I.
Introduction
Recent developments in gene transfer technology are
promising and could either supplement or potentially replace the classic
recombinant protein or vaccination regimens, eventually leading to the
development of effective and efficient therapeutic or preventive strategies (Herweijer and Wolff, 2003). Gene delivery allows the transgene product to be
synthesized within target cells using host expression machinery taking
advantage of host secretory or antigen presentation mechanisms. An ideal delivery system for in vivo gene transfer should have well-characterized
pharmaceutical properties; have few, if
any, safety issues; provide prolonged expression of transgenes at therapeutic
levels if delivered into tissues with low turnover time, with the ability to be
re-dosed; avoid auto-antibody responses following re-administration (Jiao et al, 1992); and last, but
not least, be easy to manufacture at a reasonable cost (Hebel et al, 2005). Plasmids can fulfill all these requirements.
Furthermore, plasmids can be naturally taken up into undamaged organs and
tissues (Wolff and Budker 2005). Nevertheless, when compared to viral or liposomal
vectors, there are disadvantages such as extracellular degradation and binding
and poor nuclear uptake (Wolff et al, 1997; Hebert, 2003). For instance, less than 0.01% of plasmid directly
injected into the skeletal muscle will be taken up and possibly expressed (Wolff et al, 1990, 1992b).
While the target organ vastly depends on the particular gene
therapeutic application, the skeletal muscle is a preferable target tissue for
numerous reasons: there is convenient access to multiple muscles with direct intramuscular
injection resulting in a localized expression site (Jiao et al, 1992); skeletal muscle
can act as an effective platform for the long-term secretion of therapeutic
proteins for systemic distribution similar to an endocrine organ (Goldspink, 2003); the transgene expression product may have a better pharmacological profile compared to
repeated injection of recombinant proteins; a pressing need
exists to develop effective therapies for muscular dystrophies (Braun, 2004); and
introduction of DNA vaccines into skeletal muscle promotes strong humoral and
cellular immune responses (Shedlock and Weiner, 2000). The
innovative EP technique overcomes a major disadvantage of plasmid
administration: insufficient uptake in the undamaged muscle. Using this
technology, transgene products can approach levels previously achieved only
with viral vectors for intracellular and secreted products (Draghia-Akli and Fiorotto 2004;
Fattori et al, 2004; Molnar et al, 2004). Numerous
factors can impact plasmid uptake and expression after intramuscular injection
followed by EP, such as plasmid size, formulation, concentration, injection
volume, intensity of the electric field, pulse length, lag time between
injection and EP, target muscle, species and age of the treated subject. This
review will focus on recent studies analyzing these and other factors that can
increase the efficacy of plasmid transfer and lower the total amount of plasmid
and DNA vaccines required to generate targeted levels of biologically active
proteins or antibodies.
II. Factors relating to
target species and muscle
A. Species
Electroporation
has been extensively used in mice (Lucas et al, 2001; Vilquin et al,
2001; Lesbordes et al, 2002), rats (Terada et al, 2001; Yasui et al,
2001), dogs (Fewell et al, 2001), pigs (Draghia-Akli et al,
1999, 2002b), cattle (Tollefsen et al, 2003;
Brown et al, 2004), and non-human primates
(Fattori et al, 2004) to
deliver therapeutic genes that encode for a variety of hormones, cytokines,
enzymes or antigens. Nevertheless, optimal conditions of electroporation varied
in different animal models as well as in the same animal model depending upon
physiological or pathological state of the treated animal. Theoretical and
practical data also suggest that the cell size in the region perpendicular to
the electric field plays a crucial role in determining the permeabilization
parameters (Somiari et al, 2000). The larger the ÒfunctionalÓ size
of the cell, the lower is the field strength necessary (Neumann et al, 1999). For example, tissues containing
cells that communicate through tight gap junctions amplify transmembrane
potential changes. Thus, the skeletal muscle as compared to other tissues can
be electroporated at lower field intensities (Fear and Stuchly, 1998), resulting in decreased tissue
damage. Rodent muscles have been electroporated using current intensities as
low as 50-100 mA (Zampaglione et al, 2005), and as high as approximately 1 Amp,
and electric field intensities of 100-200 V/cm (Mir et al, 1999; Bettan et al,
2000), while in larger mammals, such as
pigs or cattle, lower electric field intensities, 0.4 to 0.6 Amp and voltages,
80 to 120 V/cm, are required to avoid tissue damage (Khan et al, 2003; Brown et al,
2004). In some disease models, such as in
mdx mice, higher voltages of approximately 200 V/cm are needed (Wong et al, 2005), while milder conditions have been
described for diabetic mice than for normal mice (Wang et al, 2005).
B. Target muscle
Plasmids have
been delivered to most surface muscles in different animal species, with the
transgene product directly measured in the target muscle or in the systemic
circulation. The results are difficult to compare as an entire spectrum of
conditions can directly impact the results: fiber type composition of the
muscle, which has been substantially studied in vitro, but not in vivo,
is an important factor in rodents, where muscle can have predominantly type I
or type II fibers (Cardoso et al, 2004); eventual fibrosis (Vilquin et al, 2001), collagen or fat content, or
atrophy (Alzghoul et al, 2004); the choice of promoter driving
transgene expression (most muscle-specific promoters have a fiber type
preference) (Bertrand et al, 2003); vascularization, etc. In large
animal species, where muscles have essentially mixed fiber composition and are
well vascularized, plasmid injection followed by electroporation of different
large surface muscles results in similar transgene expression levels (Draghia-Akli et al, 2002b, 2003;
Khan et al, 2003).
C. Age
Recent studies
have described a significant difference between transgene product levels in
normal adult and young animals. The pattern of expression seems to fit a
Gaussian curve, with normal younger and very old animals showing less
expression than adult ones. Intramuscular injection and EP into muscles of
young mice resulted in lower number of transduced fibers than in adults (Molnar et al, 2004), and a more rapid loss of
expression. Lower expression level was described in old rodents (Wang et al, 2005). On the other hand, the
slower rate of turnover in adult animals may contribute to the longer duration
of expression. Similar results have been described in pigs, with young animals
having higher muscle resistance, and requiring increased electric field
intensities for optimum expression when compared to older pigs (Khan et al, 2005). Muscle properties are
affected by the age of the animal and these factors may affect muscle
resistance during the course of the EP procedure. It seems that the uptake and
activity of smaller nucleic acid molecules, such as short interfering RNAs
(siRNA), are less impacted by particular variable (Golzio et al, 2005). Age differences are also
less pronounced in disease models of muscle dystrophy, such as mdx mice (Gollins et al, 2003;
Wong et al, 2005).
III. Factors relating to
plasmids and its formulation
A. Plasmid size
A general
consensus exists on this important factor: the smaller, the better. Numerous
studies found that plasmids of smaller size entered cells more efficiently than
large plasmids (Bloquel et al, 2004; Molnar et al,
2004; Wang et al, 2005). Also, small nucleic acid
fragments, such as siRNA seem to be uptaken very efficiently by this method (Kishida et al, 2004; Golzio et al,
2005).
B. Plasmid formulation
Under some conditions, EP procedures can inflict fatal
stress on some skeletal muscle cells (Fewell et al, 2001; McMahon et al, 2001) or degrade the plasmid (Hartikka et al, 2001). Polymers
such as poly-vinylpyrrolidone (PVP), poly-(L)-glutamate (PLG) at high or
low concentrations, or mild surfactants in low concentration such as poloxamer
188 have been used to demonstrate increased plasmid
uptake and reduced tissue damage. Poloxamer 188 may induce sealing of permeabilized
lipid bilayers to rescue cells that were not extensively heat-damaged, and
consequently, cause an increase in plasmid expression levels (Lee et al, 1999). Following EP of the
skeletal muscle of mice, rats, dogs or pigs (Mumper et al, 1998; Fewell et al, 2001; Nicol et al,
2002; Draghia-Akli et al, 2002a), plasmid formulated with PLG or PVP has been observed
to increase gene expression up to 10 fold compared to non-formulated plasmid.
In mice, pre-injection of the electroporated muscle with hyaluronidase, an
enzyme that hydrolyzes hyaluronic acid, a ubiquitous component of the
extracellular matrix, increases gene expression up to 5 fold with minimal tissue
damage (Mennuni et al, 2002; Molnar et al, 2004).
Further experiments in our laboratory have
elucidated that the formulation of plasmid can enhance expression in vivo. Our strategy uses low
concentrations of the transfection-facilitating anionic polymer sodium PLG of
low molecular weight. Previous studies have
demonstrated that mice receiving SEAP construct coated with PLG at a
concentration of 0.01 mg/ml had the least inflammation associated with the
delivery procedure at 3 days post-injection (Draghia-Akli and Smith, 2003). We determined that a mol/mol ratio of DNA phosphate
groups to PLG carboxyl groups yields the optimum concentration for gene
therapeutic applications to the skeletal muscle and results in increased
expression of the transgene with no damage to the target tissue in numerous
animal species. The minimum number of repeat units, as well as lot-to-lot (even
when the compound is purchased from the same vendor) variability of PLG was
also analyzed in mice (Figure 1) and
pigs (Figure 2) and found to be an
important factor in the larger animal species, but not in rodents. Other
experimental excipients, such as MgCl2 which promotes sealing of
membrane pores, was also be analyzed, and was determine to favorably impact expression
levels in rodents.
C. Plasmid concentration and dose
Studies have shown that plasmid concentration directly
correlates with expression in rodents and other species (Bettan et al, 2000; Khan et al, 2003; Wang et al,
2005) up to an optimum concentration. Mechanistic studies related to
cellular entry of plasmids after EP show that the level of transfection remains
unchanged whether electric pulses are delivered at various periods of time
after injection and at plasmid doses that vary by a factor of 100 (Bureau et al, 2004). Thus, it is possible that at low plasmid
concentrations the relative proportion of plasmid that will be rapidly cleared
will be greater when compared to the plasmid protected from DNAses which would
be available for cell entry following EP (Wolff et al, 1992a). We have shown that increasing the number of
injection sites by dividing a certain plasmid dose does not result in increased
transgene expression (Figure 3).
Despite the success of DNA technologies in rodents,
the clinical translation to larger animals and humans may be hampered by the
large quantities of plasmid required to attain therapeutic levels of the
desired transgene product (Fewell et al, 2001). Since high doses of plasmid can saturate a single
injection site, multiple injections may be required in larger animals to
achieve optimal gene transfer (Fattori et al, 2005). Various attempts have been
made to increase the effectiveness of plasmid-based expression systems,
including significant improvements to plasmid design. We have shown in our
experiments that under the proper EP conditions as little as 0.1 mg of a plasmid
encoding for a therapeutic protein
delivered can accomplish these requirements in pigs (Draghia-Akli et al, 2003) or 2.5 mg in dairy cattle (Brown et al, 2004). This plasmid quantity is 100-fold lower than a
hypothetical dose obtained by direct extrapolation of the dose used in rodents
(an average of 1 mg/kg). Therapeutic and vaccine applications differ by the
amount of protein production that is necessary, as vaccine applications usually
require lower amounts of protein production. A recent study of influenza
vaccine demonstrated that a single injection of 0.03 mg plasmid encoding
neuraminidase from influenza virus, followed by EP, was able to provide
long-term protection from influenza challenge in mice (Chen et al,
2005).
IV. Factors relating to
electroporation conditions
Existing EP
technologies that deliver square waves targeted to the skeletal muscle are
based upon either constant-voltage or constant-current concepts. Due to
variations in tissue resistance during the EP process, a predetermined voltage
pulse may cause an unregulated variation in the current flowing through the
tissue during each pulse. The result is a loss of the perfect square-wave
function (current intensity versus time), tissue damage and reduced plasmid
uptake and expression (Draghia-Akli and Smith, 2003). By contrast, constant-current EP
maintains a square wave function in the target tissue irrespective of changes
in tissue resistance (Khan et al, 2005). Electroporation parameters such as
pulse pattern, tissue resistance, momentary electric field intensity, and
voltage can be studied only in software-driven systems that are capable of
instant feed-back and recording of both current and volts during pulses (Hebel et al, 2005) as well as adapting the output to
the changing conditions in the tissue.
Thus, software-driven devices that can adapt the current intensity
output and prevent high variations in voltage and prevent tissue damage could
be better adapted to animal and human applications of this technology.
A. Pulse pattern
Pulse patterns
can impact expression levels, with lower number of pulses and no reverse
orientation during EP with needle-electrodes resulting in a higher transgene
expression (Khan et al, 2005). These results are in agreement
with previously published studies showing that optimization of cumulated pulse
duration and current intensity dramatically reduced gene electrotransfer-associated
muscle damage (Durieux et al, 2004).
B. Electric field intensity (current, voltage, resistance)
Electroporation settings as low as 0.1 Amp are able to
successfully transfect the mouse tibialis anterior muscle and ensure high
levels of SEAP expression when using a constant-current device (Khan et al, 2005). Electroporation at amperage settings of 0.2, 0.3,
and 0.4 may induce higher voltage settings and cause damage to the surrounding
muscle tissue. Analysis of the data recorded during EP of mice and young and
old pigs revealed that muscle resistance was significantly higher in mice than
in pigs, with a lower resistance in the older animals compared to younger
animals. For constant-voltage devices (Figure
4) electric field intensities can approach 1 Amp and may result in tissue
damage and pain (Rizzuto et al, 1999; Gronevik et al, 2005; Tjelle et
al, 2005). Furthermore, tissue damage may induce cellular repair processes that
could either replace damaged myocytes that were successfully transfected or
stimulate plasmid degradation (Hartikka et al, 2001; Cappelletti et al, 2003).
C. Pulse length
From the first
comprehensive studies of Mir et al, (1999) and Aihara and Miyazaki (1998), it has been determined that very efficient plasmid DNA transfer in muscle
fibers by using square-wave electric pulses requires low field strength (less
than 300 V/cm) and long duration (more than 1 ms). Most studies describe EP to
the muscle for the different applications referenced above use pulse length
from 10-400 microseconds to 20-60 milliseconds.
D. Lag time
In previous studies, we and others have used time lags
between plasmid injection and EP of approximately 120 seconds (Aihara and Miyazaki, 1998;
Bettan et al, 2000) and subsequently 80 sec (Khan et al, 2003). Very recent studies performed
in different muscle groups in mice describe procedures that allow for a minimal
(20 seconds or less) lag time (Khan et al, 2005; Wang et al,
2005). This finding indicates that
gene expression is dependent on the time needed for the plasmid solution to
disperse into surrounding tissue, but it is not as critical in smaller murine
muscles (Bureau et al, 2004). Future studies analyzing the impact of plasmid
concentration, formulation, target muscle and species on lag time are needed
for larger animal species and humans.
E. Electrode configuration and orientation
The influence of electrode configuration on the
electric field distribution has been shown by measuring 51Cr-EDTA
uptake in vivo (Gehl and Mir 1999; Gehl et al, 1999). Both plate electrodes (Hartikka et al, 2001; Draghia-Akli et al, 2002b), a pair of wire electrodes (Mathiesen 1999) or two-needle devices (Tjelle et al, 2005) have been shown to be effective. The calculated
electric field distribution is more homogenous with larger electrode diameter (Miklavcic et al, 2000; Davalos et al, 2003). In order to maintain constant-current parameters, a
multiple needle electrode array is preferred, as reviewed in (Draghia-Akli and Smith 2003). Recent studies from our laboratory have shown that
electrode orientation can impact animal-to-animal variability, with randomly
oriented electrodes resulting in higher animal-to-animal variability in plasmid
uptake and expression (Figure 5).
Also, recent studies suggested that precise delivery of the plasmid formulation
in the area delineated by the electrodes is essential for optimum expression in
the large animal and potentially humans (Khan et al,
2003).
V. Conclusions
Non-viral gene delivery using plasmids followed by EP
is widely being used. The conditions of EP as well as the characteristics and
formulation of the plasmid should be optimized to ensure high levels of
expression. This review demonstrates the need to analyze all elements involved
in this delivery process, because minor alterations can dramatically affect
plasmid expression levels. We predict that improvements to the EP methodology
in large animal models constitute a step forward to deliver hormones, enzymes
or cytokines, or vaccines to humans.
Acknowledgments
The
authors would like to particularly thank Dr. Louis C. Smith for his significant
contribution to these studies and Ms. Catherine Tone for her editorial
correction of this manuscript. We acknowledge support for this study from
ADViSYS, Inc. (The Woodlands, Texas).
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