Devices, systems and methods for electrotransport therapeutic substance delivery - Patents.com

Abstract

A device is disclosed that allows for highly efficient DNA electrotransfer into cells through a single capacitive discharge. The principle of this system involves the accumulation of a charge on a capacitor, which is then discharged through an electrode array configured to generate an electric field with a focused field potential gradient through the arrayed region, strong enough to effect efficient DNA electrotransfer into cells. The entry of this DNA or related ribonucleic acid molecules or even other charged molecules into cells can affect altered biological functions.

Description

[Technical field]

[0001] Technical Field The field of the invention is the delivery of therapeutic molecules by electric field stimulated cell transfection, and in particular the use of linear electrode arrays on a single probe. One application of the invention is the electrotransfer of DNA into cells. [Background technology]

[0002] background The transfection or electrotransduction of cells by electrical field stimulation to deliver therapeutic substances to cells is commonly known as electroporation. Electroporation generally involves the injection of a therapeutic substance (e.g., a solution of therapeutic molecules) into tissue by inserting two separate probes into the tissue, with an electric current passing through the tissue between the two probes, one acting as a cathode and the other as an anode, on either side of the target treatment area. This causes a temporary disruption of the cell membrane, allowing the therapeutic substance to enter the cell, and is also called "transfection". This is a known method for the delivery of gene therapy agents.

[0003] When the DNA encodes a gene for a protein or part of it, this electric field-based delivery of DNA into cells is sometimes called "gene electrotransfer (GET)". Introduction of DNA into cells by electroporation is routinely performed in laboratories. Electroporation / GET is also a recognized method for inducing gene expression in tissues naturally or in vivo, although the efficiency of gene expression in tissues after DNA electroporation is generally low.

[0004] Conventional electroporation requires high voltage pulses and therefore specialized equipment and clinical facilities, which may limit the applicability of such treatments.

[0005] The development of techniques for stimulating transfection in target regions adjacent to a continuous electrode array has been described in the inventors' prior patent applications International Publication Nos. WO2011 / 006204, WO2014 / 201511 and WO2016 / 205895, the disclosures of which provide background for the present disclosure and may be consulted for a better understanding of the inventors' closed-field electroporation technique. In the inventors' prior invention, a "closed-field" electrotransfer approach using a single-probe linear electrode array was used to achieve surprising electrotransfer efficiency, such that cells are transfected in tissue adjacent to the linear array. For example, the electrode array includes two or more elongated electrodes aligned in a line and spaced apart at discrete locations along the length of a single probe. The electrodes are continuous with the substrate array structure, so that no tissue directly intersects between the electrodes. Upon application of an electroporation pulse, an electric field gradient is generated in the tissue adjacent to the array, with the shape of the electric field and the gradient of the electric field being controlled by the configuration of the linear elongated electrodes.

[0006] In the system disclosed by the inventors in International Publication WO2016 / 205895, control of the area where electronically stimulated cell transfection occurs is achieved by controlling the gradient in the generated electric field based on the configuration of the electrode array and the pulse sequence applied to generate the electric field. The system of International Publication WO2016 / 205895 describes the practical application of the inventors' discovery of enhanced cell transfection in tissues in areas subjected to steeper electrical potential gradients, and application of this discovery to control the shape of the electric field to generate contours in the electric field to target areas of cell transfection adjacent to the array. Variables for controlling the shape of the electric field include the configuration of the array and the parameters of the stimulation pulse. The use of controlled electric field formation (field effect) improves the efficacy of cell transfection at a lower cumulative charge than known from conventional open field electroporation.

[0007] This closed-field electrotransfer technique has demonstrated the advantage of requiring a lower cumulative charge than conventional electroporation techniques. Another advantage is the ability to predictably control the shape and size of the area of ​​tissue in which cell transfection occurs based on a combination of the configuration of the linear electrode array and the parameters of the electroporation pulse, thereby controlling the electric field gradient of the generated electric field. However, further improvements in electrotransfer techniques are desirable. Summary of the Invention

[0008] Summary of the Invention According to a first aspect, there is provided an electroinjection system comprising at least one probe, each probe including 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 including a capacitive charge storage configured to store an amount of charge and a switch operable to cause a discharge of the stored charge through the needle electrode array, wherein the needle electrode array incorporates at least two electrodes, each electrode having an exposed surface area substantially surrounding a circumference of the needle and in direct contact with the tissue into which the needle electrode array is inserted, the surface area including a continuous straight line. The system includes a charging station having a DC power supply module, an output terminal connectable to the probe to form an electrical contact with the capacitive discharge circuit, and a charging control circuit for controlling charging of the capacitive discharge circuit of the connected probe, the exposed surface area of ​​each electrode being at a different distance from the tip of the needle electrode array, each electrode being connected to a capacitive discharge circuit for operating as an anode or a cathode during discharge of the charge quantity, and the length of the electrodes and the length of the insulating sections between adjacent electrodes being configured to create a target electric field shape in tissue adjacent to the array during discharge of the charge quantity through the array, and the system includes a charging station having a DC power supply module, an output terminal connectable to the probe to form an electrical contact with the capacitive discharge circuit, and a charging control circuit for controlling charging of the capacitive discharge circuit of the connected probe.

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

[0010] Some embodiments of the electrointroduction system further include a switch actuator mounted to the probe body configured to enable actuation of the capacitive discharge circuit switch.

[0011] In some embodiments, the charging station includes a probe platform that supports the probe during charging.

[0012] In some embodiments, the electrodes are relatively elongated and the diameter of the electrode is less than the length of the electrode, hi some embodiments, the electrodes are 1 mm or less in length and the diameter of the electrode is less than the length of the electrode.

[0013] In some embodiments, the capacitive discharge circuit parameters are matched to the resistivity parameters of the therapeutic solution for electrotransfer to minimize the total charge delivered and optimize the discharge time constant for electrotransfer. For example, the capacitance of the capacitive discharge circuit can be selected based on the predicted total resistivity during discharge and the target accumulated charge, where the predicted total resistivity is based on the resistivity of the therapeutic solution. For example, the matching may aim to achieve a discharge time constant between 20 μs and 2 seconds.

[0014] In some embodiments, the probe body further comprises a therapeutic reservoir, a lumen for holding a volume of fluid that is in fluid communication with the therapeutic reservoir through the needle, and a therapeutic delivery actuator that acts to force fluid from the therapeutic reservoir, through the lumen, and out of the needle. In some embodiments, the therapeutic delivery actuator is further configured to actuate a switch. In some embodiments, the probe is individually packaged with a therapeutic solution stored in the reservoir. In an alternative embodiment, the reservoir is filled with a therapeutic solution prior to charging the capacitive discharge circuit.

[0015] In some embodiments, the needle electrode includes a first needle that also acts as a first electrode, a first concentric insulator covering the first needle a predetermined first distance (L1) from the needle tip, a concentric second electrode covering the first concentric insulator a distance (L2) from the distal end of the first concentric insulator, and a second concentric insulator covering the second electrode a distance (L3) from the distal end of the second electrode, the first needle and the second concentric electrode being formed of a conductive material and electrically connected to positive and negative terminals of a capacitive discharge circuit.

[0016] In some embodiments, the exposed length L1 of the first needle acts as a first electrode of the linear array, and the exposed length L3 of the second concentric electrode acts 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 that is generated adjacent to the needle array when an electrical pulse is applied to drive one electrode as an anode and the other as a cathode.

[0017] According to another aspect, there is provided a probe for an electroinjection system comprising a probe body, a needle electrode array extending from the probe body configured as a needle for insertion into tissue to be treated, and a capacitive discharge circuit connected to the needle electrode array including a capacitive charge storage configured to store a charge quantity and a switch operable to cause a discharge of the stored charge quantity through the needle electrode array, the needle electrode array incorporating at least two electrodes, each electrode having an exposed surface area substantially surrounding a circumference of the needle and in direct contact with the tissue into which the needle electrode array is inserted, the surface area including an insulating section between adjacent electrodes forming a continuous linear array configuration, the exposed surface area of ​​each electrode being at a different distance from a tip of the needle electrode array, each electrode connected to the capacitive discharge circuit for operation as an anode or a cathode during discharge of the charge quantity, and the length of the electrodes and the length of the insulating section between adjacent electrodes configured to create a target electric field shape in the tissue adjacent the array during discharge of the charge quantity through the array.

[0018] In one embodiment, the needle electrode includes a first needle, the length of the needle proximate the needle tip providing the first electrode, a first concentric insulator covering the first needle a first predetermined distance (L1) from the needle tip, a conductive coating covering the first concentric insulator a distance (L2) from a distal end of the first concentric insulator to form a second electrode, and a second concentric insulator covering the second electrode a distance (L3) from a distal end of the second electrode, the first needle and second concentric electrode being formed of a conductive material and electrically connected to positive and negative terminals of a 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 electric field gradient that is generated adjacent to the needle array when an electrical pulse is applied to drive one electrode as an anode and the other as a cathode.

[0019] Some embodiments of the probe further include a therapeutic substance reservoir, a lumen that holds a volume of fluid in fluid communication with the therapeutic substance reservoir through the needle array, and a therapeutic substance delivery actuator that acts to force fluid from the therapeutic substance reservoir, through the lumen, and out of the needles, hi some embodiments, the therapeutic substance delivery actuator is further configured to actuate a switch.

[0020] In some embodiments, the probe body is comprised of two parts, a first part incorporating a reservoir and a therapeutic substance delivery actuator provided by a syringe connectable to a second part including an electrointroduction module having a housing adapted to engage the syringe and support a needle electrode array protruding outwardly from the housing, a capacitive discharge circuit is contained within the housing and connected to the needle electrode array, and a fluid communication path is formed through the housing between the reservoir of the syringe and the lumen of the needle electrode. For example, the housing of the electrointroduction module can be luer lock compatible and engage with a conventional luer syringe.

[0021] According to another aspect, a needle electrode array is provided that includes a first needle that also acts as a first electrode, a first concentric insulator covering the first needle a predetermined first distance (L1) from the needle tip, a concentric second electrode covering the first concentric insulator a distance (L2) from a distal end of the first concentric insulator, and a second concentric insulator covering the second electrode a distance (L3) from a distal end of the second electrode, the first needle and the second concentric electrode being formed of a conductive material and electrically connected to positive and negative terminals of a pulse delivery circuit.

[0022] In some embodiments, the exposed length L1 of the first needle acts as a first electrode of the linear array, and the exposed length L3 of the second concentric electrode acts as a second electrode of the linear array. In some embodiments, 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 that is generated adjacent to the needle array when an electrical pulse is applied to drive one electrode as an anode and the other electrode as a cathode.

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

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

[0025] In some embodiments, the therapeutic solution is delivered to the tissue through an opening in the tip of the first needle.

[0026] In some embodiments, the needle array includes an opening distal to the tip of the array through which the therapeutic solution is delivered to tissue adjacent to the needle array.

[0027] Another aspect provides a method of performing gene electrotransfer using the system described above, the method including charging the probe, inserting the needle array into the target tissue, delivering a therapeutic solution to the target tissue, activating a capacitive discharge, and removing the needle array from the tissue. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 is a representative diagram of an embodiment of a closed-field capacitive discharge electrical introduction system. [Diagram 2] FIG. 2 is a block diagram of an example charging unit for an embodiment of the present system. [Diagram 3] FIG. 3 is an example of a capacitive charging circuit for an embodiment of the present system. [Figure 4] FIG. 4 is an example of the potential measured for capacitive discharge. [Diagram 5] FIG. 5 is another example of a circuit diagram of a charging unit for an embodiment of the present system. [Figure 6] FIG. 6 shows a comparison of a typical electroporation pulse profile and a capacitive discharge pulse profile. [Figure 7] 7a and 7b show the results of a first (in vitro) study comparing expression of DNA encoding (transfection) using the prototype probe stimulated with a series of square waves and exponentially decaying pulses. [Figure 8] FIG. 8 shows the results of a second study investigating the feasibility of cell transfection using a single capacitive discharge pulse. [Figure 9a] FIG. 9a is bioluminescence imaging of mice 24 hours after DNA electrotransfer in the circled hindlimb region. [Figure 9bc] Figures 9b and 9c are graphs showing the peak total luminous flux emitted from each mouse hind paw at each time point after electrotransduction and the peak total luminous flux emitted from each mouse hind paw measured at time points up to 500 days after electrotransduction, demonstrating that the effects of electrotransduction are maintained for a long period of time. [Figure 10] FIG. 10 illustrates some of the effects of manipulating the gap between the anode and cathode of an electrode array. [Figure 11] FIG. 11 shows an example of an embodiment of a probe tip. [Figure 12] FIG. 12 shows an example of an embodiment of a needle array. [Figure 13a] FIG. 13a shows a model of the potential in the medium surrounding a DNA delivery probe when 240 V is applied temporarily across the electrodes for the peak voltage induced in the electric field adjacent to the probe during discharge of the built-in capacitor. [Figure 13b] FIG. 13b shows a model of the electric field strength around the DNA delivery probe when 240 V is temporarily applied across the electrodes. [Figure 14] FIG. 14 shows another example of an embodiment of the probe tip. [Figure 15] 15a and 15b are diagrams conceptually illustrating the insertion of a probe into a charging station. [Figure 16] FIG. 16 illustrates a conceptual example of an embodiment of a probe and compatible charging station configured as a syringe device. [Figure 17] FIG. 17 provides images demonstrating the utility of the prototype SCD-BaDGE in in vivo applications, showing expression of light-driven ChrimsonR channelrhodopsin ion channel protein-TDtomato fusion reporter protein in mouse soleus muscle fibers. [Figure 18] FIG. 18 illustrates an example of an embodiment of an electrointroduction module and charging unit connectable to a syringe. [Figure 19] Figures 19a and 19b show an example embodiment of an electroinjection module and compatible syringe as separate components in Figure 19a and connected in Figure 19b. [Figure 20] FIG. 20 shows an exploded view of an embodiment of an electroinjection module and compatible syringe. [Figure 21a] FIG. 21a shows an embodiment of an electrotransfer module connected to a syringe and provides a more detailed view of the components of the electrotransfer module. [Figure 21b]FIG. 21b is a diagram of an embodiment of an electrotransfer module connected to a low dead space type syringe, showing details of the components of the electrotransfer module. [Figure 21c] FIG. 21c is a detailed diagram of the components of an embodiment of the electrotransfer module. [Figure 22] FIG. 22 shows an embodiment of an electroinjection module connected to a syringe and positioned in a module for charging a capacitive discharge circuit. [Diagram 23] FIG. 23 illustrates an example of an inoculation process for delivery of a DNA vaccine using an embodiment of an electrotransfer system. [Figure 24a] FIG. 24a illustrates the process of therapeutic substance delivery via a syringe and electrotransfer module. [Figure 24b] FIG. 24b illustrates a process for stimulating tissue for electrotransfer using an electric field generated in the tissue surrounding the needle array by capacitive discharge. [Diagram 25] FIG. 25 is a photograph of a prototype embodiment of an electrotransport module, showing one housing of the electrotransport module open to reveal the internal components. [Figure 26a] FIG. 26a is a series of images showing cell transfection results comparing DNA electrotransfer using a cochlear-type implanted electrode array with individual cochlear electrodes grouped as an anode array and a cathode array (vertical configuration) using a single pulse of 100 V, and DNA electrotransfer using a concentric needle-type array using single pulses of 100 V, 40 V, and 10 V. [Figure 26b] FIG. 26b is a graph of the analysis of variance (ANOVA) of the results shown in FIG. 26a. [Figure 26c] FIG. 26c is a potential distribution map (hemisphere) of the BaDGE concentric stainless steel electrode array. [Figure 27]Figure 27 shows the results of tracking an embodiment of electrotransfer-mediated gene expression over time. (A) Representative bioluminescence images demonstrating the spread of luciferase activity 3 days after intramuscular electrotransfer-mediated luciferase gene (Luc) transfer into mouse gastrocnemius muscle. (B) Representative traces of emitted photons per second of an ROI placed in the hind limb measured at 1-minute intervals after intraperitoneal luciferin injection. (C) Time course graph of bioluminescence measurements peaking at 1, 3, and 7 days (red arrows in Figure 27B) after BaDGE®-mediated transfer of a plasmid encoding luciferase. [Figure 28] 28a-c are photographs of example embodiments of wired needle electrodes. [Figure 29a] 29a and 29b show an embodiment of a prototype test for high throughput needle electrode DNA electrotransfer in cell suspensions for autologous gene therapy and in vitro cell transfection. [Figure 29b] 29a and 29b show an embodiment of a prototype test for high throughput needle electrode DNA electrotransfer in cell suspensions for autologous gene therapy and in vitro cell transfection. [Diagram 30] FIG. 30 shows images of HEK239 cells showing expression of mCherry reporter plasmid DNA against cellular background (DAPI nuclear stain) using in vitro electrotransfer via a concentric stainless steel DNA electrotransfer electrode configuration. [Diagram 31] FIG. 31 shows another example of an electrotransfer system for use in in vitro cell therapy that is applicable to high-throughput multi-well cell transfection with plasmid DNA via a DNA delivery probe. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description One aspect disclosed herein is a device that allows highly efficient DNA electrotransfer into cells via a single capacitive discharge.

[0030] The principle of this system involves the accumulation of a charge on a capacitor, which is then discharged through an electrode array configured to generate an electric field of sufficient strength to effect efficient electrotransfer of DNA into cells, with a field potential gradient focused through the area in the array configuration, where the entry of this DNA or related ribonucleic acid molecules or even other charged molecules into cells can affect altered biological functions.

[0031] In some embodiments, this may be the discharge of a single capacitor.

[0032] 1 shows an example of an electroinduction system 100 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 that is inserted into the tissue to be treated.

[0033] The needle electrode array 130 incorporates at least two electrodes 140, each having an exposed (non-insulated) surface area that wholly or partially surrounds the circumference of the needle, with an insulating section 150 between adjacent electrodes 140, and that is in direct contact with the tissue into which the needle electrode array is inserted. This forms a continuous linear array structure, with the exposed surface area of ​​each electrode at different distances from the tip of the needle electrode array.

[0034] A capacitive discharge circuit 145 is connected to the needle electrode array and configured to store the charge. The circuit includes a switch operable to cause discharge of the stored charge through the needle electrode array 130. Each electrode 140 is connected to the capacitive discharge circuit for operation as an anode or a cathode during discharge of the charge. The length of the electrodes and the length of the insulating section between adjacent electrodes are configured to create a target electric field shape in tissue adjacent to the array during discharge of the charge through the array. A combination of elongated electrodes are arranged in a line with a gap between them such that an electric field is created adjacent to the electrode array. The gap between the electrodes focuses the electric field in the tissue in the area proximate to the gap. The width of the gap controls the rate of change of the electric field with respect to distance and therefore the electric field strength in the area proximate to the gap, defining the shape of the electric field. Research by the inventors has shown that the high electric field strength generated by the rate of change of voltage potential due to the geometry of the linear array described above can stimulate electrotransfer using lower stimulation voltages and accumulated charges than conventional open field electroporation. Thus, effective electrotransfer is possible using a single pulse. The potential gradient can be varied by adjusting the length of the gap between the electrodes to effectively focus the electric field. There can also be an effect of varying the length of the electrodes. The combination of the electrode array geometry and the stimulation pulse controls the size and shape of the region of electrotransfer.

[0035] The charging station 120 includes a DC power supply module, an output terminal connectable to the probe 110 that forms an electrical contact with the capacitive discharge circuit, and a charging control circuit that controls charging of the capacitive discharge circuit of the connected probe. FIG. 2 is a block diagram of an embodiment of the charging station 120, 200. The DC power supply 210 may be battery powered or may be powered from a DC mains supply. Some embodiments use an adjustable DC power supply 215 that includes both the DC power supply 210 and an adjustable regulator 220 to allow a user or an automatic controller to adjust the output voltage. The charging station may include a DC / DC transformer 230. The charging station may 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 the capacitive discharge circuit. The monitoring module may also be configured to control the adjustable DC power supply to disconnect the power supply when a predetermined amount of charge has accumulated in the capacitive discharge circuit. The charging station may include a sensor contact 260 to detect the presence of a probe in the charger. The monitoring module 240 may also output status signals, for example, using visual (i.e., LEDs) or audible signals. For example, the charging station electronics may incorporate factors that determine safe charging of the needle delivery probe, indicators of charging success, and timers that model slow capacitor leakage to alert with green / red LEDs or prompts or sounds if too much time has passed for efficient DNA electrotransfer. Figure 5 is a circuit diagram of an example charging station.

[0036] FIG. 3 is an example of a capacitive discharge circuit 300 of an embodiment of the system. The circuit includes a capacitive charge storage, in this example a single capacitor 310, a switch 320, and a connection to the electrodes 140 of the needle electrode array. The circuit may also include charging contacts 330, 335 that establish an electrical connection to the charging station 200 for charging the capacitive charge storage with a charge. The switch is operable to cause discharge of the stored charge through the electrodes 140 of the needle electrode array 130. Each electrode 140 is connected to the capacitive discharge circuit and is driven as an anode or a cathode during discharge of the charge. FIG. 4 shows an example of the potential 400 measured in a medium adjacent to the anode during discharge of the capacitive circuit, where 410 shows the potential 0 before discharge (open circuit condition for up to approximately 70 ms) before the capacitor discharges. 420 shows the voltage rise at a measurement point in the medium adjacent the electrode array approximately 70 ms after the switch is closed to initiate discharge to a maximum voltage peak of approximately 2V, followed by an exponential decay of potential 430 as the capacitor discharges.

[0037] In some embodiments, the capacitive discharge circuitry is housed in the probe body, so that electrical pulses (capacitive discharge) can be delivered without the need for connection to an external power source or pulse generator. The capacitive reservoir may be a single capacitor. Alternatively, the capacitive reservoir may include multiple capacitors. In some embodiments, the capacitive discharge circuitry may be external to the probe body. In additional embodiments, other charge storage media such as supercapacitors or batteries may be used in place of the capacitor. All such alternatives are considered to be within the scope of the present system. For example, for applications such as brain therapy, a priority may be minimizing the size of the probe to allow high precision and controllability of insertion, and in such embodiments, the capacitive discharge circuitry may be housed separately from the probe body with a wired connection. This may still provide the advantage of providing electroinduction therapy without the need for connection to a mains-driven pulse generator. Thus, less clinical space may be provided for the device. Also, the lack of a pulse generator reduces the risk of electrical interference and electric shock. Further exemplary embodiments are described below.

[0038] As mentioned above, electrotransfer-based delivery of DNA into cells is commonly known as electroporation. When the DNA encodes a gene for a protein, this electric field-based delivery of DNA into cells may be referred to as "gene electrotransfer (GET)". Electroporation of DNA into cells is a routine laboratory procedure. Electroporation / GET is also a recognized method to induce gene expression in tissues, either naturally or in vivo, but the efficiency of gene expression in tissues following DNA electroporation is generally low. Two relevant factors that affect GET are the electric field strength (change in voltage over distance) and duration. To create a suitable voltage gradient for GET in tissue, a current is required to flow between the electrodes. This is accomplished using an electroporator device that applies current pulses between two or more electrodes to generate a GET-compatible electric field (typically 10-100 V / cm), typically with a duration of several hundred microseconds, and repeats these electric pulses multiple times in one or more pulse trains. A single electric pulse is generally understood to be inefficient in achieving GET. Thus, the equipment required for conventional GET requires devices connected to electrodes inserted into the tissue to generate a train of electrical pulses of controlled voltage or current amplitude, duration and frequency, making such equipment costly and bulky.

[0039] In the context of targeted DNA delivery to various biological regions, where the wired connection to the electroporator controller or integrated electroporator microcontroller limits tissue targeting, GET would have a clear advantage if the electrodes could be made independent of the electroporator controller, especially with regard to electrode geometry.

[0040] The inventors have discovered a way to achieve efficient GET using a single electrical pulse without the need for microprocessor-based control of the pulse parameters. This is achieved by decoupling the source of charge from the control of the discharge, as the discharge occurs via a capacitor discharge. The source of charge is a DC power supply, which is used to charge the capacitor, so that it can be disconnected from the capacitor circuit after charging. In using a capacitor discharge for GET, the inventors have found that the capacitor discharge time is a critical factor, as will be further described below with reference to FIG. 8. The time constant (T) of a capacitor depends on the size of the capacitor (C) and the resistance (R) of the circuit, so that T=RC. It should be understood that the total resistance in the circuit during discharge includes the resistance in the target tissue (contributions from both the tissue and the DNA carrier solution) and any circuit resistance. In this disclosure, we demonstrate that in a "closed field" electroporation configuration (two or more elongated electrodes arranged in a straight line along a single probe, with one end electrode driven as an anode or an anode array and the other end electrode driven as a cathode or a cathode array) with a high resistivity DNA carrier solution used to control and enhance the resistivity around the electrode array, the combination of very small current requirements for GET, when delivered via an electrode linear array, can be matched with a specific capacitor area to enable efficient GET. We demonstrate that single-capacitive discharge GET is feasible, where an optimal (minimum) charge delivery is identified based on an unexpected relationship between the time constant of discharge (T) and GET efficiency. In cases where Q=CV and limiting the total charge delivered Q is desired to minimize possible tissue damage, we demonstrate that single-capacitive discharge GET can achieve gene expression efficiency comparable to conventional multi-pulse electroporation based on the delivered Q. In some embodiments, the total circuit resistivity R during discharge is predictable, and the capacitance C is 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 typical tissue resistivity ranges or test results for the target tissue type (i.e., muscle tissue, adipose tissue, breast tissue).This can also be predicted through testing of tissue resistivity after injection of the carrier solution. Although carrier solutions can have a significant effect on resistivity in tissues, some carrier solutions make the effect of the inherent resistance of the target tissue negligible. For example, the resistivity of high resistivity carrier solutions can range from 2 kΩ to 100 kΩ, but the resistivity value measured for a 19% sucrose solution with a typical DNA concentration of 2 μg / μl is approximately 10 kΩ. The required cumulative charge Q required for DNA electrotransfer may be determined through pre-testing and is therefore known. This may 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 may be selected within this range. Based on the resistivity and charge values, the required capacitance for the discharge circuit and, if necessary, additional resistance to be included in the discharge circuit incorporated in the electroporation module can be calculated. In this way, the capacitive discharge circuit parameters can be matched to the resistivity of the therapeutic solution. It should be understood that different types of carrier solutions have different resistivities, so the capacitance of the circuit is selected to match the desired carrier solution.

[0041] The data underlying the invention of Single Capacitive Discharge Gene Electrotransfer (SCDGET) consist of a series of in vitro studies using a monolayer model of human embryonic kidney (HEK293) cells. We have previously used this model to develop BaDGE, or Bionic Array Directed Gene Electrotransfer (BaDGET). Here, when the prototype BaDGE DNA delivery (1010 in FIG. 10) probe was connected to a conventional computer-controlled constant current source to deliver a specific square wave pulse train, or to deliver a pulse train or a single pulse, a single exponential decay occurred that reflects the time constant of the discharge of a capacitor. FIG. 6 shows a comparison of a conventional rectangular electroporation pulse with a model capacitive discharge pulse. In FIG. 6, the left panel 610 shows a unipolar square wave pulse generated using a Digitimer DS5 analog controlled constant current source (on 100 μs, off 400 μs), and the right panel 620 shows a model capacitive discharge pulse 100 μs time constant. These pulse parameters were used with the BaDGE prototype probe to model tissue delivery and deliver reporter DNA into HEK293 cell monolayers.

[0042] Figures 7a and 7b show data comparing the expression of DNA encoding the mCherry fluorescent reporter protein over a series of these pulse trains. In this study, a prototype electrode array (designated 1010 in Figure 10) and a CMVp-mCherry plasmid fluorescent reporter (2 μg / μl) were used to compare the results of square wave unipolar 710 and exponential decay 720 electrotransfer in human embryonic kidney (HEK293) cells. The fluorescence in Figure 7a was imaged 3 days after gene electrotransfer, with the top panel 710 showing the transfection outcome using a square wave and the bottom panel 720 showing the transfection outcome using an exponential decay pulse. In the results graph, Figure 7b, "5+Rect" represents 5 x 100 μs square wave constant current pulses (50 mA, pulse interval 400 μs), "5+Decay" represents 5 x exponentially decaying (capacitive) pulses, time constant 100 μs, pulse interval 500 μs, "10+Rect" represents 10 x 100 μs square wave pulses, "10+Decay" represents 10 x 100 μs decay time constant pulses. Ranked analysis of variance (ANOVA) with Holm-Sidak multiple post-hoc comparisons (* represents P<0.05, ranked ANOVA, post-hoc pairwise comparisons).

[0043] The results shown in Figures 7a and 7b show that a train of ten 120 V peak capacitor-like discharges using a time constant (T) of 100 μs achieved equivalent expression to five square-wave pulses of 50 mA amplitude × 100 μs duration (400 μs interpulse interval). Although the capacitive discharge was not as efficient as the 10 × 50 mA × 100 μs square-wave pulses, the capacitive discharge total charge delivery (Q) was nearly equivalent. These data show that the fast-decay discharge achieved significant GET with minimal total charge delivery. However, multiple discharge pulses were required, which typically requires microprocessor-based control of the pulse train.

[0044] Figure 8 shows the results of a second study investigating whether GET of a single capacitor discharge can be achieved with increasing time constants. This study compared reference gene expression obtained with 10 x 50 mA x 100 μs square wave pulses (replicates of five HEK293 cell coverslips as described above) to single capacitive discharge pulses with time constants of 400 μs, 1 ms, 4 ms, and 10 ms, charged to 120 V (based on a current amplitude of 50 mA across a resistance of approximately 2 kΩ) equivalent to the square wave pulse parameters. These data showed that a single pulse with a time constant of 1 ms (equivalent Q to a 10 x 100 μs square wave pulse) resulted in statistically equivalent gene expression (incorporated mCherry fluorescence) to the reference square wave pulse train, and that GET efficiency is highly dependent on the capacitor time constant. A non-linear dependence on the time constant was also observed for the capacitor discharge, with a 4 ms time constant resulting in gene expression significantly greater than reference levels, whereas a 1 ms decay time constant treatment and even larger time constants (up to 10 ms) did not result in any further efficiency gains, although there was an improvement in charge delivery (Q). In summary, these two experiments provide proof of concept that single capacitive discharge gene electrotransfer can be achieved with efficiencies (expression relative to charge delivered) comparable to conventional square wave electroporation pulses.

[0045] The results shown in Figure 8 indicate that the prototype BaDGE® capacitive discharge gene electrotransfer (BCDGET) (single pulse) is at least as efficient at inducing gene expression as a conventional monopolar square wave constant current pulse train. Graph 810 shows data for mCherry reporter fluorescence from coverslips of HEK293 cells (n=5 / treatment) where plasmid DNA encoding this reporter was placed into the cells and the DNA was electrotransferred into the cells using the prototype BaDGE® DNA delivery probe. Coverslips were imaged after 3 days in tissue culture. The data show that our reference 820, 820' 10 x 100 μs square wave constant current pulses (approximately 50 mA per pulse, designated "100 up 100 us square") are comparable to 830, 830' (e.g., "1 up 400 us decay constant"), 840, 840' (e.g., "1 up 1 ms decay constant"), 850, 850' (e.g., "1 up 4 ms decay constant"), and 860, 860' (e.g., "1 up 10 ms decay constant"), which are single capacitive discharges with a time constant of 400 microseconds. The single capacitive discharges with longer time constants showed significantly higher gene electrotransfer. The bottom panel of Figure 8 shows an example of imaging mCherry reporter fluorescence of HEK293 cells on coverslips using different charge delivery protocols (time constants of 400 μs = ~200 nF, 1 ms = ~500 nF, 4 ms = ~2 μF, 10 ms = ~5 μF (* indicates P < 0.05, ranked analysis of variance (ANOVA), post-hoc pairwise comparisons)).

[0046] To verify these results in a first in vivo proof-of-concept experiment, a mouse 900 hind limb model was used (Figure 9a). Under isoflurane anesthesia, DNA (1 μg / μl, 50 μl) encoding the luciferase gene was delivered to the right gastrocnemius muscle 910 using a prototype BaDGE® DNA delivery probe (1010 in Figure 10) driven with a conventional constant current pulse train (5 x 4 ms pulses, 16 ms pulse interval, approximately 60 V was recorded driven at 50 mA current amplitude for each pulse). The plasmid DNA incorporated a hybrid cytomegalovirus-chicken β-actin-rabbit β-globulin gene splice acceptor sequence (CAG promoter) and was 6604 bp in size. The left gastrocnemius muscle 920 was treated with single capacitive discharge gene electrotransfer, and the BaDGE® DNA delivery probe was connected to a 2.2 μF capacitor and charged to 120 V. This delivered a single capacitive discharge (based on a 2 kΩ resistor model) with a time constant of 4 ms, where the total charge delivered was approximately 50% of that used for the reference 5 x 4 ms square wave pulse train delivered to the right hind paw target. Expression was analyzed 24 hours later using the SpectrumCT bioluminescence imaging platform, within 30 minutes of intraperitoneal injection of the bioluminescence substrate, luciferin. Images show that in the left hind paw, stronger expression (bioluminescence) was obtained from the single capacitive discharge GET than that obtained from the conventional square wave pulse train GET.

[0047] Figure 9a shows the in vivo proof-of-concept efficacy of the prototype single capacitive discharge gene electrotransfer (SCDGET) device. Figure 9 shows spectral CT bioluminescence imaging of a mouse (ref. ms187, 250519) 24 hours after DNA electrotransfer to induce photoemission resulting from expression of recombinant luciferase by hindlimb muscle under isoflurane anesthesia within 30 minutes of intraperitoneal injection of luciferin substrate. Light emission 920 from the left hindlimb results from electrotransfer of DNA (1 μg / μl) to a gastrocnemius skeletal muscle target using a BaDGE® DNA delivery probe connected to a 2.2 μF capacitor and charged to 120 V. The right limb 910 used the same probe driven with 5×4 ms pulses (pulse interval 16 ms) (current 50 mA via a drive voltage of approximately 60 V) using a constant current source (Digitimer DS5 analog control) via software and D / A control. To obtain the image shown in Figure 9a, mice were anesthetized with isoflurane and then injected (intraperitoneally) with luciferin (i.e., a substrate for the expressed luciferase enzyme). This substrate is taken up by muscle cells and, in the presence of luciferase expression, photons of light are produced and emerge through the skin, which are counted with a highly sensitive camera. This record of emitted light reflects the intensity of expression of the recombinant luciferase protein. This output is 24 hours after expression and is shown as an intensity-coded light emission superimposed on an image of a mouse hind paw. This image shows that SCDGET utilizes approximately 50% of the charge delivery of a conventional square wave pulse train protocol, resulting in approximately twice the bioluminescent signal.

[0048] Measurements of bioluminescence signal (light flux) are shown graphically in FIG. 9b for mouse reference number 187, along with additional experiments in both hind limbs of four additional mice. For mouse 187 (diamond trace), it can be seen that there are multiple measurements at days 1, 3, 7, 90, and 150. For the other mice, there are measurements at days 1, 3, 7, 14, and 50. FIG. 9b shows a comparison of longer-term results for bionic array directed gene electrotransfer (BaDGE) of plasmid DNA (pMK175.CAGp / luciferase) into mouse calf muscles using a single discharge from a 2.2 uF capacitor integrated into the DNA delivery probe of BaDGE. After intraperitoneal injection of D-luciferin potassium salt (15 mg per kg body weight), the peak total light flux emitted from the hind limbs of each mouse at each time point was measured. In this study, mice underwent BaDGE on day 0 using a plasmid encoding the luciferase gene. DNA was suspended in 10% sucrose solution and pH adjusted with sodium hydroxide. In mice limbs receiving 10 μg DNA, 20 μl was delivered at 0.5 μg / ul, and in mice limbs receiving 20, 40 or 50 μg DNA, this was delivered at 1.0 μg / ul in a volume of 20, 40 or 50 μl. Diamonds represent the left and right limbs of mouse reference number 187, circles are mean ± SEM data from mice reference numbers 119, 120 and 121. Squares are data from mouse reference number 118 containing two DNA payloads.

[0049] Figure 9c shows bioluminescence readout of luciferase reporter gene expression (LUC) after BaDGE®-mediated DNA electrotransfer. Images show peak expression maintained through day 486 for mouse no. 187, and similar persistent expression through day 389 for mouse nos. 118, 119, 120, and 121. Luciferin substrate-mediated bioluminescence (intraperitoneal administration) was imaged at peak, with black diamonds representing 4 x 5 ms square wave pulses (5 ms) and white diamonds representing 1 x 2.2 μF capacitive discharge from 120 V (both for 1 μg / μl pMK175-CAGp-Luc plasmid DNA). The graph shows data from a total of 5 mice (n=3 at 250V (filled circles) or 120V (open circles), 2.2μF, 10μg in 20μl), open squares are single capacitive discharges of 20μg DNA at 250V, 2.2μF, 1μg / μl, and filled squares are single capacitive discharges of 40μl DNA at 1μg / μl, 250V, 2.2uF. At the time of filing, 486 days is the longest data available due to the timing of the study, however this is a positive indication of continued efficacy given that sustained effects were observed out to 486 days.

[0050] These tests demonstrated that in a prototype device incorporating a capacitor into the BaDGE®, the DNA delivery probe can be charged via a charging station (a voltage-regulated power source, typically taking less than one second to charge) and then used for DNA electrotransfer without the burden of connecting the DNA delivery probe to a regulated power source, simply by removing the circuit (switch). Separate tests showed that once the capacitor was charged, its voltage was maintained for at least 30 minutes.

[0051] The disclosed embodiment of the system stems from the discovery that a linear array of electrode elements configured within a DNA delivery probe allows for highly efficient electrotransfer of naked DNA into target tissues, with the charge transfer required to achieve in vivo gene expression being orders of magnitude smaller than conventional electroporation systems due to the focused compression of the local electric field in the vicinity of the linear probe.

[0052] Another finding applied in an embodiment of the system is that the electrode configuration can control the extent of the transfection region for a linear electrode array. Figure 10 shows an example of a prototype linear electrode array probe 1010. This prototype electrode array includes two elongated tubular platinum-iridium (Pt / Ir) electrodes connected to a capacitive discharge circuit (or pulse generator) by insulated Pt / Ir wires, and the Pt / Ir electrodes are stacked on top of an inner needle with an insulating layer between them to insulate the electrodes from the inner needle and each other. During pulse delivery, one electrode is driven as an anode and the other electrode is driven as a cathode. The electric field gradient adjacent to the center of the array has the steepest slope of the electric field potential gradient, and experimental results have shown that this region is where the most cells are transfected. The shape of the electric field depends on the combination of elongated anodes and cathodes arranged in a linear fashion, and the respective dimensions of these conductive elements.

[0053] The elongated nature of the anode and cathode contributes to controlling the shape of the generated electric field and therefore the transfection area. Varying the width of the gap between the elongated anode and cathode changes the extent of the transfection area perpendicular to the array due to the compression of the electric field and the change in the rate of change of the potential with the adjacent distance of the array close to the gap. By varying the gap, the electric field shape and therefore the transfection area can be widened or narrowed. In the example shown, increasing the gap between the electrodes widens the electric field and the transfection area, while decreasing the gap narrows the electric field. It should be noted that this control or vectoring of the electric field shape is applicable up to a gap of approximately 5 mm between the electrodes for the pulse parameters used in this example. Increasing this gap may be feasible in conjunction with increasing the corresponding current level, but the use of more electrode pairs is preferred to allow treatment of larger areas due to the potential negative side effects of high current levels. It should be noted that controlling the radial extent of the electric field can also be referred to as "vectoring".

[0054] FIG. 10 shows some of the effects of manipulating the gap between the anode and cathode, and these results were achieved using a prototype probe. Images 1020-1050 of FIG. 10 are illustrations of the expansion and contraction of the transfection area by manipulating the gap between the anode and cathode of a linear electrode array, and in the example of FIG. 10, a probe of the type shown at 1010 was used, which is linear and has two electrode arrays with different spacing between the electrodes. The effect of manipulating the electric field was obtained by decreasing the spacing between two platinum-iridium electrodes (2 mm (length) x 0.35 mm (diameter) on an insulated scaffold, as shown in the top right image), and for each example array configuration, the electrodes were driven using a train of electric pulses (constant current, pulses 100 μs x 10, pulse interval 400 μs, phase 10 mA +ve). Data show spatial control of electrotransfer of plasmid DNA (green fluorescent reporter protein - GFP - under a cytomegalovirus (CMV) promoter) delivered to a monolayer of human embryonic kidney (HEK) 293 cells using an isotonic polysaccharose carrier. The DNA and electrotransfer array were placed on the cells on a coverslip. A pulse was delivered and then the coverslip was returned to culture for 48 h before imaging. The highest electric field strength is around the null point between the two electrodes. As the electrodes move further apart, the electric field becomes wider enough for DNA electrotransfer until a cell density for optimal dispersion and expression of the recombinant DNA expression cassette is obtained (electrode spacing 1-4 mm).

[0055] In the embodiments of the capacitive discharge electroinjection probes disclosed herein, the combination of the electrode array configuration (particularly the length of the electrodes and the gap between the electrodes or between groups of electrodes driven using the same polarity) and the capacitive discharge characteristics control the area where cellular transfection can occur. Therefore, the array configuration and discharge circuitry can be designed to achieve predictable treatment outcomes. Furthermore, the tissue and the therapeutic solution injected into the area surrounding the linear array form part of the discharge circuit and therefore affect the characteristics of the capacitive discharge, and therefore the capacitive discharge circuitry can be designed to incorporate the resistance of the therapeutic solution and / or tissue in the equivalent circuit to model the discharge characteristics and electric field. In this way, the probe can be tailored for a particular therapeutic solution and target tissue. For example, the characteristics of the capacitor for the discharge circuitry can be matched to the resistivity of the therapeutic solution to provide the target initial potential, cumulative charge delivery and pulse decay profile. The physical array configuration (i.e., the length of the electrodes and the gap between the electrodes) can also be modeled for a particular application.

[0056] As a possible practical embodiment, the SCDGET may be integrated into a disposable SCDGET / DNA delivery probe that contains DNA pre-encapsulated in a carrier solution in a syringe. The SCDGET-probe is charged immediately before use by contact with the SCDGET charging station, the DNA is delivered via the syringe and needle, and the circuit is closed for the discharge of the capacitor, achieving instant GET. An indicator (e.g., LED) in the charging station confirms that the SCDGET-probe is charged and indicates how long the probe will maintain its charge based on the discharge characteristics of a similar capacitor circuit in the charging station. As mentioned above, the charging time of the probe is less than one second. The charge maintenance time is tens of minutes. The physical size of the capacitor is in the low cubic millimeter range, in the range suitable for an optimized SCDGET (typically 1-5 μF, but other values ​​are possible), and the cost per unit is a few cents. This makes it suitable for high-throughput DNA GET applications such as DNA vaccines, where the SCDGET charging stations can be battery powered for field operations and the DNA probes can be preloaded with DNA making them one-time disposable items.

[0057] FIG. 11 illustrates an example of a prototype probe tip 1100 including a needle array 1110, a needle hub 1120, and a body 1140 (only a portion of which is shown in the figure). The hub 1120 may house a capacitive discharge circuit 1130 and electrical connections to the electrodes of the needle array. In the embodiment shown in FIG. 12, the hub 1120 also includes two sections 1135 that allow electrical connections to a charging station for charging the capacitive discharge circuit. This embodiment also describes a therapeutic reservoir in fluid communication with a central lumen through the needle array 1110 that allows the probe to be pre-filled with a therapeutic solution for delivery prior to application of an electroinduction pulse. For example, the probe tip may be integrated into a conventional syringe structure with a plunger that forces fluid from the reservoir through the lumen. In such an embodiment, the plunger may also be configured to activate a switch in the capacitive discharge circuit to cause the capacitive discharge. For example, the switch may be activated at the end of the plunger's travel.

[0058] The distal (tip) portion of the needle array 1110 is shown in more detail in Figure 12, which shows a first needle 1210 (in this embodiment having a beveled tip 1215 and a central lumen for therapeutic substance delivery) which also acts as a first electrode, a first concentric insulator 1220 covering the first needle a predetermined distance (L1) 1212 from the tip 1215, a concentric second electrode 1230 covering the first concentric insulator a distance (L2) 1235 from the distal end of the first concentric insulator, and a second concentric insulator 1240 covering the second electrode 1230 a distance (L3) 1235 from the distal end 1230 of the second electrode. The first needle 1210 and the second concentric electrode are formed of a conductive material and are electrically connected to the positive and negative terminals of the capacitive discharge circuit 1130. The exposed length 1212 of the first needle acts as the first electrode of the linear array, and the exposed length 1235 of the second concentric electrode 1230 acts as the second electrode of the linear array. The lengths L1 (1212) and L3 (1235) of the first and second electrodes, respectively, and the exposed length 1225 of the first insulating portion 1220 therebetween determine the pattern of electric field gradient that is generated adjacent to the needle array when an electrical pulse is applied to drive one electrode as an anode and the other as a cathode.

[0059] Figure 13a shows a model of the potential in the medium around the DNA delivery probe when 240 V is applied across the electrodes for the peak voltage induced in the electric field adjacent to the probe during the discharge of the built-in capacitor, and Figure 13b shows a model of the electric field strength around the DNA delivery probe when 240 V is applied temporarily across the electrodes.

[0060] An embodiment of the needle array is constructed using two concentric needles, the inner needle has insulation starting a preset distance from the tip, the uninsulated tip area is the first electrode, and the outer needle is insulated a preset distance back from its insulated tip. There is also a gap between the location of the second (shorter) needle and the insulated area of ​​the inner needle (the distance of the inner needle's insulating coat before the inner needle's conductive tip is equivalent to the spacing of the electrode elements shown in Figure 10 for a "sliding trombone" effect).

[0061] Both needles are inserted into a custom designed needle "hub." This hub incorporates circuitry that couples to the proximal ends of the electrodes, which are also electrically conductive. The hub may incorporate holes for insertion of charging station pins and a switch plate that closes the discharge circuit for an on-board capacitor. In one embodiment, the switch plate is activated when the syringe barrel plunger is fully depressed or in a predetermined position of travel.

[0062] An example of prototype development uses a 34G needle (36mm long) with a beveled tip and a 28G outer needle (27mm long) cut at a right angle (thin walled needle to act as the surrounding second electrode). In some embodiments, the outer needle may also be beveled to facilitate the transition in needle diameter from the thinner inner needle to the outer needle. Alternative embodiments of needle arrays may be fabricated using deposition (e.g., sputtering, printing, etc.) of a conductive insulating layer to form the electrodes.

[0063] In an alternative embodiment, the needle array has a closed tip and ports formed along the length of the needle array (distal to the tip) for delivery of fluids to adjacent tissue. An advantage of this embodiment is the flexibility in the location of the inner and outer needle conductive regions and the location of optimal delivery of DNA and carrier solutions near the electrodes, thereby providing optimal control of capacitive discharge time.

[0064] In some embodiments, the needle electrode may include multiple electrodes along the array (e.g., to allow multiple ports for delivery of therapeutic solutions from between the electrodes). In some embodiments, the needle electrode may include multiple electrodes, either bundled together or individually connected to one or more capacitive discharge circuits. In some embodiments, when multiple discharge circuits are used, they may be configured to activate discharge at different times to change the shape of the electric field during treatment. For example, one may have different capacitive discharge circuits that activate at different positions in the syringe plunger's travel. Different discharge circuits may also have different discharge characteristics.

[0065] In one example, the probe may have a dozen electrodes and a half dozen capacitors connected to paired electrodes along a linear array, and in other embodiments, elution of the DNA solution may occur from a port or ports between the electrode pairs, not necessarily at the needle tip.

[0066] In some embodiments, the needle array may not include a lumen for therapeutic substance delivery. For example, the therapeutic substance may be delivered using another syringe. Also, in some clinical applications or procedures, it may be desirable to use separate devices for the delivery of the therapeutic substance and the delivery of the electrical pulse. The lumen may also be omitted to minimize the diameter of the needle array for physiological reasons, such as to create a very thin needle array for use in the brain or for use in young pediatric patients. In these embodiments, the therapeutic agent may be injected or otherwise applied to the target tissue prior to the placement of the DNA electrotransfer probe. Alternatively, the needle array may be coated with a hydrogel coating containing the therapeutic substance, which is then delivered to the tissue from a continuous surface of the needle array. In other embodiments, the lumen may have multiple exit ports for the therapeutic substance along its length.

[0067] FIG. 14 shows another embodiment of a probe tip similar to that shown in FIG. 11. However, in this example, the hub includes an additional port that allows contact with a sensor in a charging station to check the status of the capacitive discharge circuit within the probe as well as to check if the probe is connected to a charger. This is a conceptual representation of a concentric BaDGE® DNA electroinjection probe. The components include an inner core DNA delivery needle with a length of conductive surface including an insulating layer that extends to an internal contact within the hub. Recessed from the outer insulated surface is an outer conductive needle sheath with a length and surface area that contributes together with the intervening insulated area with the distal electrode surface to creating an electric field shape for DNA delivery. Proximate the conductive area of ​​the outer sheath is an insulated surface except for the contact within the hub. The hub consists of three ports, two of which are used to charge the capacitor and the third port provides a sensor contact to indicate charging, all ports are enabled by inserting the probe into a remote charging station. In other embodiments, the monitoring circuitry may use this method to detect insertion by evaluating the current flow between the two charging contacts. An example conceptual representation of the insertion of a probe 1510 into a charging port 1530 of a charging station 1520 is shown in Figures 15a and 15b. A protective needle cover is not shown, but this may be placed in some embodiments until just prior to DNA delivery. The hub contains a built-in capacitor that is discharged by closing an internal switch acted upon by deflection of a pin element in the hub that fully or substantially contacts a fully depressed plunger of a syringe barrel that may contain a DNA solution and be attached to the hub of the DNA delivery probe. Figure 16 provides a further conceptual view of a syringe body 1610 with a built-in needle array and discharge capacitor 1630. This example also includes a contact rod 1640 that activates a switch to discharge the capacitor through the array when the syringe plunger reaches or near the end of travel.

[0068] In some embodiments, the electrical discharge circuit includes a single capacitor, which may have the advantage of being very small and easy to embed into conventional device structures such as syringes. An example of a capacitor integrated into the needle hub of the SCDGET has a small 2.2uF, 450V multi-layer ceramic chip (MLCC) capacitor therein. At the time of filing, a commercially available component of a known MLCC has dimensions of 5.7x5.0x2.5mm.

[0069] An 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 after contact with a remote charging unit, makes SCD-BaDGE® DNA delivery virtually indistinguishable from routine vaccination procedures. Already innovative, our SCD-BaDGE® DNA delivery system is so efficient that a single electrical pulse resulting from the discharge of a miniature capacitor integrated into the hub of the needle-like DNA delivery probe provides 100% reliable gene expression in muscle. Note that 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.

[0070] Alternative embodiments not currently included in the drawings are also contemplated. For example, an embodiment may include a metallic snap-action type contact to form an electrical connection. Another embodiment may include a magnet and reed switch that forms an electrical connection when a magnet attached to the syringe plunger comes in close proximity to the reed switch when the syringe plunger reaches or near the end of its travel. Another embodiment may include (1) a metallic snap-action switch contact, and (2) a dual-shaft syringe, where the second shaft of the syringe is used to house an electrical switch that can be activated by manipulation of the plunger. For example, the switch contact may be included in the bottom of the second syringe shaft.

[0071] Figure 17 is an image demonstrating the utility of the prototype SCD-BaDGE in in vivo applications, the image shows the expression of light-driven ChrimsonR channelrhodopsin ion channel protein-TDtomato fusion reporter protein in mouse soleus muscle fibers. This demonstrates the use of single capacitance discharge BaDGE for mouse ontogeny applications. The expression of light-driven ChrimsonR channelrhodopsin ion channel protein-TDtomato fusion reporter protein in mouse soleus muscle fibers was imaged 4 days after single capacitance discharge electrotransfer (SCD-BaDGE®) of naked plasmid DNA encoding this protein under the cytomegalovirus promoter. DNA delivery parameters are 1 μg / μl DNA in an isoosmotic sucrose-based carrier solution, a BaDGE® DNA delivery probe containing a 2.2 μF capacitor charged to 250 V. The image is a 3D reconstruction of a confocal laser scanning image stack acquired using 561 nm excitation and 567-633 nm illumination. Shown in FIG. 17 are images of individual muscle fiber expression as a result of this in vivo gene electrotransfer.

[0072] FIG. 18 illustrates one possible system embodiment, including electrotransfer modules 1810, each connectable to a syringe (not shown), and a charging unit 1820. Each electrotransfer module 1810 includes a capacitive discharge circuit that is chargeable using the charging unit 1820. FIG. 19a illustrates an example of an electrotransfer module that is configured to be compatible with a syringe 1930. For example, the electrotransfer module 1810 may be luer lock compatible to engage a conventional luer syringe. FIG. 19b is an illustration of the electrotransfer module 1810 connected to a syringe 1930. It is possible and advantageous to pre-fill the syringe with the desired therapeutic agent fluid 1940, but it may also be filled in situ. It should be understood that the electrotransfer module may be utilized for electrically stimulated delivery of multiple different therapeutic agents, such as those therapeutic agents that have short shelf lives or special handling requirements. In this manner, it may be practical to provide therapeutic agents separately from the electrotransfer module, a device that does not have the same shelf life limitations. Also, having the electrotransfer module be a separate device from the therapeutic agent delivery syringe may provide greater flexibility and adaptability to changes in the therapeutic agent. For example, a batch of electrotransfer modules may be purchased in bulk for a clinical trial comparing two or more therapeutic agents (and a control), or for a clinical practice treating a number of conditions, each with a different therapeutic agent.

[0073] FIG. 20 shows an exploded view of the syringe 1930 and electrointroduction module 1810. The electrointroduction module has a needle array 1815 (as described in any of the embodiments above), which may be normally covered using a needle cover 1850 for safe handling. FIG. 21a shows an example embodiment of the electrointroduction module 1810 in more detail. The electrointroduction module 1810 has a housing with a neck portion 2120 connectable to a syringe at one end, and a needle array protruding from the opposite end with a lumen extending through the device to allow delivery of fluid from the connected syringe. The syringe may be an off-the-shelf (OTS) syringe 2110, in this example a luer lock type syringe, with the housing configured to be compatible with the luer lock 2120, although other attachments may be used. The needle array has two coaxial electrodes with insulation between them, where the first, inner electrode is longer than the second, outer electrode, and the insulation extends along a portion of the first electrode and beyond the end of the second electrode, creating an insulated gap 2150 between the two electrodes. The length of this gap 2150 affects the electric field generated by capacitive discharge, and therefore the area targeted for electric field mediated cell transfection (electrotransduction). As such, the gap may also be referred to as an electric field lens 2150, since it affects the focusing of the electric field gradient.

[0074] The housing contains 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 to store charge for delivery, and a switch is electrically connected to the electrode of the needle array. The circuit also includes a charging pin 2180 or other means for allowing charging of the capacitor to an external electrical connection. In this embodiment, the switch is a microswitch 2160 and the switch actuator is a switch plate 2170 configured to be actuated by directing the syringe plunger to an end of plunger travel and moving the switch plate 2170 to actuate the microswitch 2160 to discharge the stored charge from the capacitor. In this embodiment, the switch plate 2170 has a flat section configured to engage the microswitch and a post including a lumen connected to the needle array that extends through the neck portion 2120 of the housing to be engageable with the syringe plunger and establishes a fluid communication channel from the syringe to the needle array for delivery of fluid to tissue. In this embodiment, the switch plate 2170 is biased (or initially mechanically held) in an open (or disengaged) position. Actuation of the syringe plunger initially forces fluid through the lumen until the plunger contacts the post, and continued movement of the plunger causes corresponding movement of the switch plate toward the microswitch, actuating the microswitch and causing a capacitive discharge through the electrode array. Figure 21b shows an alternative embodiment of the electroinjection module designed to operate with a low dead space type syringe. This embodiment has a variation of the switch plate with a slightly shorter post due to the different syringe plunger geometry.

[0075] In the illustrated embodiment, the capacitive discharge switch is a microswitch actuated by a switch plate that is mechanically moved by the syringe plunger to actuate the switch. Other types of switches and actuators are also contemplated within the scope of the system embodiments. For example, the switch can be a magnet in the base plate of the switch that actuates a micro-magnetic reed switch or a piston with a conductive (metallic) ring that is actuated to close the discharge circuit. FIG. 21c is an example of a reed relay where magnetic actuation of capacitive charge delivery occurs when a magnet embedded in the switch plate 2175 comes in close proximity to the reed relay 2165, causing the switch inside the relay 2165 to close its contacts, allowing the capacitor to discharge through the needle electrode.

[0076] FIG. 22 is a diagram of the electrotransfer module (attached to a syringe filled with therapeutic fluid) inserted into a charging unit which charges the capacitive discharge circuit. The charging unit is connected to a power source. When the electrotransfer module is inserted, it electrically connects to the capacitive discharge circuit via the charging pins. A light and / or audible alert on the charging device indicates when the capacitor is fully charged. The device is then removed and the patient is treated.

[0077] As shown in the process diagram in Figure 23, a clinician first assembles the syringe and electroinjection module, charges the capacitive discharge circuit, and then delivers the treatment to the patient similar to any typical injection inoculation. Figure 24a shows the first stage of inoculation, where the needle is inserted into the patient's arm as the syringe plunger is depressed, delivering the treatment fluid into the tissue surrounding the needle array. As the plunger nears the end of its travel, it activates a switch in the capacitive discharge circuit, which discharges the charge from the capacitor via the electrodes of the needle array, creating an electric field gradient in the tissue surrounding the array that stimulates cell transfection.

[0078] Figure 25 is a photograph of a prototype embodiment of the electrotransfer module. Figures 26a and 26b show the results of DNA electrotransfer using an 8-node bionic array compared to using a concentric stainless steel electrode array using HEK293 monolayers. Fluorescence of the mCherry reporter gene was captured 4 days after BaDGE® onto HEK293 cell monolayers using a single capacitive discharge from a 2.2 F capacitor charged to 100V, 40V or 10V (shown in Figure 26a). The electrodes used for gene delivery were concentric stainless steel needle electrodes (inner needle 34G, outer needle 28G) with active electrodes 2mm long, 1mm gap between electrodes for a cochlear-shaped implanted 8-electrode array (Pt / Ir electrodes, 400μm long, diameter and spacing, "tandem" configuration (++++----)). n=5 coverslips for each experimental protocol.

[0079] Cells were seeded on 18 mm coverslips approximately 24 hours prior to transfection, which was performed when cells were approximately 50% confluent on the coverslip. 20 ul of DNA in BaDGE® at 2ug / ul (CMVp-mCherry plasmid) was suspended at neutral pH, electrodes were aligned above the cells, and then a 10% sucrose solution was applied to the cell-covered coverslip. Figure 26a is a series of images showing the results of electrotransfer using a single capacitive discharge compared to electroporation using a cochlear-shaped implanted array and a prototype electrotransfer module. The images show fluorescence proving transfection of HEK293 cell monolayers for:

[0080] 1. An 8-electrode cochlear electrode array, also known as a tandem array configuration (++++----), with four adjacent electrodes driven as an anode array and the remaining four adjacent electrodes driven as a cathode array, was driven with a single discharge using a 2.2 μF capacitor charged to 100 V. This is the electrode configuration previously demonstrated by the inventors for electrotransfer using a train of pulses, and was used for the initial demonstration of the efficacy of electrotransfer using a single capacitive pulse discharge, and as such is used here for comparison with the testing of the concentric needle array prototype.

[0081] 2. Prototype concentric needle array after electrotransfer using a single discharge, 100V, 2.2μF capacitor to drive the needle electrode array, showing a similar transfection field as the cochlear electrode embodiment driven using the same pulse.

[0082] 3. The prototype concentric needle array after electrotransfer using a single discharge, 40V 2.2μF capacitor to drive the needle electrode array. This shows a low strength transfection response, which is not surprising given the low charge.

[0083] 4. Prototype concentric needle array after electrotransfer using a single discharge of a 2.2 μF capacitor to drive the needle electrode array at 10 V. This shows minimal transfection response.

[0084] Figure 26b is a box plot graph of the results shown in Figure 26a. These results show the effectiveness of DNA delivery to HEK293 cell monolayers using a tandem configuration of 8-electrode arrays (++++----), both driven by a single discharge of a 100V charged 2.2μF capacitor, and using a prototype electrotransfer module ("BaDGE Inoculator") with an electrode array containing stainless steel concentric needles, transfecting cells in comparable areas (P=0.00016, t comparing 100V and 40V concentric electrodes, P=0.008, 40V vs. 10V, Mann-Whitney rank sum test). This also shows that at 40V, transfection focusing around the area resulted in fewer cells being transfected in the central area, and the electric field gradient was focused around the gap between the electrodes (electric field lens). In the example using 10V charging, there was minimal transfection, indicating that this voltage provided insufficient field strength for transfection with this combination of electrotransfer module prototype and therapeutic agent.

[0085] Figure 26c shows a potential map (hemisphere) of the BaDGE concentric stainless steel electrode array, and below that, a schematic of the needle electrode array (cross section) aligned with the potential map, which corresponds to the electric field used to transfect HEK293 cells at a single capacitive discharge of 40 V as described above in connection with Figure 26a.

[0086] As an extension of the studies described above, to demonstrate reliable luciferase expression using a BaDGE® inoculator (stainless steel concentric needle), a second cohort of mice was used to test DNA expression by mouse hindlimb muscle fibers using a single capacitive discharge of either 40V or 100V (1 μg / μl luciferase reporter plasmid DNA in 10% sucrose solution, total volume 50 μl).

[0087] Figure 27 shows the results of tracking BaDGE® inoculator-mediated gene expression over time. (A) Representative bioluminescence images demonstrating the spread of luciferase activity 3 days after intramuscular BaDGE®-mediated transfer of luciferase gene (Luc) into mouse gastrocnemius muscle. (B) Representative trajectories of emitted photons per second of ROIs placed in the hind limbs measured at 1-minute intervals after intraperitoneal luciferin injection. (C) Time-course graphs of peak bioluminescence measurements (red arrows in Figure 27B) on days 1, 3, and 7 after BaDGE®-mediated transfer of a plasmid encoding luciferase (alternate lower limb) using a single capacitive discharge of either a 40V or 100V charge of a 2.2μF capacitor. Delivery of DNA plasmid reporter was by pMK175-CAGp-Luc (50 μl, 1 μg / μl, carrier was 10% sucrose, pH 7.4, n=5 gastrocnemius inoculations for each capacitor charging voltage, mean ± SEM).

[0088] The methodology applied to obtain these results is as follows:

[0089] All procedures followed procedures approved by the University of New South Wales Animal Care and Ethics Committee (ACEC 18_160a). Animals were placed in an anesthesia induction chamber and anesthesia was induced using 3% isoflurane at a slow flow rate of 1-2 L / min with oxygen. Once anesthesia was established, animals were moved to a heated animal bed in prone or spinal position while anesthesia was continued with 2% isoflurane delivered via a nose cone at 1-2 L / min with oxygen. Body temperature was maintained and oxygen saturation was monitored with a MouseStat PhysioSuite (Kent Scientific). The hind limbs and lumbar area of ​​the animals were shaved and disinfected with ethanol swabs.

[0090] BaDGE®-mediated introduction of a luciferase-encoding plasmid was performed using a single capacitive discharge of a 2.2 μF capacitor charged at 40 V or 100 V (alternate limb). DNA plasmid reporter was delivered using pMK175-CAGp-Luc (50 μl, 1 μg / μl, carrier 10% sucrose, pH 7.4, n=5 gastrocnemius inoculations for each capacitor charging voltage). Animals were allowed to recover and then returned to their home cages.

[0091] Bioluminescence imaging was repeated on the computed spectrum in a tomography scanner (IVIS® SpectrumCT, Perkin Elmer). For this procedure, anesthesia was induced using 3% isoflurane with oxygen in an induction chamber as specified above. Once anesthetized, an eye ointment was applied and the mouse was given luciferin by intraperitoneal injection (150 mg / kg). This amount of luciferin is a harmless substrate that reacts with luciferase expressed from the pMK175-CAGp-Luc plasmid delivered by BaDGE® to emit photons. The animal was moved to a heated animal bed in a prone or spinal position while anesthesia was continued with isoflurane anesthesia delivered via a nose cone at 2% isoflurane in the IVIS® SpectrumCT. Bioluminescence 2720 was imaged at 1 minute intervals until sustained luminescence (emission of photons per second in a given region of interest (ROI) 2710 on each hind paw) was detected for 5 consecutive minutes. Animals were allowed to recover and then returned to their home cage. Peak luminance 2730 was determined as the maximum brightness in a given ROI.

[0092] Initial measurements taken 24 hours after electrotransfer showed reliable luciferase expression, plotted as a function of peak luminance over time (bottom graph of FIG. 27), which continued to increase over the first 72 hours. Measurements taken 7 days after electrotransfer show similar luminance to that measured in mice for DNA incorporated into mouse hindlimb muscle fibers electrotransferred using 40V and 100V stimulation.

[0093] The disclosed electrotransfer technology is typically applied in DNA processing for in vitro cell transfection for autologous gene therapy. Figure 29 shows an embodiment of a prototype test for high throughput needle electrode stimulated DNA electrotransfer in cell suspension for autologous gene therapy and in vitro cell transfection. An example of a process for in vitro continuous cell transfection via needle electrode using naked DNA and capacitive discharge DNA electrotransfer is configured to capture cells from culture (in vitro) or living organisms (tissue cell suspension after digestion, or blood) and resuspend them in a DNA carrier solution (10% sucrose, DNA typically >0.1 μg / μl to 10 μg / μl). This suspension is pumped through a reaction tube via a syringe pump, moving the suspended cells through the DNA delivery probe of the needle electrode (length approx. 5 mm) over time (depending on the diameter of the tube) to ensure that the electroporation pulse traverses that distance at least twice. DNA is delivered effectively. A typical electroporation protocol is 100V exponential decay through a 2.2μF capacitor at 2Hz. The cells in solution are then transferred to culture medium for culturing or crushed again and returned to the organism (autologous cell gene therapy applications). DNA electrotransfer is instantaneous. For in vitro readout, cells can be counted using FACS (fluorescence activated cell sorting) from about 10 hours after transfection. Figure 30 is an image of cells showing expression of mCherry reporter plasmid DNA against the cellular background (DAPI nuclear stain) following continuous flow DNA electrotransfer.

[0094] In this cell flow experiment, the media was removed and 1 ml of TryplE was added to a single 10 cm diameter dish of HEK293 cells approximately 90% confluent. After 3 minutes, 9 ml of 10% sucrose was added to the dish at 37°C to resuspend the cells. Half of this suspension (5 ml) was pelleted at 800 rpm for 5 minutes. The 5 ml sucrose solution was removed and the cell pellet was resuspended in 0.5 ml of pH neutral 10% sucrose containing 1 ug / ul of 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 (250 μl in 90 seconds) every 6 minutes. The syringe delivered the cell-DNA suspension around concentric needle electrodes (inner needle 34G, outer needle 28G) with active electrodes 2mm long and 1mm gap between electrodes (total array length 5mm). Pulses modeling capacitive discharge were delivered to the electrodes via a microprocessor-controlled DigitimerDS5 constant current simulator to model the discharge of a 2.2μF capacitor. Pulses were applied at 2Hz, 50mA / 120V with a decay constant of 10ms. Cells that passed through the electrodes were harvested in 5cm culture dishes containing fresh medium and incubated at 37°C, 5% CO2 for 6 days.

[0095] Six days after DNA delivery by BaDGE®, cells were fixed with 4% paraformaldehyde for 20 minutes and counterstained with DAPI to label all cell nuclei. mCherry reporter fluorescence was imaged using a 561 nm DPSS laser and DAPI using a 790 nm Ti:Sapphire multiphoton laser. The results are shown in Figure 30.

[0096] FIG. 31 shows another example of an electrotransfer system utilized in in vitro cell therapy. The DNA delivery probe described above in a stainless steel concentric needle configuration (or Pt / Ir or other electrode materials) is ideal for high-throughput DNA electrotransfer to cells attached or suspended in a multi-well plate configuration. Pulse parameters such as capacitive discharge or traditional monopolar square wave pulses, or other pulse parameters or complex waveform profiles at constant current or voltage provide high-efficiency cell transfection using naked (plasmid) DNA, while minimizing well-to-well contamination / carry-over without requiring current return paths that are not efficient or convenient for rapid expansion between wells that are wired to the transfection wells or inserted separately, which is advantageous for (robotic) multi-well high throughput. Cells can be cultured in situ or extracted from the wells immediately after DNA electrotransfer.

[0097] DNA probes on individual needle electrodes can be delivered with different pulse / pulse train parameters, providing rapid optimization of DNA transfection parameters to achieve the most efficient DNA (or other molecule type) delivery to cells.

[0098] In an alternative embodiment, e.g., in a minimum viable commercial product (MVP) embodiment to allow for mass production with minimal time lag, it is envisioned to utilize a disposable needle array that can be connected to a syringe and an external charge delivery circuit. For example, a (sterile packaged) needle array may be provided with an external, wired electrical connector for connecting the needle array to a pulse delivery circuit. An example prototype of this embodiment is shown in Figures 28a-c.

[0099] In the fabrication of the needle array of this embodiment, a Parylene C coating may be used to cover the needles, shielding the electrode surface from this insulation. As the inner needle electrode is pushed into the hub, the top end enters the connector cone, but a continuity check is required as this may scrape off the insulation, and the outer needle is pushed past the inner needle and inserted into the larger diameter cone. The cone is configured within the needle hub, which in turn provides a standard connection to the syringe, so that the lumen of the inner needle electrode is in fluid communication with the syringe reservoir for fluid delivery of the DNA vaccine. Leads from both (cone) connectors may extend to external wires with connectors on the ends.

[0100] The pulse delivery circuit may be a known pulse delivery device (suitably programmed) or a simplified capacitive discharge unit incorporating a capacitive discharge circuit and a charging circuit dedicated to the required charge delivery. As described above, the capacitive discharge circuit and charging unit of the present electrotransfer system embodiment are effectively arranged in one unit that can be wired to the disposable needle array. The capacitive discharge pulse may be initiated manually by the clinician, for example, using a foot-activated manual switch. In this embodiment, the capacitor is in the charging box, allowing the charging circuit to be reused. This is an advantage of requiring less charging for circuits manufactured for large-scale supply of electrotransfer DNA vaccine delivery than is required for the disposable electrotransfer module described above. This is also advantageous for capacitor suppliers, as it allows flexible options for manufacturing scale-up (e.g., the manufacturing model of the disposable BaDGE inoculator is up to 1 million per month (2 per second), but suppliers can choose to order in the order of about 100,000). For example, in one embodiment, the concentric needle array may be robotically manufactured with the concentric insulated needles inserted into the hub under high-throughput manufacturing conditions. The hub may be configured to connect to an off-the-shelf syringe (i.e., a Luer Lock syringe). The hub may be formed with two concentric metal (i.e., stainless steel) contact cones with insulation between them (i.e., plastic), arranged so that each cone provides an electrical connection between one of the needle electrodes and a lead for connection to an external charge delivery circuit. The needles may be coated with an electrically insulating material (e.g., coated with a parylene coating) (or partially coated, leaving the electrode tip intact). The ends that enter the hub may then be inserted by the robot armature, with the first (smaller, inner) needle passing through the opening in the first contact cone, past its distal end, into the hub and contacting the smaller second contact cone nested within the first cone. The press fit scrapes the edge of the insulation (e.g., 25 μm) to make contact, which can be verified by continuity testing from the armature to the lead soldered to the connector.A second needle is then inserted into the hub beyond the smaller, inner needle, but due to its larger diameter it makes contact with the larger diameter contact cone to form an electrical connection. The needles may be secured in place using heat or other mechanisms (i.e., adhesive or pressure).

[0101] The needle array units are sterile packaged and disposable, including the wires and connectors, and the controller (e.g., two per clinic) allows for tens of thousands of cycles. Thus, the components of the needle electrode array require a very large manufacturing scale-up just to meet the emergency demands of the pandemic. This may allow for production from readily available sources of raw materials. The needles used in the concentric needle electrode array may be of standard gauge, readily available from multiple suppliers.

[0102] It should be appreciated that this naked DNA delivery platform technology presents a significant advantage in the context of the unprecedented demand for mass vaccination resulting from the spread of COVID-19. Any solution requires a rapid path for large-scale deployment. DNA vaccines work by producing antibodies without directly injecting actual viral protein fragments. Last year, a DNA vaccine designed as a treatment to prevent MERS coronavirus infection was confirmed to produce clinically significant levels of antibodies in a Phase 1 clinical trial. The COVID-19 virus is structurally similar to MERS, and clinical trials adapting the MERS vaccine to target COVID-19 are currently underway in the United States and are expected to perform similarly 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 demonstrates efficacy, the DNA sequence will be publicly available for anyone to manufacture. One clear advantage of these plasmid DNA vaccines over serum vaccines is that the DNA chemical sequence can be electronically transmitted and manufactured anywhere. Being naked DNA, COVID-19 DNA vaccines require an electroporation delivery system for administration (using a brief electrical pulse to cause gene transfection). However, current commercial delivery units are expensive and complex, making it difficult to scale up for mass production and the low costs required for mass vaccination in this pandemic context.

[0103] The applicant's single pulse electroporation technology designed for DNA vaccine delivery described herein has the advantages of being more efficient than current clinical electroporators, offering lower manufacturing costs, using less DNA (allowing for more doses of DNA vaccine), and being painless. Critically, this technology is scalable for mass production with minimal components, facilitating rapid deployment. Once a DNA vaccine for COVID-19 is identified and produced, the electrotransfer technology disclosed herein can be utilized to address the delivery needs.

[0104] Advantages of the above-described system embodiments include the following. 1. Enabling disposable DNA gene electrotransfer devices 2. DNA gene delivery system suitable for low-cost mass production 3. Charge delivery in an intrinsically safe setting 4. Standalone charging station suitable for field operation 5. Not only is it a completely innovative technology for DNA vaccine applications, but it can also be applied more broadly to realize various DNA therapeutic applications. 6. Decoupling the DNA electrotransfer process from the power source, resulting in low-cost technology immediately suitable for development of medical device / drug combinations for tissue targeting of DNA therapeutics (e.g., DNA vaccines, oncological targeting of solid tumors (melanoma, prostate cancer), nerve and muscle repair (neuroregeneration), neuromodulation of targeted brain regions), and field applications for trauma treatment.

[0105] Those skilled in the art will recognize that many modifications can be made to the invention without departing from its spirit and scope.

[0106] In the appended claims and in the above description of the invention, unless the context requires otherwise by express language or necessary implication, the terms "comprises" or variations such as "comprising" are used in their inclusive sense, i.e., to specify the presence of stated features but not to exclude the presence or addition of further features in various embodiments of the invention.

[0107] Where other documents are referred to in this specification, it is understood that the reference is not an admission that those documents form part of the general public knowledge in the art in Australia or anywhere else.

Claims

1. 1. An electroinjection system, the system including at least one probe; Each probe is A probe body; a needle electrode array extending from the probe body and configured as a needle for insertion into tissue to be treated; a capacitive charge storage configured to store an amount of charge and a capacitive discharge circuit coupled to the needle electrode array, the capacitive charge storage configured to store an amount of charge and a switch operable to cause a discharge of the amount of charge through the needle electrode array, the needle electrode array incorporating at least two electrodes, each electrode having an exposed surface area substantially surrounding the circumference of the needle and in direct contact with tissue into which the needle electrode array is inserted, the surface area having an insulating section between adjacent electrodes forming a continuous linear array structure, the exposed surface area of ​​each electrode being at a different distance from the tip of the needle electrode array, each electrode being connected to the capacitive discharge circuit for operation as either an anode or a cathode during discharge of the charge quantity, and the length of the electrodes and the length of the insulating section between adjacent electrodes being configured to create a target electric field shape in tissue adjacent the array during discharge of the charge quantity through the array, and the system comprising: a charging station having a DC power supply module, an output terminal connectable to a probe to make electrical contact with the capacitive discharge circuit, and a charging control circuit for controlling charging of the capacitive discharge circuit of the connected probe; Electrical introduction system.

2. The electrointroduction system of claim 1 , wherein the capacitive discharge circuit is housed in the probe body.

3. 3. The electroinduction system of claim 1 or claim 2, further comprising a switch actuator mounted to the probe body configured to enable actuation of a switch of the capacitive discharge circuit.

4. The electrointroduction system of any one of claims 1 to 3, wherein the charging station includes a probe platform that supports the probe during charging.

5. The electrotransfer system of any one of claims 1 to 4, wherein the electrodes are relatively elongated, the diameter of the electrodes being less than the length of the electrodes.

6. 6. The electrointroduction system of claim 5, wherein said electrode is 1 mm or less in length and said electrode has a diameter less than said length of said electrode.

7. 7. The electrotransfer system of claim 1, wherein capacitive discharge circuit parameters are matched to the resistivity parameters of the therapeutic solution for electrotransfer to minimize the total charge delivered and optimize the discharge time constant for the electrotransfer.

8. 8. The electrotransfer system of claim 7, wherein the capacitance of the capacitive discharge circuit is selected based on a predicted total resistivity and a target accumulated charge during discharge, the predicted total resistivity being based on the resistivity of the therapeutic solution.

9. 9. The electroinduction system of claim 7 or 8, wherein said matching achieves a discharge time constant between 20 μsec and 2 sec.

10. 10. The electroinduction system of claim 1, wherein the probe body further comprises a therapeutic substance reservoir, a lumen for holding a volume of fluid that passes through the needle and is in fluid communication with the therapeutic substance reservoir, and a therapeutic substance delivery actuator operative to force the fluid from the therapeutic substance reservoir, through the lumen, and out of the needle.

11. The electrotransfer system of claim 10 , wherein the therapeutic substance delivery actuator is further configured to actuate the switch.

12. 12. The electrotransfer system of claim 10 or claim 11, wherein the probe is individually packaged with a therapeutic solution stored within the reservoir.

13. 12. The electrotransfer system of claim 10 or claim 11, wherein the reservoir is filled with a therapeutic solution prior to charging of the capacitive discharge circuit.

14. 14. The electrointroduction system of claim 10, wherein the probe body is in two parts, a first part incorporating the reservoir and therapeutic substance delivery actuator provided by a syringe connectable to a second part including an electrointroduction module having a housing adapted to engage a syringe and support the needle electrode array protruding outwardly from the housing, the capacitive discharge circuitry is contained within the housing and connected to the needle electrode array, and a fluid communication path is formed through the housing between the reservoir of the syringe and the lumen of the needle electrode.

15. 15. The electrotransfer system of claim 14, wherein the housing of the electrotransfer module is a luer lock compatible for engaging a conventional luer syringe.

16. The needle electrode is a first needle that also acts as a first electrode; a first concentric insulator covering the first needle a first predetermined distance (L1) from the needle tip; a concentric second electrode covering the first concentric insulator a distance (L2) from a distal end of the first concentric insulator; and a second concentric insulator covering the second electrode a distance (L3) from the distal end of the second electrode, the first needle and the second concentric electrode being formed of a conductive material and electrically connected to the positive and negative terminals of the capacitive discharge circuit. The electrical introduction system of any one of claims 1 to 15.

17. 17. The electrointroduction system of claim 16, wherein the exposed length L1 of the first needle acts as a first electrode of a linear array and the exposed length L3 of the second concentric electrode acts as a second electrode of a linear array.

18. 20. The electrointroduction system of claim 17, wherein the length of each of the first and second electrodes and the exposed length of the first insulating portion therebetween determine a pattern of electric field gradient that is generated adjacent to the needle array when an electrical pulse is applied to drive one electrode as an anode and the other electrode as a cathode.

19. 1. A probe for an electrical introduction system, comprising: A probe body; a needle electrode array extending from the probe body and configured as a needle for insertion into tissue to be treated; a capacitive charge storage configured to store an amount of charge and a capacitive discharge circuit coupled to the needle electrode array, the capacitive charge storage configured to store an amount of charge and a switch operable to cause a discharge of the amount of charge through the needle electrode array, the needle electrode array incorporates at least two electrodes, each electrode having an exposed surface area substantially surrounding the circumference of the needle and in direct contact with the tissue into which the needle electrode array is inserted, the surface area having insulating sections between adjacent electrodes forming a continuous linear array structure, the exposed surface area of ​​each electrode being at a different distance from the tip of the needle electrode array, each electrode being connected to the capacitive discharge circuit for operation as either an anode or a cathode during discharge of the charge quantity; and a length of the electrodes and a length of the insulating section between adjacent electrodes configured to create a target electric field shape in tissue adjacent to the array during discharge of the charge quantity through the array. Probe for electrical introduction system.

20. The needle electrode is a first needle, the length of the needle proximate the needle tip providing a first electrode; a first concentric insulator covering the first needle a first predetermined distance (L1) from the needle tip; a conductive coating covering the first concentric insulator a distance (L2) from a distal end of the first concentric insulator and forming a second electrode; and a second concentric insulator covering the second electrode from a distal end of the second electrode toward a distance (L3), the first needle and the second concentric electrode being formed of a conductive material and electrically connected to positive and negative terminals of the capacitive discharge circuit.

20. A probe of the electrointroduction system of claim 19.

21. 21. The electrointroduction system probe of claim 20, wherein the length of each of the first and second electrodes and the exposed length of the first insulating portion therebetween determine a pattern of electric field gradient generated adjacent to the needle array when an electrical pulse is applied to drive one electrode as an anode and the other electrode as a cathode.

22. 22. The electroinduction system probe of any one of claims 19 to 21, further comprising a therapeutic substance reservoir, a lumen holding a volume of fluid that passes through the needle array and is in fluid communication with the therapeutic substance reservoir, and a therapeutic substance delivery actuator operative to force the fluid from the therapeutic substance reservoir, through the lumen, and out of the needles.

23. 23. The electrotransfer system probe of claim 22, wherein the therapeutic substance delivery actuator is further configured to actuate the switch.

24. 22. The probe of claim 19, further comprising a lumen extending through the needle array and configured to connect a syringe to form a fluid communication channel from a fluid reservoir in the syringe through the lumen, whereby the syringe provides a therapeutic substance reservoir to hold a volume of fluid, and further comprising a therapeutic substance delivery actuator operative to force the fluid from the therapeutic substance reservoir, through the lumen, and out of the needles, and further configured to actuate the switch.

25. A needle electrode array, a first needle that also acts as a first electrode; a first concentric insulator covering the first needle a first predetermined distance (L1) from the needle tip; a concentric second electrode covering the first concentric insulator a distance (L2) from a distal end of the first concentric insulator; and a second concentric insulator covering the second electrode a distance (L3) from the distal end of the second electrode, the first needle and the second concentric electrode being formed of a conductive material and electrically connected to positive and negative terminals of a pulse delivery circuit; the length of each of the first and second electrodes and the exposed length of a first insulating portion therebetween determine a pattern of an electric field gradient that is generated adjacent to the needle array when an electrical pulse is applied to drive one electrode as an anode and the other electrode as a cathode. Needle electrode array.

26. 26. The needle electrode of claim 25, wherein the exposed length L1 of the first needle acts as a first electrode of a linear array and the exposed length L3 of the second concentric electrode acts as a second electrode of a linear array.

27. The needle electrode according to any one of claims 25 to 26, wherein the needle tip is cut at an angle.

28. A needle electrode according to any one of claims 25 to 27, wherein the first needle has a hollow lumen through which a therapeutic solution can be delivered.

29. 30. The needle electrode of claim 28, wherein the therapeutic solution is delivered to tissue through an opening in the tip of the first needle.

30. 30. The needle electrode of claim 29, wherein the needle array includes an opening distal to a tip of the needle array, whereby a therapeutic solution is delivered to tissue adjacent to the needle array.

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