U.S. patent application number 11/985825 was filed with the patent office on 2008-09-25 for electroporation device and injection apparatus.
This patent application is currently assigned to INOVIO AS. Invention is credited to Bjorn David-Andersen, Iacob Mathiesen, Knut Arvid Sorensen Rekdahl, Torunn Tjelle.
Application Number | 20080234655 11/985825 |
Document ID | / |
Family ID | 30117093 |
Filed Date | 2008-09-25 |
United States Patent
Application |
20080234655 |
Kind Code |
A1 |
Mathiesen; Iacob ; et
al. |
September 25, 2008 |
Electroporation device and injection apparatus
Abstract
An apparatus is provided for injecting a fluid into body tissue,
the apparatus comprising: a hollow needle; and fluid delivery
means, wherein the apparatus is adapted to actuate the fluid
delivery means in use so as to automatically inject fluid into body
tissue during insertion of the needle into the said body
tissue.
Inventors: |
Mathiesen; Iacob; (Oslo,
NO) ; Tjelle; Torunn; (Oslo, NO) ; Rekdahl;
Knut Arvid Sorensen; (Tarnasen, NO) ; David-Andersen;
Bjorn; (Oslo, NO) |
Correspondence
Address: |
Biotechnology Law Group;c/o Portfolioip
P.O. Box 52050
Minneapolis
MN
55402
US
|
Assignee: |
INOVIO AS
San Diego
CA
|
Family ID: |
30117093 |
Appl. No.: |
11/985825 |
Filed: |
November 16, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10612304 |
Jul 3, 2003 |
7328064 |
|
|
11985825 |
|
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Current U.S.
Class: |
604/506 ;
604/256 |
Current CPC
Class: |
A61N 1/325 20130101;
A61N 1/327 20130101; A61B 8/0841 20130101; A61M 5/46 20130101 |
Class at
Publication: |
604/506 ;
604/256 |
International
Class: |
A61M 5/20 20060101
A61M005/20 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 4, 2002 |
GB |
GB 0215523.2 |
Jul 4, 2002 |
GB |
GB 0215529.9 |
Claims
1. Apparatus for injecting a fluid into body tissue, the apparatus
comprising: a hollow needle; and fluid delivery means, wherein the
apparatus is adapted to actuate the fluid delivery means in use so
as to concurrently inject fluid into body tissue during insertion
of the needle into the said body tissue.
2. Apparatus as claimed in claim 1 adapted to automatically inject
fluid into body tissue during insertion.
3. Apparatus as claimed in claim 1 or claim 2, further comprising
needle insertion means for guiding insertion of the needle into the
body tissue.
4. Apparatus as claimed in any of claim 1 or claim 2, further
comprising means for sensing when the needle has been inserted to a
sufficient depth for injection of the fluid to commence.
5. Apparatus as claimed in any of claim 1 or claim 2, further
comprising means for presetting the depth to which the needle is
inserted prior to injection of the fluid being commenced.
6. Apparatus as claimed in claim 4, wherein the sensing means
comprises an ultrasound probe.
7. Apparatus as claimed in claim 4, wherein the sensing means
comprises means for sensing a change in impedance or
resistance.
8. Apparatus as claimed in claim 1 or claim 2, further comprising:
a base for supporting the needle; and a housing for receiving the
base therein, wherein the base is moveable relative to the housing
such that the needle is retracted within the housing when the base
is in a first rearward position relative to the housing and the
needle extends out of the housing when the base is in a second
forward position within the housing.
9. Apparatus as claimed in claim 1 or claim 2, wherein the fluid
delivery means comprise piston driving means adapted to inject
fluid at a controlled rate.
10. Apparatus as claimed in claim 9, wherein the piston driving
means are actuated by the base being moved in the axial direction
relative to the housing.
11. Apparatus as claimed in claim 1 or claim 2, further comprising
means for applying a voltage to the needle.
12. Apparatus as claimed in claim 1 or claim 2, further comprising
means for recording the identity of the subject to be treated and
data from a treatment process.
13. A fluid dispense vessel for use in the apparatus as claimed in
claim 1 or claim 2, wherein a bar-code is provided on the vessel to
identify the contents thereof.
14. A method of injecting a fluid into body tissue, the method
comprising: injecting the fluid into the body tissue through a
hollow needle while the said needle is being inserted into the said
body tissue.
15. A method as claimed in claim 14, wherein the needle tip is
inserted into the skin and injection is then carried out while the
needle is inserted further into the body tissue.
16. A method as claimed in claim 14 or 15, wherein the injection is
commenced when the needle reaches a first desired depth in the body
tissue and is stopped when the needle reaches a second desired
depth in the body tissue.
17. A method as claimed in claim 14 or 15, wherein a change in
impedance or resistance is measure to determine when the needle has
reached a desired depth in the body tissue.
18. A method as claimed in claim 16, wherein the depth of the
needle in the body tissue is sensed using an ultrasound
transducer.
19. A method of electroporation wherein fluid is injected into body
tissue by the method of claim 14 or 15 and a voltage is then
applied to the needle.
20. A method of electroporation wherein fluid is injected into body
tissue by the method of claim 14 or 15, the needle is withdrawn
from the body tissue, an electrode is inserted in the place of the
needle, and a voltage is applied to the electrode.
21. A method of determining when a needle has been inserted to a
desired depth in body tissue comprising measuring a change in
impedance as the needle is inserted into the body tissue.
22. A method as claimed in claim 21, wherein two needles are
inserted into the body tissue adjacent one another and the
impedance between the needles is measured.
Description
[0001] The present invention relates to the injection of substances
into tissue and, in one preferred application, to delivery by
electroporation, i.e. the process of introducing substances into
cells during or after the application of an electric field. More
particularly, the present invention relates to a device which may
be used in delivery by electroporation.
[0002] Electroporation is used for example in the treatment of
cancer or in gene therapy. Electroporation provides a method of
delivering pharmaceuticals or nucleic acids (e.g. DNA) into cells,
e.g. skeletal muscle cells. Thus for example the muscle may be
electrically stimulated at the same time or shortly after the
pharmaceutical or DNA is injected. This method works on the
principle that cells act as an electrical capacitor generally
unable to pass current. Subjecting the cells to an electric field
creates transient permeable structures or micropores in the cell
membrane. The permeability or the pores are large enough to allow
the pharmaceuticals and/or DNA to gain access to the cells. With
time, the pores in the cell membrane close and the cell once again
becomes impermeable.
[0003] Various devices for effecting electroporation have been
suggested. U.S. Pat. No. 6,208,893 discloses an electrode template
apparatus having a plurality of bores through which a plurality of
needle electrodes extend, each bore being separately connected to a
conductor so that each of the electrodes can be connected to a
power supply in use. An insulating portion can be provided along
the midportion of each electrode so as to isolate the body tissue
adjacent the insulated part of the needle from the electric field
produced by the electrode in use. Further, one or more of the
needle electrodes may be hollow and can include openings through
which medicinal substances can be injected into the body
tissue.
[0004] EP0693951B discloses a device for the implementation of
electrochemotherapy. The device comprises electrode needles through
which electric pulses are applied. The electrode needles are hollow
so as to allow active substances to be injected locally into the
body tissue to be treated. Holes can be provided along the length
of the needles as well as at the ends thereof to improve the
distribution of injected substances. An insulating sheath can also
be provided over a part of the needle lengths as a means of
preventing the application of electrical pulses to certain
zones.
[0005] The present invention at least in its preferred embodiments
seeks to provide a device which can be used in electroporation in
vivo, in particular in gene therapy.
[0006] One problem in electroporation is that DNA is injected
intramuscularly and may become trapped between muscle bundles or in
adipose tissue between muscle cells. Further, the DNA can be
stopped by tendons or other connective tissue barriers. This will
make it difficult to obtain an even distribution of DNA over the
entire area of tissue to which an electric field is to be applied.
It is important to match the volume covered by the electric field
applied during electroporation to the site of DNA injection to
limit the distribution of the electrical field or volume of DNA. An
additional problem is that when carried out on human beings,
injections of large volumes of fluid at one site may cause
considerable pain to the patient.
[0007] From a first aspect, the present invention provides an
apparatus for injecting a fluid into body tissue, the apparatus
comprising: a hollow needle; and fluid delivery means, wherein the
apparatus is adapted to actuate the fluid delivery means in use so
as to concurrently (preferably automatically) inject fluid into
body tissue during insertion of the needle into the said body
tissue. This has the advantage that the ability to inject the fluid
gradually while the needle is being inserted leads to a more even
distribution of the fluid through the body tissue. It is also
believed that the pain experienced during injection is reduced due
to the distribution of the volume of fluid being injected over a
larger area.
[0008] In addition, the automatic injection of fluid facilitates
automatic monitoring and registration of an actual dose of fluid
injected. This data can be stored by a control unit for
documentation purposes if desired.
[0009] It will be appreciated that the rate of injection could be
either linear or non-linear and that the injection is preferably
carried out after the needles have been inserted through the skin
of the subject to be treated and while they are inserted further
into the body tissue.
[0010] Suitable tissues into which fluid may be injected by the
apparatus of the present invention include tumour tissue, skin or
liver tissue but will preferably be muscle tissue.
[0011] Preferably the apparatus further comprises needle insertion
means for guiding insertion of the needle into the body tissue.
Still more preferably, the rate of fluid injection is controlled by
the rate of needle insertion. This has the advantage that both the
needle insertion and injection of fluid can be controlled such that
the rate of insertion can be matched to the rate of injection as
desired. It also makes the apparatus easier for a user to
operate.
[0012] If desired means for automatically inserting the needle into
body tissue could be provided.
[0013] A user could choose when to commence injection of fluid.
Ideally however, injection is commenced when the tip of the needle
has reached muscle tissue and the apparatus preferably includes
means for sensing when the needle has been inserted to a sufficient
depth for injection of the fluid to commence. This means that
injection of fluid can be prompted to commence automatically when
the needle has reached a desired depth (which will normally be the
depth at which muscle tissue begins). The depth at which muscle
tissue begins could for example be taken to be a preset needle
insertion depth such as a value of 4 mm which would be deemed
sufficient for the needle to get through the skin layer.
[0014] In one preferred embodiment the sensing means comprises an
ultrasound probe.
[0015] In an alternative preferred embodiment the sensing means
comprises means for sensing a change in impedance or resistance. In
this case, the means may not as such record the depth of the needle
in the body tissue but will rather be adapted to sense a change in
impedance or resistance as the needle moves from a different type
of body tissue into muscle. Either of these alternatives provide a
relatively accurate and simple to operate means of sensing that
injection may commence. The depth of insertion of the needle can
further be recorded if desired and could be used to control
injection of fluid such that the volume of fluid to be injected is
determined as the depth of needle insertion is being recorded.
[0016] The apparatus preferably further comprises: a base for
supporting the needle; and a housing for receiving the base
therein, wherein the base is moveable relative to the housing such
that the needle is retracted within the housing when the base is in
a first rearward position relative to the housing and the needle
extends out of the housing when the base is in a second forward
position within the housing. This is advantageous for a user as the
housing can be lined up on the skin of a patient, and the needles
can then be inserted into the patient's skin by moving the housing
relative to the base.
[0017] As stated above, it is desirable to achieve a controlled
rate of fluid injection such that the fluid is evenly distributed
over the length of the needle as it is inserted into the skin.
Preferably therefore, the fluid delivery means comprise piston
driving means adapted to inject fluid at a controlled rate.
[0018] The piston driving means could for example be activated by a
servo motor. Preferably however, the piston driving means are
actuated by the base being moved in the axial direction relative to
the housing.
[0019] It will be appreciated that alternative means for fluid
delivery could be provided. Thus, for example, a closed container
which can be squeezed for fluid delivery at a controlled or
non-controlled rate could be provided in the place of a syringe and
piston system.
[0020] The apparatus described above could be used for any type of
injection. It is however envisaged to be particularly useful in the
field of electroporation and so it preferably further comprises
means for applying a voltage to the needle. This allows the needle
to be used not only for injection but also as an electrode during,
electroporation. This is particularly advantageous as it means that
the electric field is applied to the same area as the injected
fluid. There has traditionally been a problem with electroporation
in that it is very difficult to accurately align an electrode with
previously injected fluid and so user's have tended to inject a
larger volume of fluid than is required over a larger area and to
apply an electric field over a higher area to attempt to guarantee
an overlap between the injected substance and the electric field.
Using the present invention, both the volume of fluid injected and
the size of electric field applied may be reduced while achieving a
good fit between the electric field and the fluid.
[0021] As an aid to medical staff who may treat a large number of
patients in a day, the apparatus may further comprise means for
recording the identity of a subject to be treated and data from a
treatment process.
[0022] Further, a fluid dispense vessel may be provided for use in
the apparatus of the invention, in which a bar-code is provided on
the vessel to identify the contents thereof. This barcode could be
recognised by a pulse generator used in electroporation which would
be programmed to automatically set up the required injection speed
and electroporation conditions for the bar code.
[0023] From a further aspect, the present invention provides a
method of injecting a fluid into body tissue, the method
comprising: injecting the fluid into the body tissue through a
hollow needle while the said needle is being inserted into the said
body tissue. The injection of fluid gradually while the needle is
being inserted leads to a more even distribution of the fluid
through the body tissue. It is also believed that the pain
experienced during injection is reduced due to the distribution of
the volume of fluid being injected over a larger area.
[0024] Preferably, the needle tip is first inserted into the skin
and injection is then carried out while the needle is inserted
further into the body tissue.
[0025] Still more preferably, the injection is commenced when the
needle reaches a first desired depth in the body tissue and is
stopped when the needle reaches a second desired depth in the body
tissue.
[0026] The method of injection described above may advantageously
be used in conjunction with a method of electroporation wherein
fluid is injected into body tissue by the method of injection of
the invention and a voltage is then applied to the needle.
[0027] The method of injection described above may advantageously
be used in conjunction with an alternative method of
electroporation wherein fluid is injected into body tissue by the
method of injection of the invention, the needle is withdrawn from
the body tissue, an electrode is inserted in the place of the
needle, and a voltage is applied to the electrode.
[0028] Gene therapy by electroporation involves administering a
dose of between about 10.mu.L and 10 ml (e.g. between 10.mu.L and 1
ml, preferably between 100.mu.L and 1 ml) of DNA solution. DNA is
toxic if too much is incorporated into cells and so the quantity of
DNA in solution must not be too high. Thus, the quantities of
solution are relatively small and, especially in larger animals
such as human beings, it is difficult to administer both DNA and
electric field to the right place in the muscle. Further, as the
cells being treated should not be damaged, the electroporation
device should be much gentler than the prior art devices whose
primary use is in the treatment of cancer where the treated cells
are killed. Ideally therefore, the electroporation device should
not produce undue fields and should also not include any relatively
blunt or bulky tissue piercers.
[0029] From a first aspect, the present invention provides an
electroporation device comprising: a needle for injecting a
substance into body tissue; and an insulating sheath adapted to
surround the needle and having one or more apertures formed along
the length thereof through which the electric field may propagate
in use, wherein the needle is axially moveable relative to the
sheath.
[0030] The device of the invention has the advantage that if the
needle is also used as an electrode, as the needle is axially
moveable relative to the sheath, the needle can be withdrawn so
that the insulating sheath completely surrounds the needle after
the device has been inserted into the body tissue and before the
electric field generating means are activated. Thus in use, the
electric field propagates through the apertures in the sheath, and
so the formation of uneven electric field strengths in the body
tissue to be treated is avoided as no edge effects are created.
[0031] Preferably, the needle for injecting a substance into body
tissue also constitutes an electrode via which an electric field is
propagated in use. Thus, in this preferred embodiment, the needle
is connectable to a voltage source. It will of course be
appreciated that, in one embodiment, the needle could remain
connected to the voltage source at all times.
[0032] However if necessary, the device may be adapted to allow the
needle to be removed from the insulating sheath after injection of
the substance into the body tissue so that the needle can be
replaced by an electrode rod prior to activation of the electric
field. This would be advantageous for example to avoid the release
of unwanted metal ions by the needle which could be caused by the
provision of an electric charge on the needle. In this embodiment,
the electrode rod would be arranged so as to be completely
surrounded by the sheath in use so that again, no edge effects
would be produced by the electric field in use.
[0033] The sheath could be formed of any electrically insulating
and biologically compatible material. Preferably however, the
sheath is formed from polytetrafluoroethylene
(Teflon.sup..RTM.).
[0034] Any number of apertures could be provided in the insulating
sheath. In one preferred embodiment, the apertures are provided
along one axially extending line on the sheath only. In an
alternative preferred embodiment, the apertures are provided so as
to be spaced around the circumference of the sheath. The actual
number and arrangement of apertures provided in the sheath will
depend on the electric field patterns required in the tissue to be
treated.
[0035] The apertures in the insulating sheath could be formed in a
number of ways such as but not limited to: cutting through the
sheath, pushing the apertures out or laser ablation. Where
apertures are required on one side only of the sheath, during
aperture formation a rod can be provided within the sheath to
prevent holes forming on both sides.
[0036] The electroporation device of the invention could be used
alone. Preferably however, two or more electroporation devices are
used together and if required, any number of the devices could be
used Thus for example, a group of four, six or eight devices could
be used. Where one or more devices are used, the needles and
sheaths can be mounted to extend downwardly through a block in
which they are arranged adjacent to one another. Consequently, it
will be appreciated that any number of needles (i.e 1 or more could
be used).
[0037] Preferably, means are provided such that in use the depth of
insertion of a needle is determined and injection of a substance
into the body tissue to be treated is commenced when the needle has
reached a desired depth.
[0038] This is believed to be novel and inventive in its own right
and so from a further aspect the present invention provides a
device comprising a needle for injection of a substance into body
tissue, and means for sensing the depth of insertion of the needle
and commencing injection of a substance via the needle when a
desired depth has been reached.
[0039] Various means could be provided to determine that the needle
has reached a desired depth for injection to commence. For example,
means for determining the electrical resistance of the tissue which
will vary depending on tissue type (dermis, fat or tissue) could be
provided. Preferably however, a moveable contact can be provided on
the device such that in use, the contact determines when the needle
has been inserted to a sufficient depth into the body tissue to be
treated and then causes injection of a substance to commence. This
allows automatic injection of a substance to commence when the
needle reaches the correct depth in the body tissue to be treated.
The injection can be carried out either while the needle is
stationary or while it is continuing to be inserted.
[0040] Still more preferably, the moveable contact further
determines when the needle has been inserted to the maximum depth
at which injection should be carried out and then causes injection
of the substance to stop. In this way it is possible for the
substance to be automatically injected over the height of tissue
over which an electric field will be produced in use.
[0041] Viewed from a further aspect the invention provides a method
of electroporetic treatment of a human or nonhuman animal (e.g. a
mammal, bird or reptile), said method comprising inserting the
needle of a device according to the invention into tissue (e.g.
muscle tissue) in said animal, injecting an active agent (e.g. a
pharmaceutical or nucleic acid) through the needle into the tissue,
withdrawing the needle such that the tip thereof is within the
sheath, and applying an electric field between the needle and an
electrode.
[0042] It will be appreciated that the electrode could be provided
by the needle of a second device according to the invention
disposed inside a further sheath. Alternatively, the electrode
could be a different type of electrode which had been inserted into
the body tissue or an electrode which had been applied to the skin
surface.
[0043] Viewed from a still further aspect the invention provides a
method of electroporetic treatment of a human or non-human animal
(e.g. a mammal, bird or reptile), said method comprising inserting
the needle of a device according to the invention into tissue (e.g.
muscle tissue) in the animal, injecting an active agent (e.g. a
pharmaceutical or nucleic acid) through the needle into the tissue,
withdrawing the needle from the sheath, inserting a first electrode
into the sheath such that the tip of the first electrode does not
extend out of the sheath into the tissue, and applying an electric
field between the first electrode and a second electrode.
[0044] It will be appreciated that the second electrode could be
provided by the needle of a second device according to the
invention disposed inside a sheath. Alternatively, the electrode
could be a different type of electrode which had been inserted into
the body tissue or an electrode which had been applied to the skin
surface.
[0045] The device according to the invention could for example be
used in the method of WO98/43702, the contents of which are herein
incorporated by reference. Preferably, the device would be used
with a square bipolar electric pulse.
[0046] In the device of U.S. Pat. No. 6,208,893 as discussed above,
the needle electrodes are inserted axially from above into the
respective bores in use and are removed by being drawn axially
outward after use. The present inventors have identified a problem
with the use of such a device in which the bores become
contaminated with the blood of an animal or person when the needles
are withdrawn after use as the tips of the needles pass through the
bores. Thus, the apparatus can only be reused after very thorough
disinfection which is time consuming and expensive.
[0047] From a further aspect, the present invention seeks to
provide a device which overcomes this problem. In a first aspect,
the present invention provides a device for use in electroporation
comprising a housing formed in two or more parts, wherein the parts
are moveable relative to one another to open and close the housing,
and a groove is formed in a surface of at least one of said parts
in such a way as to form a bore extending through the housing when
the housing is closed. Preferably the bore is adapted to receive a
needle in use and the needle can be inserted and removed from the
bore by opening the housing.
[0048] Thus, as the needle can be removed from the bore by opening
the housing and so lifting it out of an open groove, there is no
need to remove the needle from the bore by pulling it out in the
axial direction Consequently blood and any other bodily fluids left
on the tip of the needle after use need not be brought through the
bore and so the housing will not be contaminated as in the prior
art devices.
[0049] The parts of the housing could for example be held together
in the closed position by a removable belt extending around the
outside of the housing. Preferably however, the parts are hingedly
attached to one another. This has the advantage of making the
housing particularly easy to open and close.
[0050] The housing could for example be formed in four parts which
make up the quarters of a cuboid, each part having a groove with
the cross section of a quadrant formed at the inner corner thereof.
Alternatively, the housing could be formed in two parts, with a
groove having for example a semi-circular or square cross section
formed on the inner surface of one part while the surface of the
other part is flat. Preferably however the housing is formed of two
parts, a groove of semicircular cross section being provided on the
inner surface of each part and being positioned to form a bore of
circular section from the two grooves when the housing is closed.
It will be appreciated that in this arrangement, the parts of the
housing can be hingedly attached together at one end thereof in a
manner allowing simple manufacture and use of the device. Further,
the circular cross section of the bore is particularly advantageous
as the needles to be held therein are normally circular in cross
section.
[0051] Still more preferably, the housing is formed to receive two
needles in two respective bores. Although the device could be used
with any number of needles, two needles are often required to carry
out electroporation and so this is a particularly preferred
arrangement.
[0052] The needles could be connected to an electric power supply
by standard means such as cables attached to an end of the needle
extending out of the housing. Preferably however an electrical
contact is provided for or within the or each bore so that a needle
within the bore is brought into contact with an electrical power
supply when the housing is closed. This has the advantage that a
user need not spend time connecting a needle to a power supply by
attaching cables etc. and so is much quicker and simpler to
use.
[0053] Still more preferably, the device is configured so as to
lock the needle in position within the bore when the housing is
closed in use. Thus, no additional means need be provided to stop
the needle from moving relative to the housing during insertion of
the needle into the body tissue to be treated and the subsequent
electroporation process.
[0054] In one preferred embodiment, a foot pedal could be provided
to activate the power supply when required for electroporation.
This has the advantage that a user would have their hands free at
all times to hold the device and the needle(s) in place in an
animal or person being treated. It will be appreciated however that
alternative means such as a switch provided on the needle holder
could be provided for activating and deactivating the power
supply.
[0055] The device of the invention could be used with any standard
known, approved needles and injection assemblies or syringes.
[0056] In one preferred embodiment, the device could be used with
one or more needles, wherein each said needle is surrounded by an
insulating sheath, the sheath having one or more apertures formed
along the length thereof. The use of such insulated needles has the
advantage of reducing the production of edge effects when the
needle is used as an electrode.
[0057] Preferably, the same needle is used for injecting a
substance into the body tissue to be treated and applying an
electric field. Where necessary however, the needle could be
withdrawn front the sheath arranged within a bore of the housing
after injection of a substance into the body tissue to be treated
and substituted by an electrode rod for carrying out the
electroporation. This would be advantageous for example to avoid
the release of unwanted metal ions by the needle which could be
caused by the provision of an electric charge on the needle. In
this embodiment, the electrode rod could be arranged to be
completely surrounded by an insulating sheath to avoid the
production of edge effects by the electric field in use. Further,
the insulating sheath arranged within the bore would protect the
bore from contamination by blood and/or other bodily fluids as the
needle was withdrawn axially from within the bore and sheath.
[0058] Preferably, even if the needle is not completely withdrawn
from the sheath after injection of a substance into the body
tissue, the needle is still axially moveable relative to the
sheath. This allows the needle to be withdrawn inside the sheath
after injection so that it is fully surrounded by the sheath before
the application of an electric field. This has the advantage of
further reducing the production of edge effects by the electric
field in use.
[0059] The sheath could be formed of any electrically insulating
and biologically compatible material. Preferably however, the
sheath is formed from polytetrafluoroethylene
(Teflon.sup..RTM.).
[0060] Preferably, the needles used for injection of a substance
into the body tissue to be treated are attached to syringe devices
via which injection is carried out. It would also be possible
however for the needles to be provided separately for attachment to
injection means at an appropriate time.
[0061] Preferably, the device is provided with means for
determining the depth of insertion of a needle into the body tissue
to be treated in use and for automatically commencing injection of
a substance into the body tissue to be treated when a desired depth
of the needle has been reached.
[0062] Preferably a moveable contact can be provided on the device
such that in use, the contact determines when the needle has been
inserted to a sufficient depth into the body tissue to be treated
and then causes injection of a substance to commence. This allows
automatic injection of a substance to commence when the needle
reaches the correct depth in the body tissue to be treated. The
injection can be carried out either while the needle is stationary
or while it is continuing to be inserted.
[0063] Still more preferably, the moveable contact further
determines when the needle has been inserted to the maximum depth
at which injection should be carried out and then causes injection
of the substance to stop. In this way it is possible for the
substance to be automatically and accurately injected over the
height of tissue over which an electric field will be produced in
use.
[0064] Viewed from a further aspect, the present invention provides
a method of electroporation treatment of a human or non-human
animal (e.g. a mammal, bird or reptile), said method comprising
inserting a needle held in a device according to the invention into
tissue (e.g. muscle tissue) in said animal, injecting an active
agent (e.g. a pharmaceutical or nucleic acid) through the needle
into the tissue, applying an electric field between the needle and
an electrode, removing the needle from the tissue and opening the
housing of the device to remove the needle therefrom.
[0065] Preferably, the needle could be pushed further into the
tissue after injection and before the application of an electric
field to enable the electric field to be applied over the full
height of injected fluid.
[0066] It will be appreciated that the electrode could be provided
by a second needle held in a or the device according to the
invention. Alternatively, the electrode could be a different type
of electrode which had been inserted into the body tissue or an
electrode which had been applied to the skin surface.
[0067] It will further be appreciated that the needle could be any
known approved form of needle or any other type of needle described
herein.
[0068] In an alternative preferred method of treatment, the needle
is removed from the device according to the invention after
injection and replaced by an electrode, an electric field being
applied between the two electrodes before the electrode is
removed.
[0069] The device according to the invention could for example be
used in the method of WO 98/43702, the contents of which are herein
incorporated by reference. Preferably, the device would be used in
an electroporation method in which a square uni or bipolar electric
pulse is applied to the electrode.
[0070] From a further aspect, the present invention provides a
method of determining when a needle has been inserted to a desired
depth in body tissue comprising measuring a change in impedance as
the needle is inserted into the body tissue.
[0071] Although this could be achieved in various ways, two needles
are preferably inserted into the body tissue adjacent one another
and the impedance between the needles is measured.
[0072] Preferred embodiments of the invention will now be
described, by way of example only, and with reference to the
accompanying drawings in which:
[0073] FIG. 1 is a schematic side elevation view of an
electroporation device according to a first embodiment of the
invention;
[0074] FIGS. 2a to 2c are schematic side elevation views showing
three stages in the operation of an electroporation device
according to the first embodiment of the invention including a skin
contact device;
[0075] FIG. 3 is a perspective view of an electroporation device
according to a second embodiment of the invention in an open
position;
[0076] FIG. 4 is a perspective view of the device of FIG. 3 in the
closed position;
[0077] FIG. 5 is a schematic plan view of a part of the device of
FIG. 3 holding a needle and injection device;
[0078] FIG. 6 is a schematic elevational view of an alternative
needle and injection device for use with the device of FIG. 3;
[0079] FIG. 7 is a side perspective view of an electroporation
device according to a third embodiment of the invention;
[0080] FIG. 8 is an underneath perspective view of the device of
FIG. 7;
[0081] FIG. 9 is a side perspective view of the base of the device
of FIG. 7;
[0082] FIG. 10 is a side elevational view of the base of the device
of FIG. 7;
[0083] FIG. 11 is a top plan view of the base of the device of FIG.
7;
[0084] FIG. 12 is a side elevational view of the base of the device
of FIG. 7 from the opposite side to that shown in FIG. 10;
[0085] FIG. 13 is a cross sectional side view of the cover of the
device of FIG. 7;
[0086] FIG. 14 is a side view of the device of FIG. 7 when fully
assembled ready to start the process;
[0087] FIG. 15 is a side view of the device of FIG. 7 at the point
at which the needles have penetrated the skin, ready to start the
injection and needle insertion process;
[0088] FIG. 16 is a side view of the device of FIG. 7 halfway
during needle insertion;
[0089] FIG. 17 is a side view of the device of FIG. 7 when needle
insertion and injection have been completed (i.e. when the device
is ready for electroporation to be carried out, before the needles
are withdrawn);
[0090] FIG. 18a is an exploded view of the gear mechanism of the
device of FIG. 7 for driving the needle insertion and injection
process;
[0091] FIG. 18b is a view of the gear mechanism of FIG. 18a mounted
on the base unit;
[0092] FIG. 18c is a view of the base unit showing the gear
mechanism of FIG. 18a and the rack member in place.
[0093] FIG. 19 shows the amount of SEAP used in serum in a test
using a device according to the invention; and
[0094] FIGS. 20a and 20b shows the results of the test using a
beta-galactosidase expressing vector introduced using a device
according to the invention.
[0095] As shown in FIG. 1, an electroporation device according to a
first embodiment of the invention comprises two separate needle
assemblies 2 mounted adjacent to one another in a support block 4.
Each needle assembly 2 comprises a hollow needle 6 having a sharp
end 8 which is open to allow the injection of fluids via the
opening. The other end of each of the needles 6 is connected to a
fluid holding chamber 10 having a piston 12 arranged therein so as
to form a syringe arrangement for injecting fluid via the needles
in use. These syringes may be standard single-use syringes.
[0096] First and second electrically insulating sheaths 14 made of
Teflon.sup..RTM. and having a greater cross sectional diameter than
that of the needles 6 are arranged to extend around the needles 6.
Three apertures 16 spaced apart in the axial direction are provided
along the length of each sheath 14. The device is configured so as
to allow axial movement of the needles 6 relative to the sheaths
14.
[0097] A voltage supply 18 is provided on the support block 4 which
can be connected and disconnected from the needles 6 of the
electroporation device.
[0098] In use, a required dose of DNA (which could for example be
100.mu.L) is provided in each of the fluid holding chambers 10 and
the needles 6 are inserted into the skin of an animal or person to
be treated. It is advantageous that the volume of fluid for
injection should be small as this will insure that the injected
fluid is kept close to the shaft of the needle (i.e. will be kept
within a high electric field strength zone during electroporation).
At this stage, the sharp ends 8 of the needles 6 extend beyond the
Teflon sheaths 14 and so provide a sharp point for piercing the
skin and penetrating into the muscle or body tissue to be treated.
During insertion, the relative position of the needles 6, sheaths
14 and support block 4 does not vary as the elements are locked
into place relative to one another. The needles are then inserted
further until they reach the correct depth in the muscle or other
body tissue to be treated. Once they have reached this depth and
while still being inserted, the DNA is injected into the muscle by
pushing downwardly on the pistons 12 to empty the fluid holding
chambers 10. If necessary, the needles can then be pushed further
down into the muscle after injection. This ensures that the needles
acting as electrodes cover the area into which the fluid has been
injected.
[0099] After insertion of the needles and once the DNA has been
injected, the needles 6 are withdrawn slightly (i.e. moved axially
towards the support block 4) relative to the Teflon sheaths 14
which remain in their original inserted position. Thus, the sharp
ends 8 of the needles 6 are retracted to locate within the Teflon
sheaths 14. Once the needles 6 have been retracted as described,
the voltage source 18 is activated and electroporation proceeds
with each of the needles 6 acting as an electrode. The electric
field produced by the needles 6 acting as electrodes propagates
into the muscle or body tissue to be treated via the apertures 16
formed along the length of the Teflon shields 14. This has the
advantage that no unwanted edge effects are created in the muscle
or body tissue to be treated.
[0100] In a further improvement to the device of FIG. 1 (as shown
in FIGS. 2a to 2c), means are provided to sense when during
insertion the needles 6 are at the correct depth in the muscle or
body tissue for injection of the DNA to begin and to automatically
move the pistons 12 to effect the injection. These means comprise a
moveable skin contact 20 which contacts the skin S as shown in
FIGS. 2a to c. As the needles 6 are inserted into the muscle or
body tissue to be treated, the contact 20 is pushed upwardly
towards the support member 4. The contact member 20 is attached to
a lever mechanism consisting of a substantially vertical link 22
extending upwardly from the contact member 20 and a lever 24 which
is attached at a first end to the vertical link 22. The lever 24 is
attached at its other end to means 26 for causing the pistons 12 to
move downwardly. The lever is adapted to pivot about a point 28 on
the support member 4 located between the two ends of the lever 24.
Thus, as the contact 20 moves upwardly relative to the support
member 4 in use, the lever 24 pivots causing the piston moving
means 26 to push the pistons down gradually so as to effect
injection of the fluids over the height of the needles being
inserted. As shown, the piston moving means comprise a vertical
member 27 attached to the lever 24 so as to move downwardly as the
lever pivots and a cross piece 30 attached to the other end of
vertical member 27 which acts to push the pistons down as it moves
downwardly with the vertical member.
[0101] The relative location of the skin contact 20 and lever
mechanism can be adjusted to ensure injection of the fluids once
the needles have reached the muscle tissue and while they are being
inserted further into the tissue to ensure a uniform distribution
of sample in the area around the electrodes in the muscle.
[0102] FIG. 2a shows the device before the pistons have been pushed
down with the tips of the needles just inserted into the skin. FIG.
2b shows the device when the needles are fully inserted to the
required depth in the muscle tissue and the pistons 12 have been
fully depressed by the action of the lever mechanism. FIG. 2c shows
the device once the needles have been attached to a power supply 18
after injection of the fluids. As shown, the syringes have been
removed although this is not essential.
[0103] In alternative embodiments, lasers or sensors could be used
to detect the depth of insertion of the needles and automatically
initiate injection of the fluids at a desired depth instead of the
mechanical skin contact arrangement described above.
[0104] The contact or sensors can be further adapted to sense when
the needles 6 have reached a depth in the body tissue at which
injection of the fluids should stop so as to ensure that fluid is
only injected into the height of body tissue to which an electric
field will be applied in use.
[0105] It will be appreciated that one advantage of the embodiment
of the invention described above is that known cannula devices
which are already on the market and so have marketing approval can
be used to provide the needle and sheath assemblies of the device,
the only modification which is required being the formation of the
apertures 16 in the sheaths. Thus, the use of such commercially
available cannulas can ensure rapid and inexpensive regulatory
clearance. One example of a known cannula device which could be
used is the 0.8/25 mm diameter Venflon.sup..RTM. sold by BOC Ohmeda
AS of Helsingborg, Sweden.
[0106] In an alternative embodiment of the invention (not shown)
the needles 6 can be withdrawn from the muscle or body tissue to be
treated after the DNA has been injected into it and electrodes
having a similar shape but made of an alternative metal such as
stainless steel can be inserted before electroporation is carried
out. This could be useful for example in a situation where
biologically incompatible metal ions would be emitted if the
needles 6 were also used as the electrodes.
[0107] As shown in FIG. 3, a device according to a second
embodiment of the invention comprises a housing 41 made up of two
halves 42, 44 which are joined together by a hinge 46 Each half 42,
44 of the housing is a rectangular solid and the hinge 46 is
provided between adjacent end faces thereof so that the upper plane
rectangular surfaces of each half of the housing can be pivoted
towards each other until the upper surface 48 of the first half 42
lies directly above the upper surface 50 of the second half 44. In
this position, the housing is said to be closed and this is shown
in FIG. 4.
[0108] From FIG. 3, it can be seen that recesses or grooves are
formed in the upper surfaces 48, 50 of each of the two halves 42,
44. Each groove is semi-circular in cross section and has a wider
portion 52 extending from a first side 54 of the housing half which
leads into a narrower portion 56 which extends to the other side 58
of the housing half. Thus, in use the needle 60 of a syringe device
fits into the narrower portion 56 while the syringe or injection
part 62 adjacent the needle fits into the wider portion 52 as shown
in FIG. 5.
[0109] The upper surface 48 of the first half 42 of the housing 41
has two recesses of the type described above formed therein which
are laterally spaced from one another. Two recesses are also formed
in the upper surface 50 of the second half 44 at corresponding
locations such that, when the housing is closed so that the first
48 and second 50 surfaces are arranged one above the other, the
recesses in the first and second surfaces join to form two bores 63
within which respective needles and syringe or injection devices
may be held.
[0110] Also as shown in FIG. 3, an electrical contact element 64 is
provided in the narrower part 56 of each recess in the first half
42 of the housing. The electrical contact elements 64 are connected
to an electrical power source V and arranged so that a needle
placed within the recess will automatically be brought into contact
with the electrical contact element when the housing is closed.
[0111] The device shown and described with reference to FIGS. 3 and
4 can be used with any standard approved needle and syringe device
such as for example the Sterile EO CE0123, Sterican 0.40.times.40
mm BL/LB, 27G.times.11/2.
[0112] In an alternative embodiment, the device can be used with
syringe devices including needles 6 which are surrounded by
insulating sheaths 14 such as those shown in FIG. 1 for use with
the device of the first embodiment of the invention. A syringe
device of this type for use in the second embodiment of the
invention is shown in FIG. 6. As can be seen, the device includes a
needle 6 and a Teflon.sup..RTM. sheath 14. As shown in FIG. 6, the
insulating sheath 14 which surrounds the needle has three apertures
16 spaced apart from one another in the axial direction and
provided along the length of the sheath. A fluid container 10
including a piston 12 is provided at one end of the needle for
injecting fluid therethrough. In one embodiment, the needle is
axially moveable relative to the sheath so that after it has been
inserted into the body tissue to be treated, the needle is
withdrawn into the sheath. This avoids the formation of harmful
edge effects when an electric field is applied to the needle. Known
cannula devices which are already on the market and so have
marketing approval can be used to provide the needle and sheath
assemblies of the device, the only modification which is required
being the formation of the apertures 16 in the sheaths. Thus, the
use of such commercially available cannulas can ensure rapid and
inexpensive regulatory clearance. One example of a known cannula
device which could be used is the 0.8/25 mm diameter
Venflon.sup..RTM. sold by BOC Ohmeda AB of Helsingborg, Sweden.
[0113] If desired, means may be provided with the device of the
second embodiment of the invention to sense when the needles 6, 60
are at the correct depth in the muscle or body tissue for injection
of the DNA to begin and to automatically move the pistons 12 to
effect the injection in the same way as for the first embodiment of
the invention as shown in FIGS. 2a to 2c. When used with the device
of the second embodiment however, the lever 24 pivots about point
28 on the housing 41 rather than support block 4.
[0114] A method of electroporation treatment using the device of
FIGS. 3 and 4 will now be described. This method could be carried
out on any human or non-human animal. A required dose of DNA (which
could for example be 100.mu.l) is provided in each fluid container
12, 62. Then the syringe devices are inserted into respective
recesses 52, 56 in one half 42 of the housing 41 and the housing is
closed so that the needles are held firmly in place in the
respective bores formed by the recesses. The needles are then
inserted into the body tissue as shown at FIG. 2a. The needles are
pushed down to the correct depth for injection of the DNA and this
is then carried out. After the injection, the needles are then
pushed slightly further down into the body tissue and the electric
power supply V is activated by a foot pedal (not shown) to apply an
electric field via the needles.
[0115] After the electric field has been applied, the needles are
removed from the body tissue and the housing is opened so that the
needles can be lifted out of the recesses. The housing is then
ready to be reused with new needles.
[0116] A third and most preferred embodiment of the invention will
now be described with reference to FIGS. 7 to 13. As shown in FIG.
7, the device comprises a base 70 which holds two syringe devices
72, 74 and a cover 76. The base 70 is capable of sliding relative
to the cover 76. This motion simultaneously inserts both the
needles 78, 80 of the syringe devices and drives a gear mechanism
(see FIG. 16) to cause injection of fluid via the needles. This
will be described in greater detail below.
[0117] The base 70 is shown in FIG. 9. It is formed from plastic
(for example polyvinyl chloride) although it could also be produced
in other suitable materials such as stainless steel (or mouldable
plastics). The base 70 has a long solid body which is substantially
rectangular in plan view. A bottom surface 82 thereof is
substantially flat and is adapted to rest slidably on an inner
surface of the cover 76. A first end 84 of the base 70 is adapted
to slide forwardly into engagement with the cover 76 and so
comprises a contact surface 86 extending upwardly at an acute angle
(45.degree. in the embodiment shown) from an end of bottom surface
82. A chamfer 88 is provided between the angled contact surface 86
and the upper surface 90 of the base 70.
[0118] The upper surface 90 of the base 70 is adapted to receive
syringe devices 72, 74. The first part 92 of the upper surface 90
extends rearwardly from chamfer 88 to form a first planar surface
which is parallel to bottom surface 82 and extends a short distance
(preferably about 16 mm or about 6% of the total length of the base
70) rearwardly of the chamfer 88 end. Contacts 91 for providing
electrical power to each needle are provided on the base, and power
may be supplied to these via wires connected to any standard plug
and socket arrangement. The contacts also form a stability
arrangement 91 for holding and supporting the needles during
electroporation.
[0119] The combined contact and stability arrangement 91 is
provided by two hooked metal plates attached to the angled contact
surface 86. The hooked metal plates are electrically connected to
wires (not shown) which may supply electrical power from any
suitable power supply via the above-mentioned pulg and socket
arrangement (not shown). Furthermore, at chamfer 88, springs 89 are
provided, the springs also being electrically connected to the
above-mentioned wires. The springs 89 serve to press the needles 78
and 80 against their respective contacts 91, thereby ensuring
electrical connection.
[0120] Beyond first part 92, a pair of parallel syringe holding
grooves 94, 96 extending in the direction of the longitudinal
extent of base 70 are provided. The grooves 94, 96 have external
side walls which are coplanar with and form part of the side walls
96, 100 of base 70 and have a central wall 102 separating the two
grooves. The external side walls and central wall have straight
sides and extend above the level of first part 92 of upper surface
90 (preferably by about 9 mm). Further the grooves 94, 96 are
formed with semi-circular bases having a radius of curvature of 3.3
mm and the lowest part of the grooves is located above the first
part 92 of upper surface 90 (preferably by about 2 mm). The grooves
94, 96 extend over a distance of about 2 to 3 times the length of
first part 92 of upper surface 90 (preferably over about 16% of the
total length of the base or about 41 mm).
[0121] Rearwardly of the parallel syringe holding grooves 94, 96, a
second planar surface 104 extends parallel with the bottom surface
82 and on the same level as the lowest part of grooves 94, 96. The
second planar surface has a similar length to the parallel syringe
holding grooves 94, 96 (and preferably extends over about 13% of
the total length of the base or about 33 mm).
[0122] Rearwardly of the second planar surface 104, a notch 106 is
cut out of the base 70 extending across the base (i.e.
perpendicular to the longitudinal extent thereof). The notch 106
has straight side edges 108, 110 and is cut out to a level below
the second planar surface 104 (preferably by about 7.5 mm). The
notch preferably has a dimension of about 3 mm in the longitudinal
extent of the base 70).
[0123] At the side of notch 106 facing away from the second planar
surface 104, a third planar surface 112 extending parallel to the
bottom surface 82 is provided at a level above the base of notch
106 but below second planar surface 104. (The third planar surface
112 is preferably at a level about 3 mm below second planar surface
104). The third planar surface 112 preferably extends over about 31
of the total length of the base or over a distance of about 79
mm.
[0124] Immediately rearwardly of the third planar surface 112 a
fourth planar surface 114 extends parallel to the bottom surface 82
and above the third planar surface (preferably about 14.3 mm above
the third planar surface). A straight edge 116 extending
perpendicular to the longitudinal direction joins the third and
fourth planar surfaces to each other.
[0125] The second end 118 of the base 70 comprises a planar surface
extending perpendicular to the longitudinal extent and joining the
fourth planar surface 114 to the bottom surface 82.
[0126] A groove 120 with straight edges is cut out from the upper
surface 90 of the body of the base 70, the groove extending
longitudinally along the centre of the base from the second end 118
thereof to a point within the third planar surface 112 close to the
notch 106 The groove 120 has a flat bottom which is about 4 mm
below the level of the third planar surface 112. The groove is
about 4.1 mm wide.
[0127] An aperture 122 is cut through one side of the base 70
underneath the fourth planar surface 114 to the groove 120 to form
a longitudinally extending guide in which a pin may slide. The
aperture is preferably 4.2 mm high and about 29 mm long, is centred
about 4.7 mm below the fourth planar surface 114 and extends from
about 8 mm from the second end 118 of the base 70.
[0128] A circular aperture 124 is cut through the base 70 to the
groove 120 and is located on the same side of the base 70 as
aperture 122 underneath the fourth planar surface 114. The aperture
124 is centred on a point about 8 mm from the straight edge 116
joining the third 112 and fourth 114 planar surfaces arid about 5.3
mm below the fourth planar surface 114. The aperture 124 has a
diameter of about 3 mm.
[0129] A second circular aperture 126 is cut through the base 70 on
the other side from and centred on the same point as the circular
aperture 124. The second circular aperture 126 has a diameter of
about 10 mm.
[0130] A gear wheel 148 on an axle 150 is mounted externally of the
base 70 by passing the axle through the first circular aperture 124
and then through the second circular aperture 126 and securing the
axle using a nut on the other side of the base 70. In use, the base
is moved forwardly relative to the cover and the gear wheel 148
engages on a rack 146 provided on toothed member 170, or on a
toothed track provided on the cover to cause the gear wheel 148 to
rotate. The gear wheel is adapted to engage with a smaller gear
wheel 149 also mounted on the axle 150 which drives injection of
fluid from the two syringes mounted on the base by a further
gear-rack mechanism 171. As shown in FIG. 18a, a spring 151 is
mounted on the axle 150 between the large gear wheel 148 and the
smaller gear wheel 149. The spring 151 enables a one-way gear
mechanism by virtue of which large gear 148 drives small gear wheel
149 when rotating in a first direction but does not drive the small
gear wheel when rotating in the opposite direction. This will be
described in further detail below.
[0131] A lever, 159 is provided on base 70 at the end 118 thereof
which can be pulled out from the base to shorten the length by
which the needles can project beyond base 70.
[0132] As stated above, the base 70 is adapted to be received
within a cover 76 as shown in FIG. 7. The cover 76 is shown in
greater detail in the cross sectional side view of FIG. 13. The
cover is again a solid body which could for example be made of
polyvinyl chloride.
[0133] The cover 76 has a first side wall 128 shaped to cover
substantially all of the base 70. The side of the cover opposite
the first side wall 128 is open to allow access to the base 70 when
it is mounted in the cover. A first end 134 of the cover is shaped
to cooperate with the first end 86 of the base 70, i.e. it extends
upwardly at an acute angle (45.degree. in the embodiment shown)
away from the bottom of the cover. The opposite end of the cover is
open such that the base 70 projects beyond the open end when
inserted in the cover in use.
[0134] On the bottom of the cover 76 extending outwardly from the
first side wall 128 is a planar support surface 130 which extends
across the full length and width of the cover so as to receive the
bottom surface of the base thereon. An L-shaped guide groove 132 is
provided in the support surface 130 extending from the open side of
the cover across the support surface perpendicular to the
longitudinal direction approximately to the centre of the support
surface and then extending in the longitudinal direction towards
the first end of the cover. This guide groove 132 is adapted to
receive a pin 136 attached to the bottom surface 82 of base 70 in
use and a user moves the base forwards and backwards relative to
the cover by manually moving this pin 136 in the groove 132. The
pin 136 and guide groove 132 arrangement has the advantage that the
base cannot fall out of the cover in use.
[0135] Further supports which hold the base 70 in place within the
cover 76 in use are provided projecting from the first side wall
128 to the other side of the cover. These supports project both
over the first end of the cover and along the top or upper edge
thereof (forming parts 134 and 138 respectively). These are
dimensioned so that gaps are left between the upper support 138 of
the cover and various parts of the base 70 in use. A flat portion
140 extends perpendicular to the longitudinal extent of the cover
between the sloping part of the first end 134 and the upper edge
138 of the cover. This flat portion is provided to be easily placed
on the skin of a subject for injection and two apertures 142, 144
are formed through it to allow two needles supported adjacent one
another above the base 70 to pass through the cover for
insertion.
[0136] A toothed track 146 is provided on the upper support 138 to
engage with the gear wheel 148 mounted on base 70 in use.
[0137] A stopping member 164 including a projection for engaging
with the open end of cover 76 is mounted on base 70 by a screw 166
engaging in the longitudinal aperture 122. The distance that the
base can move within the cover (and hence the maximum achievable
depth of needle insertion in use) can be adjusted by moving the
stopping member 164 relative to the base 70 by sliding the screw
166 in the aperture 122. The longitudinal aperture 122 may be
provided with a scale to indicate the maximum depth of needle
insertion enabled at respective positions of screw 166.
Alternatively, the scale could be provided on the base 70 itself to
be read off against a point on the stopping member 164.
[0138] In use, the base 70 and cover 76 are separated. The gear
wheel 148 is then pushed right back on the toothed track or rack
146 until it disengages therefrom. This enables the later placement
of full syringes into the base without any fluid being spilled.
Either one or both syringes are then filled with fluid (this
depending on the treatment desired). The two syringes 72 and 74
having barrels 152, 154 are the mounted in base 70 such that the
needle ends 156, 158 extend beyond the end of the base and the ends
of their piston rods 160, 162 abut against a pushing mechanism 171
driven by the small gear wheel 149.
[0139] One of the two syringes contains DNA or another substance
for injection into the person or animal to be treated. The other
syringe may be empty and be used solely to act as an electrode
during the subsequent electroporation process or it may be full of
DNA or other fluid for injection in the same manner as the first
syringe. The syringes are held against axial movement relative to
the base 70 by annular projections 157 provided on the syringes
which are received in the notch 106 in base 70 in use. The syringes
are held against movement in the direction perpendicular to the
axial direction by the grooved 96, 98 which extend upwardly on
either side of each syringe when fitted in the base.
[0140] The base 70 is inserted into cover 76 through the open side
thereof, the pin 136 in the bottom of base 70 sliding along the
groove 132 in a direction perpendicular to the longitudinal extent
of the base until it reaches the bend in groove 132. Four
adjustments are then made. Firstly, the lever 159 is adjusted so
that the needles only stick out of the cover by a distance
corresponding to the fat thickness of the subject to be treated
(i.e. to the depth of initial needle insertion before fluid
injection commences). Next, the base 70 is pushed forward within
the cover 76 to reach the maximum desired needle insertion depth
and the screw 166 is locked within aperture 122 at this point. The
base is then pushed back towards the lever 159 and the further
gear-rack mechanism 171 is pushed forward against the syringe
pistons ready for injection The device is then ready to start the
injection process as shown in FIG. 14.
[0141] Next, the flat portion 140 of the cover 76 is placed on the
skin of a subject to be treated and the base 70 is moved towards
the first end 134 of the cover by pushing the base in that
direction using the pin 136. By moving the base 70 forward, the
needles are moved towards and then through the apertures 142, 144
in the cover 76 so that they penetrate the skin of the subject to
be treated. The device at this position is shown in FIG. 15 and as
can be seen, the gear wheel 148 engages toothed track 146.
[0142] To cause synchronised needle insertion and fluid injection,
the pin 136 is then manually pushed further forward in the groove
132 thus moving the cover 76 back towards the stopping member 164
and hence inserting the needles to a depth determined by the
relative position of the stopping member while causing gear wheel
148 to rotate. The rotation of gear wheel 148 causes the smaller
gear wheel 149 to rotate also thus pushing in the piston rods into
the syringes such that fluid is injected gradually through the
needles over the depth of insertion of the needles. FIGS. 16 and 17
respectively show the device halfway through needle insertion and
when insertion has been completed.
[0143] After injection has been completed, an electric field is
activated through a current supplied through the needles. The
device includes, or is used in conjunction with, a power supply or
pulse generator and a control box (not shown) through which the
level of the voltage supplied for electroporation can be varied.
Further, the amount of current delivered through the needles during
electroporation can be measured. Similarly, other characteristics
such as electrical resistance can also be measured and recorded
either before or after the application of the voltage pulses. The
needles are subsequently withdrawn from the subject being treated,
by moving pin 136 back in groove 132 to pull the base back from the
cover such that the needles are clear of the cover and the base is
then removed from the cover through the open side thereof. The
needles can then be lifted away from the base and replaced by new
syringe devices when a new treatment is required. In an alternative
where the device is set up for multiple injections with a
multi-dose syringe, the needles are retained in the base and
further injections can then be carried out.
[0144] In an alternative embodiment of the device, automatic needle
insertion and injection can be achieved by respective servo motors.
This has the advantage that the depth of needle insertion can be
varied using a control for the servo motors.
[0145] When treating a human or animal subject, it is important
that injection of fluid is commenced and stopped at suitable needle
depths. The depths at which injection should be started and stopped
will vary from subject to subject depending on the thickness of the
superficial fat layer and muscle of the subject. Thus, the device,
power supply or control box may include means for measuring the
change in impedance between the needles of the two syringes during
insertion. This change in impedance provides an indication of when
the needles have moved into the desired type of body tissue for
fluid injection to commence as the impedance measured between the
needles will be different for different types of body tissue. In an
alternative embodiment of the device, an ultrasound transducer can
be provided on the tip of a needle to measure the depth of the
muscle below the tip of the needle and so determine when injection
should be commenced.
[0146] The device described above could be used with standard
syringes as are known in the art. However, it could alternatively
be used with prefilled vials or barrels containing the treatment
fluid in single or multiple doses and adapted to be connected to
injection needles. This has the advantage that the user does not
need to fill a syringe with the appropriate dose from a bottle of
medicament/solution.
[0147] A single-dose barrel could be used for treating humans but a
multiple dose barrel could, for example be used to treat a whole
herd of farm animals with a single needle.
[0148] The syringes or barrels for use with the device according to
the invention could be identified by unique bar-codes or other
identifiers. The bar-codes could be stored in an electronic
controller for the device and could be linked to the patient
protocol or animal number. Ideally, an iris-scan or ID tag could be
used to identify a patient and a DNA ID code could be provided on
the fluid vessel (normally in the form of a bar-code). The patient
protocol could be automatically retrieved from a computer when the
bar-code on the fluid vessel was read prior to use, leading to
great savings in time and effort in clinical situations. Data such
as the level of current applied during electroporation, and the
amount of DNA or fluid injected could also be stored electronically
with the patient protocol. This would enable improved tracking of
patient records.
[0149] A test of the device of the third embodiment has been
carried out on sheep. The device of FIG. 7 was used to distribute
DNA encoding SEAP or beta-galactosidase in body tissue.
Electroporation was carried out immediately after insertion of the
needles and injection. To administer SEAP for measurement in serum,
three sheep were sedated and shaved at one side of the rear. Local
anaesthetics were applied in a half circle around the site of
treatment. The device was loaded with syringes containing DNA
encoding human serum alkaline phosphatase (SEAP). One dose
consisted of 33.mu.g DNA in a total of 200.mu.l. After insertion
and injection, current was applied through the needles (400.mu.sec
pulses, 1000 Hz, repeated 7-10 times, 35-60 V/cm). Serum samples
were collected 7 days later and measured for SEAP expression by the
method described by Chastain in Journal of Pharmaceutical Science
90 474-484 (2001).
[0150] To transfect muscle tissue with cDNA encoding
beta-galactosidase (.beta.-gal), in order to assess .beta.-gal
expression, one sheep was treated as described above. The device
was loaded with syringes containing DNA encoding
beta-galactosidase, and one dose consisted of 40.mu.g DNA in a
total of 200.mu.l. Muscle biopsies were taken 3 days later and
beta-galactosidase activity was visualised by the method of Sanes
et al. Development 113 1181-91 (1991).
[0151] The results of the test are shown in FIG. 19 which shows the
amount of SEAP in serum and FIGS. 20a and 20b which show the
beta-galactosidase in muscle. The sheep were given 3 different
doses of DNA encoding SEAP as shown in FIG. 19. As shown in FIGS.
20a and 20b, the method gave even distribution of DNA which in turn
gives better and more reproducible accessibility to target cells
and thereby better transfection.
[0152] As a further test of the third embodiment of the invention,
experiments were conducted to measure the resistance between the
needles following insertion and optionally injection. Sheep were
used for the purpose. The syringes were filled with saline, mounted
on the base unit of the device and the cover applied. The needles
of the device were inserted into the muscle with or without
injection of saline and the resistance measured by use of a control
box.
[0153] The resistance in muscle without saline injected was
measured at 332 ohms, with a total of 100 microliter saline
injected the resistance was 291 ohms and resistance in muscle with
a total of 400 microliter saline injected was 249 ohms.
[0154] In a yet further test, the third embodiment was also tested
upon a human volunteer in order to assess whether the use of this
device would be tolerable in humans and whether local anaesthesia
would be necessary.
[0155] The syringes were filled with saline and mounted in the
device. The device was pre-set to allow penetration through the
skin (3 mm) and a further 1 cm of needle insertion with concomitant
injection of saline.
[0156] The skin of the leg muscle was disinfected and the needles
were inserted into the skin. Then the needles were further
inserted, and saline injected, into the muscle by pushing the knob
(136). When the needles were in place, the electroporation was
performed. The pulse given lasted for 20 ms. The voltage was
changed successively from 10 V to 70 V (in 10 V steps), with new
insertions and injections of saline each time.
[0157] At the highest voltage the current delivered was around 240
mA. The resistance in the muscle tissue was around 300 ohms (within
the same range as seen in sheep).
[0158] The description from the volunteer was that the injection
and insertion were without any pain. The electrical stimulation was
rated as unpleasant but not painful. Some stiffness in the treated
area was experienced 1-3 hours after the treatment. The stiffness
was less pronounced than after physical exercise. No anesthesia was
used or considered necessary in this case although a local
anesthesia may be beneficial if larger areas of the muscle are to
be treated.
[0159] The embodiments of the electroporation device described
above are preferred embodiments only to which various modifications
could be made. For example, the sheaths in the first embodiment
could be made of a material other than Teflon and the apertures in
them could be provided in a different pattern. Further, although
the device has been described as including a syringe arrangement to
which the needles are connected, it will be appreciated that this
need not be an integral part of the device. Thus, in an alternative
embodiment, the needles in the device could be left free to be
connectable to a fluid delivery system such as a syringe in
use.
[0160] Further, although the needles of the device of the second
embodiment have been described as being attached to a syringe
arrangement, it will be appreciated that the needles and syringe
part could be provided separately. Further, although the housing
has been described as being formed in two halves each having two
recesses formed therein, it will be appreciated that it could be
formed by any number of parts which allowed the needles to be
removed from the housing without pulling out in the axial
direction. Further, it could be adapted to hold any desired number
of needles. Thus, the scope of the invention is not limited by the
embodiments of the device as described above but rather is defined
by the scope of the appended claims.
* * * * *