Electrotransfer therapy delivery devices, systems and methods

Abstract

A device capable of efficient electroporation of DNA into cells by single-capacitor discharge is disclosed. The principle of the system involves storing a charge quantum on a capacitor, which is then discharged through an electrode array configured to produce an electric field having an electric field potential gradient focused on an area by the array configuration and of sufficient strength for efficient electroporation of DNA into cells. The DNA or associated ribonucleic acid molecules or even other charged molecules once inside the cells affect changes in biological function.

Description

TECHNICAL FIELD

[0001] The field of the invention is electric field stimulated cell transfection for the delivery of therapeutic molecules, particularly using a linear electrode array on a single probe. One application of the invention is for the electroporation of DNA into cells. BACKGROUND

[0002] Electric field stimulated cell transfection or electroporation for the delivery of therapeutic agents to cells is commonly known as electroporation. Electroporation is typically achieved by injecting a tissue with a therapeutic agent (e.g. a solution of therapeutic molecules), inserting two separate probes into the tissue on either side of the target treatment area, one to act as a cathode and the other as an anode, and passing an electric current through the tissue between the two probes. This causes temporary disruption of the cell membrane, allowing the therapeutic agent to penetrate into the cell, also known as transfection. This is a known method for the delivery of gene therapy.

[0003] When the DNA encodes a gene for a protein or part thereof, this electric field based delivery of DNA to cells can be referred to as “gene electroporation” (GET). Electroporation of DNA into cells is a routine laboratory procedure. Electroporation / GET is also a recognised means for achieving gene expression in situ or in vivo in tissues, although the efficiency of gene expression in tissues following DNA electroporation is typically low.

[0004] Traditional electroporation requires high voltage pulses and therefore requires specialised equipment and a clinical environment. This can limit the accessibility of such therapies.

[0005] The inventors’ previous patent applications with publication numbers WO2011 / 006204, WO2014 / 201511 and WO2016 / 205895 describe the development of techniques for stimulating transfection within a target area adjacent to a contiguous electrode array, the disclosures of which provide background to the present disclosure and can be referred to for a better understanding of the inventors’ near field electroporation techniques. The inventors’ previous work has shown that surprising efficiencies of electroporation have been achieved using a “near field” electroporation technique which uses a single probe linear electrode array such that cells are transfected in tissue adjacent to the linear array. For example, the electrode array comprises two or more elongate electrodes which are arranged linearly and are spaced apart at discrete positions along the length of a single probe. The electrodes are adjacent to a substrate array structure such that no tissue will be located directly between the electrodes. When an electroporation pulse is applied, an electric field gradient is generated in the tissue adjacent to the array, the shape of the electric field and the gradient of the electric field being controlled by the configuration of the linear elongate electrodes.

[0006] In WO 2016 / 205895, the inventors disclose a system in which the area of cell transfection occurring by electrical stimulation is controlled by controlling the gradient within the electric field generated by the electrode array configuration and the pulse sequence applied to generate the electric field. The system of WO 2016 / 205895 illustrates the practical application of the inventors' finding of improved cell transfection within the area of tissue subjected to a steeper electric field potential gradient, and the application of this finding to control the shape of the electric field to generate a curve in the electric field for the target area of cell transfection adjacent to the array. The variables for controlling the electric field shape include the array configuration and the stimulation pulse parameters. The use of controlled electric field shaping (electrofield effect) enables to improve the efficiency of cell transfection with lower cumulative charge than previously known open field electroporation.

[0007] This near field electrotransfer technique has the advantage over conventional electroporation techniques in requiring lower cumulative charge. Another advantage is that the shape and size of the area of tissue in which cell transfection occurs can be predictably controlled based on the combination of linear electrode array configuration and electroporation pulse parameters controlling the electric field gradient of the generated electric field. However, it is desirable to further improve the electrotransfer technique. SUMMARY

[0008] According to a first aspect, there is provided an electrotransfer system comprising: at least one probe, each probe comprising: a probe body; an array of needle electrodes extending from the probe body configured to be inserted into tissue to be treated; and a capacitive discharge circuit connected to the array of needle electrodes, comprising a capacitive charge storage configured to store a charge quantum, and a switch actuatable to discharge the stored charge quantum through the array of needle electrodes, the array of needle electrodes comprising at least two electrodes each having a surface area substantially surrounding a needle and exposed to directly contact tissue into which the array of needle electrodes is inserted, with an insulating portion between adjacent electrodes to form a contiguous linear array structure, the exposed surface area of each electrode being different from a distance of the needle electrode array tip, each electrode being connected to the capacitive discharge circuit for driving as an anode or cathode during discharge of the charge quantum, and wherein the electrode length and the length of the insulating portion between adjacent electrodes are configured to generate a target electric field shape in tissue adjacent to the array during discharge of the charge quantum through the array; and a charging station having a DC power module, output terminals connectable to a probe to form electrical contact with the capacitive discharge circuit, and a charging control circuit for controlling charging of the capacitive discharge circuit of a connected probe.

[0009] In one embodiment, the capacitive discharge circuit is housed in the probe body.

[0010] Some embodiments of the electrical transfer system also include a switch actuator carried on the probe body configured to actuate a capacitance discharge circuit switch.

[0011] In some embodiments, the charging station includes a probe mount to support the probe during charging.

[0012] In some embodiments, the electrodes are relatively elongate, where the electrode diameter is less than the electrode length. In some embodiments, the electrodes have a length of 1 mm or less and an electrode diameter that is less than the electrode length.

[0013] In some embodiments, the capacitance discharge circuit parameters are matched to the resistivity parameters of the treatment solution used for electrical transfer to minimize the total charge delivered and optimize the discharge time constant for electrical transfer. For example, the capacitance of the capacitance discharge circuit can be selected based on a predicted total resistivity during discharge and a target cumulative charge, and the predicted total resistivity is based on the treatment solution resistivity. For example, a target for the fit can be used to achieve a discharge time constant between 20 ps and 2 s.

[0014] In some embodiments, the probe body also includes a treatment reservoir and a lumen through the needle in fluid communication with the treatment reservoir to hold a volume of fluid, and a treatment delivery actuator operable to force fluid out of the treatment reservoir and through the lumen and out of the needle. In some embodiments, the treatment delivery actuator is also configured to activate the switch. In some embodiments, the probe is packaged with a treatment solution stored within the reservoir. In alternative embodiments, the reservoir is filled with treatment solution prior to charging the capacitance discharge circuit.

[0015] In some embodiments, the needle electrode includes a first needle that also serves as a first electrode, a first concentric insulator that wraps the first needle to a predetermined first distance (LI) from the needle tip, a second electrode that is concentric that wraps the first concentric insulator to a distance (L2) from the end of the first concentric insulator, and a second concentric insulator that wraps the second electrode to a distance (L3) from the end of the second electrode, where the first needle and second concentric electrode are formed of an electrically conductive material and are electrically connected to the positive and negative terminals of the capacitance discharge circuit.

[0016] In some embodiments, the exposed length LI of the first needle serves as a first electrode of a linear array, and the exposed length L3 of the second concentric electrode serves as a second electrode of the linear array. The respective lengths of the first and second electrodes and the exposed length of the first insulating portion therebetween can determine the pattern of the electric field gradient generated in the vicinity of the needle array when an electrical pulse is applied to drive one electrode as an anode and the other electrode as a cathode.

[0017] According to another aspect, there is provided an electrical transfer system probe comprising: a probe body; a needle electrode array extending from the probe body, configured as a needle to be inserted into tissue to be treated; and a capacitive discharge circuit connected to the needle electrode array, comprising a capacitive charge store configured to store a charge quantum, and a switch actuatable to cause the stored charge quantum to discharge through the needle electrode array, wherein the needle electrode array comprises at least two electrodes each having a surface area substantially surrounding the needle and exposed to directly contact tissue into which the needle electrode array is inserted, with an insulating portion between adjacent electrodes to form a contiguous linear array structure, the exposed surface area of each electrode being different from a distance of the needle electrode array tip, each electrode being connected to the capacitive discharge circuit for driving as an anode or cathode during discharge of the charge quantum, and wherein the electrode lengths and lengths of the insulating portions between adjacent electrodes are configured to generate a target electric field shape in tissue adjacent to the array during discharge of the charge quantum through the array.

[0018] In one embodiment, the needle electrode comprises: a first needle providing a first electrode for a length of the needle proximate to a needle tip; a first concentric insulator wrapping the first needle to a predetermined first distance (LI) from the needle tip, a conductive sheath cladding the first concentric insulator to a distance (L2) from a distal end of the first concentric insulator and forming a second electrode, and a second concentric insulator wrapping the second electrode to a distance (L3) from a distal end of the second electrode, wherein the first needle and second concentric electrode are formed of a conductive material and electrically connected to positive and negative terminals of the capacitive discharge circuit. The respective lengths of the first and second electrodes and the exposed length of the first insulating portion therebetween determine the pattern of the electric field gradient generated in the vicinity of the needle array when an electrical pulse is applied to drive one electrode as an anode and the other electrode as a cathode.

[0019] Some embodiments of the probe further comprise a treatment reservoir and a lumen passing through the needle array in fluid communication with the treatment reservoir to hold a volume of fluid, and a treatment delivery actuator operable to cause the fluid to be forced out of the treatment reservoir and through the lumen from the needle. In some embodiments, the treatment delivery actuator is further configured to activate the switch.

[0020] In some embodiments, the probe body has a two-part form, where a first part comprising the reservoir and the treatment delivery actuator is provided by a syringe, which is connectable to a second part comprising an electrotransfer module having a housing adapted to engage with the syringe and support an array of needle electrodes projecting outwardly from the housing, where the capacitive discharge circuit is housed within the housing, connected to the array of needle electrodes, and forms a fluid communication path between the syringe reservoir and the lumens of the needle electrodes through the housing. For example, the electrotransfer module housing can be luer lock compatible to engage with a conventional luer syringe.

[0021] According to another aspect, there is provided a needle electrode array comprising: a first needle also serving as a first electrode; a first concentric insulator wrapping the first needle to a predetermined first distance (LI) from the needle tip, a second concentric electrode wrapping the first concentric insulator to a distance (L2) from the end of the first concentric insulator, and a second concentric insulator wrapping the second electrode to a distance (L3) from the end of the second electrode, where the first needle and the second concentric electrode are formed of an electrically conductive material and are electrically connected to positive and negative terminals of a pulse delivery circuit.

[0022] In some embodiments, the exposed length LI of the first needle serves as a first electrode of a linear array, and the exposed length L3 of the second concentric electrode serves as a second electrode of the linear array. In some embodiments, when an electrical pulse is applied to drive one electrode as an anode and the other electrode as a cathode, the respective lengths of the first and second electrodes and the exposed length of the first insulating portion therebetween determine the pattern of the electric field gradient generated in the vicinity of the needle array.

[0023] In some embodiments, the needle tip is beveled.

[0024] In some embodiments of the needle electrode, the first needle has a hollow lumen through which a treatment solution can be delivered.

[0025] In some embodiments, the treatment solution is delivered from an aperture in the tip of the first needle to the tissue.

[0026] In some embodiments, the needle array comprises an aperture in the array distal to the tip, whereby a treatment solution is delivered to tissue adjacent to the needle array.

[0027] Another aspect provides a method of gene electrotransfer using a system as described above, comprising the steps of: charging the probe; inserting the needle array into a target tissue; delivering a treatment solution to the target tissue; actuating the capacitive discharge; and removing the needle array from the tissue. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a representative diagram of an embodiment of a near field capacitive discharge electrical transfer system.

[0029] Figure 2 is a block diagram of one example of a charging unit of an embodiment of the system.

[0030] Figure 3 is an example of a capacitive charging circuit of an embodiment of the system.

[0031] Figure 4 is an example of a measured potential for capacitive discharge.

[0032] Figure 5 is another example of a circuit diagram for a charging unit of an embodiment of the system.

[0033] Figure 6 Contrastively illustrates a typical electroporation pulse profile and a capacitive discharge pulse profile.

[0034] Figure 7a and 7b illustrates results of a first study comparing DNA encoding (transfection) expression using a prototype probe stimulated by a square wave sequence and exponential decay pulse.

[0035] Figure 8 illustrates results of a second study investigating the feasibility of cell transfection using a single capacitive discharge pulse.

[0036] Figure 9a illustrates bioluminescence imaging of a mouse in the area of the encircled hind limb 24 hours after DNA electrical transfer.

[0037] Figure 9b and 9c is a chart showing the peak total luminous flux emitted from each mouse leg at each time point after electrical transfer, showing the peak total luminous flux emitted from each mouse leg sampled at time points up to 500 days after electrical transfer, demonstrating the long term retention effect of electrical transfer.

[0038] Figure 10 illustrates some effects of manipulating the gap between the anode and cathode of an electrode array.

[0039] Figure 11 illustrates one example of an embodiment of a probe tip.

[0040] Figure 12 illustrates an example of an embodiment of a needle array.

[0041] Figure 13aA model showing the electric potential in the medium surrounding the DNA delivery probe when 240V is applied on the electrodes, with respect to the peak voltage induced in the field near the probe during discharge of the integrated capacitor.

[0042] Figure 13b A model showing the electric field strength around the DNA delivery probe when a 240V voltage is applied instantaneously on the electrodes.

[0043] Figure 14 Another example of an embodiment of a probe tip is shown.

[0044] Figure 15a And 15b A conceptual rendering of a probe being inserted into a charging station is illustrated.

[0045] Figure 16 A conceptual example of an embodiment of an embodiment of a probe configured as a syringe device and compatible charging station is illustrated.

[0046] Figure 17 An image showing the utility of a prototype SCD-BaDGE in vivo application, showing expression of a light-activatable ChrimsonR channel rhodopsin ion channel protein - TD tomato fusion reporter protein in a mouse pectoral fin fiber.

[0047] Figure 18 An example of an embodiment of an electrotransfer module and charging unit that can be connected to a syringe is illustrated.

[0048] Figure 19a And 19b An example of an embodiment of an electrotransfer module and compatible syringe is shown in Figure 19a as separate components and connected in Figure 19b .

[0049] Figure 20 An exploded view of an embodiment of an electrotransfer module and compatible syringe is shown.

[0050] Figure 21a An example of an embodiment of an electrotransfer module connected to a syringe is illustrated, providing a more detailed view of the electrotransfer module components.

[0051] Figure 21b An example of an embodiment of an electrotransfer module connected to a low dead band syringe is illustrated, and details of the electrotransfer module components are shown.

[0052] Figure 21c The components of an embodiment of an electrotransfer module are illustrated in detail.

[0053] Figure 22An embodiment of an electroporation module connected to a syringe that is placed in the module to charge a capacitor discharge circuit is illustrated.

[0054] Figure 23 An example of an inoculation process for delivering a DNA vaccine using an embodiment of an electroporation system is illustrated.

[0055] Figure 24a Steps for therapeutic delivery by a syringe and an electroporation module are illustrated.

[0056] Figure 24b Steps for electroporation using an electric field generated in tissue around a needle array by a capacitor discharge to stimulate tissue are shown.

[0057] Figure 25 A photograph of a prototype embodiment of an electroporation module showing the housing of one electroporation module opened to show the internal components.

[0058] Figure 26a A set of images showing comparative cell transfection results for electroporating DNA using a single pulse of 100 V and a cochlear electrode array and a concentric needle type array using single pulses of 100 V, 40 V, and 10 V, where each cochlear electrode was grouped into an anode array and a cathode array (series configuration).

[0059] Figure 26b A bar graph of the results shown in FIG. 16B. Figure 26a

[0060] A plot of the potential of a BaDGE concentric stainless steel electrode array (hemisphere). Figure 26c

[0061] Time course results for an embodiment of electroporation-mediated gene expression are shown. (A) Representative bioluminescence images demonstrating the spread of luciferase activity three days after intramuscular electroporation-mediated transfer of a luciferase gene (Luc) to the gastrocnemius muscle of a mouse. (B) Representative trace of photons emitted per second in the ROI placed on the hind limb measured at one minute intervals after intraperitoneal injection of luciferin. (C) Time course plot of bioluminescence measurements at the peak (red arrow in B) one day, three days, and seven days after electroporation-mediated transfer of a luciferase-encoding plasmid. Figure 27

[0062] Figures 28a-28c A photograph of an example of a wired needle electrode embodiment.

[0063] Figures 29a-29b A prototype test embodiment of high-throughput needle electrode DNA electroporation in a cell suspension for autologous gene therapy and ex vivo cell transfection is illustrated. ​​

[0064] Figure 30 An image of HEK 239 cells showing expression of mCherry reporter plasmid DNA under cell background (DAPI nuclear stain) achieved using in vitro electroporation with a concentric stainless steel DNA electroporation electrode configuration.

[0065] Figure 31 Another example of an electroporation system used in in vitro cell therapy is illustrated, which is suitable for high-throughput multi-well cell transfection with plasmid DNA by DNA delivery probes. DETAILED DESCRIPTION

[0066] One aspect disclosed herein is a device capable of high efficiency electroporation of DNA into cells by single capacitance discharge.

[0067] The principle of the system involves storing a quantum of charge on a capacitor, which is then discharged through an electrode array configured to produce an electric field having an electric field potential gradient focused on an area by the array configuration, and of sufficient strength for efficient electroporation of DNA into cells. This DNA or related ribonucleic acid molecule or even other charged molecules once inside the cell affect changes in biological function.

[0068] In some embodiments, this can be a discharge of a single capacitor.

[0069] Figure 1 An example of an electroporation system 100 is shown having at least one probe 110 and a charging station 120. Each probe 110 includes a probe body 170, a needle electrode array 130, and a discharge circuit 145. In the simplest embodiment, the discharge circuit includes a capacitor and a switch. The needle electrode array 130 extends from the probe body 170 and is configured as a needle to be inserted into tissue to be treated.

[0070] The needle electrode array 130 includes at least two electrodes 140, each having a surface area fully or partially surrounding the needle and exposed to directly contact tissue into which the needle electrode array is inserted, with an insulating portion 150 between adjacent electrodes 140. This forms a contiguous linear array structure with the exposed surface area of each electrode being different from the distance of the needle electrode array tip.

[0071] A capacitance discharge circuit 145 is connected to the needle electrode array and is configured to store a charge quantum. The circuit includes a switch that is actuable to cause the stored charge quantum to discharge through the needle electrode array 130. Each electrode 140 is connected to the capacitance discharge circuit for driving as an anode or cathode during discharge of the charge quantum. The electrode length and the length of the insulating portion between adjacent electrodes are configured to produce a target electric field shape in tissue adjacent to the array during discharge of the charge quantum through the array. The combination of the elongated electrodes arranged in a straight line with gaps between them causes an electric field to be produced in the vicinity of the electrode array. The gaps between the electrodes focus the electric field in the tissue in the region close to the gaps. The width of the gaps controls the rate of change of the electric field with distance, and thereby the electric field strength in the region near the gaps, thereby defining the shape of the electric field. Research by the inventors has shown that the high electric field strength resulting from the rate of change of voltage potential resulting from the linear array geometry described can stimulate electrical transfer with lower stimulation voltages and cumulative charge than conventional open field electroporation. Thus, effective electrical transfer is achieved using a single pulse. The potential gradient can be varied by adjusting the length of the gaps between the electrodes. The electric field is effectively focused. Changing the electrode length also has some effect. The combination of the electrode array geometry and the stimulation pulse controls the size and shape of the electrical transfer region.

[0072] The charging station 120 has a DC power supply module, output terminals connectable to the probe 110 to make electrical contact with the capacitance discharge circuit, and a charge control circuit for controlling the charging of the capacitance discharge circuit of the connected probe. Figure 2 is a block diagram of an embodiment of the charging station 120, 200. The DC power supply 210 can be a battery powered or mains powered DC power supply. Some embodiments use an adjustable DC power supply 215, including the DC power supply 210 and an adjustable regulator 220, to enable user or automatic control of the output voltage. The charging station can include a DC-DC voltage converter 230. The charging station can also include an output monitoring module 240 configured to determine whether a probe is connected to the charger, and the status of the connected probe and capacitance discharge circuit. The monitoring module can also be configured to control the adjustable DC power supply to disconnect power once a predetermined charge quantum is stored in the capacitance discharge circuit. The station can include a sensor contact 260 to detect the presence of a probe at the charger. The monitoring module 240 can also output a status signal, for example using a visual signal (i.e. LED) or an audio signal. For example, the electronics of the charging station can include elements for determining safe charging of a needle delivery probe, an indicator of successful charging, and a timer to simulate the slow leak of a capacitor, with a green / red LED or a perspiration prompt or a chime to indicate whether the effective DNA electrotransfer has been too long. Figure 5 is a circuit diagram illustrating an example of the charging station.

[0073] Figure 3is an example of a capacitive discharge circuit 300 that is an embodiment of the system. The circuit includes a capacitive charge store, in this example a single capacitor 310, a switch 320 and connections to the electrodes 140 of the needle electrode array. The circuit can also include charge contacts 330, 335 to establish an electrical connection to the charging station 200 for charging the capacitive charge store with a quantum of charge. The switch can be actuated to discharge the stored quantum of charge through the electrodes 140 of the needle electrode array 130. Each electrode 140 is connected to the capacitive discharge circuit to be driven as an anode or cathode during discharge of the quantum of charge. Figure 4 An example of the potential 400 measured in the medium adjacent to the anode during discharge of the capacitive circuit is shown, 410 shows the potential at zero prior to discharge of the capacitor (open circuit condition for up to approximately 70ms). 420 shows the rapid rise in voltage at the sampling point medium close to the electrode array, reaching a maximum voltage peak of approximately 2V at approximately 70ms when the switch is closed and discharge begins, and the potential 430 decays exponentially as the capacitor discharges.

[0074] In one embodiment, the capacitive discharge circuit is housed in the probe body, thus enabling delivery of the electrical pulse (capacitive discharge) without the need to connect to an external power supply and pulse generator. The capacitive store can be a single capacitor. Alternatively, the capacitive store can comprise more than one capacitor. In some embodiments, the capacitive discharge circuit can be external to the probe body. In further embodiments, other charge storage media such as supercapacitors and batteries can be used in place of capacitors. All of these alternatives are considered to be within the scope of the present system. For example, for applications such as brain therapy, the priority can be to minimise the size of the probe to enable high precision and insertion control, in such embodiments the capacitive discharge circuit can be housed separately from the probe body, connected by a wire. This would still provide the advantage of providing electrical transfer therapy without the need to connect to a power supply to power the pulse generator. Thus, the equipment footprint in the clinical space can be reduced. As the pulse generator is not required, the risk of electrical interference or electric shock is also reduced. Examples of further embodiments are discussed below.

[0075] As noted above, DNA delivery to cells based on electrotransfer is commonly referred to as electroporation. When the DNA encodes a protein’s gene, this DNA delivery to cells based on an electric field can be referred to as “genetic electrotransfer” (GET). Electroporation of DNA into cells is a routine laboratory procedure. Electroporation / GET is also a recognized means for achieving gene expression in situ or in vivo in tissue, although the efficiency of gene expression in tissue following DNA electroporation is typically low. Two related factors that affect GET are electric field strength (voltage as a function of distance) and duration. Creating a voltage gradient within a tissue suitable for GET requires current flow between electrodes. This is achieved using an electroporation device that delivers a pulse of current between two or more electrodes to create a GET-compatible electric field (typically 10-100 V / cm) for a duration of typically hundreds of microseconds to hundreds of milliseconds, with multiple iterations of these electric pulses within one or more pulse sequences. A single electric pulse is generally considered to be inefficient in achieving GET. Thus, the instrumentation required for conventional GET includes a device for generating a sequence of electric pulses of controlled voltage or current amplitude, duration, and frequency, connected to electrodes inserted into tissue. Such instrumentation is both expensive and bulky.

[0076] GET has a distinct advantage if the electrodes can be controlled independently of the electroporator controller, particularly in terms of electrode geometry, where a wired connection to the electroporator controller or an integrated electroporator microcontroller imposes limitations on the tissue target in the context of targeted delivery of DNA to different body regions.

[0077] The inventors have discovered how to achieve efficient GET using a single electric pulse that does not require microprocessor-based control of pulse parameters. This is achieved by separating the charge source from the discharge control, where the latter occurs through the discharge of a capacitor. The charge source is a DC power source used to charge the capacitor, which can then be decoupled from the capacitor circuit. In using the discharge of the capacitor for GET, the inventors have found that the discharge time of the capacitor is a key factor—see below Figure 8Further explanation. The capacitor time constant (tau) depends on the size of the capacitor (C) and the resistance of the circuit (R), so Tau = R.C. It will be appreciated that during discharge, the total resistance in the circuit includes the resistance in the target tissue (affected by both the tissue and the DNA carrier solution) and any circuit resistance. In the present disclosure, we show that the combination of the minimal current requirements of GET, enhanced by the use of a high resistivity DNA carrier solution to control the resistivity around the electrode array, in a "near field" electroporation configuration (two or more elongated electrodes arranged in a line sequentially along a single probe, with the electrode at one end of the single probe driven as an anode or anode array and the electrode at the other end driven as a cathode or cathode array), enables efficient GET when matched to a specific capacitor range. We show the implementation of single capacitor discharge GET, where the optimal (minimal) charge delivery is determined based on the unexpected relationship of the discharge time constant (tau) to GET efficiency. Where Q = C.V., and desiring to limit the total charge Q delivered to minimize potential tissue damage, we show that single capacitor discharge GET can achieve gene expression efficiency equivalent to conventional multi-pulse electroporation based on the delivered Q. In some embodiments, the total circuit resistivity R during discharge can be predicted, as well as the capacitor C selected based on the target charge delivery Q. The total circuit resistivity R during discharge can be predicted based on the resistivity of the carrier solution and the typical tissue resistivity range or test results for the target tissue type (i.e. muscle tissue, fat tissue, breast tissue). This can also be predicted by testing the resistivity of the tissue after injection of the carrier solution. The carrier solution can have a significant impact on the resistivity in the tissue, and some carrier solutions can make the impact of the inherent resistance of the target tissue negligible. For example, the resistivity of a high resistivity carrier solution can be in the range of 2 kOhms to 100 kOhms, with a measured resistivity value of approximately 10 kOhms for a 19% sucrose solution with a typical 2 pg / pl DNA concentration. The cumulative charge Q required for the DNA electrotransfer can be determined by previous testing and is therefore known. This can be a range indicating the minimum cumulative charge to achieve electrotransfer and the maximum cumulative charge to avoid tissue damage, and the target cumulative charge value can be selected within this range. Based on the resistivity and the charge value, the capacitor required for the discharge circuit can be calculated, and if needed, additional resistors included in the discharge circuit packaged in the electroporation module can also be calculated. Thus, the capacitor discharge circuit parameters can be matched to the resistivity of the treatment solution. It will be appreciated that different types of carrier solutions can have different resistivities, and therefore the circuit capacitance selected is matched to the intended carrier solution.

[0078] The foundational data for the invention of Single Capacitor Discharge Gene Electrotransfer (SCDGET) includes a series of in vitro studies using a human embryonic kidney (HEK293) cell monolayer model. We previously used this model to develop BaDGE-Bionic Array Directed Gene Electrotransfer (BaDGET). Here, the prototype BaDGE DNA delivery (… Figure 10 The 1010 probe is connected to a computer-controlled conventional constant current power supply to deliver a specified square wave pulse sequence, or a pulse sequence or a single pulse followed by a single exponential decay, reflecting the time constant of capacitor discharge. Figure 6 A comparison is shown between a conventional square electroporation pulse and a simulated capacitor discharge pulse. Figure 6 In the middle, left pane 610 shows a unipolar square wave pulse generated using a Digitimer DS5 to simulate a constant current power supply (100 μs on, 400 μs off), while right pane 620 shows the simulated capacitor discharge pulse with a 100 μs time constant. These pulse parameters, along with the BaDGE prototype probe, were used to deliver pf reporter gene DNA into a HEK293 cell monolayer to simulate delivery to the tissue.

[0079] Figure 7a and 7b Data comparing DNA expression encoding the mCherry fluorescent reporter protein across a series of these pulse sequences are presented. This study used a prototype electrode array (e.g., Figure 10 (See Figure 1010) to compare the results of electrotransfer in human embryonic kidney (HEK293) cells with CMVp-mCherry plasmid fluorescent reporter gene (2 μg / μl) using square wave monopolar 710 and exponential decay 720 functions. Figure 7a The fluorescence shown was imaged 3 days after gene electrotransfer; the upper pane 710 shows the transfection results using a square wave, while the lower pane 720 shows the transfection results using an exponentially decaying pulse. In the results... Figure 7b In the diagram, 5+square represents a 5×100μs square wave constant current pulse (50mA; 400μs pulse interval), 5+attenuation represents a 5×exponentially decaying (capacitive) pulse, 100μs time constant, and 500μs pulse interval; 10+square represents a 10×100μs square wave pulse; 10+attenuation represents a 10×100μs decay time constant pulse. Holm-Sidak rank-based ANOVA with multiple post-hoc comparisons was used (* indicates P<0.05; rank-based ANOVA, post-hoc pairwise comparisons).

[0080] Figure 7a and 7bThe results show that, using a time constant (tau) of 100 μs, ten capacitor discharge sequences at a peak value of 120 V achieve an equivalent expression to five square wave pulses with an amplitude of 50 mA and a duration of 100 μs (400 μs pulse interval). Capacitor discharge is inefficient with 10 × 50 mA × 100 μs square wave pulses, while the total charge delivery (Q) is ~equivalent. These data demonstrate that rapidly decaying discharges achieve significant GET with minimal total charge delivery. However, multiple discharge pulses are required, which typically necessitates microprocessor-based pulse sequence control.

[0081] Figure 8 The results of a second study are presented, which explored whether increasing the time constant could achieve single-capacitor discharge GET. This study compared reference gene expression induced by a 10 × 50 mA × 100 μs square wave pulse (as above, copies of five HEK293 cell coverslips) with reference gene expression induced by single-capacitor discharge pulses with time constants of 400 μs, 1 ms, 4 ms, and 10 ms; charged to 120 V, which corresponds to the square wave pulse parameters (based on a 50 mA current amplitude across a ~2 kΩ resistance). These data indicate that GET efficiency is highly dependent on the capacitor time constant, where a single pulse with a 1 ms time constant (equivalent to the Q of a 10 × 100 μs square wave pulse) produced statistically equivalent gene expression (integrated with mCherry fluorescence), as the reference square wave pulse sequence. Furthermore, there was a non-linear dependence on the capacitor discharge time constant, where a 4 ms time constant produced significantly higher gene expression than the reference level, as did a 1 ms decay time constant treatment, and further increasing the time constant (to 10 ms) did not achieve further efficiency gains, although charge delivery (Q) increased. In summary, these two experiments demonstrate that single-capacitor discharge gene electrotransfer can be achieved with comparable efficiency (relative to the amount of charge delivered) to conventional square wave electroporation pulses.

[0082] Figure 8 The results shown indicate that the prototype Capacitor discharge gene electrotransfer (BCDGET) (single pulse) is at least as effective as conventional monopolar square wave constant current pulse sequences in inducing gene expression. Figure 8 Figure 810 shows fluorescence data of the mCherry reporter gene from a coverslip of HEK293 cells (n=5 per treatment), where the plasmid DNA encoding the reporter gene is placed on the cell, and the prototype... DNA delivery probes were used to electrotransfer DNA to cells. Coverslides were imaged 3 days after tissue culture. The data showed equivalence between our reference 10 × 100 μs square wave constant current pulses 820, 820' (~50 mA per pulse – designated “100Up 100µs square”) and single-capacitor discharges with a time constant of 400 µs 830, 830' (e.g., “1Up 400µs decay constant”), 1 ms 840, 840' (e.g., “1Up 1ms decay constant”), 4 ms 850, 850' (e.g., “1Up 4ms decay constant”), and 10 ms 860, 860' (e.g., “1Up 10ms decay constant”). Single-capacitor discharges with longer time constants showed significantly greater gene electrotransfer. Figure 8 The lower panel shows examples of fluorescence imaging of the mCherry reporter gene in HEK293 cells on coverslips using different charge delivery protocols (400 μs time constant = ~200 nF; 1 ms = ~500 nF, 4 ms = ~2 μF, 10 ms = ~5 μF (* indicates P < 0.05; ranked ANOVA, post-hoc pairwise comparisons)).

[0083] To validate these findings in the first in vivo proof-of-concept study, a mouse 900 hindlimb model was used. Figure 9a Under isoflurane anesthesia, a prototype was used, driven by a standard constant current pulse sequence (5 × 4 ms pulses, 16 ms pulse intervals, ~60 V, recorded as 50 mA current amplitude for each pulse). DNA delivery probe ( Figure 10 In step 1010, DNA encoding the luciferase gene (1 μg / μl; 50 μl) was delivered to the right gastrocnemius muscle (910). The plasmid DNA included a hybrid cytomegalovirus-chicken β-actin-rabbit β-globin gene splice acceptor sequence (CAG promoter, 6604 bp in size). The left gastrocnemius muscle (920) was treated with single-capacitor discharge gene electrotransfer, in which… The DNA delivery probe was attached to a 2.2 μF capacitor charged to 120 V. This delivered a single-capacitor discharge (based on a 2 kΩ resistance model) with a time constant of 4 ms, where the total charge delivered was approximately ~50% of the charge used by a reference 5 × 4 ms square wave pulse sequence delivered to the right hind limb target. Expression was analyzed 24 hours later using a SPECTRUM CT bioluminescence imaging platform within 30 minutes of intraperitoneal injection of the bioluminescent substrate fluorescein. Images showed stronger expression (bioluminescence) in the left hind limb—induced by the single-capacitor discharge GET, rather than by the conventional square wave pulse sequence GET.

[0084] Figure 9a The illustration shows the efficacy of the in vivo proof-of-concept for the prototype single-capacitor discharge gene electrotransfer (SCDGET) device. Figure 9a This study presents spectral-CT bioluminescence imaging of mice (reference ms187,250519) within 30 minutes after intraperitoneal injection of a luciferin substrate under isoflurane anesthesia 24 hours following DNA electrotransfer to induce photoemission induced by recombinant luciferase expression in hind limb muscles. Emission 920 from the left hind limb was obtained using... The DNA delivery probe electrotransfers DNA (1 μg / μl) to the gastrocnemius skeletal muscle target, coupled to a 2.2 μF capacitor charged to 120 V. The right leg 910 uses the same probe, driven by a constant current power supply (Digitimer DS5 analog control) via software and DA control at 5 × 4 ms pulses (16 ms pulse intervals) (50 mA current through a ~60 V drive voltage). To obtain... Figure 9a The image shown depicts mice anesthetized with isoflurane and then injected (intraperitoneally) with luciferin—a substrate for expressed luciferase. This substrate is absorbed by muscle cells, and in the presence of luciferase expression, the resulting photons travel through the skin and are counted by a highly sensitive camera. This recording of emitted light reflects the expression intensity of the recombinant luciferase protein. Readings are taken 24 hours post-expression and overlaid on images of the mouse hind limbs, showing intensity-encoded light emission. This image demonstrates that SCDGET used a standard square-wave pulse sequence protocol for approximately 50% charge delivery and elicited approximately twice the bioluminescent signal.

[0085] For additional experiments on the legs of mouse reference number 187 and four other mice, the measurement of bioluminescent signal (luminous flux) is shown graphically. Figure 9b In the study, multiple readings were observed in mouse 187 (diamond-shaped trace) at days 1, 3, 7, 90, and 150. Other mice had readings at days 1, 3, 7, 14, and 50. Figure 9bThe illustration shows long-term comparative results of biomimetic array-directed gene electrotransfer (BaDGE) of plasmid DNA (pMK175.CAGp-luciferase) into mouse calf muscles via a single discharge using a 2.2 uF capacitor integrated into the BaDGE DNA delivery probe. The peak total luminous flux emitted by each mouse leg was measured at each time point following intraperitoneal injection of potassium D-luciferin (15 mg / kg body weight). In this study, mice underwent BaDGE on day 0 using a plasmid encoding the luciferase gene. The DNA was suspended in a 10% sucrose solution pH equilibrated with sodium hydroxide. For mouse legs receiving 10 μg of DNA, 20 μl was delivered at a volume of 0.5 μg / μl; for mouse legs receiving 20, 40, or 50 μg of DNA, 20 μl was delivered at volumes of 1.0 μg / μl, 20, 40, or 50 μl. The diamond symbol represents the left and right legs of mouse reference number 187; the circles represent the mean ± SEM data from mice reference numbers 119, 120, and 121. The squares represent data from mouse reference number 118, with two DNA payloads.

[0086] Figure 9c It shows Bioluminescent readings of luciferase reporter gene expression (LUC) after luciferase-mediated DNA electrotransfer. Images show peak expression in mouse #187 maintained until day 486, while similar sustained expression was observed in mice #118, 119, 120, and 121 at day 389. In luciferase substrate-mediated bioluminescence (intraperitoneal administration) peak imaging, dark rhombuses represent 4 × 5 ms square wave pulses (5 ms), while hollow rhombuses represent discharges from a 1 × 2.2 μF capacitor at 120 V (both 1 μg / μl pMK175-CAGp-Luc plasmid DNA). The chart shows a total of data from 5 mice (n=3, 250V (black circles) or 120V (hollow circles), 2.2μF, 10μg in 20μl); hollow squares represent 20μg DNA at 1μg / μl at 250V 2.2μF; black squares represent 40μl DNA at 1μg / μl at 250V, 2.2μF single-capacitor discharge. At the time of data submission, the longest available period was 486 days due to the timing of the tests, but given the observed sustained effects lasting up to 486 days, this provides a positive indication of a continuous effect.

[0087] These studies demonstrate a prototype device in which, integrated into The capacitor in the DNA delivery probe can be charged via a charging station (regulated power supply—charging typically takes <1 second). The DNA delivery probe is then unaffected by a controlled power supply and is used for DNA electrotransfer by simply short-circuiting the circuit (switch). Individual tests show that once the capacitor is charged, its voltage is maintained for at least 30 minutes.

[0088] Embodiments of the disclosed system stem from the discovery that a linear array of electrode elements configured as a DNA delivery probe is capable of efficiently electroporating naked DNA into a target tissue. The charge transfer required to achieve gene expression in vivo is orders of magnitude lower than conventional electroporation systems; due to the focused compression of the local electric field near the linear probe.

[0089] Another discovery applied in embodiments of the system is that the electrode configuration can control the extent of the transfection region relative to the linear electrode array. Figure 10 An example of a prototype linear electrode array probe 1010 is illustrated. The prototype electrode array includes two elongated tubular platinum-iridium (Pt-Ir) electrodes connected by an insulated Pt-Ir wire to a capacitor discharge circuit (or pulse generator), the Pt-Ir electrodes are coated on an inner needle with an insulating layer between them to insulate the electrodes from the inner needle from each other. During pulse delivery, one electrode is driven as the anode and the other electrode is driven as the cathode. The electric field gradient near the middle of the array has the steepest electric field potential gradient and experimental results show that this region is where the most cells are transfected. The field shape is due to the combination of the linear arrangement of the elongated anode and cathode and the respective dimensions of these conductive elements.

[0090] The elongated nature of the anode and cathode helps control the shape of the electric field generated and thus the transfection region. Changing the width of the gap between the elongated anode and cathode changes the extent of the transfection region orthogonal to the array because the compression of the electric field and the rate of change of the potential changes with distance near the array as one approaches the gap. By changing the gap, the electric field shape can be expanded or contracted, thus expanding or contracting the transfection region. In the illustrated example, increasing the gap between the electrodes expands the electric field and the transfection region, while decreasing the gap limits the field. It should be noted that this field shape control or vectoring is applicable to a gap between the electrodes of up to about 5 mm for the pulse parameters used in this example. Increasing this gap can be feasible in combination with a corresponding increase in the current level, but using more sets of electrodes can be more preferable to achieve treatment of a larger region due to potential negative side effects from higher current levels. Controlling the radial spread of the electric field can also be referred to as vectoring.

[0091] Figure 10 Images 1020-1050 of FIG. 1 illustrate the expansion and contraction of the transfection region with manipulation of the gap between the anode and cathode of the linear electrode array, in this case using the prototype probe. Figure 10 Figure 10 ​In the examples shown, probes 1010 of the type shown with two electrode arrays with linear spacing between the electrodes were used. The effect of electric field manipulation - achieved by reducing the separation between two platinum-iridium electrodes (2 mm long x 0.35 mm diameter on insulating support - as shown in the upper right image) - was implemented for each example array configuration using an electrical pulse sequence (constant current, 100 μs x 10 pulses, 400 μs pulse interval, 10 mA +ve phase) to drive the electrodes. The data shows spatial control of electroporation of plasmid DNA (green fluorescent reporter protein - GFP - under a cytomegalovirus (CMV) promoter) into a monolayer of human embryonic kidney (HEK) 293 cells using an isotonic polysaccharide carrier. The DNA and electroporation array were placed on the cells on a coverslip. The pulses were delivered and the coverslip was then returned to culture for 48 hours before imaging. The highest electric field strength is near the zero point between the two electrodes. As the electrodes are separated, the electric field sufficient for DNA electroporation expands until an optimal dispersion of expression of the recombination DNA expression cassette and cell density is achieved (electrode separation between 1 - 4 mm).

[0092] In embodiments of the capacitive discharge electroporation probes disclosed herein, the combination of electrode array configuration (particularly electrode length and gap between electrodes or groups of electrodes driven with the same polarity) and capacitive discharge characteristics control the region in which cell transfection can occur. Thus, the array configuration and discharge circuit can be designed to achieve a predictable therapeutic outcome. Furthermore, the tissue and therapeutic solution injected into the region surrounding the linear array forms part of the discharge circuit, thus affecting the characteristics of the capacitive discharge, and thus the capacitive discharge circuit can be designed to incorporate the therapeutic solution and / or tissue resistance into the equivalent circuit modeling the discharge characteristics and electric field. Thus, the probe can be designed for a particular therapeutic solution and target tissue. For example, the capacitor characteristics of the discharge circuit can be matched to the resistivity of the therapeutic solution to provide a target initial voltage potential, cumulative charge delivery, and pulse decay curve. The physical array configuration - length of the electrodes and the gap between them - can also be modeled for a particular application.

[0093] For one practical embodiment, it is envisioned that the SCDGET will be integrated into a disposable SCDGET-DNA delivery probe within a syringe, including pre-packaged DNA in the carrier solution. The SCDGET probe will be charged prior to use by contact with a SCDGET charging station, the DNA will be delivered through the syringe and needle, and the circuit closed to discharge the capacitor, thus instantiating the GET. An indicator on the charging station (e.g. LED) will confirm that the SCDGET probe has been charged, and indicate how long the charge will remain in the probe based on the discharge characteristics of a similar capacitor circuit within the charging station. As previously described, the probe charge time is less than a second. The charge retention time is on the order of tens of minutes. The physical size of the capacitor (typically 1 - 5 μF, but can be other values) appropriate to optimize the range of the SCDGET is on the order of mm3 The unit cost is a few cents within the range. Thus, this is suitable for high-throughput DNA GET applications such as DNA vaccines, where the SCD GET charging station can be battery powered for field work, and the DNA probe will be pre-loaded with DNA and single-use items.

[0094] Figure 11 An example of a prototype probe tip 1100 is illustrated, including a needle array 1110, a needle hub 1120, and a body 1140 (only a portion of which is shown). The hub 1120 can house a capacitor discharge circuit 1130 and electrical connections to the electrodes of the needle array. In Figure 12 In the illustrated embodiment, the hub 1120 also includes two ports 1135 to enable electrical connections to a charging station to charge the capacitor discharge circuit. The embodiment also illustrates a therapeutic reservoir in fluid communication with the central lumen through the needle array 1110, enabling the probe to be pre-loaded with a therapeutic solution for delivery prior to application of the electrical transfer pulse. For example, the probe tip can be integrated into a conventional syringe structure with a plunger to force fluid from the reservoir through the lumen. In such an embodiment, the plunger can also be configured to actuate a switch of the capacitor discharge circuit to cause the capacitor discharge. For example, the switch can be activated at the end of the plunger stroke.

[0095] The distal (tip) portion of the needle array 1110 is shown in more detail in Figure 12 and illustrates: a first needle 1210 that also serves as a first electrode (in this embodiment, with a beveled tip 1215 and a central lumen for therapeutic delivery); a first concentric insulator 1220 that wraps the first needle to a predetermined distance (LI) 1212 from the tip 1215; a concentric second electrode 1230 that wraps the first concentric insulator to a distance (L2) 1235 from the end of the first concentric insulator; and a second concentric insulator 1240 that wraps the second electrode 1230 to a distance (L3) 1235 from the end of the second electrode 1230. The first needle 1210 and the second concentric electrode are formed of an electrically conductive material and are electrically connected to the positive and negative terminals of the capacitor discharge circuit 1330. The exposed length 1212 of the first needle operates as the first electrode of the linear array, and the exposed length 1235 of the second concentric electrode 1230 operates as the second electrode of the linear array. The respective lengths LI 1212 and L3 1235 of the first and second electrodes, and the exposed length 1225 of the first insulating portion 1220 between them, determine the pattern of the electric field gradient that is generated in the vicinity of the needle array when an electrical pulse is applied to drive one electrode as an anode and the other as a cathode.

[0096] Figure 13aA model showing the electric potential in the medium surrounding the DNA delivery probe when a 240V voltage is applied on the electrodes, with respect to the peak voltage induced in the field near the probe during discharge of the integrated capacitor. Figure 13b A model showing the electric field strength around the DNA delivery probe when a 240V voltage is applied instantaneously on the electrodes.

[0097] One embodiment of the needle array is configured using two concentric needles, where the inner needle is insulated from a preset distance from the tip, the uninsulated tip region being the first electrode, and the outer needle is insulated from a set distance back from its tip. There is also a gap between the position of the second (shorter) needle and the insulated region of the inner needle (the distance of the insulating coating on the inner needle before its conductive tip corresponds to Figure 10 The spacing between the electrode elements shown, for the "slide tube" effect.

[0098] Both needles are inserted into a custom designed needle "hub". The hub includes circuitry coupled to the proximal end of the electrodes - this is also conductive. The hub can include holes for insertion of charging station pins, as well as a switch plate that closes the discharge circuit of the integrated capacitor. In one embodiment, the switch plate is activated when the plunger of the syringe barrel is fully depressed or at a predetermined point of travel.

[0099] One example of a prototype development involves using a 34G needle with a beveled tip (36mm long) and a 28G outer needle (27mm long) square cut thin walled needle as the circumferential second electrode. In one embodiment, the outer needle can also be beveled to facilitate the transition in needle diameter from the thinner inner needle to the outer needle. Alternative embodiments of the needle array can be fabricated using deposition (e.g. sputtering, printing, etc.) of conductive and insulating layers to form the electrodes.

[0100] In an alternative embodiment, the needle array has a closed tip and a port formed along the length of the needle array (away from the tip) for delivery of fluid to the adjacent tissue. One advantage of this embodiment is the flexibility in the location of the conductive regions of the inner and outer needles and optimal delivery of the DNA and carrier solution near the electrodes, enabling optimal control over the capacitor discharge time.

[0101] In some embodiments, the needle electrodes can include multiple electrodes along the array. For example, to allow multiple ports for delivery of a therapeutic solution from between the electrodes. In some embodiments, the needle electrodes can include multiple electrodes combined together or individually connected to one or more capacitor discharge circuits. In embodiments using multiple discharge circuits, these can be configured to be driven to discharge at different times, thereby changing the shape of the electric field during the treatment. For example, different capacitor discharge circuits are actuated at different travel positions of the syringe plunger. Different discharge circuits can also have different discharge characteristics.

[0102] In one example, the probe can have a dozen electrodes and six capacitors, which are connected to pairs of electrodes along a linear array. In another embodiment, elution of the DNA solution can also come from a port or ports between pairs of electrodes, rather than necessarily from the tip of the needle.

[0103] In some embodiments, the needle array can not include a lumen for treatment delivery. For example, another syringe can be used to deliver a therapeutic agent. For certain clinical applications or treatments, it can be desirable to use a separate device to deliver a therapeutic agent and to deliver electrical pulses. For physiological reasons, the lumen can be omitted to minimize the diameter of the needle array, for example to create an extremely thin needle array for use in the brain or for use in young pediatric patients. In these embodiments, the therapeutic agent can be injected or otherwise applied to the target tissue prior to placement of the DNA electroporation probe. Alternatively, the needle array can be coated with a hydrogel coating containing the therapeutic agent, delivering it from the outer surface of the needle array to the tissue. In another embodiment, the lumen can have multiple exit ports to distribute the therapeutic agent along its length.

[0104] Figure 14 Another embodiment of a probe tip is shown, similar to that shown in Figure 11 However, in this example, the hub includes additional ports to enable contact with sensors of a charging station to check that the probe is connected to the charger, as well as to verify the status of the capacitive discharge circuit within the probe. This is a concentric Conceptual diagram of a DNA electroporation probe. Features include an inner core DNA delivery needle with a predetermined length of conductive surface, and an insulating layer extending to the internal contact points within the hub. Away from the outer insulating surface is an outer conductive needle sheath with length and surface area that, in conjunction with the distal electrode surface and intermediate insulating region, contribute to the creation of a shaped electric field for DNA delivery. Near the conductive region of the outer sheath, the surface is insulating, except for the contact points within the hub. This hub is comprised of three ports—two of which are used to charge capacitors, while the third port provides a sensor contact to indicate charging, all of which are enabled by insertion of the probe into a remote charging station. In another embodiment, a monitoring circuit can evaluate the current flow between the two charging contacts and utilize this measurement to detect insertion. Figure 15a and 15b An example of a conceptual rendering of the insertion of a probe 1510 into a charging port 1530 of a charging station 1520 is shown in FIGS. 15 and 16. Not shown is a protective needle cover, which in one embodiment would be placed up until immediately prior to DNA delivery. The hub contains an integrated capacitor that is discharged by closing an internal switch that is acted upon by deflection of a pin element within the hub that contacts a fully or substantially fully depressed plunger of a syringe barrel that will contain a DNA solution and is attached to the hub of the DNA delivery probe. Figure 16A further conceptual diagram of a syringe body 1610 with an integrated needle array and discharge capacitor 1630 is provided. This example also includes a contact rod 1640 to cause switch actuation to discharge the capacitor through the array as the syringe plunger reaches or nears the end of its stroke.

[0105] In some embodiments, the discharge circuit includes a single capacitor. This can have the advantage of being extremely small and easy to embed into a conventional device structure such as a syringe. An example of a capacitor that could be integrated into an SCDGET needle hub is a small 2.2uF 450V multilayer ceramic chip (MLCC) capacitor. A size of a known at the time of filing MLCC commercial part is 5.7 x 5.0 x 2.5mm.

[0106] One embodiment of the proposed system incorporates a disposable concentric needle type DNA delivery probe that looks and feels like a conventional hypodermic needle, is compatible with conventional syringes, and once contacted with a remote charging unit, causes DNA delivery indistinguishable from conventional vaccination procedures. Our The DNA delivery system has been revolutionary in that it is so efficient that a single electrical pulse generated by the discharge of a small capacitor integrated into the hub of the needle-like DNA delivery probe provides 100% reliable gene expression in muscle tissue. However, the use of this technology is not limited to muscle tissue, for example, the system can also be used to transfect brain tissue, retina, or other tissue types.

[0107] Alternative embodiments not currently illustrated in the drawings are also contemplated. For example, one embodiment can include metal snap-type contacts to make the electrical connection. Another embodiment can include a magnet and a reed switch to make the electrical connection when the magnet attached to the syringe plunger is in close proximity to the reed switch as the syringe plunger reaches or nears the end of its stroke. Another embodiment can include (1) metal snap-type switch contacts and (2) a dual shaft syringe, where the second shaft of the syringe is used to house an electrical switch that can be actuated by operation of the plunger. For example, the switch contacts can be included in the bottom of the second syringe shaft.

[0108] Figure 17 is an image demonstrating the utility of the prototype SCD-BaDGE for in vivo applications, showing expression of the light-activated ChrimsonR channelrhodopsin ion channel protein - TD Tomato fusion reporter protein in mouse soleus muscle fibers. This demonstrates the use of the single capacitor discharge BaDGE in mouse optogenetics applications. Expression of the light-activated ChrimsonR channelrhodopsin ion channel protein - TD Tomato fusion reporter protein in mouse soleus muscle fibers was performed by single capacitor discharge electroporation of naked plasmid DNA encoding the protein under the cytomegalovirus promoter Imaging was performed 4 days later. DNA delivery parameters: 1 pg / pl DNA in an isotonic sucrose carrier solution, The DNA delivery probe included a 2.2 pF capacitor charged to 250 V. The image is a 3D reconstruction of a confocal laser scan image stack acquired using 561 nm excitation and 567-633 nm emission. Figure 17 The image shown illustrates single muscle fiber expression resulting from this in vivo gene electroporation.

[0109] Figure 18 An example of a possible system embodiment is illustrated, including: electroporation modules 1810, each of which can be connected to a syringe (not shown); and a charging unit 1820. Each electroporation module 1810 includes a capacitor discharge circuit, which can be charged using the charging unit 1820. Figure 19a An example of an electroporation module is shown, which is configured to be compatible with a syringe 1930. For example, the electroporation module 1810 can be luer lock compatible to interface with a conventional luer syringe. Figure 19b An electroporation module 1810 is shown connected to a syringe 1930. This can be advantageous because it allows the syringe to be pre-loaded with the desired therapeutic fluid 1940 or it can be loaded on site. It will be appreciated that the electroporation module can be used for the delivery of electrical stimulation of a variety of different therapeutic agents, which can have a short shelf life or special handling conditions. It can therefore be practical to supply the therapeutic substance separately from the electroporation module, which is a device that does not have similar shelf life limitations. Having the electroporation module as a separate device from the therapeutic agent delivery syringe can also provide greater flexibility and the ability to adapt to changing therapeutic agents. For example, a batch of electroporation modules can be purchased in bulk for use in a clinical trial comparing two or more therapeutic agents (and a control), or for use in clinical practice treating a variety of diseases, each using a different therapeutic agent.

[0110] Figure 20 An exploded view of the syringe 1930 and electroporation module 1810 is shown. The electroporation module has a needle array 1815 (as described in any of the embodiments described above), which can typically be covered for safe handling using a needle shield 1850. Figure 21aAn example of an embodiment of the electrotransfer module 1810 is shown in more detail. The electrotransfer module 1810 has a housing with a neck 2120 at one end that can be connected to a syringe, and a needle array protruding from the opposite end with a lumen extending through the device to enable delivery of fluid from the connected syringe. The syringe can be an off-the-shelf (OTS) syringe 2110, in this example a luer lock type syringe, and the housing is configured to be compatible with the luer lock 2120. However, other attachments can also be used. The needle array includes two coaxial electrodes, insulated from each other, with the first, inner electrode longer than the second, outer electrode, and the insulation extending along a portion of the first electrode beyond the end of the second electrode to create an insulating gap 2150 between the two electrodes. The length of this gap 2150 affects the electric field generated by the capacitive discharge, and thus the target zone for electric field-mediated cell transfection (electrotransfer). This gap can therefore also be referred to as an electric field lens 2150 due to its effect on focusing the electric field gradient.

[0111] The housing houses a capacitive discharge circuit and a mechanism for actuating the capacitive discharge. The capacitive discharge circuit is carried on a printed circuit board and includes a capacitor 2130 for storing charge for delivery, a switch, and electrical connections to the electrodes of the needle array. The circuit also includes a charging pin 2180 or other means for enabling an external electrical connection to charge the capacitor. In this embodiment, the switch is a microswitch 2160, and the switch actuator is a switch plate 2170 configured to be acted upon by the syringe plunger towards the end of the plunger stroke to move the switch plate 2170 to actuate the microswitch 2160 and cause the stored charge to be released from the capacitor. In this embodiment, the switch plate 2170 has a planar portion configured to engage with the microswitch, and a post extending through the neck 2120 of the housing to be engageable with the syringe plunger, and the post includes a connection to the needle array to establish a fluid communication path from the syringe to the needle array for delivery of fluid to the tissue. In this embodiment, the switch plate 2170 is biased (or otherwise initially mechanically held) to an open (or disengaged) position. Actuation of the syringe plunger will first force fluid through the lumen, eventually the plunger will contact the post, and continued travel of the plunger causes corresponding movement of the switch plate towards the microswitch to actuate the microswitch and trigger the capacitive discharge through the electrode array. Figure 21b An alternative embodiment of the electrotransfer module is shown, designed to operate with a low dead zone type syringe. This embodiment has a variation of the switch plate, with the post slightly shorter due to the different syringe plunger shape.

[0112] In the illustrated embodiment, the capacitor discharge switch is a microswitch driven by a switch plate that is mechanically moved to actuate the switch by the syringe plunger. Other types of switches and actuators are also contemplated in embodiments of the system. For example, the switch can be a magnet in the switch plate to activate a micro magnetic reed switch or a piston with a conductive (metal) ring that is driven down to close the discharge circuit. Figure 21c is an example of a reed relay that when a magnet embedded on the switch plate 2175 is brought near the reed relay 2165, a magnetic actuation of the capacitor charge delivery occurs such that the switch within the relay 2165 closes its contacts allowing the capacitor to discharge through the needle electrodes.

[0113] Figure 22 illustrates the electrotransfer module (attached to a syringe filled with therapeutic fluid) being inserted into the charging unit to charge the capacitor discharge circuit. The charging unit is connected to a power source. When the electrotransfer module is inserted, an electrical connection is made with the capacitor discharge circuit through the charging pins. A light and / or sound alarm on the charging device can indicate when the capacitor is fully charged. The device can then be removed and the patient treated.

[0114] As shown in the flowchart of Figure 23 , the clinician first assembles the syringe and electrotransfer module, then charges the capacitor discharge circuit, and after that delivers the treatment to the patient, which is similar to any typical vaccination injection. Figure 24a illustrates the first stage of the vaccination where the needle is inserted into the patient's arm and as the syringe plunger is depressed, the therapeutic fluid is delivered to the tissue surrounding the array of needles. As the plunger approaches the end of its travel, it actuates the capacitor discharge circuit switch, which causes the charge from the capacitor to discharge through the electrodes of the needle array and create an electric field gradient in the tissue surrounding the array, thus stimulating cell transfection.

[0115] Figure 25 is a photo of a prototype embodiment of the electrotransfer module. Figure 26a and 26b show the results of DNA electrotransfer using an 8 node biomimetic array and a concentric stainless steel electrode array using HEK293 monolayers. Fluorescence of the mCherry reporter gene was captured 4 days after electrotransfer to HEK293 cell monolayers (as shown in ). The electrodes used for gene delivery were artificial cochlea 8 electrode arrays (Pt / Ir electrodes, 400 pm length, diameter and pitch, "tandem" configuration (+++----), in comparison, the active electrodes of the concentric stainless steel needle electrodes (34G inner needle and 28G outer needle) were 2 mm long with a 1 mm gap between electrodes. n = 5 coverslips for each experimental protocol. Figure 26a ​

[0116] Approximately 24 hours prior to gene transfer, cells were seeded onto 18mm coverslips, this was done when the cells were approximately 50% confluent on the coverslips. For After aligning the electrode pair over the cells, 20ul of 2ug / ul DNA (CMVp-mCherry plasmid) suspended in a pH neutral 10% sucrose solution was applied to the cell-coated coverslips. Figure 26a Figure 1 is a set of images showing results of electroporation using single capacitance discharge for electroporation, comparing results using a cochlear array and a prototype electroporation module. The images show fluorescence demonstrating transfection of a monolayer of HEK293 cells for:

[0117] 1. An 8 electrode cochlear electrode array driven with a single discharge of a 2.2pF capacitor charged to 100V, where four adjacent electrodes are driven as an anode array and the remaining four adjacent electrodes are driven as a cathode array, also referred to as a series array configuration (+++ - - -). This is the electrode configuration previously demonstrated by the inventors to be effective for electroporation using a pulse sequence, and was used to initially demonstrate efficacy of electroporation using a single capacitance pulse discharge, and is therefore used here for comparison with the concentric needle array prototype testing.

[0118] 2. Prototype concentric needle array after electroporation of the needle electrode array with a single discharge of a 2.2pF capacitor 100V. This shows a similar field of transfection to the cochlear electrode embodiment using the same pulse drive.

[0119] 3. Prototype concentric needle array after electroporation of the needle electrode array with a single discharge of a 2.2pF capacitor 40V. This shows a lower intensity transfection response, which is not surprising given the lower charge.

[0120] 4. Prototype concentric needle array after electroporation of the needle electrode array with a single discharge of a 2.2pF capacitor 10V. This shows a minimal transfection response.

[0121] Figure 26b Figure 1 is a set of images showing results of electroporation using single capacitance discharge for electroporation, comparing results using a cochlear array and a prototype electroporation module. The images show fluorescence demonstrating transfection of a monolayer of HEK293 cells for: Figure 26aA box plot of the results shown. These results indicate that the efficiency of DNA delivery to HEK293 cell monolayers using an 8 electrode array in a series configuration (++++— —) driven by a single discharge of 2.2 μF capacitor charged to 100 V and a prototype electrotransfer module ('BaDGE-Inoculator') with an electrode array comprising stainless steel concentric needles was to transfect cells within the equivalent area. (P = 0.00016, t-test comparing 100 V and 40 V concentric electrodes; P = 0.008, 40 V vs 10 V, Mann-Whitney rank sum test). This also indicates that for 40 V fewer cells were transfected, with the transfection concentrated around the area within the middle region where the electric field gradient is concentrated around the gap between the electrodes, i.e. the electric field lens. In the example using 10 V charge, there was little transfection, indicating that the electric field strength provided by this voltage was insufficient for transfection of this combination of electrotransfer module prototype and therapeutic agent.

[0122] Figure 26c A potential plot (hemisphere) of the BaDGE concentric stainless steel electrode array is shown, as is a schematic of the needle electrode array (cross-section) and aligned with the potential plot. This matches the electric field achieved at 40 V single capacitor discharge to transfect HEK293 cells as described above in relation to Figure 26a

[0123] An extension of the above study was performed on a second group of mice to show reliable luciferase expression, here using DNA expression from mouse hind limb muscle fibres using single capacitor discharge at 40 V or 100 V - 1 μg / μl luciferase reporter plasmid DNA in 10% sucrose solution, total volume 50 μl.

[0124] Figure 27 A time course of gene expression mediated is shown. (A) shows the spread of luciferase activity three days after intramuscular (B) representative traces of photons emitted per second in the ROI placed on the hind limb measured at one minute intervals after intraperitoneal injection of luciferin. (C) time course of luciferase gene (Luc) transfer to mouse gastrocnemius muscle after intramuscular injection of 1 μg / μl luciferase reporter plasmid DNA in 10% sucrose solution, total volume 50 μl. ​Time-course graph of bioluminescence measurements at peak (red arrow in B) at 1, 3, and 7 days after luciferase-encoded plasmid transfer. DNA plasmid reporter gene delivery was pMK175-CAGp-Luc (50 μl; 1 μg / μl; vector was 10% sucrose @ pH 7.4; gastrocnemius muscle inoculation n = 5 for each capacitor charging voltage; mean ± sem).

[0125] The methods used to obtain these results are as follows:

[0126] All procedures were performed in accordance with those approved by the New South Wales Animal Care and Ethics Commission (ACEC 18_160a). Animals were placed in an induction chamber and anesthesia was induced with 3% isoflurane at a flow rate of 1–2 L / min. After anesthesia, animals were transferred to a heated animal bed in a prone or supine position while maintaining anesthesia with 2% isoflurane administered via a nasal cone at a flow rate of 1–2 L / min. Eye ointment was applied. Body temperature was maintained, and oxygen saturation was monitored using a MouseStat PhysioSuite (Kent Scientific). The hind limbs and lower back were shaved and disinfected with ethanol swabs.

[0127] The mediated transfer of the luciferase-encoded plasmid utilized single-capacitor discharge when a 2.2 μF capacitor (alternate branch) was charged to 40 V or 100 V. DNA plasmid reporter gene delivery was pMK175-CAGp-Luc (50 μl; 1 μg / μl; vector was 10% sucrose @ pH 7.4; for each capacitor charging voltage, gastrocnemius muscle inoculation n = 5; allowing animals to recover before returning to their home cages).

[0128] In spectral computed tomography (CT) Bioluminescence imaging was repeated on a SpectrumCT (Perkin Elmer). For this procedure, as described above, anesthesia was induced in the induction chamber with 3% isoflurane and oxygen. Following anesthesia, eye ointment was applied, and mice were intraperitoneally injected with fluorescein (150 mg / kg). At this dose, fluorescein is a harmless substrate, exhibiting good bioluminescence properties with... The luciferase expressed on the delivered pMK175-CAGp-Luc plasmid emits photons during the reaction. The animals are then transferred to a heated animal bed in a prone or supine position, while simultaneously... Spectrum CT was used to maintain anesthesia by delivering isoflurane anesthetic through the nose cone at 2% isoflurane. Bioluminescence 2720 was imaged at 1 minute intervals until there was a drop in sustained radiation (photons emitted per second in a defined region of interest (ROI) 2710 on each hind limb) detected for 5 consecutive minutes. After this, the animals were allowed to recover before being returned to their home cage. Peak luminescence 2730 was determined as the maximum radiation in a given ROI.

[0129] The plot of luminescence peak over time (lower plot in FIG. 27) shows that reliable luciferase expression is increased within the first 72 hours. Measurements taken 7 days after electroporation show similar luminescence in mice stimulated for DNA uptake using 40V and 100V to drive mouse hind limb muscle fibers for electroporation. Figure 27

[0130] One alternative application of the disclosed electroporation technology is DNA therapy for ex vivo cell transfection for autologous gene therapy. Figures 29a-29b A prototype test embodiment of high-throughput needle electrode stimulation of DNA electroporation in cell suspensions for autologous gene therapy and ex vivo cell transfection is illustrated. An example of the process of ex vivo serial cell transfection with naked DNA by needle electrode and capacitor discharge DNA electroporation is configured to capture cells from culture (ex vivo) or from the body (tissue cell suspension after digestion) or from blood and resuspend in a DNA vector solution (10% sucrose, DNA typically > 0.1 μg / μl - 10 μg / μl). This suspension is pumped through a reaction tube by a syringe pump such that the suspended cells migrate through a needle electrode DNA delivery probe (about 5 mm long) at a through time (dependent on tube diameter) that ensures that the electroporation pulse is delivered at least twice over that distance. A typical electroporation protocol is 100V exponential decay at 2Hz through a 2.2 μF capacitor. The cells in solution are then transferred to culture media for incubation, or re-granulated for return to the body (autologous cell gene therapy application). DNA electroporation is transient. For ex vivo readout - cells can be counted starting about 10 hours after transfection using FACS (fluorescence activated cell sorting). Figure 30 Cell images showing expression of mCherry reporter plasmid DNA under cell background (DAPI nuclear stain) after serial flow DNA electroporation are shown.

[0131] ​In this cell flow experiment, culture medium was removed and 1 ml TryplE was added to a single 10 cm diameter petri dish where HEK293 cells were approximately 90% confluent. After 3 minutes at 37°C, 9 ml of 10% sucrose was added to the petri dish and the cells were resuspended. Half of the cells from this suspension (5 ml) were pelleted at 800 rpm for 5 minutes. The 5 ml of sucrose solution was removed and the cell pellet was resuspended in 0.5 ml of pH neutral 10% sucrose containing 1 ug / ul reporter plasmid DNA (pJLP17-CMVp-mCherry). The cell-DNA suspension was collected in a 5 ml syringe and attached to an infusion syringe pump set to deliver 1 ml every 6 minutes (250 ul in 90 seconds). The syringe delivered the cell DNA solution around a concentric needle electrode (34G inner needle and 28G outer needle) with an active electrode 2 mm long and a 1 mm gap between the electrodes (total array length 5 mm). Capacitive discharge analog pulses were delivered to the electrode by a Digitimer DS5 constant current stimulator controlled by a microprocessor to mimic a 2.2 uF capacitor discharge. The pulses were delivered at 2 Hz at 50 mA / 120 V with a decay constant of 10 ms. Cells were collected after passing through the electrode into a 5 cm petri dish with fresh culture medium and incubated for 6 days at 37°C 5% C02.

[0132] In Six days after DNA delivery, cells were fixed in 4% paraformaldehyde for 20 minutes and counterstained with DAPI to label all nuclei. mCherry reporter fluorescence was imaged using a 561 nm DPSS laser and DAPI, using a 790 nm titanium sapphire multi-photon laser. Results are shown in Figure 30 .

[0133] Figure 31 Another example of an electroporation system used in in vitro cell therapy is illustrated. The DNA delivery probe in the described stainless steel concentric needle configuration (or Pt / Ir or other electrode material) is well suited for high throughput DNA electroporation into cells adhered to or suspended in a multi-well plate configuration. Pulse parameters such as capacitive discharge or regular monopolar square wave pulses, or constant current or constant voltage, or complex waveform profiles provide efficient DNA electroporation with the advantage of (robotic) multi-well high throughput in providing efficient cell transfection using naked (plasmid) DNA, and without the need for any hardwired or separately inserted current return paths into the transfection wells (which is neither efficient nor convenient to quickly deploy between wells), while minimizing cross-well contamination / carryover. Cells can be cultured in situ, or extracted from the wells immediately after DNA electroporation.

[0134] Single needle electrode DNA probes can be provided with different pulse / pulse sequence parameters to provide rapid optimization of DNA transfection parameters to achieve the most efficient delivery of DNA (or other molecular types) to cells.

[0135] In alternative embodiments, such as a minimum viable product (MVP) - to enable mass production with minimal time delay - embodiments are envisioned in which a disposable needle array is used that can be connected to a syringe and external charge delivery circuit. For example, the needle array can be provided (in a sterile package) with external wired electrical connectors to connect the needle array to the pulse delivery circuit. An example of a prototype of this embodiment is shown in FIG. 8. Figure 28a - c.

[0136] Production of the needle array for this embodiment can involve coating the needles with a parylene C coating, where the electrode surface is masked by this insulator, the inner needle electrode will be pushed into the hub so that the upper end enters the connector taper which will scrape off the insulator and continuity is checked, then the outer needle is pushed onto the inner needle and inserted into the larger diameter taper. The taper is configured within the needle hub which provides a standard connection to a syringe so that the inner needle electrode lumen is in fluid communication with the syringe reservoir to deliver the DNA vaccine fluid. The leads of the two (tapered) connectors will be connected to the external wires with connectors at the ends.

[0137] The pulse delivery circuit can be a known pulse delivery device (appropriately programmed) or a simplified capacitor discharge unit, comprising a capacitor discharge circuit and a charging circuit specifically configured for the required charge delivery. As noted above, the capacitor discharge circuit and charging unit of one embodiment of the electrical transfer system are effectively placed in one unit, which can be connected to the disposable needle array by wires. The capacitor discharge pulse can be manually triggered by the clinician, for example, using a hand switch, which can be foot actuated. In this embodiment, the capacitor will be in the charging box, enabling the reuse of the charging circuit. This has the advantage of requiring fewer charging circuits to be produced for large scale deployment of electrical transfer DNA vaccine delivery. This also facilitates production that expands the flexibility of capacitor vendor selection (e.g., vendors can be selected with orders on the order of 100,000, whereas the production mode for disposable BaDGE vaccinators can be up to 1 million per month (2 per second...)). For example, in one embodiment, the concentric needle array can be manufactured with a robot, where the concentric insulated needles are inserted into a hub under high yield manufacturing. The hub can be configured to connect to an off-the-shelf syringe (i.e., a luer lock syringe). The hub can be formed of an insulating material (i.e., plastic) and two concentric metal (i.e., stainless steel) contact cones with an insulating layer between them, each contact cone arranged to provide an electrical connection between one of the needle electrodes and a lead wire for connection to an external charge delivery circuit. The needles can be coated (or partially coated to leave the electrode tip portion clean) with an electrically insulating material (e.g., coated with parylene). The end of the hub that enters can then be inserted by a robotic armature, the first (smaller inner) needle entering the hub most distally through a hole in the first contact cone to contact the smaller second contact cone nested within the first cone. The crimp fitting peels off the edge of the insulating layer (e.g., 25 pm) and forms a contact, which can be confirmed by continuity testing from the armature to the lead wire soldered to the connector. After that, the second needle is inserted into the needle hub on the smaller inner needle, the second needle being larger in diameter and thus making contact with the larger diameter contact cone to form an electrical connection. The needles can be secured in place using heat staking or other mechanisms (i.e., adhesive or pressure).

[0138] The needle array unit will be sterile packaged, including the wires and connectors, and is disposable; the controller (e.g., 2 per clinic) will be enabled for tens of thousands of cycles. Thus, only to meet the urgent needs of a pandemic, the needle electrode array component requires a massive production scale. This can make manufacturing from readily available raw materials possible. The needles for the concentric needle electrode array can use standard gauges, which are readily available from multiple vendors.

[0139] It is understood that this naked DNA delivery platform technology offers significant advantages in situations such as the unprecedented need for mass vaccination to help stop the spread of COVID-19. Any solution requires a fast path to mass deployment. DNA vaccines work by having the body produce antibodies without directly injecting real viral protein fragments. Last year, a DNA vaccine designed to treat to prevent infection with the MERS coronavirus was found to produce clinically significant levels of antibodies in a Phase 1 clinical trial. The COVID-19 virus is structurally similar to MERS, and a clinical trial is currently underway in the US to adapt the MERS vaccine to address COVID-19, with performance expected to be similar to the MERS vaccine. In addition to this MERS-COVID-19 DNA vaccine derivative, several other groups around the world are also developing DNA vaccines against COVID-19. Once any group proves effective, the DNA sequence will be universally available for anyone to manufacture. A significant advantage of these plasmid DNA vaccines over serum vaccines is that the DNA chemical sequence can be distributed electronically and produced anywhere. As naked DNA, COVID-19 DNA vaccines require an electroporation delivery system to administer (genetic transfection using a brief electrical pulse). However, the current commercial delivery units are both expensive and complex, making them difficult to scale to the low cost required for mass vaccination in this pandemic outbreak.

[0140] The single pulse electroporation technology described herein, designed by the Applicant for DNA vaccine delivery, has the following advantages: it is more efficient, less costly to produce, uses less DNA than the current clinical electroporator (releasing more DNA vaccine dose) and is painless. It is of paramount importance that this technology is highly scalable in mass manufacturing, with minimal components, facilitating fast deployment. Once a DNA vaccine for COVID-19 is identified and produced, the disclosed electroporation technology can be leveraged to address the delivery need.

[0141] Advantages of embodiments of the presently described system include:

[0142] 1. This enables a disposable DNA gene electroporation device

[0143] 2. A DNA gene delivery system suitable for low cost mass manufacturing

[0144] 3. The setup charge delivery is intrinsically safe 4. A standalone charging station suitable for field operations

[0145] 5. A complete “game changer” for DNA vaccine applications, but also more broadly applicable to enable a wide range of DNA therapeutic applications

[0146] 6. Separating the DNA electrotransfer process from the power source reduces the cost of the technology, making it immediately suitable for development into medical devices / drug combinations for tissue targeting in DNA therapies—such as DNA vaccines, solid tumor oncology targets (melanoma, advanced cancer), nerve and muscle repair (nerve reinnervation), and neuromodulation targeting brain regions. Field applications in trauma treatment are also possible.

[0147] Those skilled in the art will understand that many modifications can be made without departing from the spirit and scope of the invention.

[0148] In the following claims and the foregoing description of this invention, unless the context requires otherwise due to the language of expression or necessary meaning, the word "comprising" or variations such as "comprising" or "including" are used in the sense of inclusion, that is, specifying the presence of the said feature, but not excluding the presence or addition of further features in various embodiments of the invention.

[0149] It should be understood that any reference to prior art publications in this document does not constitute an admission that such publication constitutes part of common general knowledge in the art, in Australia, or in any other country.

Claims

1. An electrical transfer system, comprising: At least one probe, each probe comprising: Probe body; A needle electrode array, extending from the probe body, is configured as a needle to be inserted into the tissue to be treated; and A capacitor discharge circuit, connected to the needle electrode array, includes a capacitor charge memory configured to store charge quanta, and a switch actuated to discharge the stored charge quanta through the needle electrode array. The needle electrode array includes at least two electrodes, each electrode having a surface area substantially surrounding the needle and exposed to directly contact the tissue into which the needle electrode array is inserted. Insulating portions are present between adjacent electrodes to form a continuous linear array structure. The exposed surface area of ​​each electrode is at a different distance from the tip of the needle electrode array. Each electrode is connected to the capacitor discharge circuit for driving as an anode or cathode during charge quantum discharge. The electrode length and the length of the insulating portions between adjacent electrodes are configured to generate a target electric field shape in the tissue adjacent to the array during charge quantum discharge through the array. A charging station having a DC power module, an output terminal that can be connected to a probe to form an electrical contact with the capacitor discharge circuit, and a charging control circuit for controlling the charging of the capacitor discharge circuit connected to the probe.

2. The electrical transfer system according to claim 1, wherein, The capacitor discharge circuit is housed within the probe body.

3. The electrotransfer system according to claim 1 further includes a switch actuator, which is mounted on the probe body and configured to actuate a capacitor discharge circuit switch.

4. The electrical transfer system according to claim 2, wherein, The charging station includes a probe mount to support the probe during charging.

5. The electrical transfer system according to claim 1, wherein, The electrode is relatively long and thin, wherein the electrode diameter is smaller than the electrode length.

6. The electrical transfer system according to claim 5, wherein, The electrode has a length of 1 mm or less and an electrode diameter smaller than the electrode length.

7. The electrical transfer system according to claim 1, wherein, The parameters of the capacitor discharge circuit are matched with the resistivity parameters of the treatment solution used for electrotransfer to minimize the total charge delivered and optimize the discharge time constant for electrotransfer.

8. The electrical transfer system according to claim 7, wherein, The capacitor of the capacitor discharge circuit is selected based on the predicted total resistivity and the target accumulated charge during discharge, and the predicted total resistivity is based on the resistivity of the treatment solution.

9. The electrical transfer system according to claim 7 or 8, wherein, The matching is to achieve a discharge time constant between 20 μs and 2 s.

10. The electrical transfer system according to claim 1, wherein, The probe body also includes a treatment reservoir and a lumen passing through the needle, as well as a treatment delivery actuator. The lumen is in fluid communication with the treatment reservoir to maintain a volume of fluid, and the treatment delivery actuator is operable to force fluid out of the treatment reservoir and out of the needle through the lumen.

11. The electrical transfer system according to claim 10, wherein, The treatment delivery actuator is also configured to activate the switch.

12. The electrical transfer system according to claim 10, wherein, The probe is packaged together with the treatment solution stored in the reservoir.

13. The electrical transfer system according to claim 10, wherein, The reservoir is filled with the treatment solution before the capacitor discharge circuit is charged.

14. The electrical transfer system according to claim 10, wherein, The probe body has a two-part form, wherein a first part, including the reservoir and the treatment delivery actuator, is provided by a syringe, the syringe being connectable to a second part including an electrotransfer module having a housing adapted to engage with the syringe and support a needle electrode array projecting outward from the housing, wherein a capacitor discharge circuit is housed within the housing, connected to the needle electrode array, and forms a fluid communication path between the lumen of the syringe reservoir and the needle electrode array through the housing.

15. The electrical transfer system according to claim 14, wherein, The electrotransfer module housing is Luer lock compatible for engagement with standard Luer injectors.

16. The electrical transfer system according to claim 1, wherein, The needle electrode array includes: The first needle, which also serves as the first electrode; A first concentric insulator wraps around the first needle at a predetermined first distance L1 from the needle tip. A concentric second electrode, which wraps around the first concentric insulator at a distance L2 from the end of the first concentric insulator, and The second concentric insulator wraps around the concentric second electrode at a distance L3 from the end of the concentric second electrode, wherein the first needle and the second concentric electrode are formed of conductive material and are electrically connected to the positive and negative terminals of the capacitor discharge circuit.

17. The electrical transfer system according to claim 16, wherein, The predetermined first distance L1 defines the exposure length of the first needle, which functions as the first electrode of the linear array, and the distance L3 from the end of the concentric second electrode to the second concentric insulator defines the exposure length of the concentric second electrode, which functions as the second electrode of the linear array.

18. The electrical transfer system according to claim 17, wherein, When an electric pulse is applied to drive one electrode as the anode and the other electrode as the cathode, the corresponding exposure lengths of the first and second electrodes, as well as the exposure length of the first concentric insulator between them, determine the pattern of the electric field gradient generated near the needle electrode array.

19. A probe for an electrical transfer system, comprising: Probe body; A needle electrode array, extending from the probe body, is configured as a needle to be inserted into the tissue to be treated; as well as A capacitor discharge circuit, connected to the needle electrode array, includes a capacitor charge memory configured to store charge quanta, and a switch actuated to discharge the stored charge quanta through the needle electrode array. The needle electrode array comprises at least two electrodes, each electrode having a surface area substantially surrounding the needle and exposed to directly contact the tissue into which the needle electrode array is inserted. Insulating portions are present between adjacent electrodes to form a continuous linear array structure. The exposed surface area of ​​each electrode is at a different distance from the tip of the needle electrode array. Each electrode is connected to the capacitor discharge circuit for driving as an anode or cathode during charge quantum discharge. The electrode length and the length of the insulating portion between adjacent electrodes are configured to generate a target electric field shape in the tissue adjacent to the array during the discharge of charge quanta through the array.

20. The probe for the electrotransfer system according to claim 19, wherein, The needle electrode array includes: The first needle, whose length near the needle tip provides the first electrode; A first concentric insulator surrounds the first needle at a predetermined first distance L1 from the needle tip. A conductive sheath that covers the first concentric insulator at a distance L2 from the end of the first concentric insulator and forms a second electrode, and The second concentric insulator wraps around the second electrode at a distance L3 from the end of the second electrode, wherein the first needle and the second concentric electrode are formed of conductive material and are electrically connected to the positive and negative terminals of the capacitor discharge circuit.

21. The probe for the electrotransfer system according to claim 20, wherein, When an electric pulse is applied to drive one electrode as the anode and the other electrode as the cathode, the corresponding lengths of the first and second electrodes, as well as the exposed length of the first concentric insulator between them, determine the pattern of the electric field gradient generated near the needle electrode array.

22. The electrotransfer system probe of claim 19, further comprising a treatment reservoir and a lumen through the needle electrode array, and a treatment delivery actuator, the lumen being in fluid communication with the treatment reservoir to maintain a volume of fluid, the treatment delivery actuator being operable to force the fluid out of the treatment reservoir and out of the needle through the lumen.

23. The probe for the electrotransfer system according to claim 22, wherein, The treatment delivery actuator is also configured to activate the switch.

24. The electrotransfer system probe according to any one of claims 19 to 21, further comprising a lumen through the needle electrode array and a treatment delivery actuator, wherein, The probe is configured to connect to a syringe to form a fluid communication channel from the syringe's fluid reservoir through the lumen, whereby the syringe provides a treatment reservoir to hold a volume of fluid, the treatment delivery actuator is operable to force the fluid out of the treatment reservoir and through the lumen from the needle, and the treatment delivery actuator is further configured to activate the switch.

25. A needle electrode array, comprising: The first needle, which also serves as the first electrode; A first concentric insulator surrounds the first needle at a predetermined first distance L1 from the needle tip. A concentric second electrode, which wraps around the first concentric insulator at a distance L2 from the end of the first concentric insulator, and A second concentric insulator surrounds the second electrode at a distance L3 from its end. The first needle and the concentric second electrode are formed of a conductive material and are electrically connected to the positive and negative terminals of a pulse delivery circuit. The predetermined first distance L1 defines the exposed length of the first needle, which functions as the first electrode of a linear array. The distance L3 from the end of the concentric second electrode to the second concentric insulator defines the exposed length of the concentric second electrode, which also functions as the second electrode of a linear array. When an electric pulse is applied to drive one electrode as the anode and the other electrode as the cathode, the corresponding lengths of the first and second electrodes, as well as the exposed length of the first concentric insulator between them, determine the pattern of the electric field gradient generated near the needle electrode array.

26. The needle electrode array according to claim 25, wherein, The needle tip is beveled.

27. The needle electrode array according to claim 25, wherein, The first needle has a hollow lumen through which a treatment solution can be delivered.

28. The needle electrode array according to claim 27, wherein, The treatment solution is delivered into the tissue through an opening in the tip of the first needle.

29. The needle electrode array according to claim 28, wherein, The needle electrode array includes holes in the array away from the tip, thereby delivering a treatment solution to tissue adjacent to the needle electrode array.

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