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What you always needed to know about electroporation based DNA vaccines

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Abstract and Figures

Vaccinations are increasingly used to fight infectious disease, and DNA vaccines offer 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 electrotransfer to muscle and skin, may greatly augment efforts 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.
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©2012 Landes Bioscience. Do not distribute.
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Human Vaccines & Immunotherapeutics 8:11, 1–9; November 2012; © 2012 Landes Bioscience
REVIEW
REVIEW
Introduction
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.gehl@regionh.dk
Submitted: 06/12/12; Revised: 08/24/12; Accepted: 09/02/12
http://dx.doi.org/10.4161/hv.22062
Vaccinations are increasingly used to ght infectious disease,
and DNA vaccines oer 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 eorts
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
structures.
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
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
DNA vaccination.
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
remaining E-coli.
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 dierently
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
membrane.30
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
being explored.28
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 dierent pulse
combinations that have been used for gene electrotransfer to skin. There is no consensus regarding
which electrical parameter is the most eective, but eciency 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
by-passed.
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
ref. 37).
©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 (modied from ref. 45).
©2012 Landes Bioscience. Do not distribute.
6 Human Vaccines & Immunotherapeutics Volume 8 Issue 11
Table1. Comparison of gene electrotransfer to muscle and skin, from a clinical point of view
Muscle Skin
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
of proteins
Contains many different cell types, including
antigen presenting cells
Cell turn-over Muscle fibers are terminally differentiated and
long-living
Rapid cell turn-over, particularly of the
keratinocytes
Injection technique Intramuscular dif fusion of plasmid from the injection site, particularly
along the length of the fibers
The injected volume stays intradermally for
minutes
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
muscles
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)
Safety
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
Table2. Clinical trials using electroporation and gene transfection
Tissue Gene or vaccine Patients Study
Skin CEA Colorectal cancer Phase 1
Phase 2
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
Phase 2
Leukemia CML and AML Phase 2
Malaria (Plasmodium Falciparum) Healthy adults Phase 1
Misc, DNA vaccines Malignant melanoma Phase 1
Phase 2
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
be appropriate.
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
Pitfalls
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
minutes.
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
tumor.
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.
Future Perspectives
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
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... De nombreux essais cliniques basés sur les immunothérapies anti-cancer existent mais peu impliquent une vaccination ADN et moins encore avec une injection ID (Gothelf and Gehl, 2012 ...
... L'EGT permet d'augmenter l'immunogénicité des vaccins ADN par l'amélioration de leur transfert dans les cellules cibles favorisant la prise en charge antigénique et ainsi l'efficacité de la vaccination (Gothelf and Gehl, 2012 ;Kutzler and Weiner, 2008 ;Saade and Petrovsky, 2012 Néanmoins, l'aspect primordial à évaluer pour l'optimisation de cette procédure reste tout de même l'induction des réponses immunitaires cellulaires suite à une immunisation avec INVAC-1. En effet, l'induction des réponses CTLs et CD4 Th1 est cruciale pour l'immunité anti-tumorale (Mittal et al., 2014 ;Rice et al., 2008 ;Vesely et al., 2011). ...
... Néanmoins, la peau étant un tissu en renouvellement, l'expression du transgène est donc régie par le type de cellules transfectées au site d'injection, e.g. la transfection des kératinocytes provenant des cellules souches de la membrane basale limite l'expression du transgène à quelques semaines(Elnekave et al., 2011 ;Gothelf and Gehl, 2012).La peau est divisée en deux couches principales, l'épiderme et le derme (Figure 22). L'épiderme représente une barrière physique constituant la première ligne de l'immuno-surveillance et est séparé en quatre strates qui permettent le renouvellement des cellules de l'épiderme(Peachman et al., 2003). ...
Thesis
Le cancer est l’une des principales causes de morbidité et de mortalité dans le monde. L’étude de ses mécanismes a mis en évidence des interactions particulières entre la tumeur et l’immunité adaptative. Les cellules cancéreuses suite à leur transformation maligne expriment des antigènes tumoraux reconnus par les LTs. Ce fondement constitue la base des immunothérapies ciblant les antigènes associés aux tumeurs (TAAs). Parmi les TAAs identifiés, la hTERT apparaît comme un antigène universel, par son implication dans le processus d’oncogenèse et sa surexpression dans 80 à 90% des cancers. Les réponses anti-hTERT trouvées chez des individus sains et des patients cancéreux, témoin d’un répertoire T spécifique préexistant et d’une cassure naturelle de la tolérance, ont orienté nos stratégies vaccinales sur ce TAA. Durant ce doctorat, des stratégies d’immunothérapies basées sur différents produits codant une forme inactive de la hTERT ont été développées. Le premier axe a constitué au développement de vaccins ADN thérapeutiques optimisés à la fois dans leur construction (délétions, réarrangements) et dans leur mode de délivrance. Une procédure d’électroporation a été mise au point afin de les délivrer efficacement dans le derme. Deuxièmement, dans l’optique d’augmenter l’immunogénicité de l’ADN par la réalisation de vaccinations hétérologues ou de créer un produit dérivé, un vecteur rougeole recombinant la hTERT a aussi été développé. Au cours de ce projet, l’immunogénicité et la cytotoxicité des réponses induites par la vaccination pour l’ensemble des constructions ont été démontrées in vivo dans des souris conventionnelles ou transgéniques HLA. De plus, un effet anti-tumoral a aussi été démontré pour le premier produit clinique d’Invectys.
... DNA vaccination consists of injecting a DNA sequence coding for a pathogenic cell epitope (e.g., cancerous cell), leading to the activation of the immune system against the cells exposing that epitope. With the possibility of multi-potent vaccines, the high level of safety due to the absence of viral elements and the stability of the DNA molecule in lack of need for cold storage, DNA vaccines offer considerable advantages and the possibility to produce good manufacturing practices (GMP)grade plasmid DNA (GMP-DNA) for direct gene transfer into humans (Gothelf et al., 2012). For example, a recent study done in our laboratory by Calvet et al. used a plasmid that encodes a STATE OF THE ART Biophysical-based treatments, except for cancer treatment -44 -modified form of the human telomerase reverse transcriptase gene (hTERT) and its intradermal electrotransfection in mice. ...
... Since LV pulses application is favourable for the delivery of DNA vaccines (André, 2008;Gothelf, 2012;Mir, 2009), it is worth highlighting the most recent news related to EGT and the ongoing COVID-19 pandemic. On November 24 th , 2020 -a week before the end of this thesis writing, when the world is facing the SARS-CoV-2 virus that has already infected ca. ...
Thesis
Electrochemotherapy (ECT) – an established method for the local eradication of cutaneous tumours based on electroporation technology – is widely used in Europe in human and veterinary medicine with very few systemic side effects. Non-thermal plasmas (NTP) are recently gaining momentum for application in cancerology based on their rich content of short-lived reactive species as well of long-lived reactive oxygen and nitrogen species (RONS) which can be transferred to liquids being treated with NTP. We sought to combine those technologies in order to enhance the ECT capabilities and to better understand the contribution of RONS in those anticancer strategies. As a multidisciplinary study at the crossroads of physics, chemistry, biotechnologies and medicine, this thesis aimed at emphasising the potent beneficial outcome of the combination of ECT and NTP medicine. A novel, stable source of helium plasma multi-jet (PMJ) has been utilised for in vitro investigations on three different cell lines (the DC-3F, B16-F10 and LPB cells), and for in vivo studies on nude and C57Bl/6 mice bearing sub-cutaneous tumours. We demonstrated that the pH of the plasma-treated PBS (pPBS) and the stability of the three main RONS (H₂O₂, NO₂- and NO₃-) under various storage conditions were crucial for the storage of plasma-treated liquids while maintaining their anti-cancer capabilities. In combination with ECT at an electric field amplitude lower than the one usually applied, those stable pPBS partially restored the ECT efficacy, delaying tumour growth and hence prolonging mice survival.
... Electroporation Electroporation is usually employed as a non-viral gene delivery technique in vitro and in vivo. The technique involves the application of short high-voltage pulses to cells to form pores within its cell membrane to create a transient state of permeability [94,95]. This state of permeability allows the entry of drugs and fluorochrome compounds and even large-sized cargo such as nucleotides, which is optimized by varying the electric pulse and its duration. ...
... Electroporation is usually employed as a non-viral gene delivery technique in vitro and in vivo. The technique involves the application of short high-voltage pulses to cells to form pores within its cell membrane to create a transient state of permeability [94,95]. This state of permeability allows the entry of drugs and fluorochrome compounds and even large-sized cargo such as nucleotides, which is optimized by varying the electric pulse and its duration. ...
Article
Full-text available
Numerous factors need to be considered to develop a nanodrug delivery system that is biocompatible, non-toxic, easy to synthesize, cost-effective, and feasible for scale up over and above their therapeutic efficacy. With regards to this, worldwide, exosomes, which are nano-sized vesicles obtained from mammalian cells, are being explored as a biomimetic drug delivery system that has superior biocompatibility and high translational capability. However, the economics of undertaking large-scale mammalian culture to derive exosomal vesicles for translation seems to be challenging and unfeasible. Recently, Bacterial Membrane Vesicles (BMVs) derived from bacteria are being explored as a viable alternative as biomimetic drug delivery systems that can be manufactured relatively easily at much lower costs at a large scale. Until now, BMVs have been investigated extensively as successful immunomodulating agents, but their capability as drug delivery systems remains to be explored in detail. In this review, the use of BMVs as suitable cargo delivery vehicles is discussed with focus on their use for in vivo treatment of cancer and bacterial infections reported thus far. Additionally, the different types of BMVs, factors affecting their synthesis and different cargo loading techniques used in BMVs are also discussed.
... In the case of mRNA-based vaccines, there is the problem of temperature-sensitive lipid nanoparticles containing mRNA and their limitations for production in large scale [92]. In DNA vaccines, the use of electroporation to deliver the vaccine to recipients increases the immune response, but this technology has made the use of these vaccines difficult and complicated [111]. There is also risk of vaccine-enhanced disease for inactivated vaccine candidates, specially vaccine-associated enhanced respiratory disease (VAERD), that needs to be considered [112,113]. ...
Article
The world has been suffering from COVID-19 disease for more than a year, and it still has a high mortality rate. In addition to the need to minimize transmission of the virus through non-pharmacological measures such as the use of masks and social distance, many efforts are being made to develop a variety of vaccines to prevent the disease worldwide. So far, several vaccines have reached the final stages of safety and efficacy in various phases of clinical trials, and some, such as Moderna/NIAID and BioNTech/Pfizer, have reported very high safety and protection. The important point is that comparing different vaccines is not easy because there is no set standard for measuring neutralization. In this study, we have reviewed the common platforms of COVID-19 vaccines and tried to present the latest reports on the effectiveness of these vaccines.
... The delivery of foreign material to the target tissue (skeletal muscle, skin, tumor) is crucial for the effective expression of the transgene. Gene transfer of different molecules using conventional electroporation was shown to be an attractive and efficient approach for gene therapy in a variety of normal tissues (muscle, skin) and tumor, as well as for DNA vaccination in preclinical and clinical studies [26,31,[44][45][46][47][48][49][50][51][52][53][54][55]. Depending on the targeted molecule or specific gene (K-ras, CD105, MCAM, IL-12, AMEP, erithropoetin, p53, CpG or GpC oligonucleotides, bacterial purine nucleoside phosphorylase (ePNP)) [31,45,46,49,51,53,[56][57][58][59], good antitumor, antimetastatic and/or vascular targeted effects were demonstrated. ...
Article
Full-text available
High-Intensity Pulsed Electromagnetic Fields (HI-PEMF) treatment is an emerging noninvasive and contactless alternative to conventional electroporation, since the electric field inside the tissue is induced remotely by external pulsed magnetic field. Recently, HI-PEMF was applied for delivering siRNA molecules to silence enhanced green fluorescent protein (EGFP) in tumors in vivo. Still, delivered siRNA molecules were 21 base pairs long, which is 200-times smaller compared to nucleic acids such as plasmid DNA (pDNA) that are delivered in gene therapies to various targets to generate therapeutic effect. In our study, we demonstrate the use HI-PEMF treatment as a feasible noninvasive approach to achieve in vivo transfection by enabling the transport of larger molecules such as pDNA encoding EGFP into muscle and skin. We obtained a long-term expression of EGFP in the muscle and skin after HI-PEMF, in some mice even up to 230 days and up to 190 days, respectively. Histological analysis showed significantly less infiltration of inflammatory mononuclear cells in muscle tissue after the delivery of pEGFP using HI-PEMF compared to conventional gene electrotransfer. Furthermore, the antitumor effectiveness using HI-PEMF for electrotransfer of therapeutic plasmid, i.e., silencing MCAM was demonstrated. In conclusion, feasibility of HI-PEMF was demonstrated for transfection of different tissues (muscle, skin, tumor) and could have great potential in gene therapy and in DNA vaccination.
... Unlike alternative ablative therapies, electroporation does not affect the structural integrity of the surrounding tissue, thereby enabling the treatment of tumors in the vicinity of vital structures. This technique has also led to new research into DNA vaccine delivery and gene therapy [22,23]. In addition to the direct antitumor function of the drug, ECT has several mechanisms of action, which may involve vascular effects and an immune response. ...
Article
Full-text available
Despite recent advances in the development of chemotherapeutic drug, treatment for advanced cancer of the head and neck cancer (HNC) is still challenging. Options are limited by multiple factors, such as a prior history of irradiation to the tumor site as well as functional limitations. Against this background, electrochemotherapy (ECT) is a new modality which combines administration of an antineoplastic agent with locally applied electric pulses. These pulses allow the chemotherapeutic drug to penetrate the intracellular space of the tumor cells and thereby increase its cytotoxicity. ECT has shown encouraging efficacy and a tolerable safety profile in many clinical studies, including in heavily pre-treated HNC patients, and is considered a promising strategy. Efforts to improve its efficacy and broaden its application are now ongoing. Moreover, the combination of ECT with recently developed novel therapies, including immunotherapy, represented by immune checkpoint inhibitor (ICI)s, has attracted attention for its potent theoretical rationale. More extensive, well-organized clinical studies and timely updating of consensus guidelines will bring this hopeful treatment to HNC patients under challenging situations.
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Interleukin-12 has been a promising candidate for cancer treatment for over 20 years. And from its infamous start, the gene electrotransfer delivery has regained assurance of the safe and effective use of interleukin-12 in cancer treatment. Here, basic facts about interleukin-12 are presented with a focus on its antitumor action and the benefit of electrotransfer delivery. In veterinary oncology, interleukin-12 gene electrotransfer has been used in preclinical and clinical studies on dogs for over 10 years. In this chapter studies of interleukin-12 immunotherapy alone or in combination with electrochemotherapy or surgery in different types of dogs’ tumors are presented with emphasis on the antitumor effect on primary tumors as well as on distant metastasis. Also, the impact of interleukin-12 gene electrotransfer on the immune system is discussed as well as the safety of the procedure and possible improvement of the procedure.
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A novel coronavirus, which has been designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected in December 2019 in Wuhan China and causes the highly infectious disease referred to as COVID-19. COVID-19 has now spread worldwide to become a global pandemic affecting over 24 million people as of August 26th, 2020 and claimed the life of more than 800,000 people worldwide. COVID-19 is asymptomatic for some individuals and for others it can cause symptoms ranging from flu-like to acute respiratory distress syndrome (ARDS), pneumonia and death. Although it is anticipated that an effective vaccine will be available to protect against COVID-19, at present the world is relying on social distancing and hygiene measures and repurposed drugs. There is a worldwide effort to develop an effective vaccine against SARS-CoV-2 and, as of late August 2020, there are 30 vaccines in clinical trials with over 200 in various stages of development. This review will focus on the eight vaccine candidates that entered Phase 1 clinical trials in mid-May, including AstraZeneca/Oxford's AZD1222, Moderna's mRNA-1273 and Sinovac's CoronaVac vaccines, which are currently in advanced stages of vaccine development. In addition to reviewing the different stages of vaccine development, vaccine platforms and vaccine candidates, this review also discusses the biological and immunological basis required of a SARS-CoV-2 vaccine, the importance of a collaborative international effort, the ethical implications of vaccine development, the efficacy needed for an immunogenic vaccine, vaccine coverage, the potential limitations and challenges of vaccine development. Although the demand for a vaccine far surpasses the production capacity, it will be beneficial to have a limited number of vaccines available for the more vulnerable population by the end of 2020 and for the rest of the global population by the end of 2021.
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L'électroporation est une méthode physique utilisant le champ électrique pour perméabiliser transitoirement la membrane plasmique afin de faciliter l'entrée de molécules d'intérêt thérapeutique dans les tissus ciblés. La principale application clinique est l'électrochimiothérapie (ECT), un traitement anticancéreux local utilisé pour traiter les tumeurs primaires et métastases. La seconde application est l'électrotransfert de gène (EGT), une méthode pour introduire des acides nucléiques à l'intérieur des cellules. Enfin, l'électroporation irréversible (IRE) est une méthode utilisée pour tuer les cellules grâce à la perméabilisation permanente des membranes plasmiques. Même si les mécanismes in vitro sont de mieux en mieux compris, l'efficacité in vivo est variable selon le tissu cible. En effet, bien que l'électrotransfert d'ADN soit très efficace in vitro sur des cultures en deux dimensions (2D), il est souvent beaucoup moins efficace in vivo, ce qui limite ses applications cliniques. L'organisation du tissu in vivo est plus complexe que la culture cellulaire in vitro car les cellules développent des jonctions intercellulaires et une matrice extracellulaire (MEC). Des études in vivo ont démontré que la composition de la MEC module la biodistribution de l'ADN dans le tissu et donc l'efficacité de l'électrotransfert de gène. La réponse de la MEC à l'application de champ électrique dans ce processus reste encore à définir afin d'améliorer l'efficacité de cette méthode. Les modèles classiques en 2D ne possèdent pas cette organisation architecturale tridimensionnelle (3D) leur permettant d'être physiologiquement comparable au tissu natif. Afin d'étudier les mécanismes d'électrotransfert d'ADN à l'échelle des tissus, nous avons utilisé un modèle de peau humaine 3D pour imiter et prédire les situations in vivo. Les objectifs de ce travail étaient d'étudier le rôle et la réponse de la MEC cutanée lors de l'électrotransfert de gène à l'échelle du tissu. La première partie de ce projet a été de caractériser la MEC du substitut dermique reconstruit par ingénierie tissulaire. La MEC a été caractérisée par microscopie électronique, coloration histologique et génération de seconde harmonique (SHG). Pour évaluer si ce modèle imite efficacement la réponse in vivo observée lors de l'électroporation, une gamme de voltage utilisant des paramètres électriques ECT ou EGT a été appliquée et la perméabilisation cellulaire ainsi que l'expression du plasmide ont été analysées sur tissu frais. Dans la deuxième partie, nous avons étudié les effets directs et indirects du champ électrique pulsé sur les collagènes fibrillaires.[...]
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Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles. In this study, IMEP was used to express the DUX4 gene into mouse tibialis anterior muscle. DUX4 is normally expressed in germ cells and early embryo, and silenced in adult muscle cells where its pathological reactivation leads to Facioscapulohumeral muscular dystrophy. DUX4 encodes a potent transcription factor causing a large deregulation cascade. Its high toxicity but sporadic expression constitutes major issues for testing emerging therapeutics. The IMEP method appeared as a convenient technique to locally express DUX4 in mouse muscles. Histological analyses revealed well delineated muscle lesions 1-week after DUX4 IMEP. We have therefore developed a convenient outcome measure by quantification of the damaged muscle area using color thresholding. This method was used to characterize lesion distribution and to assess plasmid recirculation and dose–response. DUX4 expression and activity were confirmed at the mRNA and protein levels and through a quantification of target gene expression. Finally, this study gives a proof of concept of IMEP model usefulness for the rapid screening of therapeutic strategies, as demonstrated using antisense oligonucleotides against DUX4 mRNA.
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Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme β-galactosidase (β-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, “X-gal,” in an oxidation reaction. LacZ is the “reporter gene” most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully—with adequate optimization and individualization of various parameters (e.g., pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls—then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial β-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have emphasized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli β-gal activity.
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Over the last decade a new cancer treatment modality, electrochemotherapy, has emerged. By using short, intense electric pulses that surpass the capacitance of the cell membrane, permeabilization can occur (electroporation). Thus, molecules that are otherwise non-permeant can gain direct access to the cytosol of cells in the treated area.A highly toxic molecule that does not usually pass the membrane barrier is the hydrophilic drug bleomycin. Once inside the cell, bleomycin acts as an enzyme creating single- and double-strand DMA-breaks. The cytotoxicity of bleomycin can be augmented several 100-fold by electroporation. Drug delivery by electroporation has been in experimental use for cancer treatment since 1991.This article reviews 11 studies of electrochemotherapy of malignant cutaneous or subcutaneous lesions, e.g., metastases from melanoma, breast or head- and neck cancer. These studies encompass 96 patients with altogether 411 malignant tumours. Electroporation was performed using plate or needle electrodes under local or general anaesthesia. Bleomycin was administered intratumourally or intravenously prior to delivery of electric pulses. The rates of complete response (CR) after once-only treatments were between 9 and 100% depending on the technique used. The treatment was well tolerated and could be performed on an out-patient basis.
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Purpose: To evaluate and confirm efficacy and safety of electrochemotherapy with bleomy- cin or cisplatin on cutaneous and subcutaneous tumour nodules of patients with malig- nant melanoma and other malignancies in a multicenter study. Patients and methods: This was a two year long prospective non-randomised study on 41 patients evaluable for response to treatment and 61 evaluable for toxicity. Four cancer cen- ters enrolled patients with progressive cutaneous and subcutaneous metastases of any his- tologically proven cancer. The skin lesions were treated by electrochemotherapy, using application of electric pulses to the tumours for increased bleomycin or cisplatin delivery into tumour cells. The treatment was performed using intravenous or intratumoural drug injection, followed by application of electric pulses generated by a Cliniporator TM using plate or needle electrodes. Tumour response to electrochemotherapy as well as possible side- effects with respect to the treatment approach, tumour histology and location of the tumour nodules and electrode type were evaluated. Results: An objective response rate of 85% (73.7% complete response rate) was achieved on the electrochemotherapy treated tumour nodules, regardless of tumour histology, and drug used or route of its administration. At 150 days after the treatment (median follow up was 133 days and range 60-380 days) local tumour control rate for electrochemotherapy was 88% with bleomycin given intravenously, 73% with bleomycin given intratumourally and 75% with cisplatin given intratumourally, demonstrating that all three approaches were
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For physicians or researchers from the biological sciences, it is usually not necessary to deal with the in-depth scientific theory of the electric field in order to handle procedures involving electrotransfer. On the other hand, it is very often of utmost importance to know the basic concept, from a pragmatic point of view, to understand simple electric field distributions. In this chapter, we initially address the basic physics and definitions of electrostatics in order to prepare the reader for the practical topics that follow. The electric field strength and the electric vector field are explained in connection with tissue characteristics, especially heterogeneity and anisotropy. The role of the pulse generator and the electrode geometry is addressed in relation to electric field distributions and electric field strength. The reader is provided with electric field visualization methods, which are important for both comprehension and interpretation of otherwise complicated data. Finally, critical steps for ensuring the intended delivery of the appropriate electric field for treatment procedures are proposed. It is stressed that numerical derivation of the electric field is the only reasonable way of handling individual nonstandardized electrotransfer procedures. KeywordsElectrostatics-Electric field-Electric potential-Electrode geometry-Tissue heterogeneity-Treatment planning-Electric field plot
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This chapter provides an introduction to the equipment available for treatments based on electroporation. The purpose is to acquaint the reader with the equipment available for the performance of electroporation-based treatments. To that end, pulse generators and applicators are presented along with details on their use. Particular features of relevance to the clinician are described, and manufacturers of the different types of equipment are introduced. KeywordsPulse generator-Applicator-Electrode-Electrochemotherapy-Gene electrotransfer-DNA vaccination-Irreversible electroporation
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Gene electrotransfer to skin is achieving increasing interest and is likely to gain considerable clinical application due to the ease with which it is performed and the safety of the procedure. There is a potential use of gene electrotransfer to skin in e.g., DNA vaccinations, local production of therapeutic molecules as well as production of molecules for systemic therapy. More than 30 preclinical studies concerning gene electrotransfer to skin have been reported in the literature and this chapter aims at creating an overview of plasmids injected, electrical parameters used, and duration and level of transgene expression. KeywordsSkin-Gene electrotransfer-Electroporation
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Cutaneous metastases can present themselves in many ways and adversely affect self-esteem and body image. Management and palliative treatment of cutaneous metastases remain as clinically challenging problems. Electrochemotherapy as a palliative treatment for smaller metastases (size < 3 cm) is well investigated, whereas experience with electrochemotherapy for larger metastases (size >3 cm) mostly relies on case reports and small clinical studies.The present chapter focuses on the knowledge obtained from case reports, clinical studies, and authors’ experience. Tumor depth and the ability to cover the whole area, evaluation and benefit for the patient, repeated treatment, and penetration of the skin are some of the challenges that will be discussed.In conclusion, electrochemotherapy for larger malignant tumors seems promising as a palliative treatment and could be a good supplement to surgery, but further investigation is needed in order to make evident indications and guidelines for such treatment. KeywordsElectrochemotherapy-Clinical-Metastases-Large tumors-Bleomycin
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Infections with certain viruses, bacteria, and parasites have been identified as strong risk factors for specific cancers. An update of their respective contribution to the global burden of cancer is warranted. We considered infectious agents classified as carcinogenic to humans by the International Agency for Research on Cancer. We calculated their population attributable fraction worldwide and in eight geographical regions, using statistics on estimated cancer incidence in 2008. When associations were very strong, calculations were based on the prevalence of infection in cancer cases rather than in the general population. Estimates of infection prevalence and relative risk were extracted from published data. Of the 12·7 million new cancer cases that occurred in 2008, the population attributable fraction (PAF) for infectious agents was 16·1%, meaning that around 2 million new cancer cases were attributable to infections. This fraction was higher in less developed countries (22·9%) than in more developed countries (7·4%), and varied from 3·3% in Australia and New Zealand to 32·7% in sub-Saharan Africa. Helicobacter pylori, hepatitis B and C viruses, and human papillomaviruses were responsible for 1·9 million cases, mainly gastric, liver, and cervix uteri cancers. In women, cervix uteri cancer accounted for about half of the infection-related burden of cancer; in men, liver and gastric cancers accounted for more than 80%. Around 30% of infection-attributable cases occur in people younger than 50 years. Around 2 million cancer cases each year are caused by infectious agents. Application of existing public health methods for infection prevention, such as vaccination, safer injection practice, or antimicrobial treatments, could have a substantial effect on the future burden of cancer worldwide. Fondation Innovations en Infectiologie (FINOVI) and the Bill & Melinda Gates Foundation (BMGF).
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Gene electrotransfer is a non-viral technique using electroporation for gene transfection. The method is widely used in the preclinical setting and results from the first clinical study in tumours have been published. However, the preclinical studies, which form the basis for the clinical trials, have mainly been performed in rodents and the body of evidence on electrode choice and optimal pulsing conditions is limited. We therefore tested plate and needle electrodes in vivo in porcine skin, which resembles human skin in structure. The luciferase (pCMV-Luc) gene was injected intradermally and subsequently electroporated. Simultaneously, studies with gene electrotransfer to porcine skin using plasmids coding for green fluorescent protein (GFP) and betagalactosidase were performed. Interestingly, we found needle electrodes to be more efficient than plate electrodes (p<0.001) and electric field calculations showed that penetration of the stratum corneum led to much more homogenous field distribution at the DNA injection site. Furthermore, we have optimised the electric pulse regimens for both plate and needle electrodes using a range of high voltage and low voltage pulse combinations. In conclusion, our data support that needle electrodes should be used in human clinical studies of gene electrotransfer to skin for improved expression.