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Electroporation device for use in skeletal muscle; Clinical evaluation of pain and muscle damage

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Title: Electroporation device for use in skeletal muscle; Clinical evaluation of pain and muscle damage


1
Electroporation device for use in skeletal
muscleClinical evaluation of pain and muscle
damage
Torunn Elisabeth Tjelle1), Rune Kjeken1), Ragnar
Salte2), Dag Kvale3) and Iacob Mathiesen1) 1)
INOVIO AS, Forskningsveien 2a, 0319 Oslo, Norway
t.e.tjelle_at_inovio.com 2) Agricultural University
of Norway (NLH), 1432 Ås, Norway 3) Ullevål
University Hospital, 0407 Oslo, Norway
Introduction Background Intramuscular injection
of plasmid DNA followed by electrical stimulation
(electroporation, EP) is an efficient method for
non viral delivery of genes in vivo in mice and
rats. In larger animals, the problem of achieving
a uniform distribution of the injected DNA in
relation to the applied electrical field is
normally tried solved using multiple electrodes
in combination with a large injection volume and
a high electrical field. As an alternative
approach we have developed a new device for the
combined DNA injection and electroporation in
which the injection needles also serve as
electrodes. Since the electrical field is highest
close to a needle electrode, a near perfect match
between the DNA and the electric field is
achieved. Hence, milder electroporation
conditions might be used to obtain efficient
transfection. Methods DNA encoding
?-galactosidase or mouse IgG was injected into
sheep muscle using the newly designed electrode.
At different time points the muscle was either
dissected to assay for ?-galactosidase activity
or blood samples were taken to assay for mouse
IgG. For evaluation of pain, 6 volunteers
received multiple injections with saline followed
by electroporation with different field
strengths. Muscle damage was assessed by the
release of LDH and CK in blood. Results DNA
transfection using the new device showed an even
distribution of transfected cells along the
entire needle path. Also, the dose of mouse IgG
in sheep serum was linear with numbers of
injection sites. Electroporation in humans was
painful but tolerable and only led to negligible
muscle damage. Conclusions This first clinical
safety trial to assess pain and muscle damage
induced by electroporation of human skeletal
muscle showed that the method is tolerated
without anesthesia using voltage range and pulse
patterns effective for DNA delivery and for
eliciting immune responses in larger animals.
Materials and Methods
Fig 1b
Animals Sheep and rabbits DNA Plasmid DNA
encoding beta-galactosidase (pLacZ) and mouse
anti-NIP antibodies (pLNOgamma2bpLNOlambda)
Electroporation 5-6 pulses of 20 ms, either
unipolar or bipolar, of low amplitude (250 mA),
with 250 msec interval, were applied at the site
of injection. Analysis GFP and beta-gal were
analyzed 4-3 days after transfection. Mouse IgG
and sheep anti-mouse IgG was analyzed in serum
collected at different time points using antigen
specific ELISA. Design of electrode The
injection/electroporation device consists of an
outer "housewith an inner sledge" carrying two
syringes with DNA and needles/electrodes. A
gearing system presses the piston of the syringes
when the sledge moves forward. The device inject
DNA during the insertion of the needles. Clinical
study design The study was an open label, single
centre, non-controlled, non-randomized pilot
study to evaluate the potential pain/discomfort
and muscle damage associated with electroporation
of human skeletal muscles. Endpoints 1)
Monitoring pain and/or discomfort. 2)
Determination of muscle damage. Procedures for
the clinical study Needle electrodes where
inserted 1.3 cm into the Quadriceps of the
participant followed by EP and subsequently, the
needle electrode was withdrawn. Judgement of pain
and discomfort were performed right after each
treatment. Four different treatmens were done
(all pulses was 20 ms) 1) 1x 20V (40-70 mA) 2)
1x 250 mA 3) 6x 250 mA (approx. 70V), 250 msec
interval, 4) 6x 250 mA bipolar pulses, 250 ms
intervals. Volunteers were monitored by ECG for
arrhythmia during the whole electroporation
procedure. Creatinine kinase (CK) and lactate
dehydrogenase (LDH) was analyzed from blood
samples to determine muscle damage.
Fig 1a
1a) The injection of DNA through the electrodes
allowes a complete mach between the electrical
field (red) and the DNA (blue). 1b) The
injection/electrode device pushes the syringe
piston as the needles are inserted into the
tissue.
Results
Transfection along the entire needle path
Expression of LacZ
Increasing number of injections increases the
expression of the encoded protein linearly
Fig 3
25 ug DNA encoding ?-gal in 50 ul saline was
injected into quadriceps of anesthetized sheep
through each needle during insertion followed by
electroporation using the same needles as
electrodes. Tissue samples from the transfection
site are shown. Figure 2a shows the barrel of
transfected muscle fibers with electroporation.
Note two parallel paths, and that transfected
fibers both above and below the tendon which only
could have been achieved with injection during
insertion. Figure 2b illustrates the same device
used to inject with electroporation (EP) or
without electroporation (-EP).
The electroporation treatment was repeated two
(total of 100 ug DNA) or four times (total of 200
ug DNA) and sera were assayed after 7 days for
the encoded protein (n5 in each group). The
results show that increasing the number of
injections increases the expression
correspondingly (fig 3).
Electroporation associated pain is dependent on
voltage and pulse patterns
Electroporation does not lead to heart arrhythmia
or long lasting muscle damage
Pain associated with needle insertion was
neglible. Intensity of pain associated with
electroporation varied, but a general trend was
observed a single pulse with 20V of 20 ms
duration was less painful than a pulse with 70V.
Also single pulses were better tolerated than
treatment with 6 pulses. While single unipolar
pulses lead mostly to mild or moderate pain and
discomfort, treatment with 6 unipolar or bipolar
pulses were reported to be severely painful
(fig.1). However, all participants noted that the
short duration of the treatment (less than 1.5
second) made it tolerable in spite of the
intensive pain since after the treatment only a
minor feeling of soreness in the muscle were
reported. Since all volunteers were subjected to
the same sequence of treatments, it can not be
ruled out that the successive increase in
reported pain was caused by an increase in
sensitivity to the electroporation and not only
by differences in the procedure.
All volunteers were monitored by ECG and found
normal for heart arrhythmia both before and after
electroporation. During the electroporation
procedure and shortly after it was difficult to
monitor heart arrhythmia since the volunteers
moved as a result of muscle contraction and pain
(not shown). Electroporation lead to moderate
increases in CK and LDH activity (ltWHO grade 1).
All values except one were within the normal
range (200) 18 h after the treatment. No
correlation was observed between the increase in
CK or LDH level and the number of treatments or
pulse patterns used.
Fig 4
Fig 5
Conclusions
Injection of DNA during insertion of the needle
electrodes results in efficient transfection
giving a linear correlation between the number of
injections and level of expressed protein .
Electroporation is applicable without anesthesia
using voltage range and pulse patterns effective
for DNA delivery and for eliciting immune
responses in larger animals.
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