Method and system for controlling molecular electrotransfer

Abstract

A system and method for controlling electrotransfer delivery of therapeutic molecules to a target cell population. The system has an array of two or more physically contiguous electrodes configured to be inserted into biological tissue and a pulse generator configured to drive the two or more electrodes. A first selection of electrodes is determined, and for the selected electrodes, electrical pulse parameters are determined for generating a first shaped electric field for a target treatment area adjacent to the array. A second selection of electrodes is determined, and for the selected electrodes, electrical pulse parameters are determined for generating a second shaped electric field for the target treatment area adjacent to the array. The pulse generator is controlled to apply a first sequence of monopolar pulses using the first selection of electrodes to generate the first shaped electric field, and to apply a second sequence of monopolar pulses using the second selection of electrodes to provide the second shaped electric field.

Description

Technical Field

[0001] The present invention relates to a system for electrotransferring (also known as electroporating) therapeutic molecules, such as naked plasmid DNA or RNA, to a target cell population within a tissue. An example of the application of the system is the delivery of DNA to cells using a near-field electrotransfer (electroporation) system that uses a continuous electrode array, wherein the electrodes are integrated into the array inserted into the tissue, and the cells targeted by electroporation are adjacent to the electrodes rather than between the electrodes. Background Art

[0002] Electroporation is a technique used in molecular biology in which an electric field is applied to cells to increase the permeability of the cell membrane, thereby allowing the introduction of chemicals, drugs, DNA or RNA into the cell. The basic principle is that the electric field generated by a high-voltage pulse between two electrodes causes a transient dielectric breakdown of the plasma membrane of the cell within the high-intensity electric field, allowing DNA or other molecules to enter the cell.

[0003] Conventional electroporation uses an electric field between physically separated electrodes, resulting in a direct current path through the tissue between the electrodes. The inventors have discovered that using an electric field adjacent to a physically continuous linear electrode array can achieve transfection of cells near the array using a lower cumulative charge than required to achieve the same number of cell transfections using open field electroporation, where the target cells or tissues are placed in the direct current path between the electrodes. The development of techniques for stimulating transfection in a target area adjacent to a continuous electrode array is described in the inventors' previous patent application publications No. WO2011 / 006204, No. WO2014 / 201511, and No. WO2016 / 205895, the disclosures of which provide the background of the present disclosure, and reference can be made to them to better understand the inventors' near-field electroporation techniques. In WO2016 / 205895, the inventors disclose a system in which the area in which electrostimulated cell transfection occurs is controlled by controlling the gradient within the generated electric field based on the electrode array configuration and the pulse sequence used to generate the electric field. The system of WO2016 / 205895 shows the practical application of the inventor's discovery of improved cell transfection in tissue areas subjected to steeper electric field gradients. The present invention drives the array by configuring the electrode array geometry and pulse sequence to control the shape of the electric field to produce a profile in the electric field passing through the target area, and applies this discovery to the target area for cell transfection adjacent to the array. The variables used to control the shape of the electric field include physically continuous electrode array configurations and stimulation pulse parameters. Compared with previously known open field electroporation, the use of controlled electric field shaping can achieve improved cell transfection efficacy with lower cumulative charge. However, there is a need for further improved electrotransfer technology. Summary of the invention

[0004] According to a first aspect, there is provided a method for controlling electrotransfer delivery of a therapeutic molecule to a target cell population using a system, the system comprising: an array of two or more physically continuous electrodes configured to be inserted into biological tissue; and a pulse generator configured to selectively drive the two or more electrodes as one or more anodes and one or more cathodes for applying an electrical pulse, the method comprising:

[0005] determining a first selection of electrodes to be driven as anodes and cathodes using one or more electrical pulses, and determining electrical pulse parameters of the one or more electrical pulses for the selected electrodes to produce a first shaped electric field for a target treatment area adjacent to the array, wherein the physical configuration of the electrodes, the selection of the electrodes and anodes and cathodes, and the applied electrical pulse parameters control a gradient profile within the electric field adjacent to the target treatment area of ​​the array;

[0006] determining a second selection of electrodes to be driven as anodes and cathodes using the one or more electrical pulses, and determining electrical pulse parameters of the one or more electrical pulses for the selected electrodes to produce a second shaped electric field for a target treatment area adjacent the array;

[0007] controlling a pulse generator to apply a first sequence of one or more unipolar pulses using a first selection of electrodes driven as anodes and cathodes to produce a first shaped electric field; and

[0008] The pulse generator is controlled to apply a second sequence of one or more unipolar pulses using a second selection of electrodes driven as anodes and cathodes to provide a second shaped electric field.

[0009] According to another aspect, there is provided an electrotransfer delivery system comprising:

[0010] an array of two or more physically contiguous electrodes configured for insertion into biological tissue;

[0011] a pulse generator electrically connected to the electrodes of the array and configured to apply one or more electrical pulses to selectively drive two or more electrodes as one or more anodes and one or more cathodes to generate an electric field in biological tissue adjacent to the array, wherein the electric field is shaped to provide a controlled profile of gradients within the electric field based on the physical configuration of the electrodes, the selection of the electrodes and the anodes and cathodes, and the parameters of the applied electrical pulses; and

[0012] A controller is configured to control a pulse generator, the controller being configured to control the pulse generator to apply a first sequence of one or more monopolar pulses using a first configuration of electrodes driven as anodes and cathodes to provide a first shaped electric field, and to provide a second sequence of one or more monopolar pulses using a second configuration of electrodes driven as anodes and cathodes to provide a second shaped electric field.

[0013] In one embodiment of the system, one or more additional electrodes are selected to be driven using one or more electrical pulses as anodes and cathodes, and for the selected electrodes, electrical pulse parameters of the one or more electrical pulses are determined to produce a second shaped electric field for a target treatment area adjacent to the array; and for each additional selection of electrodes, the controller controls the pulse generator to apply an additional sequence of two or more monopolar pulses using each additional selection of electrodes driven as anodes and cathodes to produce each additional shaped electric field, wherein each additional selection produces a controlled electric field gradient within the target treatment area that is different from the electric field gradient of the previous electric field. The selection of electrodes and the sequence of pulses can be selected to produce a sequence of electric fields, wherein subsequent electric fields each have an electric field gradient that passes through the target treatment area at an increasing angle relative to the previous electric field.

[0014] In one embodiment, the first selection of electrodes comprises a linear configuration of one or more anodes and one or more cathodes, and the second selection of electrodes comprises the same electrodes with the anodes and cathodes switched so that the polarity is reversed.

[0015] In one embodiment, the electrode array is a two-dimensional array, and wherein a first selection of electrodes comprises a configuration of one or more anodes and one or more cathodes, and a second selection of electrodes comprises an electrode configuration that includes electrodes different from the first selection, selected to produce a change in electric field gradient within the target treatment area.

[0016] The method may also include the steps of determining one or more additional selections of electrodes to be driven as anodes and cathodes using the one or more electrical pulses, and determining electrical pulse parameters of the one or more electrical pulses for the selected electrodes to generate a second shaped electric field for a target treatment area of ​​an adjacent array; and for each additional selection of electrodes, controlling the pulse generator to apply an additional sequence of one or more monopolar pulses using each additional selection of electrodes driven as anodes and cathodes to generate each additional shaped electric field, wherein each additional selection generates a controlled electric field gradient within the target treatment area that is different from the electric field gradient of the previous electric field. In one embodiment, a sequence of electrode selections and pulses is selected to generate a sequence of electric fields in which subsequent electric fields each have an electric field gradient through the target treatment area at an incremental angle relative to the previous electric field.

[0017] In some embodiments, increasing or decreasing the spacing between the anode and cathode is used to control the radius of the treatment area. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Embodiments incorporating all aspects of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0019] Figure 1 is an exemplary block diagram of an electrotransfer system according to an embodiment of the present invention,

[0020] Figure 2 shows the polarized electrotransfer of DNA into cells,

[0021] Figure 3 HEK293 cells expressing reporter plasmid DNA driven by the same 2-electrode linear array using different pulse sequences,

[0022] Figure 4 One-way ANOVA is shown for Figure 3 Results shown in Figure 2, n = 4 per treatment group,

[0023] Figure 5a-5d shows the mapped electric field and cell transformation results using a tandem array configuration,

[0024] Figure 6a-6c shows the mapped electric field and cell transformation results using an alternating array configuration,

[0025] Figure 7a-7b shows the mapped electric field and cell transformation results using a 1+2 array configuration,

[0026] Figure 8a-8b shows the mapped electric field and cell transformation results using a 1+5 array configuration,

[0027] Figure 9a-9b shows the mapped electric field and cell transformation results using a 1+8 array configuration,

[0028] Fig.10 shows some of the effects of manipulating the gap between the anode and cathode,

[0029] Fig.11 is a block diagram of an alternative system configuration,

[0030] Fig.12a -c shows the first example of comparative analysis of monophasic and biphasic electrotransfer using an 8-electrode linear array,

[0031] Fig.13 A second example of comparative analysis of monophasic and biphasic electrotransfer using an 8-electrode linear array is shown, and

[0032] Fig.14 is a graph showing that extending the inter-pulse interval using alternating biphasic pulses enhances gene expression. DETAILED DESCRIPTION

[0033] An electrotransfer system is disclosed that utilizes changes in the direction of an electric field gradient to assist in the delivery of molecules, such as naked plasmid DNA, into cells. Figure 1 ) includes an array 110 of two or more physically continuous electrodes configured to be inserted into biological tissue, a pulse generator 120, and a controller 130. The pulse generator 120 is electrically connected to the electrodes of the array 110 and is configured to apply one or more electrical pulses to selectively drive the two or more electrodes as one or more anodes and one or more cathodes to generate an electric field in the biological tissue adjacent to the array. The physical configuration of the electrodes, the selection of the electrodes and the anodes and cathodes, and the parameters of the applied electrical pulses control the shape and profile of the gradient within the electric field generated in the tissue adjacent to the electrode array. The controller 130 is configured to control the pulse generator 120 to apply a first sequence of one or more monopolar pulses using a first configuration of the electrodes driven as anodes and cathodes to provide a first shaped electric field, and to apply a second sequence of one or more monopolar pulses using a second configuration of the electrodes driven as anodes and cathodes to provide a second shaped electric field. The effect achieved by using the shaped electric field and the different first and second electric field gradients is to increase the amount of cell surface area, where molecules can be stimulated by the electric field gradient to contact the cell and enter the cell by electrotransfer. Increasing the cell membrane area coated with molecules increases the likelihood that the cell will take up the molecule (transfection). If there is a waiting period between two pulses of different polarities, the efficiency of electrotransfer is improved when only one pulse of each polarity is used. In one embodiment, the waiting period is 15 ms or longer, such as 15-400 ms, optionally 10-600 ms. In the case of using two (or more) sequences of two or more monopolar pulses, a waiting period may also be used between polarity changes.

[0034] Array 110 can be configured to be temporarily inserted into tissue, for example, the array can be incorporated into a probe for inserting into tissue for treatment and then removed. The array can be configured as a linear array of electrodes. Alternatively, a two-dimensional or three-dimensional array of continuous electrodes can be used. The array can be configured to allow some electrodes to be driven as anodes and cathodes, and the electrodes are selected from a large number of electrodes (so not all are driven at once), which can realize different configurations of anodes and cathodes based on the selection of electrodes and polarity. Optionally, the array can be preconfigured as two electrodes (or multiple electrodes are combined to operate as a single electrode) and selectively driven as anodes or cathodes. In some embodiments, the array can be implantable, such as a cochlear implant array, which can be configured for an electrical transfer mode other than an auditory stimulation mode, or a biomimetic electrode array compatible with deep brain stimulation can be developed similarly using this dynamic electric field shaping mode of molecular electrotransfer. Other types of implantable arrays can be configured to be implanted in a target area over an extended period of time so that electrotransfer-based therapy can be applied multiple times over time (e.g., weeks, months, or years). All these changes are considered to be within the scope of the present disclosure.

[0035] It is well known that electrotransfer of DNA to cells is polarized. When sufficient voltage is applied to generate an electric field suitable for delivering naked DNA to cells, such DNA accumulates on the side of the cell facing the cathode (e.g., electrically mediated formation of DNA complexes with cell membranes and their impact on gene delivery, such as Figure 2 shown. Figure 2 The schematic diagram shown in shows that cellular DNA is occupied only from one side into the target cell. In order to improve the efficiency of electrotransfer, it is necessary to improve the coverage of the cell by DNA. Therefore, it is expected that alternating polarity of electroporation pulses will improve the efficacy of cell transfection - however, the inventor's work has demonstrated that the use of alternating pulses actually reduces the transfection efficacy.

[0036] Figure 3 Images 310, 330, 350 show the same 2-electrode array driven using different pulse sequences with reporter plasmid DNA expressed by HEK293 cells. Figure 3The images are examples of mCherry fluorescent reporter expression in a HEK293 cell monolayer following electrotransfer of naked plasmid DNA encoding the reporter under a cytomegalovirus (CMV) promoter; 10x100μs pulses x10mA in a pulse train were delivered in monophasic mode using a prototype current stimulator developed by the inventors (a proprietary constant current stimulator or constant voltage stimulator may also be used) and a linear array of two continuous elongated electrodes, one driven as an anode (+ve) and one driven as a cathode (-ve). The applied stimulus sequence was applied in a monophasic mode 320, where all ten pulses had the same polarity, and in a biphasic mode 340, where five pulses had the same polarity and then a second set of five pulses had the opposite polarity (biphasic). The third pulse train configuration had alternating positive and negative going pulses (alternating biphasic) 360. The inter-pulse interval was 400 microseconds. The DNA and electrotransferred array were placed on the cells on a coverslip. The pulses were delivered and the coverslips were then returned to culture for 48 hours before imaging. This example demonstrates that the DNA electrotransfer efficiency 330 is significantly improved in the biphasic mode 340 . Figure 4 One-way ANOVA is shown, n=4 per treatment group.

[0037] Figure 3 The images in show an area where DNA cell transfection is performed by near-field electrotransfer using a linear array electroporation probe, with electroporation occurring in an area adjacent to the linear electrode array. The area where most cells are transfected is the area where the electric field generated by the linear array is concentrated. The illustration of 310 shows cells transfected in response to the application of a series of monopolar electroporation pulses 320 (referred to as a monophasic pulse sequence), whereas 350 shows cells transfected using the same electrode array driven with alternating polarity electroporation pulses (alternating biphasic), and surprisingly, this charge-balanced alternating biphasic pattern results in a significant reduction in DNA electrotransfer and reporter gene expression compared to the monophasic result 310. From the effect of DNA being driven to the side of the cell facing the negative electrode, one might expect the alternating pulses to have the effect of driving DNA to both sides of the cell and improving transfection efficacy by increasing the cell area exposed to the DNA, but surprisingly, the use of alternating polarity provides very poor results.

[0038] In case of electrotransfer using a sequence of several monopolar pulses followed by another sequence of several monopolar pulses of opposite polarity (called biphasic), the transfection efficiency is significantly improved, e.g. Figure 3 These results suggest that transfection efficiency is enhanced by prolonged electrotransfer of DNA to cells and maintaining contact (adhesion) with the cell membrane. Figure 3 and 4This is demonstrated by comparison of the single phase result 310 shown in Figure 3 to the alternating biphasic result 350. The further improvement in efficacy of the biphasic result provides evidence that after driving DNA toward the cell in one direction for a long period of electrotransfer, changing to the opposite polarity and driving the DNA toward the cell from the opposite direction and covering the opposite side further improves transfection efficacy.

[0039] The present disclosure describes a method for improving the efficiency of delivering DNA into cells using a technique the inventors call "switching mode electrotransfer". This is a mode of electrotransfer that uses a continuous array of electrodes to generate an electric field with a controlled electric field gradient in adjacent arrays. The switching mode operation uses a first monopolar pulse sequence to generate a first shaped electric field with a controlled electric field gradient through the target area, and uses a second monopolar pulse sequence to generate a second shaped electric field, changing the direction of the electric field gradient through the treatment area. This change in the electric field gradient causes molecules (DNA) to be driven toward the cell from different directions, and thereby covers different areas of the cell. An embodiment of the present system uses multiple monopolar pulses to generate and maintain each shaped electric field for a period of time sufficient to establish functional contact between the DNA molecule and the cell to achieve transfection. It should be noted that during the application of the monopolar pulse sequence, complete transfection or uptake of the DNA molecule by the cell may not occur. Establishing functional contact should be understood to include any stage of cellular uptake of DNA: it enables uptake to continue despite the stopping or change of direction of the driving electrotransfer electric field. This may involve the uptake of DNA (or RNA) bound to the cell's plasma membrane through processes such as endocytosis.

[0040] exist Figure 3 In, the inventors provide proof of concept data showing the enhanced expression of reporter plasmid DNA by HEK293 cells in a monolayer mode using a continuous array of electrodes (in this example, a linear array) when the electrode polarity in the gene electrotransfer array is switched during the pulse train. It is the nature of this switch that provides an improvement in the efficacy of DNA (or RNA) electrotransfer. The inventors have shown that switching pulse polarity according to specific requirements can achieve an unexpected increase in DNA electrotransfer efficiency. In one embodiment of the method and system, the electrotransfer pulse train includes a series of pulses of one polarity, followed by a series of pulses of opposite polarity. The inventors have shown that the potentially obvious and advantageous strategy of alternating between positive and negative polarity on the electrode (commonly used for continuous electrical stimulation as a charge recovery strategy) is actually counterproductive, resulting in a lower efficiency of DNA electrotransfer. Therefore, by sequentially constructing DNA transfer using a series of unipolar current transfer pulses, followed by pulses of opposite polarity, the unexpected gain in efficiency provides a significant transfection efficacy advantage of the system.

[0041] Applicant's prior publication No. WO2016 / 205895 discloses a system for controlling the electric field gradient to thereby control the area where transfection occurs. The current development of this technology disclosed herein exploits this ability to shape the electric field to control the electrotransfer coating of cells over a considerable area of ​​the cell by changing the direction of the electric field gradient. This essentially enables controlled "painting" of the cell with DNA molecules for electrotransfer by driving the molecules toward the cell from different directions. Furthermore, by using several monopolar pulses, functional contacts can be formed between the DNA molecule and the cell before changing the direction of the electric field gradient, and possibly driving the DNA molecule away from the cell surface.

[0042] The electrotransfer technology of the present invention targets cells near an electrode array, where an electric field is formed around the array by a combination of a current source (anode) and a current return (cathode), and the gradient within the shaped electric field controls the area in which cell transfection occurs. By changing the direction of the electric field gradient, the coverage of the treated DNA molecules on the cells can be increased, thereby improving the transfection efficacy. Examples of mapped electric fields are shown in Figures 5-9, showing changes in the treatment area based on changing the array configuration, and thus showing the resulting electric field. Figure 5b , 6a , 7a, 8a, and 9a show examples of mapped electric field potentials produced by different anode and cathode configurations of an eight-electrode array, and Figure 5c , 5d , 6b, 6c, 7b, 8b and 9b show the distribution of cell transformation results after electroporation for each array configuration. The 8 electrodes in the array are configured as anodes and cathodes in the following combinations:

[0043] Series – four juxtaposed cathodes followed by four juxtaposed anodes, all separated by 300 μm and with a total length of 5 mm (e.g. Figure 5b-5d );

[0044] Alternating – Alternating cathode and anode at 300 μm intervals, total length 5 mm (e.g. Figure 6a-6c );

[0045] A single anode and a single cathode within a 1+2–300 μm interval (e.g. Figure 7a-7b );

[0046] 1+5 – single anode and single cathode, separated by 2.45 mm (e.g. Figure 8a-8b ); and

[0047] 1+8 – single anode and single cathode, separated by 4.55mm (e.g. Figure 9a-9b ).

[0048] Figure 5b , 6bFigures 7b, 8b, and 9b show a comparison of the effect of array configuration on electroporation-mediated gene delivery, all driven using a pulse train with the following parameter set: 40 V, 10 pulses, 50 ms duration, and 1 pulse / sec. Although all array configurations produced significant cell transduction, there were significant effects on transformation efficiency due to differences in array configuration, spacing between anodes and cathodes, and the number and pattern of anodes and cathodes. The 1+2 array drive configuration produced a spherical cell field of ~1 mm in diameter, with the active electrode located in the center ( Figure 7b ). The alternating array drive configuration produced a linear deviation of the transfected cell field, extending the length of the array (~5 mm; 81.8 ± 11.3 GFP-positive cells), as Figure 6b and 6c The 1+5 and 1+8 array drive configurations produced smaller average transformed cell numbers with a low-density distribution ( Figure 8b and 9b The transfection efficiency of the tandem configuration is significantly higher than any other configuration ( Figure 5c and 5d ), and the pattern shape is spherical, centered at the midpoint of the array, which is the junction between the four anodes and the four cathodes. The inventors' testing showed that when the array is configured to combine the anodes and cathodes together as a bipolar (a "tandem" configuration), the charge transfer required to achieve efficient cell transduction is minimized.

[0049] For comparison, Figure 5b , 6a , 7a, 8a and 9a show the electric field mapped for each array configuration, with the field potential measured at the end of a 100 ms 4 V pulse 300 applied to the array, as shown in Figure 5a shown. Figure 5a Also shown is a trace 310 of field potentials recorded at 0.5V to 4V (100ms duration) using a tandem array configuration. The "tandem" configuration allows for significantly higher transduction efficiencies than an equal number of electrodes wired in an "alternating" configuration. The study also showed that smaller bipolar electrode configurations were less efficient (1+2, 1+5, 1+8). The reason the "tandem" array configuration showed unexpected cell transduction efficiencies is attributed to the geometry of the electric field focusing ( Figure 5b ). The series array 400 exhibits the highest electric field profile density, starting with zero tracking from the junction between the anode and cathode 410 ( Figure 5b ). In contrast, despite using the same number of electrodes, the alternating array configuration 500 has a lower electric field density gradient distributed along the array with a peak at the end of the array 510 ( Figure 6a ). Given that the spherical GFP positive field of the cell is centered at the zero point of the “tandem” array ( Figure 5c and 5d;and Figure 5b The data indicate that it is the electric field gradient across the cell, rather than an absolute step change in potential, that drives electroporation and DNA uptake. The cell distributions for the other array configurations show similar associations with the measured electric field, and the decrease in the number of GFP-positive cells in 1+2>1+5>1+8 correlates with the broadening of the electric field relative to the electrodes.

[0050] The electric field around the array (and in particular the gradient within the field) is closely related to the spatial mapping of transformed cells. Therefore, cell transduction depends on the potential gradient across the cell, rather than the absolute voltage. This is most evident in the contourmap of the tandem configuration using 0.9% saline solution, where the zero region in the field is orthogonal to the array migration between electrodes 4 and 5 ( Figure 5b ). The field contour lines are steepest near this line and maintain a spherical shape corresponding to the transduced cell map. The potential measurements at either end of the series array are largest but more uniform. As the distance separating the bipolar electrodes increases, the field density decreases, as can be seen by comparing Figure 7b , 8b This is demonstrated by the results for the 1+2, 1+5 and 1+8 arrays in 9a and 9b, respectively.

[0051] It should be apparent from the above description that a combination of electrode configurations and pulse parameters can be selected to achieve a shaped electric field with a controlled electric field gradient. A key element in generating these controlled electric fields is the array configuration. One embodiment is envisioned as an array comprising a continuous sheet of electrodes, for example arranged in a two-dimensional grid (i.e., a rectangular, hexagonal, triangular, etc. grid configuration) and connected to allow the selective use of a combination of electrodes to effectively provide a variety of electrode configurations, and when the selected electrodes are driven as anodes and cathodes, the associated changes in the shape and gradient of the electric field. For example, a row of electrodes can be selected and driven in the following manner: a first group of adjacent electrodes acts as a combined cathode, and another group of adjacent electrodes acts as a combined anode-series configuration. Then another group of aligned electrodes angled relative to the first row can be selected and driven to change the direction of the electric field gradient. For example, selecting a different set of electrodes can result in the generation of an electric field through a target area, with an electric field gradient angled relative to the previous electric field. In the case of a simple reversal of polarity, electric fields of similar shape have opposite electric field gradients. Selecting different electrode groups can change the relative electric field gradient angle. Selecting different electrode groups can also change the shape of the electric field. Any electrode configuration can be selected based on the target electric field shape (or shapes). A sequence of different electrodes can be selected and driven with electrotransfer pulses to optimally target the treatment area to deliver charged molecules to the cell surface via electric field gradient, shape and polarity.

[0052] Some embodiments extend this transfection targeting capability based on linear arrays of electrodes to provide a "switched mode" electrotransfer strategy, in which both the polarity of the pulses and the electrode combinations within the electrode array that transfer current can be switched within a pulse train or within multiple pulse trains to rotate the focused electric field around target cells within the tissue; effectively coating the DNA on all surfaces of the cell; conceptually, enabling "vortex electrotransfer" DNA coating of cells.

[0053] In some embodiments, the electrode configuration also controls the extent of the transfection area relative to the linear electrode array. Figure 5b As shown, the electric field gradient near the middle of the array has the steepest electric field gradient, and Figure 5c and 5d The results indicate that this area is the area where the most cells are transfected. The field shape is attributed to the combination of linearly arranged elongated anodes and cathodes (in this configuration, each of the anodes and cathodes includes multiple adjacent linearly arranged electrodes). The elongated nature of the anode and cathode helps to control the shape of the electric field generated, thereby controlling the transfection area. Changing the width of the gap between the elongated anode and cathode changes the range of the transfection area orthogonal to the array. By changing the gap, the electric field shape can be expanded or reduced, thereby expanding or reducing the transfection area. In the example shown, increasing the gap between the electrodes expands the electric field and the transfection area, while reducing the gap limits the field. It should be noted that for the pulse parameters used in this example, this field shape control or vectorization applies to gaps of up to about 5 mm between the electrodes. Increasing this gap may be feasible in conjunction with a corresponding increase in the current level, but due to potential negative side effects caused by higher current levels, using additional groups of electrodes to enable treatment of larger areas may be preferred. Controlling the radial expansion of the electric field is also called vectorization. Fig.10 The effect of manipulating the gap between the anode and cathode is shown.

[0054] Fig.10 Images 1020 to 1050 show the expansion and contraction of the transfection area when the gap between the anode and cathode of the linear electrode array is manipulated. Fig.10In the example of , a probe of the type shown as 1010 was used, which has a linear two-electrode array with different spacing between the electrodes. The effect of manipulating the electric field - achieved by reducing the spacing between two platinum-iridium electrodes (2mm x 0.35mm diameter on an insulating support - as shown in the upper right figure) with a train of electric pulses (constant current, 100μsx10 pulses, 400μs pulse interval, 10mA +ve phase). The data show spatial control of electrotransfer of plasmid DNA (green fluorescent reporter protein - GFP - under the cytomegalovirus (CMV) promoter) delivered to a monolayer of human embryonic kidney (HEK) 293 cells using an isotonic polysaccharide carrier. The DNA and electrotransfer array were placed on the cells on a coverslip. Pulses were delivered and then the coverslips were returned to culture for 48 hours before imaging. The highest electric field strength was near the zero point between the two electrodes. As the electrodes separated, the electric field sufficient for DNA electrotransfer expanded until optimal dispersion of the recombinant DNA expression cassette and expression cell density was achieved (electrode spacing between 1–4mm).

[0055] In further embodiments, manipulation of pulse polarity can be combined with dynamic changes in the shape of the electric field by switching between electrodes within a near-field electrode array (e.g., expanding or contracting the electric field). This switching can occur during a pulse train sequence, or between pulse trains, so that both the polarity and vectorization of the field are rotated around the target cell. This is used to "paint" DNA onto the cell surface membrane. The effect of moving the spacing of two main electrodes (each 2 mm long and 350 μm in diameter) along a linear array can be seen in the vectorization method of changing the electric field - as shown in FIG. Fig.10 as shown in .

[0056] System embodiments may use separate components for the array, pulse generator, and controller, or may integrate these functions into a comprehensive system. Fig.11 , a block diagram of an embodiment of a system including a controller is shown in FIG. 1500 , the system 1500 including an electrode array 1510 as described above and a controller 1530. In the block diagram, the controller is shown to include a pulse generator 1520, however, the pulse generator can be a separate device. The illustrated embodiment also includes optional features, such as a configuration module 1540 to enable control of the array configuration as described above; an agent delivery module 1550 to control the delivery of a therapeutic agent through the probe; a user interface 1560; and a memory 1570 for storing data such as treatment parameters 1580 and a treatment log 1590.

[0057] In one embodiment, the controller is implemented using a computer system that can include the full functionality of the system or control dedicated hardware components through a data connection. Any possible configuration of the controller hardware, firmware, and software is foreseen within the scope of the present invention. In one embodiment, the controller function can be implemented using a dedicated hardware device, such as a pulse generator version or a dedicated hardware device modified to provide the controller function, such as a hardware logic ASIC (application-specific integrated circuit) or FPGA (field programmable gate array), firmware and software that implement the controller function. The advantage of a dedicated system is that it can be independent of commercial software platforms and operating systems, which may be beneficial to obtain regulatory approval and limit the use of the system to the intended purpose. However, the disadvantage of this embodiment may be the increased system development and ongoing maintenance costs, and the lack of flexibility in utilizing new technologies or developments in the field of technology.

[0058] Alternatively, the controller functionality may be provided as a software program executable on a computer system such as a personal computer, server, or tablet computer and configured to provide control instructions to a stand-alone pulse generator and other optional hardware such as a drug delivery actuator. It is contemplated that a software-based controller embodiment that can be executed using commercially available computer hardware provides a user interface 1560 for a specialist to input parameters for an electroporation treatment 1580, which are then stored in a memory 1570 for use in driving the pulse generator 1520 and optional configuration module 1540 and drug delivery module 1550 during electroporation treatment delivery. In one embodiment, this may include the controller analyzing target treatment area parameters and carrier solution parameters; determining appropriate electrode array configuration(s); and defining at least one pulse sequence calculated based on the above relationships to achieve a target treatment field for each appropriate array configuration. In embodiments where drug delivery may be controlled by the controller, the controller may also calculate and define drug delivery actuations in the treatment pulse sequence. In embodiments where array configuration may be controlled by the controller, the controller also defines the array configuration implemented by the configuration module. In embodiments where the array configuration is dependent upon selection of a fixed array configuration, the expert / surgeon / clinician may input the selected probe array configuration into the controller, or the controller may output a probe selection suggestion. If more than one combination of array and pulse sequence is determined to be suitable to meet the treatment requirements, the possible combinations may be output to the surgeon / expert / clinician for selection.

[0059] Some embodiments of the controller may provide software to model the treatment. In some embodiments, the data used for modeling may include patient imaging data (i.e., MRI or CT scans) to enable the modeled treatment field to be considered in conjunction with the patient data. The modeling data may also include options for therapeutic agent carrier solutions. For example, feasible or optimal carrier solution parameters are determined to safely achieve the expected treatment outcome through modeling. Optionally, in the case of a limited number of carrier solution options available, the potential outcomes of each option are modeled to help experts / surgeons / clinicians make decisions.

[0060] It will be appreciated that the system functionality provided in connection with planning a treatment may be complex and utilize complex software models to determine the data required to drive the physical therapy device. However, the actual data required to actually perform the treatment may be very simple - a defined array configuration (which may even be a fixed array), a sequence of timed pulses, and optionally a signal for controlling the delivery of a selected agent solution. Thus, the controller may simply output sequence data for driving the physical therapy device.

[0061] Embodiments of the pulse generator 1520 can operate in either voltage or current drive mode. In one embodiment, the pulse generator is an autonomous stand-alone unit having its own power supply independent of the controller. The pulse generator can be in data communication with the controller to enable the controller to program the pulse generator with a pulse sequence. Alternatively, the pulse sequence can be calculated by the controller and loaded into the pulse generator by manual programming or data transmission (e.g., by direct connection, wireless connection, or by physical media such as a portable solid-state storage device), and the pulse generator is operated independently of the controller for therapeutic delivery. Isolating the pulse generator from the controller may be a safety requirement for certain systems, particularly enabling disposable DNA (or RNA) delivery probes to operate using pulse generators that have been approved and available for human clinical use. In some countries, this may enable the probe to obtain independent regulatory approval for use in conjunction with a selected commercially available and regulatory-approved pulse generator.

[0062] Embodiments of the present system and method can be advantageously applied in the field of DNA therapy, molecular therapy involving electrotransfer of nucleic acids, such as naked plasmid DNA and RNA, or other charged molecules. Embodiments can be used in the pharmaceutical and biotechnology industries, and can be applicable to any industry that engages in DNA-based therapies and requires an efficient non-viral DNA delivery platform.

[0063] Figures 12-14 show examples of prototype test results.

[0064] Example 1: Effect of electric pulse polarity on the expression of fluorescent reporter protein after biomimetic array-directed gene electrotransfer (BaDGE) of mCherry reporter plasmid at 1 μg / μl concentration - monophasic and biphasic pulse trains.

[0065] Figure 12a-12c A pulse sequence using unidirectional pulses of only one polarity is shown ( Fig.12a ) and a biphasic pulse train with the same pulse number and amplitude but altered polarity ( Figure 12b ) results of an electrotransfer comparison example. The test results in Figure 12 show the effect of switching electrode polarity on mCherry expression in HEK293 cells. Four days after DNA electrotransfer using an 8-electrode animal model cochlear implant array, mCherry fluorescence was imaged using a confocal microscope. The array was wired in a series configuration (four adjacent Pt / lr electrode strips were combined as anodes, and the next four electrodes were combined as cathodes). Circular coverslips (18 mm in diameter) were inoculated ~24 hours before electrotransfer, when the HEK293 cell confluency was approximately 50%. After 20 μl of DNA (CMVp-mCherry and pFAR4-BDNF-NT3 each 1 ug / ul) was transferred in 9% sucrose, 0.09% NaCl, and 500uM NaOH, electrotransfer was performed using a 10x40mA 100μs pulse transmitted by a gene delivery array. The electrotransfected coverslips were then placed in a 30 mm dish with 4 ml of Dulbecco's Modified Eagle's Medium (DMEM) cell culture medium containing 5% fetal bovine serum (FBS). Monophasic refers to 5 pulses of fixed polarity. Biphasic refers to 5 pulses applied with electrodes of one polarity followed by 5 pulses of opposite polarity. After 4 days, live cells were imaged and 2x0.5 ml aliquots of the culture medium were then snap frozen for BDNF and NT-3 ELISA. Fig.12a Shown is an example of a coverslip transfected with 10 pulses, each pulse having the same combination of 4 electrodes as active and the opposite 4 as return. Figure 12b Shown is an example of a coverslip transfected with 5 pulses, with 4 combination electrodes active and 4 opposite electrodes as return, followed by 5 pulses after switching the electrode polarity. Fig.12c Box plots of data from individual coverslips superimposed are shown, with boundaries representing the 25% and 75% limits of the data and bars representing the 95% limits of the distribution. Solid lines are medians and dashed lines are means. Statistical comparisons were performed by t-tests.

[0066] Example 2: Biomimetic array-directed gene electrotransfer (BaDGE) of mCherry reporter plasmid at 4 μg / μl concentration - Effect of electric pulse polarity on the expression of fluorescent reporter protein after monophasic and biphasic pulse trains.

[0067] Fig.13 An example of the effect of switching electrode polarity on nuclear localized mCherry reporter gene expression in HEK293 cells is shown. mCherry fluorescence was imaged by confocal microscopy 4 days after DNA electrotransfer using an 8-electrode animal model cochlear implant array. The biomimetic array was wired in a series configuration (four adjacent Pt / Ir electrode strips were combined as anodes, and the next four electrodes were combined as cathodes). Circular coverslips (18 mm in diameter) were inoculated with HEK293 cells ~24 hours before BaDGE, when the HEK293 cell confluency was approximately 50%. After applying 20 ul of DNA to the coverslip (4 ug / ul CMVp-mCherry) in a carrier solution of 9% sucrose, 0.09% NaCl, and 500 μM NaOH (total plasmid DNA concentration of 4 μg / μl), BaDGE was performed using 10x40 mA 100 μs pulses transmitted through the gene delivery array. The electrotransfected coverslips were then placed in a 30 mm culture dish with 4 ml of Dulbecco's modified Eagle's medium (DMEM) cell culture medium containing 5% fetal bovine serum (FBS). Monophasic refers to all 10 pulses with fixed electrode polarity. Biphasic refers to 5 pulses applied with electrodes of one polarity followed by 5 pulses of opposite polarity. Box plots are of data from the individual coverslips superimposed. The boundaries represent the 25% and 75% limits of the data, and the bars represent the 95% limits of the distribution. The solid line is the median; the dashed line is the mean. Statistical comparisons were performed by the Mann-Whitney rank sum test.

[0068] Example 3: Effect of inter-pulse intervals with alternating polarity - driven fluorescent reporter protein expression following biomimetic array-directed gene electrotransfer (BaDGE) of mCherry reporter plasmid.

[0069] Fig.14 Results are shown, illustrating that extending the interpulse interval with alternating biphasic pulses enhances gene expression. The test uses alternating biphasic polarity of the BaDGE electrode (changing from (+) to (-) between each pulse; duration of 10x4ms) to result in increased transfection of HEK293 cells when the interpulse interval exceeds 16ms. 2μg / μl of CMVp-mCherry reporter plasmid in 9% sucrose, 0.09% NaCl and 500μM NaOH carrier solution (20μl total volume plasmid DNA applied to HEK293 cells on 18mm coverslips). n=5 under various conditions; total pixel intensity on coverslips represents integration of mCherry-derived fluorescence (nuclear localization) by confocal imaging. Kruskal-Wallis one-way ANOVA was performed on ranks using Tukey's test for multiple paired comparisons.

[0070] Those skilled in the art of the invention will appreciate that many modifications can be made without departing from the spirit and scope of the invention.

[0071] In the following claims and the foregoing description of the invention, unless the context requires otherwise due to the express language or necessary implication, the word "comprise" or variations such as "comprising" or "including" are used in an inclusive sense, i.e. specifying the presence of stated features but not excluding the presence or addition of further features in various embodiments of the invention.

[0072] It should be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms part of the common general knowledge in the art in Australia or any other country.

Claims

1. An electrotransfer delivery system (100), comprising: An array (110) of two or more physically contiguous electrodes configured to be inserted into biological tissue; a pulse generator (120) electrically connected to the electrodes of the array and configured to apply one or more electrical pulses to selectively drive the two or more electrodes as one or more anodes and one or more cathodes to generate an electric field in biological tissue adjacent to the array, wherein the electric field is shaped to provide a controlled profile of a gradient within the electric field based on the physical configuration of the electrodes, the selection of the electrodes and the anodes and cathodes, and the parameters of the applied electrical pulses; and A controller (130) configured to control the pulse generator (120), the controller being configured to control the pulse generator (120) to apply a first sequence of one or more monopolar pulses using a first configuration of electrodes driven as anodes and cathodes to provide a first shaped electric field adjacent to the array (110), and after a waiting period with a duration ranging from 10 ms to 600 ms, to apply a second sequence of one or more monopolar pulses using a second configuration of electrodes driven as anodes and cathodes to provide a second shaped electric field adjacent to the array (110).

2. The electrotransfer delivery system of claim 1 , wherein the first selection of electrodes comprises a linear configuration of one or more anodes and one or more cathodes, and the second selection of electrodes comprises the same electrodes with the anodes and cathodes switched so as to reverse polarity.

3. An electrotransfer delivery system as described in claim 1, wherein the electrode array (110) is a two-dimensional array, and wherein the first selection of electrodes includes a configuration of one or more anodes and one or more cathodes, and the second selection of electrodes includes an electrode configuration as follows: it includes electrodes different from the first selection, and the electrodes are selected to produce a change in the electric field gradient within the target treatment area.

4. The electrotransfer delivery system of claim 1 , wherein one or more additional electrodes are selected to be driven as anodes and cathodes using one or more electrical pulses, and electrical pulse parameters of the one or more electrical pulses are determined for the selected electrodes to generate a second shaped electric field for a target treatment area adjacent to the array; and For each additional selection of electrodes, the controller (130) controls the pulse generator (120) to apply an additional sequence of two or more monopolar pulses using each additional selection of electrodes driven as anodes and cathodes to produce each additional shaped electric field, Each additional selection produces a controlled electric field gradient within the target treatment area that is different from the electric field gradient of the previous electric field.

5. An electrotransfer delivery system as described in claim 4, wherein the selection of electrodes and the sequence of pulses are selected to produce an electric field sequence, wherein subsequent electric fields each have an electric field gradient across the target treatment area at incremental angles relative to the previous electric field.

6. The electrotransfer delivery system of claim 1, wherein increasing or decreasing the spacing between the anode and cathode is used to control the radius of the treatment area.

7. The electrotransfer delivery system according to any one of claims 1 to 6, wherein: The duration of the waiting period ranges from 15 ms to 400 ms.

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