld strength the shorter pulse
Figure 2. Electric pulses for gene electrotransfer to skin. The graph depicts the dierent pulse
combinations that have been used for gene electrotransfer to skin. There is no consensus regarding
which electrical parameter is the most eective, but eciency may be correlated with electrode
type and electric eld distribution.
©2012 Landes Bioscience. Do not distribute.
4 Human Vaccines & Immunotherapeutics Volume 8 Issue 11
immunogenic themselves, attract immune active cells, which
can stimulate an immune response after e.g., transfection with
a DNA vaccine. The muscle is thus a frequently used organ for
clinical trials involving vaccination protocols.
An important issue regarding gene transfection to muscles is
the question of delivering the electric pulses properly. The muscle
consists of muscle ﬁbers running throughout the length of the
muscle and since the grade of permeabilization after electropora-
tion is dependent on the shape of the cells,34 there is a signiﬁcant
impact of the direction of the electric ﬁeld on the muscle ﬁbers.
The damage to the cells can be minimized if the electric ﬁeld is
perpendicular to the direction of the muscle ﬁbers.35
As opposed to the skin where there is a tendency toward the
higher dose of DNA transfected the higher expression, the muscle
cells only need few μg of plasmid DNA to create a systemic rel-
evant level of expression. There is on the contrary a risk of over-
dosing, since too high a dose of DNA seems to have a toxic effect
on the muscle cells.5,36
A disadvantage with gene transfection to the muscle is that
one has to overcome the barrier created by the skin in order to
permeabilize the muscle ﬁbers underneath. In the preclinical set-
ting the plate electrodes are often used in small animals and
the electric pulses are adjusted to reach a proper result, whereas
the needles have shown to be effective in larger animals and
in the clinical setting. The ﬁeld strength can consequently be
lower since the insulating properties of the skin then has been
Gene electrotransfer to skin. The skin is the largest organ in
the body and there are many future possibilities for gene therapy
delivered through that organ, summarized in Figure 3.37 Many
of these are being explored both in the preclinical and the clinical
setting. Due to the presence of antigen presenting cells, the skin
is an obvious target for DNA vaccinations and electroporation
is a safe and efﬁcient means for improving the antigen response.
Besides DNA vaccinations, the skin has the capability to pro-
duce cytokines and hormones, which can have a systemic effect,
and can thus be a target for gene therapy where the purpose is
protein production rather than immune reactions.6,38
The skin is divided in three layers, the epidermis, the dermis
and the subcutaneous layer and each of them contains different
cell types, which in theory can be targeted by either different
electrical parameters or cell speciﬁc promoters.39 This means
that it could be a possibility to obtain differentiated responses
after a gene transfection.
Compared with muscle cells, the skin is a more changeable
organ, where cell renewal is constantly occurring. The most
superﬁcial layer, the epidermis, consists mainly of keratinocytes,
which are created from stem cells on the basal layer but then
grow more and more superﬁcially and end up being ﬂat keratin-
rich, but empty membrane shells and create the corniﬁed layer.
It is thus envisioned that transfecting the keratinocytes may
result in a short-term expression of only few weeks compared
with the muscle, where an expression can be measured for sev-
eral months, Figure 4.5,6
In theory, a longer expression must be expected if other
cell types, such as ﬁbroblasts in the dermis, are transfected.
delivery of the electric pulses, which creates a short contraction of
the muscle treated (muscle transfection) or the muscle below (skin
and tumor treatment). The contraction is reported by patients to
be a discomfort; however, healthy volunteers have tended to have
no objections to repeated treatments.32
Furthermore, gene electrotransfer in combination with DNA
vaccines is a safe technique, since no viral components are neces-
sary to create the immunological response. Naked plasmid DNA
can easily be manufactured and distributed and the procedure is
in fact very simple and easy to perform. The equipment needed
is relatively inexpensive, and it can easily be transported and dis-
seminated around the world. Furthermore it has been shown that
it is possible, in a safe and non-toxic way, to turn off the expression
of a transfected gene after gene electrotransfer, if the same tissue is
re-electroporated in combination with local injection of calcium.33
It is important to create the best conditions for gene trans-
fection with any type of gene. The pulses must be delivered at
the exact spot, where the plasmid has been injected. The lack of
consistency and preciseness can be one of the reasons for a lack of
electroporation mediated enhancement of responses.
Gene electrotransfer to muscle. The organ most often used
for gene electrotransfer, apart from tumor, is the muscle.31 The
qualities the muscle cells possess render them an obvious target
for gene transfection. The muscles are under normal conditions
readily available and can be reached either through the skin or
directly with minimal surgical intervention, both in the pre-
clinical and the clinical setting. Muscle cells are post-mitotic
and thus capable of creating a long-term expression after a gene
transfection. The blood supply is abundant and they are indeed
adequate for producing proteins and releasing them to act sys-
temically. After gene transfection with the use of electroporation
it is thought that the electric pulses, even though they are not
Figure 3. Therapeutic groups for gene electrotransfer to skin. Gene
therapy to skin can be divided in three therapeutic groups: local treat-
ment, systemic treatment and DNA vaccination. The borders between
the groups are arbitrary and will depend on the transfected gene (from
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Human Vaccines & Immunotherapeutics 5
a plasmid encoding Katushka, a ﬂuorescent molecule in the far
red area, could be an option.45,4 6
Gene electrotransfer to tumors. Gene electrotransfer to
tumors are mostly aimed at the production of proteins with anti-
cancer effect such as e.g., the antiangiogenic plasmid AMEP47 or
transfection with immune-active products such as IL-12.48 The
differences, advantages and disadvantages between muscle and
skin for gene transfection, are summarized in Table 1.
Clinical trials with electroporation and DNA plasmids.
The ﬁrst clinical studies with gene electrotransfer to muscle and
tumor have been published and more are underway.48 The clini-
cal setting mimics the preclinical and particularly the muscle
is the object for DNA vaccinations. Different devices are used
for application of the electrical pulses, but mutual for them all
is the use of needle electrodes. The muscle is the organ most
often used for gene transfection and the choice of this organ for
clinical trials is based on robust data. The muscle is a natural
‘protein-factory’ and is able to produce a large amount of protein
after transfection with small amount of plasmid.5 The next few
years will clarify many difﬁculties regarding DNA vaccination
and help ﬁnding the optimal parameters for clinical trials. It
is still too early to state, whether the muscle or the skin is the
optimal organ for DNA vaccinations; it could be that a com-
bined approach will prove to be the most efﬁcient in eliciting
an immune response. Table 2 summarizes the clinical studies
currently running with DNA transfection and electroporation.
Unfortunately there is still no clear evi-
dence of which cells are in fact transfected
and thus responsible for the expression
seen after transfection. This is probably
due to the existence of many different elec-
troporation protocols and hence the tar-
geting of different cell types. If the focus
is protein production after gene therapy
the level and the duration of the expression
achieved is naturally important. However,
if the purpose is DNA vaccination instead
the concerns about level and duration of an
expression may not be so crucial as long as
the expected immune response is achieved.
The mouse is the animal most often
used in the preclinical studies of gene
electrotransfer to skin.26 However when
the skin is the focus of transfection, other
animals, particular the pig, are better
choices. There are two reasons for choos-
ing the porcine model over the mouse: (1)
Mouse skin contains many hair follicles
and that may in theory have an impact
on the expression after electroporation.
One reason for the success of transfect-
ing mice skin compared with human skin
could be the fact that cells in the hair fol-
licles are very suitable for gene transfec-
tion and hence production of the expected
response.40 (2) Porcine skin is more similar to human skin, both
in texture and composition. As in human skin, which only has
a subcutaneous muscle layer in the platysma under the chin and
in the male scrotum, the porcine skin does not contain the pan-
niculus carnosus, which is a subcutaneous muscle layer present
in many types of animal skin, including rodents. Furthermore
porcine skin is thicker and the risk of injecting the plasmid sub-
cutaneously is smaller.
In the preclinical setting the plasmids most often transfected
have been encoding reporter proteins such as luciferase, green
ﬂuorescent protein (GFP) and β-galactosidase. There are how-
ever drawbacks related to all three plasmids: (1) Luciferase is
easily measured both in vitro and in vivo but it displays a rather
high level of variance, often several logs, which makes it difﬁ-
cult to compare different studies.41 (2) GFP is a ﬂuorescent mol-
ecule in the green spectrum, but since the skin itself contains a
signiﬁcant level of autoﬂuorescence, it can be very difﬁcult to
distinguish between transfected cells and background ﬂuores-
cence.25,42 (3) Beta-galactosidase is an enzyme, which converts a
substrate, X-gal, to a blue color. There have been reports of false
positive staining in porcine skin,25 bone43 and neural tissue,44
which must have an impact on interpreting the results.
The drawbacks of GFP and β-galactosidase are to our knowl-
edge the main reasons for the lack of certainty about which cells
are responsible for the expression after gene transfection to skin.
Other new markers have to be developed, and studies using e.g.,
Figure 4. Duration of expression after gene electrotransfer to skin. Repor ter plasmids encoding
luciferase are often used in gene transfection studies. In this graph the duration of luciferase
expression is compared with transfection of two other compounds, pTagFP-635 encoding a
red uorescent protein, Katushka and a plasmid encoding the hormone erythropoietin, EPO.
It is seen that the duration of expression peaks after two days (luciferase), nine days (Katushka)
and two weeks (EPO). This is short compared with expression in muscles, which can be several
months (modied from ref. 45).
©2012 Landes Bioscience. Do not distribute.
6 Human Vaccines & Immunotherapeutics Volume 8 Issue 11
Table1. Comparison of gene electrotransfer to muscle and skin, from a clinical point of view
Accessibility Accessible, but invasive techniques may be necessary in order to secure
future localization of the injection site with e.g., sutures Accessible
Cell types Mainly muscle fibers, which are effective in producing large amounts
Contains many different cell types, including
antigen presenting cells
Cell turn-over Muscle fibers are terminally differentiated and
Rapid cell turn-over, particularly of the
Injection technique Intramuscular dif fusion of plasmid from the injection site, particularly
along the length of the fibers
The injected volume stays intradermally for
Injection volume Dependent on muscle size, but limited by the diffusion
Max 100 µl per injection, if larger amount is
injected there is risk of pain and oozing of
plasmid back through the needle hole
Electrodes Needle electrodes Preferably needles, but plates can be used
Electrical parameters Lower field strength compared with skin Higher field strength compared with
Duration of expression Long (months) Short (weeks)
Level of expression,
measured in protein Few µg DNA can produce a high level of expression
Much more DNA is required to achieve the
same level of expression (e.g., 20 times more
or even higher)
Transfected muscle tissue can be removed provided the exact
localization of the area is known. Calcium electroporation may also be
used to terminate gene expression (see text)
Transfected skin is easily removed
Table2. Clinical trials using electroporation and gene transfection
Tissue Gene or vaccine Patients Study
Skin CEA Colorectal cancer Phase 1
Influenza virus Healthy adults Phase 1
Muscle Avian influenza virus Healthy adults Phase 1
Hemorrhagic fever Healthy adults Phase 1
Hepatitis C virus HCV infected adults Phase 2
HIV HIV-1 infected adults Phase 1
Healthy adults Phase 1
HIV and IL-12 plasmid HIV-1 infected adults Phase 1
Healthy adults Phase 1
Human papilloma virus CIN 2 and 3 Phase 1
Leukemia CML and AML Phase 2
Malaria (Plasmodium Falciparum) Healthy adults Phase 1
Misc, DNA vaccines Malignant melanoma Phase 1
Tum or AMEP plasmid Malignant melanoma Phase 1
IL-2 plasmid Malignant melanoma Phase 1
IL-12 plasmid Merkel cell cancer Phase 1
Malignant melanoma Phase 2
The purpose of this table is to supply an overview of the types of diseases and gene targets that currently are being explored. The website w ww.
clinicaltrials.gov was searched for the terms “electroporation” and studies involving gene transfection, either active-not recruiting, active, recruiting or
completed and these were included in the list provided above.
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Human Vaccines & Immunotherapeutics 7
must encompass the area of the injected plasmid, (2) a proper
contact between electrodes and skin must be secured and (3) a
homogenous electric ﬁeld distribution, which ensures that all the
cells in the intended area get reversibly electroporated with a low
risk of induction of cell death.
The pulse parameters. The pulse parameters are closely
related to the electrodes. There is, however, a wide range of pos-
sible pulse parameters which can be efﬁcient for gene transfec-
tion. For DNA vaccination the main purpose is to transfect the
cell and secure that the cell membrane is able to restore the equi-
librium afterwards and the electric pulse parameters must hence
Level of expression and evaluation of the response. With
respect to protein production it is crucial that the level of circulat-
ing protein is high enough to have the intended effect. Quite dif-
ferent is the situation with DNA vaccination. As long as a certain
threshold for provoking an immune response has been achieved,
there is no need for further production of expressed proteins/anti-
gens. One question is; for how long time is the expression needed
to last before a relevant immune reaction has been reached. Also
can there be differences between the muscle and skin both in
terms of cells transfected (e.g., antigen presenting cells) and
The gene electrotransfer procedure con-
sists of several steps, each of which can
result in issues that may decrease the
efﬁciency of the method (summarized in
Figure 5). The steps of the procedure can
be divided in the following parts:
Preparation of the plasmid/gene.
Present laboratory facilities and the capa-
bility to produce GMP DNA for clinical
use secure a high level of consistence and
stability in the production of the actual
gene. It should thus be possible to pro-
duce uniform batches and minimize the
risk of inter-batch-variability.
Injection of DNA. DNA is a highly
viscous and hydrophilic molecule and to
obtain an efﬁcient expression it must be
injected prior to the electric pulse. For
gene transfection different issues exist in
different organs. For gene electrotransfer
to the skin, in rodents or where the skin
is particularly thin, the injection must be
performed with utmost care in order to
prevent subcutaneous leakage. However,
if the injection is made properly and is
located in the skin, it is visible as an intra-
dermal bleb and stays there for several
In the muscle conditions are quite dif-
ferent. The muscle consists of muscle cells
or ﬁbers situated inside the muscle fascia.
Once a liquid or DNA is injected into the muscle it has the pos-
sibility of dispersing in the muscle by the length of the ﬁbers. In
small animals such as mice it does not have a signiﬁcant impact
on the result, since the muscles are small and can be encompassed
by the electrodes. In larger animals and humans the conditions
are different with the muscle ﬁbers being much longer. This is
why it is suggested that the electrodes are inserted into the tissue
initially, followed by injection of the plasmid with the subsequent
delivery of the electric pulses.31
There are several commercial products available where the
substance, e.g., the plasmid for gene transfection, is transferred
into the organ by air or a jet stream.
Injection of a plasmid into e.g., a cutaneous or subcutaneous
tumor may present other challenges. Some tumors have a soft
structure while others are very hard and difﬁcult to penetrate.
The important factor is to use the correct type of needle suit-
able for the tumor in question and not to inject with too much
force, since the plasmid can leak out instead of being inside the
Electrodes for delivery of the electric pulses. As mentioned
previously the electrodes for electroporation consist of differ-
ent types. However three important issues must be taken into
account in order to get optimal transfection: (1) The electric ﬁeld
Figure 5. The importance of consistency in the gene elec trotransfer procedure. Gene electrotrans-
fer is simple and easy to perform, but is in many aspects a complicated process with many interac-
tions. There are thus many aspects, which need to be evaluated when electroporative delivery fails
to induce a response.
©2012 Landes Bioscience. Do not distribute.
8 Human Vaccines & Immunotherapeutics Volume 8 Issue 11
same time it is a reasonable assumption that further optimiza-
tion of DNA injection, pulse conﬁguration and electrode geom-
etry may further improve efﬁcacy and at the same time decrease
va ri a bility.
Looking at vaccination coverage on a global scale, there is
still much to be done in order to prevent preventable infectious
diseases. In addition, it was recently estimated that one in six
cancers worldwide is caused by preventable infectious disease,
principally of viral etiology such as hepatitis B and C as well as
human papilloma virus.49
Electroporation delivered DNA vaccines may offer the pos-
sibility to vaccinate with multiple epitopes, while being relatively
cost-effective and not requiring a cold chain. This may, in effect,
suggest that one of the most economical and effective prophy-
lactic and therapeutic weapons against a number of diseases may
be DNA-based vaccines and therapeutics that are administered
through delivery enhancement technologies.
duration of expression? Future studies will hopefully provide
information on these important questions.
In the preclinical setting differences often exist in how expres-
sion is evaluated, whether in terms of protein production or in
terms of measuring an immune response. As a consequence it
can be difﬁcult to make valid comparisons between studies.
Instead of ﬁnding unusual ways of analyzing results it is more
convenient to make use of solid methods, which make the results
more reliable and comparable. The same is valid for the clinical
setting, where international guidelines and recommendations for
responses must be followed.
It has been stated, and reasonably so, that DNA vaccines are one
of the future technologies for vaccination. As well electroporation
will likely have an important role as a technology to boost DNA
vaccine efﬁcacy. Important progress has been made, but at the
1. Tang DC, DeVit M, Johnston SA. Genetic immu-
nization is a simple method for eliciting an immune
response. Nature 1992; 356:152-4; PMID:1545867;
2. Rochard A, Scherman D, Bigey P. Genetic immuniza-
tion with plasmid DNA mediated by electrotransfer.
Hum Gene Ther 2011; 22:789-98; PMID:21631165;
3. Kutzler MA, Weiner DB. DNA vaccines: ready
for prime time? Nat Rev Genet 2008; 9:776-88;
4. Bodles-Brakhop AM, Heller R, Draghia-Akli R.
Electroporation for the delivery of DNA-based vaccines
and immunotherapeutics: current clinical develop-
ments. Mol Ther 2009; 17:585-92; PMID:19223870;
5. Hojman P, Gissel H, Gehl J. Sensitive and precise
regulation of haemoglobin after gene transfer of eryth-
ropoietin to muscle tissue using electroporation. Gene
Ther 2007; 14:950-9; PMID:17410179; http://dx.doi.
6. Gothelf A, Hojman P, Gehl J. Therapeutic levels of
erythropoietin (EPO) achieved after gene electro-
transfer to skin in mice. Gene Ther 2010; 17:1077-
84; PMID:20410932; http://dx.doi.org/10.1038/
7. Agerholm-Larsen B, Iversen HK, Ibsen P, Moller
JM, Mahmood F, Jensen KS, et al. Preclinical valida-
tion of electrochemotherapy as an effective treatment
for brain tumors. Cancer Res 2011; 71:3753-62;
8. Marty M, Sersa G, Garbay JR, et al.
Electrochemotherapy - An easy, highly effective
and safe treatment of cutaneous and subcutaneous
metastases: Results of ESOPE (European Standard
Operating Procedures of Electrochemotherapy) study.
Eur J Cancer, Suppl 2006; 4:3-13; http://dx.doi.
9. Matthiessen LW, Muir T, Gehl J. Electrochemotherapy
for larger malignant tumors. In: Kee S, Gehl J, Lee
EW, editors. Clinical aspects of electroporation. 1st ed.
Springer 2011; 103-14.
10. Gehl J. Electroporation: theory and methods, perspec-
tives for drug delivery, gene therapy and research. Acta
Physiol Scand 2003; 177:437-47; PMID:12648161;
11. Vernier PT, Ziegler MJ, Sun Y, Chang WV, Gundersen
MA, Tieleman DP. Nanopore formation and phospha-
tidylserine externalization in a phospholipid bilayer
at high transmembrane potential. J Am Chem Soc
2006; 128:6288-9; PMID:16683772; http://dx.doi.
12. Gabriel B, Teissié J. Direct observation in the millisec-
ond time range of fluorescent molecule asymmetrical
interaction with the electropermeabilized cell mem-
brane. Biophys J 1997; 73:2630-7; PMID:9370457;
13. Mahmood F. Understanding electric fields for clinical
use. In: Kee S, Gehl J, Lee EW, editors. Clinical aspects
of electroporation. 1st ed. Springer 2011; 31-44.
14. Gothelf A, Mir LM, Gehl J. Electrochemotherapy:
results of cancer treatment using enhanced delivery
of bleomycin by electroporation. Cancer Treat Rev
2003; 29:371-87; PMID:12972356; http://dx.doi.
15. André FM, Gehl J, Sersa G, Préat V, Hojman P, Eriksen
J, et al. Efficiency of high- and low-voltage pulse
combinations for gene electrotransfer in muscle, liver,
tumor, and skin. Hum Gene Ther 2008; 19:1261-
71; PMID:19866490; http://dx.doi.org/10.1089/
16. Kinosita K Jr., Tsong TY. Voltage-induced pore forma-
tion and hemolysis of human erythrocytes. Biochim
Biophys Acta 1977; 471:227-42; PMID:921980;
17. Rols MP, Teissié J. Electropermeabilization of mam-
malian cells. Quantitative analysis of the phenomenon.
Biophys J 1990; 58:1089-98; PMID:2291935; http://
18. Lee RC, River LP, Pan FS, Ji L, Wollmann RL.
Surfactant-induced sealing of electropermeabilized
skeletal muscle membranes in vivo. Proc Natl Acad Sci
USA 1992; 89:4524-8; PMID:1584787; http://dx.doi.
19. Saulis G. Pore disappearance in a cell after elec-
troporation: theoretical simulation and comparison
with experiments. Biophys J 1997; 73:1299-309;
20. Gehl J, Skovsgaard T, Mir LM. Vascular reactions to in
vivo electroporation: characterization and consequences
for drug and gene delivery. Biochim Biophys Acta
2002; 1569:51-8; PMID:11853957; http://dx.doi.
21. Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D,
Caillaud JM, et al. High-efficiency gene transfer into
skeletal muscle mediated by electric pulses. Proc Natl
Acad Sci USA 1999; 96:4262-7; PMID:10200250;
22. Joergensen M, Agerholm-Larsen B, Nielsen PE, Gehl
J. Efficiency of cellular delivery of antisense peptide
nucleic acid by electroporation depends on charge
and electroporation geometry. Oligonucleotides
2011; 21:29-37; PMID:21235293; http://dx.doi.
23. Staal LG, Gilbert R. Generators and applicators;
equipment for electroporation. In: Kee S, Gehl J, Lee
EW, editors. Clinical aspects of electroporation. 1st ed.
Springer 2011; 66-202.
24. Khan AS, Smith LC, Abruzzese RV, Cummings
KK, Pope MA, Brown PA, et al. Optimization of
electroporation parameters for the intramuscu-
lar delivery of plasmids in pigs. DNA Cell Biol
2003; 22:807-14; PMID:14683591; http://dx.doi.
25. Gothelf A, Mahmood F, Dagnaes-Hansen F, Gehl J.
Efficacy of transgene expression in porcine skin as
a function of electrode choice. Bioelectrochemistry
2011; 82:95-102; PMID:21724474; http://dx.doi.
26. Gothelf A, Gehl J. Gene electrotransfer to skin; review
of existing literature and clinical perspectives. Curr
Gene Ther 2010; 10:287-99; PMID:20557284; http://
27. Mir LM, Moller PH, André F, Gehl J. Electric pulse-
mediated gene delivery to various animal tissues. Adv
Genet 2005; 54:83-114; PMID:16096009; http://
28. Donate A, Coppola D, Cruz Y, Heller R. Evaluation of
a novel non-penetrating electrode for use in DNA vacci-
nation. PLoS ONE 2011; 6:e19181; PMID:21559474;
29. Drabick JJ, Glasspool-Malone J, King A, Malone RW.
Cutaneous transfection and immune responses to
intradermal nucleic acid vaccination are significantly
enhanced by in vivo electropermeabilization. Mol
Ther 2001; 3:249-55; PMID:11237682; http://dx.doi.
30. Favard C, Dean DS, Rols MP. Electrotransfer as
a non viral method of gene delivery. Curr Gene
Ther 2007; 7:67-77; PMID:17305529; http://dx.doi.
31. Hojman P. Basic principles and clinical advance-
ments of muscle electrotransfer. Curr Gene Ther
2010; 10:128-38; PMID:20222860; http://dx.doi.
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com Human Vaccines & Immunotherapeutics 9
46. Hojman P, Eriksen J, Gehl J. In Vivo Imaging of Far-
red Fluorescent Proteins after DNA Electrotransfer to
Muscle Tissue. Biol Proced Online 2009; 11:253-62;
47. Trochon-Joseph V, Martel-Renoir D, Mir LM,
Thomaïdis A, Opolon P, Connault E, et al. Evidence
of antiangiogenic and antimetastatic activities of the
recombinant disintegrin domain of metargidin. Cancer
Res 2004; 64:2062-9; PMID:15026344; http://dx.doi.
48. Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI,
Sondak VK, et al. Phase I trial of interleukin-12 plasmid
electroporation in patients with metastatic melanoma. J
Clin Oncol 2008; 26:5896-903; PMID:19029422.
49. de Martel C, Ferlay J, Franceschi S, Vignat J, Bray F,
Forman D, et al. Global burden of cancers attributable
to infections in 2008: a review and synthetic analysis.
Lancet Oncol 2012; 13:607-15; PMID:22575588;
50. Golzio M, Teissie J, Rols MP. Direct visualization
at the single-cell level of electrically mediated gene
delivery. Proc Natl Acad Sci USA 2002; 99:1292-7;
40. Gupta S, Domashenko A, Cotsarelis G. The hair fol-
licle as a target for gene therapy. Eur J Dermatol 2001;
41. Pavselj N, Préat V. DNA electrotransfer into the
skin using a combination of one high- and one low-
voltage pulse. J Control Release 2005; 106:407-15;
42. Tam JM, Upadhyay R, Pittet MJ, Weissleder R,
Mahmood U. Improved in vivo whole-animal detec-
tion limits of green fluorescent protein-expressing
tumor lines by spectral fluorescence imaging. Mol
Imaging 2007; 6:269-76; PMID:17711782.
43. Odgren PR, MacKay CA, Mason-Savas A, Yang M,
Mailhot G, Birnbaum MJ. False-positive beta-galactosi-
dase staining in osteoclasts by endogenous enzyme: stud-
ies in neonatal and month-old wild-type mice. Connect
Tissue Res 2006; 47:229-34; PMID:16987755; http://
44. Sanchez-Ramos J, Song S, Dailey M, Cardozo-Pelaez F,
Hazzi C, Stedeford T, et al. The X-gal caution in neural
transplantation studies. Cell Transplant 2000; 9:657-
45. Gothelf A, Eriksen J, Hojman P, Gehl J. Duration
and level of transgene expression after gene electro-
transfer to skin in mice. Gene Ther 2010; 17:839-
45; PMID:20376097; http://dx.doi.org/10.1038/
32. Vasan S, Hurley A, Schlesinger SJ, Hannaman D,
Gardiner DF, Dugin DP, et al. In vivo electroporation
enhances the immunogenicity of an HIV-1 DNA
vaccine candidate in healthy volunteers. PLoS ONE
2011; 6:e19252; PMID:21603651; http://dx.doi.
33. Hojman P, Spanggaard I, Olsen CH, Gehl J, Gissel H.
Calcium electrotransfer for termination of transgene
expression in muscle. Hum Gene Ther 2011; 22:753-
60; PMID:21470044; http://dx.doi.org/10.1089/
34. Teissié J, Eynard N, Gabriel B, Rols MP.
Electropermeabilization of cell membranes. Adv Drug
Deliv Rev 1999; 35:3-19; PMID:10837686; http://
35. Mathiesen I. Electropermeabilization of skeletal muscle
enhances gene transfer in vivo. Gene Ther 1999; 6:508-
14; PMID:10476210; http://dx.doi.org/10.1038/
36. Durieux AC, Bonnefoy R, Busso T, Freyssenet D.
In vivo gene electrotransfer into skeletal muscle:
effects of plasmid DNA on the occurrence and extent
of muscle damage. J Gene Med 2004; 6:809-16;
37. Gothelf A, Gehl J. Gene electrotransfer to skin. In: Kee
S, Gehl J, Lee EW, editors. Clinical aspects of electro-
poration. 1st ed. Springer 2011; 189-202.
38. Katz AB, Taichman LB. Epidermis as a secretory tissue:
an in vitro tissue model to study keratinocyte secretion.
J Invest Dermatol 1994; 102:55-60; PMID:8288911;
39. Vandermeulen G, Richiardi H, Escriou V, Ni J,
Fournier P, Schirrmacher V, et al. Skin-specific promot-
ers for genetic immunisation by DNA electroporation.
Vaccine 2009; 27:4272-7; PMID:19450641; http://