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.

Claims

1. A method for delivering an active agent into a body tissue, comprising: inserting at least first and second hollow needles in the body tissue to a first desired depth, wherein first and second syringes are connected respectively to the first and second hollow needles; advancing the at least the first and second hollow needles from the first desired depth to a second desired depth in the body tissue; wherein the advancing step comprises rotating a gear engaged with a rack, thereby causing the rack to advance first and second pistons, respectively into the first and second syringes, thereby injecting fluid from the first and second syringes and through the first and second hollow needles and gradually into the body tissue at a rate that is uniform over time while the first and second needles are being inserted into the body tissue from the first desired depth to the second desired depth; and electroporating cells of the body tissue during or after the fluid has been injected, thereby delivering the active agent into the body tissue.

2. The method of claim 1, wherein the injection commences when the at least the first and second hollow needles reach the first desired depth in the body tissue and stops when the at least the first and second hollow needles reach the second desired depth in the body tissue.

3. The method of claim 1, further comprising: coupling the first and second hollow needles and the first and second syringes to a base prior to the inserting step; wherein the advancing step comprises moving the base forward relative to a cover, such that a pin of the base travels along a guide groove defined in the cover.

4. The method of claim 3, wherein the advancing step comprises driving the pin along the guide groove.

5. The method of claim 4, wherein driving the pin is performed manually.

6. The method of claim 4, wherein the coupling step comprising positioning the first and second syringes within respective parallel grooves formed in the base.

7. The method of claim 6, wherein: the cover has a bottom, a flat portion extending from the bottom, and a support frame, wherein the guide groove is defined by the support frame and extends from the flat portion; and during the advancing step, the base slideably engages an inner surface of the cover to move laterally between the bottom and the support frame.

8. The method of claim 7, further comprising contacting the flat portion against a tissue surface prior to the inserting step.

9. The method of claim 7, wherein an additional rack is disposed on the cover, and an additional gear is rotatably connected to the gear, such that rotating the additional gear during the advancing step while the additional gear is engaged with the additional rack rotates the gear, thereby causing the rack to advance the first and second pistons.

10. The method of claim 9, wherein the gear and the additional gear are rotatably coupled in one-way fashion such that the additional gear drives the gear when rotating in a first direction and does not drive the gear when rotating in a second direction.

11. The method of claim 7, wherein the at least the first and second hollow needles advance through apertures defined in the flat portion during the advancing step.

12. The method of claim 3, further comprising adjusting a position of a stopping member mounted on the base, wherein a distance between the stopping member and the cover defines a distance that the base can move laterally within the cover.

13. The method of claim 12, wherein the distance between the stopping member and the cover corresponds to the second desired depth.

14. The method of claim 12, wherein the adjusting step comprises rotating a drive screw coupled to the base and the stopper.

15. The method of claim 3, further comprising adjusting a position of a lever mounted to the base to set an initial distance that each of the at least the first and second needles protrudes through the apertures.

16. The method of claim 15, wherein the initial distance corresponds to the first desired depth.

17. The method of claim 1, further comprising sensing, with a moveable contact, when the at least the first and second hollow needles have been inserted to the second desired depth.

18. The method of claim 17, further comprising moving the contact relative to the at least the first and second hollow needles and the fluid delivery assembly to adjust the second desired depth.

19. The method of claim 1, further comprising measuring a change in impedance between the first and second hollow needles during the inserting step, thereby determining when the first and second hollow needles reach the first desired depth.

20. The method of claim 1, wherein the electroporating step comprises delivering electric current to the at least the first and second hollow needles.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Preferred embodiments of the invention will now be described, by way of example only, and with reference to the accompanying drawings in which:

(2) FIG. 1 is a schematic side elevation view of an electroporation device according to a first embodiment of the invention;

(3) 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;

(4) FIG. 3 is a perspective view of an electroporation device according to a second embodiment of the invention in an open position;

(5) FIG. 4 is a perspective view of the device of FIG. 3 in the closed position;

(6) FIG. 5 is a schematic plan view of a part of the device of FIG. 3 holding a needle and injection device;

(7) FIG. 6 is a schematic elevational view of an alternative needle and injection device for use with the device of FIG. 3;

(8) FIG. 7 is a side perspective view of an electroporation device according to a third embodiment of the invention;

(9) FIG. 8 is an underneath perspective view of the device of FIG. 7;

(10) FIG. 9 is a side perspective view of the base of the device of FIG. 7;

(11) FIG. 10 is a side elevational view of the base of the device of FIG. 7;

(12) FIG. 11 is a top plan view of the base of the device of FIG. 7;

(13) 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;

(14) FIG. 13 is a cross sectional side view of the cover of the device of FIG. 7;

(15) FIG. 14 is a side view of the device of FIG. 7 when fully assembled ready to start the process;

(16) 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;

(17) FIG. 16 is a side view of the device of FIG. 7 halfway during needle insertion;

(18) 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);

(19) FIG. 18a is an exploded view of the gear mechanism of the device of FIG. 7 for driving the needle insertion and injection process;

(20) FIG. 18b is a view of the gear mechanism of FIG. 18a mounted on the base unit;

(21) FIG. 18c is a view of the base unit showing the gear mechanism of FIG. 18a and the rack member in place.

(22) FIG. 19 shows the amount of SEAP used in serum in a test using a device according to the invention; and

(23) FIGS. 20a and 20b shows the results of the test using a beta-galactosidase expressing vector introduced using a device according to the invention.

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS

(24) 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.

(25) First and second electrically insulating sheaths 14 made of TEFLON® 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.

(26) 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.

(27) 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.

(28) 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.

(29) 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.

(30) 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.

(31) 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.

(32) 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.

(33) 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.

(34) 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® sold by BOC Ohmeda AS of Helsingborg, Sweden.

(35) 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.

(36) 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.

(37) 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.

(38) 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.

(39) 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.

(40) 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 rom BL/LB, 27G.times.11/2.

(41) 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® 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® sold by BOC Ohmeda AB of Helsingborg, Sweden.

(42) 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.

(43) 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.!) 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.

(44) 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.

(45) 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.

(46) 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.

(47) 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.

(48) 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 plug 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.

(49) 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).

(50) 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).

(51) 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).

(52) 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.

(53) 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.

(54) 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.

(55) 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.

(56) 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 nun 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.

(57) 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.

(58) 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.

(59) 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.

(60) 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.

(61) 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.

(62) 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.

(63) 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.

(64) 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.

(65) A toothed track 146 is provided on the upper support 138 to engage with the gear wheel 148 mounted on base 70 in use.

(66) 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.

(67) 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.

(68) 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.

(69) 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.

(70) 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 FIGURE and as can be seen, the gear wheel 148 engages toothed track 146.

(71) 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.

(72) 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.

(73) 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.

(74) 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.

(75) 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.

(76) 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.

(77) 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.

(78) 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 anesthetics 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).

(79) To transfect muscle tissue with eDNA 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).

(80) 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.

(81) 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.

(82) 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.

(83) 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.

(84) 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.

(85) 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.

(86) 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).

(87) 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.

(88) 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.

(89) 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.