WO2021158685A1

WO2021158685A1 - Hemostatic combination therapy with low voltage electroporation

Info

Publication number: WO2021158685A1
Application number: PCT/US2021/016449
Authority: WO
Inventors: John F. RODRIGUEZ; Brandon D. PHUNG; James R. NITZKORSKI
Original assignee: Oncosec Medical Incorporated
Priority date: 2020-02-04
Filing date: 2021-02-03
Publication date: 2021-08-12

Abstract

Provided herein are systems, methods, and apparatus for electroporation, which may include an applicator; an endoscope, trocar or the like; a generator; and a drug delivery device. The applicator may include a control portion, an insertion tube connected to the control portion, an actuator engaged with the control portion, and a plurality of electrodes comprising a first electrode having a first tip and a second electrode having a second tip. Various treatment methods are also provided.

Description

HEMOSTATIC COMBINATION THERAPY WITH LOW VOLTAGE

ELECTROPORATION

CROSS REFERENCE TO RELATED APPLICATIONS [001] This application claims priority from and the benefit of US 62/970,071 filed February 4, 2020, which is incorporated herein by reference.

BACKGROUND

[002] Electrical fields may be used to create pores in cells through a process known as electroporation to increase the permeability of target cells and administer various localized treatments to a patient. There is a need for electroporation therapy in difficult to reach areas of the body, such as to treat tumors within the lungs, and there is a need to provide a large treatment area while still being able to fit the electroporation devices into these difficult to reach areas. There is also a need to administer a variety of treatment agents and combination therapies with a high degree of precision and minimal invasiveness. The inventors have identified that existing treatment systems and methods may be improved by increasing the efficacy of the treatment agents and combination therapies and reducing incidental harm to the patient tissues.

[003] Through applied effort, ingenuity, and innovation, many of these identified problems have been solved by developing solutions that are included in embodiments of the present invention, many examples of which are described in detail herein.

BRIEF SUMMARY

[004] Disclosed herein are electroporation systems, applicators, associated methods of treatment and use, and associated apparatus. In some embodiments, an applicator for electroporation may be provided. The applicator may include a control portion, an insertion tube connected to the control portion, an actuator engaged with the control portion, and a plurality of electrodes comprising a first electrode having a first tip and a second electrode having a second tip.

[005] In embodiments, a method of treating a lesion or tumor in a subject, the method comprising administering to the lesion or tumor an effective dose of a hemostatic agent, administering to the lesion or tumor an effective dose of at least one treatment agent, administering electroporation therapy to the lesion or tumor; and wherein administering the electroporation therapy comprises administering an electric pulse to the lesion or tumor using an electroporation system comprising an applicator comprising, a plurality of electrodes comprising a first electrode having a first tip and a second electrode having a second tip, and a generator electrically connected to the plurality of electrodes, wherein administering the electric pulse to the lesion or tumor comprises disposing the first electrode and the second electrode into or adjacent to the lesion or tumor, and delivering the electric pulse via the first electrode and the second electrode.

[006] In embodiments, the hemostatic agent is administered after the electroporation therapy. In embodiments, the hemostatic agent is administered after the effective dose of the at least one treatment agent. In embodiments, the electroporation therapy is administered between administering the hemostatic agent and administering the effective dose of the at least one treatment agent.

[007] In embodiments, the generator is configured to output low-voltage electric pulses. In embodiments, the electric pulses have a field strength of 700V/cm or less.

[008] In embodiments the electroporation therapy is reversible electroporation therapy. [009] In embodiments, the treatment agent is a plasmid coding for a cytokine, a checkpoint inhibitor, a plasmid encoding an immunomodulatory polypeptide wherein the immunomodulatory polypeptide comprises: a cytokine, a costimulatory molecule, a genetic adjuvant, an antigen, a genetic adjuvant-antigen fusion polypeptide, a chemokine, or an antigen binding polypeptide, or a combination thereof. In embodiments, the plasmid coding for a cytokine is a plasmid coding for IL-12. In embodiments, the plasmid comprises tavokinogene telseplasmid.

[0010] In embodiments, the applicator further comprises a drug delivery channel configured to deliver at least one of the at least one treatment agent and the hemostatic agent co-locally with the electroporation therapy. In embodiments, each of the at least one treatment agent and the hemostatic agent delivered co-locally with the electroporation therapy are delivered via the drug delivery channel of the applicator.

[0011] In embodiments, the plurality electrodes are configured to move between a retracted position and a deployed position. In embodiments, the distance between the first tip of the first electrode and the second tip of the second electrode is greater in the deployed position than in the retracted position.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS [0012] Having thus described embodiments of the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein:

[0013] FIG. 1 shows a block diagram of an electroporation system in accordance with some embodiments;

[0014] FIG. 2 shows a cross sectional view of a portion of an applicator in accordance with some embodiments;

[0015] FIG. 3 shows a generator and simplified applicator in accordance with some embodiments;

[0016] FIG. 4 shows an endoscope in accordance with some embodiments;

[0017] FIG. 5 shows a portion of an insertion tube and electrodes of an applicator in a retracted position in accordance with some embodiments;

[0018] FIG. 6 shows the portion of the insertion tube and electrodes of FIG. 5 in a deployed position;

[0019] FIG. 7 shows a portion of an insertion tube, electrodes, and bladder of an applicator in a retracted position in accordance with some embodiments;

[0020] FIG. 8 shows the portion of the insertion tube, electrodes, and bladder of FIG. 7 in a deployed position;

[0021] FIG. 9 shows a portion of an insertion tube and electrodes of an applicator in a retracted position in accordance with some embodiments;

[0022] FIG. 10 shows the portion of the insertion tube and electrodes of FIG. 9 in a deployed position;

[0023] FIG. 11 shows an electrode having a nitinol sleeve in accordance with some embodiments;

[0024] FIG. 12 shows a portion of an insertion tube and electrodes of an applicator in a retracted position in accordance with some embodiments;

[0025] FIG. 13 shows the portion of the insertion tube and electrodes of FIG. 12 in a deployed position;

[0026] FIG. 14 shows a portion of an insertion tube, carrier, and electrodes of an applicator in a retracted position in accordance with some embodiments;

[0027] FIG. 15 shows the portion of the insertion tube, carrier, and electrodes of FIG. 14 in a deployed position;

[0028] FIG. 16 shows a portion of an insertion tube, carrier, and electrodes of an applicator in a retracted position in accordance with some embodiments; [0029] FIG. 17 shows the portion of the insertion tube, carrier, and electrodes of FIG. 16 in a deployed position;

[0030] FIG. 18 shows a flow chart of an example method of treatment in accordance with some embodiments;

[0031] FIG. 19 shows a side view of an applicator in accordance with some embodiments; [0032] FIG. 20 shows a perspective view of an applicator with electrodes in a deployed position in accordance with some embodiments;

[0033] FIG. 21 shows a portion of an insertion tube and electrodes of an applicator in a retracted position in accordance with some embodiments;

[0034] FIG. 22 shows a side view of an applicator with electrodes in a deployed position in accordance with some embodiments;

[0035] FIG. 23 shows a partial view of a control portion and actuator of an applicator in accordance with some embodiments;

[0036] FIG. 24 shows a portion of an insertion tube and electrodes in a deployed position in accordance with some embodiments;

[0037] FIG. 25 shows a perspective view of an applicator with electrodes in a retracted position in accordance with some embodiments;

[0038] FIG. 26 shows a portion of an insertion tube and electrodes in a deployed position in accordance with some embodiments;

[0039] FIG. 27 shows a cross sectional, top view of an applicator in accordance with some embodiments;

[0040] FIG. 28 shows a side view of an applicator with electrodes in a deployed position in accordance with some embodiments;

[0041] FIG. 29 shows a perspective view of an insertion tube, carrier, and electrodes in accordance with some embodiments;

[0042] FIG. 30 shows a partial, cross-sectional view of an insertion tube, carrier, and electrodes in a deployed position in accordance with some embodiments;

[0043] FIG. 31 shows a perspective view of an applicator with electrodes in a deployed position in accordance with some embodiments;

[0044] FIG. 32 shows a perspective view of an applicator with electrodes in a retracted position in accordance with some embodiments;

[0045] FIG. 33 shows a partial, cross-sectional view of an insertion tube, a carrier, a pushing element, a wire, and an inner member in accordance with some embodiments; [0046] FIG. 34 shows a side, cross-sectional view of an applicator in accordance with some embodiments;

[0047] FIG. 35 shows a side view of an applicator with electrodes in a deployed position in accordance with some embodiments;

[0048] FIG. 36 shows a cross-sectional view of a wire, a pushing element, an insertion tube, and a hollow mandrel in accordance with some embodiments;

[0049] FIG. 37 shows a second actuator according to some embodiments;

[0050] FIG. 38 shows a cross-sectional view of a portion of an insertion tube, a carrier, an inner member, an electrode, a pushing element, and a wire in accordance with some embodiments;

[0051] FIG. 39 shows a partial perspective view of a control portion and actuator in accordance with some embodiments;

[0052] FIG. 40 shows a perspective view of an applicator with electrodes in a retracted position in accordance with some embodiments;

[0053] FIG. 41 shows a portion of an insertion tube and electrodes in a deployed position in accordance with some embodiments;

[0054] FIG. 42 shows a portion of an insertion tube and electrodes in a deployed position in accordance with some embodiments;

[0055] FIG. 43 shows a perspective view of an applicator with electrodes in a retracted position in accordance with some embodiments;

[0056] FIG. 44 shows a cable and connector in accordance with some embodiments;

[0057] FIG. 45 shows the cable and connector of FIG. 44;

[0058] FIG. 46 shows a cross-sectional view of the connector of FIG. 44 taken along line A- A;

[0059] FIG. 47 shows a perspective view of an applicator having electrodes in a retracted position in accordance with some embodiments;

[0060] FIG. 48 shows a zoomed perspective view of the applicator of FIG. 47;

[0061] FIG. 49 shows another zoomed perspective view of the applicator of FIG. 47;

[0062] FIG. 50 shows a perspective view of the distal end of the applicator of FIG. 47; [0063] FIG. 51 shows a cross-sectional view of the applicator of FIG. 47;

[0064] FIG. 52 shows another cross-sectional view of the applicator of FIG. 47;

[0065] FIG. 53 shows a cross-sectional view of a portion of the insertion tube, electrodes, and pushing element of the applicator of FIG. 47; [0066] FIG. 54 shows the perspective view of the applicator of FIG. 47 having electrodes in a deployed position in accordance with some embodiments;

[0067] FIG. 55 shows a zoomed side view of the applicator of FIG. 54;

[0068] FIG. 56 shows a perspective view of the distal end of the applicator of FIG. 54;

[0069] FIG. 57 shows a cross-sectional view of the applicator of FIG. 54;

[0070] FIG. 58 shows a cross-sectional view of the distal end of the applicator of FIG. 54; [0071] FIG. 59 shows a pushing element capable of carrying electrical pulses in accordance with some embodiments;

[0072] FIG. 60 shows a portion of an insertion tube, electrodes, and drug delivery tube of an applicator in a deployed position in accordance with some embodiments;

[0073] FIG. 61 shows a cross-sectional view of the insertion tube, electrodes, and drug delivery tube of the applicator of FIG. 60 in a deployed position in accordance with some embodiments;

[0074] FIG. 62 shows a portion of an insertion tube, electrodes, and drug delivery tube of an applicator in a deployed position in accordance with some embodiments;

[0075] FIG. 63 shows a cross-sectional view of the insertion tube, electrodes, and drug delivery tube of the applicator of FIG. 62 in a deployed position in accordance with some embodiments;

[0076] FIG. 64 shows a portion of an insertion tube, electrodes, and drug delivery tube of an applicator in a deployed position in accordance with some embodiments;

[0077] FIG. 65 shows a cross-sectional view of the insertion tube, electrodes, and drug delivery tube of the applicator of FIG. 64 in a deployed position in accordance with some embodiments;

[0078] FIG. 66 shows a portion of an insertion tube, carrier, inner member, electrodes, and drug delivery tube of an applicator in a deployed position in accordance with some embodiments;

[0079] FIG. 67 shows another flow chart of an example method of treatment in accordance with some embodiments;

[0080] FIG. 68 shows a yet another flow chart of an example method of treatment in accordance with some embodiments;

[0081] FIG. 69 shows an example applicator and endoscope extending into a stomach to access the pancreas in accordance with some embodiments;

[0082] FIG. 70 shows a cutaway view of the applicator, endoscope, stomach, and pancreas of FIG. 69; [0083] FIG. 71 shows a zoomed perspective view of the distal ends of the endoscope and applicator of FIG. 69;

[0084] FIG. 72 shows a zoomed perspective view of the distal ends of the endoscope and applicator of FIG. 69 piercing a stomach wall;

[0085] FIG. 73 shows another zoomed perspective view of the distal ends of the endoscope and applicator of FIG. 69 piercing a stomach wall;

[0086] FIG. 74 shows a zoomed perspective view of the distal ends of the endoscope and applicator of FIG. 69 having electrodes and a drug delivery channel in the deployed position piercing the pancreas;

[0087] FIG. 75 shows an example applicator and bronchoscope extending into the lungs to access a lesion in accordance with some embodiments;

[0088] FIG. 76 shows cutaway view of the applicator, bronchoscope, and lungs of FIG. 75; [0089] FIG. 77 shows a zoomed perspective view of the distal ends of the applicator and bronchoscope of FIG. 75;

[0090] FIG. 78 shows a zoomed perspective view of the distal ends of the bronchoscope and applicator of FIG. 75 having electrodes and a drug delivery channel in the deployed position piercing the lesion;

[0091] FIG. 79 shows experimental results of tumor volume vs time for five different trials; [0092] FIG. 80 shows a plot of transfection rates for high and low voltage RFP-Luc;

[0093] FIG. 81 shows expression of mIL-12p70 by electroporation into established B16-F 10 tumors;

[0094] FIG. 82 shows LacZ staining after electroporation of a Lax Z expressing plasmid in B16-F10 tumors;

[0095] FIG. 83 shows expression of trimeric CD40L by electroporation in B16-F10 tumors; [0096] FIG. 84 shows expression of trimeric CD80 by electroporation in B16-F10 tumors; [0097] FIG. 85 shows IT expression of sdAbs by electroporation in B16-F10 tumors; and [0098] FIG. 86 shows a perspective view of an applicator in accordance with some embodiments.

DETAILED DESCRIPTION

[0099] Some embodiments of the present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, various embodiments of the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like reference numerals refer to like elements throughout.

System Overview

[00100] Disclosed herein are various electroporation systems, apparatus, and methods. In some embodiments, the electroporation systems, apparatus, and methods disclosed herein may be used in connection with minimally-invasive procedures involving inserting portions of an applicator into a patient via a narrow opening and, in some embodiments, administering various therapies and treatment agents therethrough. The systems, apparatus, and method used herein may be used to deliver any treatment agent ( e.g ., nucleic acid-based therapies) and apply any electroporation therapy viscerally. In some embodiments, the electroporation systems, apparatus, and methods disclosed herein may be used in connection with an insertion device. In some example embodiments, the electroporation systems, apparatus, and methods disclosed herein may be used to deliver a combination therapy comprising electroporation therapy, a treatment agent, and a hemostatic agent. In some of the aforementioned embodiments, the electroporation therapy may include a low-voltage, reversible electroporation. In some of the aforementioned embodiments, the treatment agent may include a plasmid coding for IL-12.

[00101] As used herein, the term “insertion device” means any apparatus or structure capable of allowing a portion of an applicator to be inserted into a patient, for example via a cannula or other working channel. In some embodiments, the electroporation systems, apparatus, and methods disclosed herein may be used in connection with endoscopic devices and procedures to reach and treat remote tissues (e.g., visceral lesions, such as tumors) within a patient. In some embodiments, various types of endoscopic devices may be used along with the electroporation systems, apparatus, and methods disclosed herein depending on the particular location of the remote tissue, such as bronchoscopic devices, laparoscopic devices or other cannulated devices suitable for providing access to such remote tissues. Such endoscopic devices may be of any type, including for example either a flexible endoscopic instrument or a rigid endoscopic instrument (e.g, a trocar, such as for use in laparoscopic procedures), which may be selected based on the anticipated procedure and/or location of the remote tissue. In some embodiments, the electroporation systems, apparatus, and methods disclosed herein may be used to access lesions anywhere in or adjacent to the alimentary canal. In some embodiments, the electroporation systems, apparatus, and methods disclosed herein may be used to access lesions in the lungs. In some embodiments, the electroporation systems, apparatus, and methods disclosed herein may be used in connection with minimally invasive electroporation, one example being in connection with any such aforementioned endoscopic instrument.

[00102] As used herein, the term “reversible electroporation” may refer to electroporation therapy that is administered below a magnitude and duration that would cause permanent damage to all or substantially all of the target tissue. The effect of the magnitude and duration of therapy may be related, such that a higher magnitude therapy may be used with lower duration and vice versa, while remaining reversible electroporation. The low voltage electroporation described herein may be considered reversible electroporation. In some embodiments, reversible electroporation may include some minor cell damage, such as damage cells immediately proximate the electrodes.

[00103] In a variety of medical treatments, electroporation may be used to increase the permeability of cells by using electrical fields to create pores in biological cells without causing permanent damage ( e.g ., reversible electroporation). In some instances, the increased permeability of reversible electroporation may enable a contemporaneous treatment, such as drug administration or gene therapy, to be more effective because the treatment is better able to permeate the cells. During electroporation, a voltage may be applied across two or more electrodes to create an electric field therebetween. In some examples, the electrodes may be disposed on either side of, embedded within, or otherwise be positioned relative to, cell tissue that is then subjected to the electric field. The electric field creates the pores within the cell tissue which then allow the cell to be permeated by one or more treatment agents. Performance of electroporation with a low voltage generator as described herein is particularly advantageous in satisfying the conditions necessary to achieve reversible electroporation. Although tissue around the target site may have varying electric field thresholds, the application of low voltage is intended to, even among the extant range of threshold values, apply a voltage amount that is below such a threshold in order to minimize or avoid damage to the tissue during the electroporation procedure.

[00104] With reference to FIGS. 1-3, an example electroporation system 10 is shown. In the embodiment as illustrated, the system 10 includes a generator 12 for generating and delivering electrical signals to at least two electrodes 100 and an applicator 14 including the at least two electrodes. The applicators 14 described herein using reference numeral 14 may be generally representative of each of the embodiments of specific applicators 14, 60, 70,

110, 1000 described herein as if each applicator were discussed individually. The electrodes 100 described herein using reference numeral 100 may be generally representative of each of the embodiments of electrodes 100, 200, 300, 400, 500, 600, 700, 800 described herein as if each electrode were discussed individually. To the extent there are differences among the various embodiments of the applicators and electrodes in the various embodiments of the present disclosure, such differences are described as applicable. In some embodiments, the electrodes 100 may include two or more electrodes, which may each define a pointed tip at a distal end for piercing the tissue at the target site. In some embodiments, the electrodes 100 may include four or more electrodes. In some embodiments, the electrodes 100 may include six or more electrodes. The electrodes may be disposed about a central axis. In some embodiments, the tips of the electrodes are exposed while adjacent surfaces of the electrodes are insulated so that current passes through the tips only. In some embodiments, a region on the respective electrode away from the tip is exposed while surrounding surfaces are insulated and current is directed only through these exposed surfaces between the electrodes. The location of exposure may be close enough to the tip, and/or at the tip, so that the exposed portion of the electrode is outside of the insertion tube 15 of the applicator, described below, when the electrodes are in the deployed position. In some embodiments, as discussed in further detail below, the tips of the electrodes 100 may be closer together in a retracted position for insertion into the patient ( e.g ., via the working channel), and once in position, the electrodes may be deployed to a deployed position in which the tips of the electrodes are spread farther apart for administering electroporation to a larger treatment area. In some embodiments, the electrodes are included as part of an applicator with a predetermined spacing such that whether the electrodes are in the retracted or deployed position, the spacing remains constant. In one example, the spacing of the electrodes in such embodiments is about 4 mm. The electrodes may still be housed in a tube or other delivery structure in these embodiments. In yet another example, the applicator may only include a single electrode 100, while a second electrode can be constituted by, for instance, a distal -most portion of a housing tube or other portion of an applicator body or like structure. In such an example, the applicator would only have a single needle (which could thus be fixed or deployable) which need only be spaced a sufficient distance from the structure constituting the second electrode to be effective in providing a voltage to the desired tissue and to prevent arcing.

[00105] In some embodiments, the applicator 14 of the system 10 may be used to administer one or more treatment agents (e.g., a drug and/or plasmid). For example, the applicator may include an insertion tube 15 serving as a delivery path for the treatment agent(s). In some examples, and as described in greater detail elsewhere in the application, a designated drug delivery channel 18 may be included within the insertion tube 15 for administration of treatment agents ( e.g ., as shown in FIGS. 47-67). The drug delivery channel 18 may extend through the applicator 14 for co-localization of the electrodes and treatment agent(s). The drug delivery channel 18 may terminate at the electrodes 100 adjacent the electroporation site to administer the one or more treatment agents adjacent to or as close as possible to the cells being electroporated. In some examples, the drug delivery channel may terminate slightly proximal to the electrode tips. In still other examples, the delivery channel may also have a shape suitable for insertion into the tissue to be electroporated, such as a needle, such that the delivery channel extends at or distal to the electrode tips.

[00106] In some embodiments, the electroporation system 10 may further include a drug delivery device 16 for administering one or more treatment agents (e.g., a drug and/or plasmid) to the electroporation site. FIG. 1 illustrates some examples of how drug delivery device 16 may be positioned in the system, and in a larger context includes dashed arrows to indicate fluid flow paths and solid arrows to indicate electrical connections. With reference to FIG. 1, the drug delivery device 16 may define a syringe having a distal tube or needle for administering the treatment agent. In some embodiments, the drug delivery device 16 may include at least one reservoir, configured to receive the one or more treatment agents, and at least one pump configured to deliver the treatment agents to the electroporation site. In some embodiments, the drug delivery device 16 administers the one or more treatment agents directly to the target site while the applicator 14 is used to perform electroporation at the target site. In some embodiments, the drug delivery device 16 administers the one or more treatment agents to the applicator 14, which in turn, directly administers the treatment agent to the target site. In this manner, the applicator 14 is used for treatment agent administration and for performance of electroporation. In some examples, the treatment agent is delivered through a drug delivery channel 18 within the applicator 14.

[00107] In some embodiments, and as discussed elsewhere herein, the one or more treatment agents may be administered via a separate drug delivery applicator 19 (e.g, a long distal needle, a conduit passing through an endoscopic instrument, or the like) instead of being administered through the applicator 14 itself, as shown in FIG. 1. Still further, the drug delivery applicator 19 may deliver at least one of the treatment agent(s) systemically rather than directly to the electroporation site. The separate drug delivery applicator 19 (or other administration device) may be used sequentially with the electroporation applicator 14 to administer the one or more treatment agents to the electroporation site. In some examples, the drug delivery applicator 19 alone is used to administer the one or more treatment agents.

In other examples, the drug delivery device 16 is used in conjunction with the drug delivery applicator 19 to administer the one or more treatment agents, as shown in FIG. 1. In these examples, the applicator 14 separately performs electroporation.

Example System Architecture

[00108] In some embodiments, the generator 12 and applicator 14 are controlled by one or more controllers 24, which includes at least a processor 30 and memory 36. In some embodiments, the controller 24 may be disposed in the generator 12 and may control the applicator 14 therewith. In embodiments in which the drug delivery device 16 requires electronic control, one or more controllers may operate the drug delivery device, and in embodiments in which the drug delivery device 16 has no electronic control, the drug delivery device may be manually operated ( e.g ., by depressing a syringe). In some embodiments, electronic control may be in the form of robotics, described elsewhere herein.

In some embodiments, each of the generator 12, applicator 14, and drug delivery device 16 may have its own controller. In some embodiments, one or more of the controllers may be controlled by another controller (e.g., in a master-slave relationship). In some embodiments, each controller 24 may be embodied as a single device or as a distributed processing system, some or all of which may be remote from the respective device that it controls. Examples of an electroporation system and corresponding electronic control methods, signals, and apparatus; treatment agents; and therapies are described in U.S. Patent Nos. 7,412,284 and 9,020,605 and International Application No. W02016/161201, each of which is incorporated by reference herein in its entirety.

[00109] With continued reference to FIG. 1, in some embodiments, the generator 12 may be a low-voltage generator for administering the electroporation therapy and/or performing electrochemical impedance spectroscopy (EIS) as described herein. In some embodiments, the generator 12 may include pulse circuitry 33 configured to generate waveforms for excitation of the electrodes during electroporation. In some embodiments, the generator 12 is configured solely to perform electroporation therapy. In some embodiments, the generator 12 may include sensing circuitry 31 configured to receive signals from the electrodes 100 (e.g, EIS signals described herein) and facilitate analysis of the properties of the target tissue. As described herein, in some embodiments, the generator 12 may control the pulses output from the pulse circuitry 33 in response to the sensed parameters of the target tissue and the treatment agent determined by the sensing circuitry 31. In embodiments of the system with sensing circuitry 31, the circuitry may be toggled to activate or deactivate control of the parameters of the electroporation therapy based on the analysis of the EIS signals received by the system. In this manner, if the circuitry is toggled off, the therapy will maintain a preset voltage and pulse duration (or a predetermined voltage and pulse duration pattern) irrespective of any variation in impedance reported to the system by the sensors. [00110] With reference to FIG. 3, an example generator 12 and simplified applicator 14 are shown. The generator may generate electrical signals to electroporate the target tissues. The generator 12 may regulate the properties of the electrical signals (e.g, voltage, amplitude, frequency, duration, and the like) to cause reversible electroporation of the tissues without damaging the target tissues. In some embodiments, the generator 12 may include a foot pedal 58 for allowing a user to actuate and operate the generator and electroporation.

The foot pedal 58 may be connected to the generator via a wired connection or via a low energy wireless connection, such as Bluetooth®. Where a wireless connection is used, each of the foot pedal 58 and the generator may include sensors to send and receive signals communicating changes in the status of the foot pedal 58. Operation of the generator may be aided or fully controlled by a robotic system. For example, a robotic arm may be configured to control the generator to achieve desired electrical parameters for electroporation.

Examples of an electroporation system and corresponding electronic control methods, signals, and apparatus are described in U.S. Patent Nos. 7,412,284 and 9,020,605 and International Application No. W02016/161201, each of which is incorporated by reference herein in its entirety.

Example Electroporation Applicator

[00111] In some embodiments, the electroporation system 10 may be operable for use with access instrumentation, such as an endoscope or the like. Endoscopy involves inserting an endoscope into a cavity of the patient and administering at least some of the treatment locally using the endoscope ( e.g ., endoscope 52 shown in FIG. 4). Endoscopes may be rigid e.g ., a trocar) or flexible, and may include imaging, illumination, or operative features to assist the surgeon with the endoscopy. One example of an endoscope that may be incorporated into the electroporation system 10 is described in U.S. Pat. No. 6,181,964, hereby incorporated by reference herein in its entirety. With reference to FIG. 4, in some embodiments, endoscopes 52 also include a working channel 54 that extends from an upper or proximal end of the endoscope (e.g., a control section that is actuated by the user) to a distal end 56 of the endoscope through which one or more instruments, such as applicator 14, may be inserted to conduct the endoscopic procedure. In some instances, a flexible endoscope may have a narrower working channel than a rigid endoscope. As is known in the art, a flexible endoscope is typically used for procedures where the access pathway is via a conduit, such as in an esophageal approach to reach the lungs, while a rigid endoscope is typically used for procedures where the access pathway is a “line of sight” into the patient and to the particular tissue, such as is used in many abdominal procedures.

[00112] Endoscopic electroporation may involve inserting at least a portion of an applicator ( e.g ., the insertion tube 15 of the applicator 60 shown in FIG. 2; the insertion tube 15 of the applicator 70 shown in FIG. 19; or the insertion tube 15 of the applicator 110 shown in FIG. 47), with the electrodes (e.g., electrodes 100) at a distal end of the applicator, through the working channel of the endoscope to apply an electric field to the tissue adjacent to the distal end of the endoscope. In some examples, the slidable connection holding the applicator and the endoscope together may be controllable such that once the endoscope is advanced to a location in the body approaching the target site for the electroporation therapy, the applicator may be controllably advanced relative to the endoscope so that a distal end of the applicator reaches the target site while the endoscope remains at a distance relative to the target site. As discussed elsewhere herein, embodiments of the applicator may be mechanically steerable such that the tip may be steered to the target site via controls at or proximate the handle of the applicator. The control mechanism may be established based on direct visualization (e.g., a camera associated with the endoscope), surgical navigation, manual guidance based on the expected friction between the applicator surface and the interior surface of the endoscope, or other parameters as may be applicable for the particular structures included in the system. This controllable advancement of the applicator relative to the endoscope is of particular advantage where access to the target site involves passage through an internal vessel that is small in diameter. In such circumstances, the smaller diameter of the applicator relative to the endoscope allows the applicator to be advanced independently at lesser risk to the patient. This circumstance may arise, for example, where a tumor to be treated is in the cerebrum and intra-cranial blood vessels must be traversed to reach the tumor.

[00113] The electroporation system 10 can be used in any endoscopic access approach desired to fulfill its use and purpose. For example, in some embodiments, the electroporation system 10 may be used with an Olympus® EBUS Bronchoscope for performing bronchoscopy. In some embodiments, a flexible laparoscopic instrument may be used with the insertion tube of the applicator disposed therein. Further, in some embodiments, the applicator may be inserted directly into a keyhole opening in the patient ( e.g ., with the laparoscopic device shown in FIG. 86). In this arrangement, the keyhole opening in the body of the patient operates as the working channel during the electroporation procedure. Thus, in some examples, the system may include an applicator with an insertion end that is configured to be advanced to the target site unenclosed by an insertion device. In some examples, the properties and structure of the insertion tube may be modified to accommodate use of the applicator as a standalone access element in the procedure. In the aforementioned examples, the system is complete without an endoscope, though it may be used with any type of endoscopic instrument desired. Further, in some examples of the aforementioned systems, applicator 14, 60, 70, 110, 1000 may be the applicator of the system.

[00114] In some examples, the electroporation system 10 may include an integral, “all- in-one” system having any combination of one or more of an endoscope, drug delivery channel or applicator, electroporation applicator, steering system, vision system, and/or imaging system (e.g., ultrasound). Embodiments of each of the foregoing components may include those discussed elsewhere herein. In such embodiments, the applicator (e.g, including electrodes and/or a drug delivery channel) may be any of the applicators 14, 60, 70, 110, 1000 disclosed herein. In some embodiments, the applicator may be a retractable portion of the all-in-one system.

[00115] Turning now to the structure of the applicator itself, with reference to FIG. 2, an example applicator 60 is shown having an insertion tube 15, an actuator 42, and a control portion 48. The insertion tube 15 may have a diameter less than an internal diameter of the working channel of an endoscope (e.g, working channel 54 of endoscope 52 shown in FIG.

4) so that the insertion tube may be inserted into the working channel and may extend from the control portion 48 outside the endoscope at the external end (e.g, the end outside the patient) to the endoscopic site within the patient at the distal end of the endoscope. The insertion tube 15 may be longer than the working channel of the endoscope. The insertion tube 15 may also include one or more channels extending therethrough to allow the various components described herein to extend into the patient for treatment. For example, the actuator 42 may be movably engaged with at least a portion of the control portion 48 and may extend through the insertion tube 15 to interact with the electrodes to allow a user to apply a force from the trigger 44 to deploy the electrodes at the distal end of the insertion tube 15 as described herein. In the embodiment depicted in FIG. 2, the actuator 42 includes a trigger 44 pivotally attached to the control portion 48 and a pushing element 46 connecting the trigger 44 to the electrodes such that pushing element 46 moves axially along the insertion tube 15, to move the electrodes, when the trigger is actuated.

[00116] In some examples, the electrodes are biased so that when no force is applied to the trigger 44, the electrodes are in a retracted position. In some examples, the trigger 44 must be held to maintain deployment of the electrodes such that anytime the trigger is released, the electrodes return to their retracted state. In some examples, the actuator may be modified to include a lock to hold the trigger in the actuated position or to include a slow release so that after the force applied to the trigger has ceased, the retraction of the electrodes is delayed and/or controlled. It is contemplated that these principles may be applied to other actuators as well, both those requiring physical movement and others that operate solely by control of an electrical connection. In some examples, each of the control portion 48, insertion tube 15 and actuator 42 are separate elements. In other examples, two or more of the control portion, insertion tube and actuator are integral with one another.

[00117] With reference to FIG. 19, another example applicator 70 is shown having an insertion tube 15, an actuator 74, and a control portion 72. The insertion tube 15 may have a diameter less than an internal diameter of the working channel of an endoscope ( e.g ., working channel 54 of endoscope 52 shown in FIG. 4) so that the insertion tube may be inserted into the working channel of the endoscope and may extend from the control portion 72 outside the endoscope at the external end (e.g., the end outside the patient) to the endoscopic site within the patient at the distal end of the endoscope. The insertion tube 15 may be longer than the working channel of the endoscope. Further, at least a portion of the insertion tube 15 may be flexible to, for example, allow for passage through a flexible endoscope already positioned through the tortuous pathway from the nose or mouth to the lungs, or may be rigid such that it is more suitable for passage through a rigid cannula, or further, for passage into the body of a patient without the need for an access instrument of any kind, or of course, for use with a rigid endoscope. These configurations of insertion tube 15 and access instrument are examples only as, of course, a configuration with a flexible insertion tube 15 could be used with a rigid cannula, such as a rigid endoscope. The insertion tube 15 may also include one or more channels extending therethrough to allow the various components described herein to extend into the patient for treatment. For example, the actuator 74 may be movably engaged with at least a portion of the control portion 72 and may extend through the insertion tube 15 to allow a user to apply a manual force from the control portion 72 (for example, via a switch 80) to deploy the electrodes at the distal end of the insertion tube 15 as described herein. The control portion 72 may include a body 90 and at least one end cap 88, which may support the insertion tube 15 and/or the cables 76 therein. In the embodiment depicted in FIGS. 19, 27, and 34, the actuator 74 includes a thumb switch 80 that is slidingly attached to the control portion 72 and engaged with a hollow mandrel 86 via a connector 84. With reference to FIG. 36, the mandrel 86 may be attached to a pushing element 92 ( e.g ., by crimping), such that when the actuator 74 is slid forward on the control portion 72 by a user sliding switch 80, the switch 80 pushes the hollow mandrel 86 axially forward, which drives the pushing element 92 axially forward to extend the electrodes 100 from the insertion tube 15 (e.g., either directly or by driving an electrode carrier 206, 602, 802 or other intermediate component, such as a balloon 302). Such a manual actuation mechanism for electrode deployment may be any structure desired other than the thumb switch 80 illustrated, for example, switch 80 could be a thumb wheel, a push button, a trigger mechanism, or the like.

[00118] With reference to FIG. 47, yet another example applicator 110 is shown having an insertion tube 15, an actuator 112, and a control portion 114. In some examples, the insertion tube 15 may have a diameter less than an internal diameter of the working channel of a cannulated access instrument, such as an endoscope (e.g, working channel 54 of endoscope 52 shown in FIG. 4), so that the insertion tube may be inserted into the working channel and may extend from the control portion 114 to a position outside the endoscope at the external end (e.g, the end outside the patient) to the endoscopic site within the patient at the distal end of the endoscope. The insertion tube 15 may be longer than the working channel of the endoscope. The insertion tube 15 may also include one or more channels extending therethrough to allow the various components described herein to extend into the patient for treatment. For example, the actuator 112 may be movably engaged with at least a portion of the control portion 114 and a portion of the actuator may extend into the insertion tube 15 to allow a user to apply a force from a switch 116 to deploy the electrodes at the distal end 118 of the insertion tube 15 as described herein. The control portion 114 may include a body 120 and at least one end cap 122, which may support the insertion tube 15 therein. In the embodiment depicted in FIGS. 47, 51, 52, and 57, the actuator 112 includes a thumb switch 116 that is slidingly attached to the control portion 114 and engaged with a hollow mandrel 124 of the actuator via a connector 126 (e.g, a lure lock). The mandrel 124 may be attached to a pushing element 128 (e.g, by crimping as shown in the embodiment of FIG. 36), such that when the actuator 112 is slid forward on the control portion 114 by a user sliding switch 116, the switch 116 pushes the hollow mandrel 124 axially forward, which drives the pushing element 128 axially forward to extend the electrodes 100 from the insertion tube 15 (e.g., either directly or by driving an electrode carrier 206, 602, 802 or other intermediate component, such as a balloon 302). In this manner, the actuator 112, including the switch 116, mandrel 124, and pushing element 128, may extend at least partially into the insertion tube 15 to drive the electrodes ( e.g . electrodes 100).

[00119] The applicator 14, 110 may further include the actuator 42, 74 structure, second actuator 94 (FIG. 35), described in greater detail elsewhere in the present disclosure, secondary button 82, and/or any of the other features from the applicators 14, 60, 70, 1000 described herein as if each individual feature had been described with respect to each embodiment, and such features may operate in accordance with their intended purpose in such combined embodiments. Similarly, in some embodiments, the features of any one applicator 14, 60, 70, 110, 1000 may be included in one of the other applicators.

[00120] With reference to FIGS. 49, 50, and 56, the applicator 110 may define a piercing tip 130 at the distal end 118 of the insertion tube 15. The piercing tip 130 may define a generally needle-shaped projection having a pointed end 132 and a hollow core through which the electrodes (e.g., electrodes 100) and/or drug delivery channel 18 may pass. The piercing tip 130 may be configured to puncture body tissue to reach a target site before deploying the electrodes (e.g, electrodes 100) and/or treatment agent. For example, the piercing tip 130 may be used to pierce a patient’s stomach liner to reach nearby organs such as the pancreas or liver. In some embodiments, the distal end 118 may comprise a flat, non piercing tip according to other embodiments discussed herein, such as is illustrated in FIGS. 5 and 6.

[00121] With reference to FIGS. 53, 58, 59, in some embodiments, the pushing element 128 may comprise a portion of the wiring for the electrodes. The generator (e.g, generator 12 shown in FIG. 1) may supply electrical impulses via a cable that enters the body 120 of the control portion 114 via a cable opening 134, as shown in FIGS. 51 and 57. With reference to FIG. 51, the cable 136 may pass through the cable opening 134 and connect to the mandrel or one or more wires therein (e.g, wires 17 shown in FIG. 36). The wires may transmit electrical impulses to the pushing element 128 from the cable 136, and to the electrodes 500 from the pushing element 128, as shown in FIG. 53.

[00122] With reference to the embodiment illustrated in FIG. 59, the pushing element 128 may comprise two coiled and electrically isolated wires 138, 140 that carry the impulses directed to two respective electrodes (e.g, the electrodes 100 discussed herein). The coiled wires 138, 140 may be insulated, for example, with an insulating casing (e.g, made of polyethylene, PVC, rubber-like polymers, etc.) and may have conductive cores passing therethrough. The coiled wires 138, 140 may be insulated so that the respective opposing signals of the electrodes ( e.g ., positive and negative electrical contacts) do not short. The pushing element 128 and mandrel 124 may define a central cavity 142 through which a drug delivery channel (e.g., drug delivery channel 18), or additional treatment-related device may pass. The ends of the coiled wires 138, 140 closest to the control portion 114 of the applicator may electrically connect to corresponding electrical wires (e.g, wires of the cable 136). These corresponding electrical wires of the cable 136 may run from the coiled wires 138, 140, along the mandrel 124 (e.g, floating outside of the mandrel), and out the applicator via cable opening 134.

[00123] Turning to FIGS. 1, 51-53 and 56-58, the applicator 110 may include a drug delivery channel 18 configured to direct fluid from a drug delivery device 16 (shown in FIG. 1) to a target site (e.g, a tumor or lesion) in the patient. The drug delivery device 16 (shown in FIG. 1) may couple to a shroud 144 of the applicator 110 (e.g, via a threaded connection 146), which shroud 144 may engage a second distal end 148 of the drug delivery channel 18. In an alternative configuration of the system, the treatment agent may be supplied directly into the drug delivery channel 18 via the second distal end 148. The drug delivery channel 18 may extend from the second distal end 148 at the shroud 144 to a first distal end 164 through which the one or more treatment agents may be delivered. The drug delivery channel 18 may be coupled to the actuator 112 at the connector 126, mandrel 124, and/or pushing element 128, and the drug delivery channel 18 may travel axially with the actuator 112 relative to the insertion tube 15. In some embodiment the drug delivery channel 18 may be bonded to the pushing element 128. In some embodiments, for example as shown in FIGS. 51-53 and 56- 58, the shroud 144 may be attached to and travel with the drug delivery channel 18. In some embodiments, the drug delivery channel 18 may be disposed in the central cavity 142 of the mandrel and pushing element 128. The drug delivery channel 18 may include a delivery channel 166 extending from the first distal end 164 to the second distal end 148 through which the one or more treatment agents may be delivered from the shroud 144 to the treatment site, as shown in FIGS. 52-53. The first distal end 164 of the drug delivery channel 18 may be pointed to pierce the tissue at the target site, or alternatively may have a blunt end for atraumatic delivery to the tissue at the target site. The drug delivery tube 18 may be flexible such that the tube can extend from the control portion 114 down into the target tissue in any direction desired.

[00124] In some embodiments, the drug delivery channel may have a non-circular cross-sectional shape. For instance, the shape may be polygonal, rectangular, oblong, elliptical, and so on. In some embodiments, the delivery channel 18 may be positioned on a periphery of the path through the insertion tube 15. In some examples, the delivery channel 18 may be positioned outside of a path of the electrodes. In some examples, the delivery channel 18 abuts an inner wall of the insertion tube 18. In some examples, the delivery channel 18 is formed with the inner wall of the insertion tube 18 and includes a further tube passing therethrough to advance out of the insertion tube for drug delivery during performance of the method. In some embodiments, the drug delivery channel 18 may be a hypotube.

[00125] In some embodiments, the drug delivery channel 18 may be made of a non- conductive material. In some embodiments, the drug delivery channel 18 may be made of a ceramic material. In some embodiments, the drug delivery channel 18 may be made of stainless steel. In a conductive embodiment ( e.g ., stainless steel), the distal end of the drug delivery channel 18, adjacent to the electrodes, may be coated in a non-conductive material (e.g., non-conductive ceramic). In some embodiments, the drug delivery channel 18 may be made of plastic. In some examples, the drug delivery channel may define a diameter of about 0.025 inches. The drug delivery channel 18 is advantageous in that it provides a protected structure within the applicator to deliver a treatment agent. Thus, the electrodes for electroporation and the treatment agent may all be safely carried within one structure, simplifying the surgical procedure.

[00126] With reference to FIGS. 53, 56, and 58, the electrodes 500 (e.g, any of the electrodes 100 discussed herein) and the drug delivery channel 18 may both be actuated simultaneously by the actuator 112. In some embodiments, the electrodes 500 (e.g, any of the electrodes 100, 200, 300, 400, 600, 700, 800 discussed herein) and the drug delivery channel 18 may move as a single unit. In some examples, the electrodes and the drug delivery channel move as a single unit where the electrodes are fixed relative to the drug delivery channel 18. In other examples, drug delivery channel may be movable independent of the electrodes and the applicator may include separate actuation mechanisms accessible to or otherwise controllable by a user for each of the drug delivery channel and the electrodes (and similarly, the electrodes can be actuated collectively and simultaneously or actuated individually by separate actuation actions). In this manner, the applicator may be configured so that deployment of the drug delivery channel may occur independently from deployment of the electrodes, such that the user can decide to actuate both simultaneously or sequentially. The first distal end 164 of the drug delivery channel 18 may be offset from the tips 501 of the electrodes, such that, given a flat planar target site, the electrodes pierce the target site before the drug delivery channel 18. In other examples, the first distal end 164 of the drug delivery channel 18 may be close to the tips 501 of the electrodes 500. In some embodiments, the first distal end 164 of the drug delivery channel 18 is positioned immediately inside an outward face of end cap 510 and remains stationary when the electrodes 500 are deployed.

[00127] In an alternative embodiment, the drug delivery channel may be integral with one of more of the electrodes, such that the electrode(s) is/are cannulated to provide a flow path for the treatment agent(s). In such an alternative configuration, the electrode(s) would be positioned in the target tissue first, and then the treatment agent(s) would be delivered to the tissue via the cannulated pathway through the electrode(s).

[00128] With reference to FIG. 61, the distal end 118 of the insertion tube 15 may include an alignment channel 168 and/or an end cap 510 comprising an alignment opening 512, in each instance for aligning and positioning the drug delivery channel 18 during operation. As shown in FIG. 53, the alignment channel 168 may engage the drug delivery channel 18 throughout its full range of travel to prevent misalignment. Similarly, the alignment channel 168 may have a length representing only a fraction of the insertion tube 15 or it may extend over a significant majority of the length. In some embodiments the alignment channel 168 and/or the end cap 510 may seal the end of the insertion tube 15 to prevent treatment agent or bodily fluid from entering the applicator 110.

[00129] Turning to FIG. 86, another example applicator 1000 is shown having a steerable insertion tube 1015. The applicator 1000 includes a steering mechanism to provide additional control of the applicator, particularly where applicator has a flexible body. For example, applicator 1000 may include one or more cables extending from the control portion 1014 to the distal end 1018 of the insertion tube 1015 to allow a user to steer the distal end 1018, the electrodes 500 and the delivery channel 18 to the target site within the patient. The insertion tube 1015 may include a flexible portion 1005 and a rigid portion 1010 to allow only the desired portions of the applicator to bend during steering ( e.g ., the cables may be offset from the axial center of the insertion tube such that applying a force to one or more cables bends the flexible portion 1005 in the direction of the cable(s)). The cables may be attached to the applicator at or near the control portion 1014 and between the rigid portion 1010 and the first distal end 1018 to bend the flexible portion 1005 upon application of a force to the cables from the control portion.

[00130] The applicator 1000 may include electrodes 500, a delivery channel 18, a control portion 1014, and an actuator 1012, which may include the features, structure, and operation of any of the electrodes, control portions, actuators, and delivery channels described herein, such as those of applicators 14, 60, 70, 110, and which may cooperate with the other components of an electroporation system disclosed herein including a generator and drug delivery device. The insertion tube 1015 and steerable components may be substituted for the insertion tubes 15 of any other embodiment discussed herein as if each individual feature had been described with respect to each embodiment, and such features may operate in accordance with their intended purpose in such combined embodiments. The insertion tube 1015 may comprise any of the dimensions or configurations of the insertion tubes 15 described herein with the addition of steerable components.

[00131] In some embodiments, the applicator 1000 may be a steerable laparoscopic applicator. As described herein, a steerable laparoscopic applicator can be used an alternative to an endoscopic applicator. For example, in some embodiments, the applicator 1000 may gain access to the interior anatomy via a trocar. The rigid portion 1010 of the insertion tube 1015 may allow for easy maneuverability, while the flexible portion 1005 enables steering via the cables. The applicator 1000 may have a knob that can be rotated which triggers movement of the tip of the applicator up and down to 120 degrees or less relative to the rigid portion 1010 in each direction. In some embodiments, the steerable tip may move 90 degrees or more in two or more directions ( e.g ., up and down).

[00132] In some embodiments, as discussed herein, the endoscope may be a trocar, flexible cannula, or other insertion instrument for insertion into a patient. In some embodiments, the applicator 14 may be a steerable device (e.g., the laparoscopic applicator 1000 shown in FIG. 86) that may be inserted into a patient without a separate insertion device. In some embodiments, the applicator may be radiopaque at its distal end.

[00133] The working channels of endoscopes used for various endoscopies (e.g, working channel 54 of endoscope 52 shown in FIG. 4) may have a limited diameter through which one or more portions of the electroporation system 10 may be inserted to reach the endoscopic site (e.g, adjacent distal end 56 of the endoscope 52 shown in FIG. 4). In embodiments that include an endoscope as part of the system, the portions of the electroporation system 10 that extend into the endoscope must fit within the working channel of the endoscope. For example, in some instances, such as with bronchoscopy, the working channel of the endoscope may be 2.2 mm or smaller in diameter, and the portions of the electroporation system 10 that enter the endoscope (e.g, the insertion tube 15) may be 2.0 mm or smaller in diameter. In some embodiments, the working channel of the endoscope may be 4 mm or smaller in diameter. In some embodiments, the insertion tube 15 is flexible to follow any curves or bends in the working channel of the endoscope. [00134] In some embodiments, the applicator 14 may include at least two electrodes 100 at the distal end of the insertion tube 15 ( e.g ., the end opposite the control portion 48, 72, 114) with one or more wires or other conductive material extending from the generator 12 (shown in FIG. 1) to the electrodes 100 via the insertion tube 15. In some embodiments (e.g., as described below with respect to FIGS. 47-67), the applicator 14 may also include other components, such as a drug delivery channel 18, that extend through the insertion tube 15 from a drug delivery device 16 (shown in FIG. 1) to the distal end of the insertion tube 15. In such embodiments, the wiring for the electrodes 100 and the drug delivery channel 18 may run parallel to each other down the insertion tube 15 from the control portion (e.g, control portion 48 shown in FIG. 2; control portion 72 shown in FIG. 19; or control portion 114 shown in FIG. 47) of the applicator 14 to the distal end. In some embodiments, applicator 60, 70, 110, 1000 may include the aforementioned features.

[00135] In some embodiments, the applicator 14 may include at least two electrodes 100 that extend through the insertion tube 15 to the distal end, and a separate drug delivery applicator 19 may deliver a plasmid, drug, and/or other treatment agent to the electroporation site. The drug delivery applicator 19 may administer the one or more treatment agents sequentially with the electroporation or concurrently through different channels or vectors.

In some embodiments, applicator 60, 70, 110, 1000 may include the aforementioned features. [00136] For example, in a system with an endoscope, once the endoscope is in position within the patient, the drug delivery applicator 19 may first be inserted into the endoscope until a distal end of the drug delivery applicator 19 reaches the target electroporation site (e.g, a tumor or other visceral lesion) at or adjacent to the distal end of the endoscope, after which the treatment agent(s) may be administered. The drug delivery applicator 19 may then be removed and replaced in the endoscope by the applicator 14 for electroporation, and the target electroporation site may be electroporated to facilitate permeation of the treatment agent(s) into the cells.

[00137] In some embodiments, one or more treatment agents may be administered through other means instead of or in addition to administering treatment agent(s) via the endoscope or drug delivery applicator 19. For example, one or more treatment agents may be administered via intramuscular (IM), intrathecal (IT), or intravenous (IV) injections before, during, or after electroporation.

[00138] With reference to FIGS. 44-46, a cable 76 and corresponding connector 78 are shown for connecting an applicator 14, 60, 70, 110, 1000 to a generator 12. [00139] In some embodiments, an applicator may include an actuator that remains physically stationary when actuated. For example, the actuator may be a button on a touchscreen display that is operable to control deployment of the electrodes within the insertion tube. The touchscreen may include a sensor (e.g, a pressure, capacitive touch, and/or gesture sensors) to detect contact with the screen and thereby control whether a circuit linked to a control element in the applicator causes the control element to move axially in response to opening and closing of the circuit. The element may be physically associated with the electrodes so that axial movement of the control element occurs with axial movement of the electrodes. In some examples, the circuit may be configured to cause the electrodes to move directly in response to opening and closing of the circuit. In some embodiments, actuation of the applicator may occur on a remote device linked to the applicator via a wireless connection. In this arrangement, a signal from the actuator is received in the applicator to control movement of the electrodes. In some examples, a drug delivery channel axially fixed relative to the electrodes may be simultaneously controlled through this electronic actuation means. In other examples, a second electrical control (e.g., touchscreen) may be included to control deployment of the drug delivery channel separately from the electrodes.

Electrode Deployment

[00140] During electroporation, the distance between electrodes ( e.g ., electrodes 100) may affect the size of the treatment area and the required amplitude, frequency, and/or wavelength of the electrical signals needed for electroporation. The working channel size in the endoscope or in the insertion tube of the applicator may limit the spacing between electrodes because the electrodes must fit within the working channel, and thus the size of the electroporation treatment area may be restricted during endoscopic therapies in ways not required in non-endoscopic methods and apparatus or non-minimally-invasive procedures. [00141] In some embodiments of the present disclosure, the applicator 14 and electrodes 100 may be structured such that the electrodes are able to be deployed to a spacing wider than the working channel in an instance in which the electrodes are able to clear the distal end of the endoscope. In some embodiments, the electrodes 100 may expand wider than an opening (e.g., a keyhole opening) at a point of access in the patient. In some embodiments, the electrodes 100 may expand wider than a distal end of the insertion tube 15. In some embodiments, the electrodes 100 may expand wider than one or more channels (e.g, channels 204, 404, etc.) in the insertion tube 15. In some embodiments, the electrodes may expand to a spacing about equal to the distal end of the insertion tube 15 or about equal to a width of the one or more channels. In some embodiments, the electrodes may expand to a spacing less than the distal end of the insertion tube 15. In some embodiments, an actuator 42, 74, 112 may extend through or onto the insertion tube 15 of the applicator 14 and may be configured to apply an axial force ( e.g ., a force having a component along the longitudinal axis of the insertion tube 15) to the electrodes 100. This axial force may cause the electrodes to extend axially and/or radially outwardly from the distal end of the insertion tube 15 of the applicator 14 to electroporate the target tissue at the electroporation site. In some examples, the manner of expansion of the electrodes may be a function of the space available in view of the cross-sectional size of the insertion tube and the electrode position within the tube in the retracted position. In one specific example, an applicator with electrodes very close together in the retracted position may include a radially expanding deployment of such electrodes so that the electrode tips reach a spacing necessary for the safe and effective operation of the applicator upon deployment (e.g., minimize the possibility of electrical arcing between the electrodes). In some other embodiments, the electrodes may be fixed or may extend axially outward with no change in the respective spacing of the electrodes.

[00142] In some embodiments, insertion tube 15 may define a diameter of about 2mm. In a retracted position, stored within the insertion tube 15, the tips of the electrodes 100 may be spaced about 1.8mm apart. In the deployed position, the tips of the electrodes 100 may be spaced about 3mm apart. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced greater than the external diameter of the distal end of the insertion tube. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced greater than the external diameter of a distal end of the insertion device (e.g., endoscope). In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced greater than 2mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced greater than 3mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced from 2mm to 3mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced about 4mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced greater than 4mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced less than 4mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced greater than 5mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced from 3mm to 5mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced from 2mm to 5mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced about 5mm. In some embodiments, in the deployed position, the tips of the electrodes 100 may be spaced less than 5mm. In one particular example, the electrode spacing may preferably be about 5 mm or less for an applicator described in conjunction with low voltage generator electroporation. In any of the above configurations, low voltage electroporation may be performed.

[00143] In some embodiments, the electrodes 100 may be made of stainless steel and coated with gold. In some embodiments, the electrodes 100 may be substantially flexible, having a similar structure to acupuncture needles. The electrodes 100 may be 0.25mm in diameter in some embodiments. The electrodes 100 may extend about 6mm in length in some embodiments. In some embodiments, the diameter and length of the electrodes may vary from the specific dimensions described herein. In some embodiments, the actuator 42, 74, 112 and remaining non-metallic components of the applicator 14, 60, 70, 110, 1000, such as the body 90, 120 and end caps 88, 122, may be made of a plastic material ( e.g ., high- density polyethylene, braided polyurethane (FEP, PEEK, etc.), etc.).

[00144] With reference to FIGS. 2, 19, 47, and 86, as detailed above, the applicator 14, 60, 70, 110, 1000 may include the insertion tube 15, 1015, the control portion 48, 72, 114, 1014, and the actuator 42, 74, 112, 1012. The actuator 42, 74, 112, 1012 may include a trigger 44, switch 80, 116 or other actuating element and a pushing element 46, 92, 128 that may be rigid in some embodiments, and sufficiently flexible to bend with a flexible endoscope in some embodiments. For example, in some laryngeal applications, the insertion tube 15 and actuator 42, 74, 112 may be rigid. With reference to FIG. 2, the trigger 44 may be pivotally attached to the control portion 48 and the pushing element 46 such that pulling the trigger forces the pushing element 46 along the insertion tube 15 of the applicator 60 towards the endoscopic site at the distal end of the insertion tube 15, and extending the trigger 44 (e.g., moving the trigger back to the position shown in FIG. 2) will retract the pushing element 46 back towards the control portion 48. With reference to FIG. 19, the switch 80 may be slidingly attached to the control portion 72 and the pushing element 92 via hollow mandrel 86 such that sliding the switch forces the pushing element 92 along the insertion tube 15 of the applicator 70 towards the endoscopic site at the distal end of the insertion tube 15, and retracting the switch 80 (e.g, moving the switch back towards the user) will retract the pushing element 92 back towards the control portion 72. With reference to FIG. 47, the switch 116 may be slidingly attached to the control portion 126 and hollow mandrel 124 such that sliding the switch forces the pushing element 128 along the insertion tube 15 of the applicator 110 towards the endoscopic site at the distal end of the insertion tube 15, and retracting the switch 116 ( e.g ., moving the switch back towards the user) will retract the pushing element 128 back towards the control portion 114.

[00145] Turning to FIGS. 5-41 and 60-66, several embodiments of the distal end assemblies of the insertion tube 15 of the applicator 14 are shown. In each embodiment, the electrodes may be driven axially and radially outwardly to create a greater spacing between the ends of the electrodes. In some embodiments, when moved to a deployed position, the ends of the electrodes are spaced farther apart than the external diameter of the insertion tube 15 of the actuator 14. In some embodiments, when moved to a deployed position, the ends of the electrodes are spaced farther apart than the internal diameter of the working channel (e.g., working channel 54 of the endoscope 52 shown in FIG. 4). In each embodiment, the electrodes may be directly or indirectly actuated by the actuator via the pushing element in both the outward (e.g, deploying) and inward (e.g, retracting) directions.

[00146] With reference to FIGS. 5, 6, 20-21, 62, 63, a pair of electrodes 200 are shown having a retracted position (FIGS. 5, 21, 63) and a deployed position (FIGS. 6, 20, 62) in accordance with some embodiments described herein. The electrodes 200 may each include a tip 201 at a distal end thereof opposite the insertion tube 15. The tip 201 of the electrodes 200 may define a pointed end configured to pierce the target tissue for electroporation. In the depicted embodiment, the applicator 14, 110 includes an end cap 202, 210 at the distal end of the insertion tube 15 having at least two angled channels 204 defined therein. The two angled channels 204 in the depicted embodiment are configured to angle the electrodes outwardly in the deployed position (FIGS. 6, 20, 62) so that the spacing between the ends of the electrodes increases. The embodiment of FIGS. 62 and 63 depicts another embodiment of the insertion tube 15 and end cap 210 through which the electrodes 200 may extend via the angled channels 204, and which also depict an alignment opening 212 and alignment channel 168 to support a drug delivery channel 18 therein. The embodiment of FIGS. 62-63 depicts the embodiment of FIGS. 5, 6, 20-21 having an insertion tube 15 with a drug delivery channel 18 extending therethrough. The drug delivery channel 18 and electrodes 200 may be operated and structured in accordance with any of the embodiments herein.

[00147] With reference to FIGS. 5 and 63, the angled channels 204 are oriented at respective angles a, b relative to the longitudinal axis 50. In some embodiments, the angles a, b may be equal, such that the electrodes 200 are oriented at substantially mirrored angles relative to the axis 50 in the deployed position. In some embodiments, the angles a, b may be slightly different, but extend in different directions relative to axis 50. In some embodiments, the angles a, b are each acute, such that when the pushing element 46 applies an axial force, directly or indirectly, on the electrodes 200 towards the end cap 202, 210, the angle of the channels 204 pivots the electrodes to angle the electrodes in the direction of the channels 204 as the electrodes extend outwardly from the end cap 202 into the deployed position shown in FIGS. 6 and 62. Similarly, when the pushing element 46, 128 retracts back towards the control portion 48, 114 as described above, the electrodes 200 may be pulled back into the end cap 202, 210 of the applicator 14, 110 and into the internal cavity of the insertion tube 15, allowing the electrodes to reorient within the insertion tube 15. Thus, in the retracted position (FIGS. 5, 21, 63), the electrodes 200 are substantially parallel to each other, and in the deployed position, at least a portion of the electrodes 200 are at an angle ( e.g ., a + b) to each other as defined by the angled channels 204 as a result of the actuator pushing the electrodes into the angled channels.

[00148] In any of the embodiments discussed herein, the electrodes (e.g., a needle) may be made of a sufficiently flexible material to allow the electrodes to bend when moving between the retracted and the deployed positions. In some embodiments, the electrodes 100 may be made of stainless steel and coated with gold. For example, in some embodiments, the electrodes may be substantially the same as acupuncture needles. With reference to FIG. 21, a carrier 206 may fixedly hold the electrodes 200 such that the electrodes protrude a predetermined distance from the carrier (e.g, 5mm). In such embodiments, the electrodes 200 may bend when passing through the end cap 202, 210 along the angled channels 204 such that the distal end of the electrodes is oriented in the direction of the angled channels while the bases (opposite the distal end) of the electrodes remain parallel. In any of the embodiments herein including an electrode carrier, the carrier may include passages for disposal of electrodes therein and a further passage for the disposal of a drug delivery channel.

[00149] In the embodiment shown in FIGS. 20-21, the carrier 206 is actuated between the retracted and deployed positions by the pushing element 92 (shown in FIGS. 33, 36), which pushing element may abut a proximate, rear surface of the carrier opposite the distal end. The electric wires 17 which supply the electric signals to the electrodes 200 may pass through a channel 208 in the carrier 206 to connect the generator to the electrodes. In some embodiments, the pushing element 92 may be fixed to the carrier 206.

[00150] With reference to FIGS. 7, 8, and 22-25, a pair of electrodes 300 are shown having a retracted position (FIGS. 7, 25) and a deployed position (FIGS. 8, 22, 24) in accordance with some embodiments described herein. The electrodes 300 may each include a tip 301 at a distal end thereof opposite the insertion tube 15. The tip 301 of the electrodes 300 may define a pointed end configured to pierce the target tissue for electroporation. In the depicted embodiment, the applicator 14, 60, 70, 110, 1000 includes an expandable bladder 302 in which ends of the electrodes 300 are embedded. In some embodiments, the bladder may be made of a flexible, elastic material such as rubber. In use, the bladder 302 may be retracted and compressed within the insertion tube 15 the retracted position (FIG. 7, 25). In the retracted position, the electrodes 300 are positioned close together at a distance less than the internal diameter of the insertion tube 15 because the bladder 302 is compressed radially inwardly by the insertion tube 15.

[00151] In operation, the pushing element 46, 92 applies an axial force, directly or indirectly, on the bladder 302 and causes the bladder to exit the distal end of the insertion tube 15. Upon clearing the distal end of the insertion tube 15, the bladder 302 may expand into a deployed shape ( e.g ., a substantially spherical shape). In some embodiments, the bladder 302 may expand by pneumatic pressure supplied from an air supply upstream of the bladder 302 (e.g., via a conduit running through the applicator). For example, with reference to FIGS. 22-23, the control portion 72 may include a secondary button 82 to activate a pneumatic supply to inflate the bladder 302. In some embodiments, the bladder 302 may expand mechanically due to the elastic restorative force of the bladder returning to its natural, expanded shape with or without pneumatic assistance. Similarly, when the pushing element 46 retracts back towards the control portion 48 as described above, the electrodes 300 may be pulled back into the insertion tube 15 of the applicator 14, causing the bladder 302 to recompress and deform and causing the electrodes 300 to move closer together.

[00152] The electrodes 300 may be parallel in both the retracted (FIG. 7) and expanded (FIG. 8) positions. In some embodiments, the electrodes 300 may be angled in either or both of the retracted and expanded positions. For example, the electrodes may be mounted at any position on the bladder 302 and at any desired orientation (e.g, angled outwardly, similar to the embodiment of FIGS. 5-6).

[00153] Turning to FIGS. 9, 10, 64, and 65, another embodiment of the electrodes 400 is shown in which the electrodes 400 are made of Nickel Titanium (Nitinol). Nitinol is a shape memory alloy capable of “remembering” a programmed shape and returning to the programmed shape under certain temperature conditions. Nitinol may be programmed to a specific shape by holding the nitinol in a predetermined position (e.g, the “S” shape shown in FIG. 10) and heating the nitinol to about 500 °C (932 °F) to set the shape of the nitinol. After shape setting, the nitinol may be cooled to room temperature and mechanically deformed into a second shape ( e.g ., the straight shape shown in FIG. 9). During use, when the nitinol is heated above a transformation temperature, the nitinol returns to its programmed shape. The electrodes 400 may each include a tip 401 at a distal end thereof opposite the insertion tube 15. The tip 401 of the electrodes 400 may define a pointed end configured to pierce the target tissue for electroporation.

[00154] By adjusting the proportions of nickel and titanium in the Nitinol, the transformation temperature (e.g. , the temperature at which 50% of the nitinol changes from the shape shown in FIGS. 9, 65 to the position shown in FIGS. 10, 64) of the nitinol may be tuned relative to human body temperature, such that the Nitinol changes shape upon coming into contact with the temperature of the patient’s body tissue. In use, nitinol may have a “start” temperature and a “finish” temperature at which the transformation begins and ends, respectively. In some embodiments, the finish temperature may be less than or equal to body temperature. For example, in some embodiments, the nitinol may include 54.5% nickel and 45.5% titanium, which may have a transformation temperature of 60° Celsius. In some embodiments, the transition temperature of the Nitinol may be human body temperature. Alternatively, rather than relying on the body temperature of the patient to warm the Nitinol, the electrodes 400 may instead change shape upon a voltage passing through it, whether it be the actual voltage being used for electroporation, or some amount of pre-voltage, such as a smaller voltage with a sole intended use of assisting the electrodes to change shape. Once the shape has been changed, the standard voltage may be passed through the electrodes.

[00155] The pushing element 46, 128 may deploy the electrodes 400 by applying an axial force, directly or indirectly, on the electrodes 400 towards the distal end and end cap 402, 420 when the electrodes 400 are in their deformed, substantially straight shape (e.g., the shape shown in FIGS. 9, 65). The pushing element 46, 128 may cause the electrodes 400 to translate axially through the channels 404 end cap 402, 420 until a portion of the electrodes extends from the distal end of the applicator 14. In some embodiments, the channels 404 may be substantially parallel to the axis 50 of the applicator 14. Upon changing temperature above the transformation temperature, the electrodes 400 may change shape to their programmed shape in which the electrodes are curved outwardly to widen the spacing between the ends of the electrodes as shown in FIGS. 10, 64. The embodiment of FIGS. 64 and 65 depicts another embodiment of the insertion tube 15 and end cap 420 through which the electrodes 400 may extend via the channels 404, and which also depict an alignment opening 422 and alignment channel 168 to support a drug delivery channel 18 therein. The embodiment of FIGS. 64 and 65 depicts the embodiment of FIGS. 9-10 having an insertion tube 15 with a drug delivery channel 18 extending therethrough. The drug delivery channel 18 and electrodes 400 may be operated and structured in accordance with any of the embodiments herein.

[00156] In some embodiments, the tips 401 of the electrodes 400 may be substantially parallel to each other in both the retracted (FIGS. 9, 65) and deployed (FIGS. 10, 64) positions, while the middle sections of the electrodes curve into an “S” shape when transitioning from the retracted position to the deployed position. Similarly, when the pushing element 46, 128 retracts back towards the control portion 48, 114 as described above, the electrodes 400 may be pulled back into the end cap 402 of the applicator 14 and into the cavity of the insertion tube 15, causing the nitinol to mechanically deform back into a substantially straight position when the nitinol is forced against the channels 404.

[00157] With reference to FIG. 11, in some embodiments, the electrodes 400 may engage an outer nitinol sleeve 410 and a wire 17 ( e.g ., separate wires 17 or wires connected to a conducting pushing element 128) running through the sleeve. For example, the electrodes 400 may be rigid needles affixed to the nitinol sleeve 410 at one end (e.g., the distal end when exiting the end cap 402) and the wire 17 may connect the electrodes to the generator (e.g, generator 12 described herein). In such embodiments, the nitinol is not required to carry the electrical signals for electroporation and instead forms a shape-changing sleeve around the conductive elements. In some embodiments, the electrodes 400 may be made of nitinol coated in a conductive material to carry an electrical signal thereon. For example, the electrodes 400 may have a nitinol structure with a nickel base coating and a gold conductive coating over the nickel coating.

[00158] FIGS. 26-30 another embodiment in accordance with the disclosure of FIG.

11. In particular, the embodiment of FIGS. 26-30 include electrodes 800 and a nitinol carrier 802 (also referred to as a sleeve) having two at least partially cylindrical halves 804 that change shape in substantially the same manner as described with respect to the embodiments of FIGS. 9-11 to position the electrodes 800 in a wider position when deployed by returning to a pre-programmed “S” shape at or above body temperature, after the actuator 74 deploys the electrodes. In some embodiments, the electrodes 800 may be attached to a straight portion of the carrier halves 804 with the wire 17 being disposed in the shape-changing portions of the carrier 802. The electrodes 800 may include tips 801 configured to extend into the target tissue.

[00159] The carrier 802 may include a cylindrical portion 806 connecting the two halves 804. With reference to FIGS. 29-30, the pushing element 92 may engage the cylindrical portion 806 of the carrier 802 to actuate the electrodes 800, which electrodes may be fixedly attached to the carrier. In some embodiments, the cylindrical portion 806 may be fixed to the pushing element 92. In the depicted embodiment, the wires 17 for supplying the electrical signals from the generator may pass through the nitinol carrier 802 and may be connected to the electrodes 800 (as shown in FIG. 30). In some embodiments, the wires 17 may not be attached to the carrier 802 such that the wires may slide relative to the carrier when the carrier halves 804 change shape. In some embodiments, the nitinol carrier 802 may be 20-25mm in length when straightened out. In some embodiments, the pushing element 92 may be fixed to the nitinol carrier 802 at a base end of the nitinol carrier.

[00160] Turning to FIGS. 12, 13, 31, 32, 60, and 61, an embodiment of the electrodes 500 is shown having substantially the same deployed shape (shown in FIGS. 13, 31, 60) as the Nitinol electrodes 400 shown in FIG. 10. The electrodes 500 may each include a tip 501 at a distal end thereof opposite the insertion tube 15. The tip 501 of the electrodes 500 may define a pointed end configured to pierce the target tissue for electroporation. In the embodiment of FIGS. 12, 13, 31, 32, 60, and 61, the electrodes 500 are made of traditional conductive materials, which may be somewhat flexible, but elastically return to their original shape when stressing forces are removed. For example, as discussed above, the electrodes 500 may be made of a flexible needle having the properties of an acupuncture needle. In the depicted embodiment, the electrodes 500 are compressed radially inward in the retracted position (FIG. 12, 32, 61) and are then able to expand outwardly in the deployed position (FIG. 13, 31, 62).

[00161] The insertion tube 15 of the applicator 14, 60, 70, 110, 1000 may include an end cap 502, 510 defining channels 504 therein through which the electrodes 500 may extend. In the depicted embodiment, the electrodes 500 have a curved, “S” shape at all times, and forcing the electrodes through the end cap 502, 510 may require some deformation of the electrodes. The pushing element 46, 92, 128 may deploy the electrodes 500 by applying an axial force, directly or indirectly, towards the distal end and end cap 502, 510 of the insertion tube 15. The pushing element 46, 92, 128 may force the electrodes 500 through the end cap 502, 510, and allow the electrodes 500 to expand to their final width in the deployed position. In some embodiments, the ends of the electrodes 500 may be substantially parallel at least in the deployed position. The pushing element 46, 92, 128 may then retract the electrodes 500 by pulling the electrodes back into the insertion tube 15. In some embodiments, a carrier ( e.g ., carrier 206 shown in FIG. 21) may engage the electrodes 500 and the pushing element 46, 92, 128 to transmit the axial force from the actuator 42, 74, 112 to the electrodes. The embodiment of FIGS. 60 and 61 depicts another embodiment of the insertion tube 15 and end cap 510 through which the electrodes 500 may extend via the channels 504, and which also depict an alignment opening 512 and alignment channel 168 to support a drug delivery channel 18 therein. The embodiment of FIGS. 60 and 61 depicts the embodiment of FIGS.

12, 13, 31, and 32 having an insertion tube 15 with a drug delivery channel 18 extending therethrough. The drug delivery channel 18 and electrodes 500 may be operated and structured in accordance with any of the embodiments herein.

[00162] In any of the embodiments of the electrodes 100 described herein, the portion of the electrodes 100 closest to the tip may be defined parallel to each other in both the deployed and retracted positions. In some embodiments, the portion of the electrodes 100 farthest from the tip may also be parallel in both the deployed and retracted positions, and at least a part of this farthest portion may remain within the insertion tube 15 in both the deployed and retracted positions. Between the farthest portion from the tip and the closest portion to the tip, the electrodes may include 100 a straight or curved portion of electrode.

For example, the “S” shaped curve may be defined between the respective end portions of the electrode. In some embodiments, the middle portion of the electrode may be straight in the retracted position and curved in the deployed position.

[00163] With reference to FIGS. 14, 15, 33-41, and 66, an embodiment of the electrodes 600 is shown disposed in an expandable center carrier 602 from which the electrodes extend. The electrodes 600 may each include a tip 601 at a distal end thereof opposite the insertion tube 15. The tip 601 of the electrodes 600 may define a pointed end configured to pierce the target tissue for electroporation. In some embodiments, in the retracted position (FIG. 14), the electrodes 600 may be withdrawn into the carrier 602 and the carrier may be withdrawn into the distal end of the insertion tube 15. In some embodiments (FIGS. 33-41), the electrodes 600 may be fixed to the carrier 602 and the carrier may be withdrawn in to the distal end of the insertion tube 15 in the retracted position (FIGS. 38, 40). With reference to FIG. 33, in some embodiments, wires 17 may pass through the carrier 602 to the electrodes 600 via channels 612.

[00164] In some embodiments, the pushing element 46, 92, 128 may apply an axial force, directly or indirectly, to an inner member 606, 610, 620, which may separate the halves 604 of the carrier 602 to spread the electrodes 600 outwardly. In some embodiments, the inner member may be a wedge 606 (shown in FIG. 15) within the carrier 602. In some embodiments, the inner member may be a cylinder 610 (shown in FIGS. 33, 38). In some embodiments, the inner member 606, 610 may translate axially 50 relative to the carrier 602, while also pushing the carrier at least partially out of the distal end of the insertion tube 15. The embodiment of FIGS. 66 depicts another embodiment of the insertion tube 15 and inner member 620 which may deploy the carrier 602 and electrodes 600. The embodiment of FIG. 66 depicts the embodiment of FIGS. 14, 15, and 33-41 having an insertion tube 15 and inner member 620 with a drug delivery channel 18 extending therethrough. The drug delivery channel 18, inner member 620, and electrodes 600 may be operated and structured in accordance with any of the embodiments herein.

[00165] In some embodiments, the inner member 606, 610 may be separately actuated by a second actuator 94 (shown in FIGS. 35, 37, 39, and 40). In operation, with reference to FIGS. 35, 37, 39, and 40, after the actuator 74 deploys the carrier 802 forwards from the distal end of the insertion tube 15, the second actuator 94 may be pressed inwardly into the body 90 of the control portion 72 to align a distal end 98 of the second actuator with an opening in the hollow mandrel 86 ( e.g ., along axis 50 shown in FIG. 14), with the second actuator having a bent portion 97 to allow the distal end to reach deeper into the hollow mandrel. The actuation of the hollow mandrel 86 by the actuator 74 may allow the second actuator 94 to fit behind the hollow mandrel in line with its opening. The inner member 606, 610 (FIGS. 15, 41) may be configured to translate relative to the hollow mandrel 86 from a position within the hollow mandrel, such that a user may actuate the second actuator 94 by sliding the second switch 96 axially forward (e.g., towards the distal end of the insertion tube 15) such that the distal end 98 of the second actuator engages a base surface 614 (shown in FIGS. 33, 38) of the inner member 606, 610. The second actuator 94 may thereby cause the halves 604 of the carrier 602 to separate (as shown in FIGS. 15 and 41) by actuating the inner member 606, 610 through the hollow mandrel 86 after the carrier 602 has been actuated by the actuator 74 (e.g., after the carrier 602 has been advanced axially from within the insertion tube 15 by actuation of the first actuator).

[00166] The relative axial movement between the inner member 606, 610 and the carrier 602 may apply a radial force on a ramped surface within two halves 604 of the carrier, to cause the halves 604 to expand radially outwardly. For example, with reference to FIG.

38, the carrier 602 may include a tapered surface 616 in its interior that, when operated on by the inner member 606, 610, causes the halves 604 of the carrier to expand outwardly. Although FIGS. 15, 35, and 41 depict a portion of the carrier 602 and electrodes 600 being articulated substantially parallel to each other in the deployed position, in some embodiments, the carrier 602 and electrodes 600 may curve radially outwardly (e.g, similar to the angles of FIG. 5) in response to the actuation of the wedge 606 with only the halves 604 of the carrier 602 being a substantially contiguous piece of material.

[00167] In some embodiments, the carrier 602 may only define two halves 604 near the distal end, and a remaining portion of the carrier may be a single, solid piece, such that the two halves are still affixed to each other ( e.g ., cylindrical portion 606).

[00168] In some embodiments, with reference to FIG. 41, the inner member 606, 610 may define a needle fluidly connected to the drug delivery device (e.g., drug delivery device 16 shown in FIG. 1), such that the inner member administers the treatment agent to the target area after the halves 604 of the carrier 602 separate. In such embodiments, the treatment agent may be delivered via a drug delivery channel (e.g, drug delivery channel 18 shown in FIG. 1) extending through the insertion tube 15 as described herein.

[00169] Turning to FIGS. 16, 17, 42, and 43, another embodiment of the electrodes 700 is shown. In the depicted embodiment, the electrodes 700, carrier 702, and applicator 14, 60, 70 may operate in substantially the same manner as the embodiment of FIGS. 14, 15, and 33-41, except that the inner member (e.g, wedge 606 or cylinder 610) and second actuator 94 are replaced with a spring 706 that expands the carrier halves 704 radially outwardly, while the pushing element 46, 92 directly or indirectly drives the electrodes 700 and carrier 702 axially out of the applicator 14, 60, 70 and into a deployed position (FIGS. 17, 42). The electrodes 700 may each include a tip 701 at a distal end thereof opposite the insertion tube 15. The tip 701 of the electrodes 700 may define a pointed end configured to pierce the target tissue for electroporation. In some examples, the spring 706 may be biased so that upon deployment from the insertion tube 15, the spring expands to its biased position and thereby spreads apart the electrodes and electrode tips 701.

[00170] Additionally, or alternatively, the pusher member may similarly be spring- biased such that, upon actuation by the user, the electrodes are forced into a deployed position by the spring-loaded actuator. Then, if present, the spring 706 may simultaneously expand the electrodes away from one another (or another mechanism as discussed above may complete this action).

[00171] While in most of the described embodiments herein, the electrodes are in the shape of needles with pointed tips, capable of piercing tissue to be treated, in other embodiments, the electrodes may take on the shape of something other than a needle which may or may not include a tip capable of piercing tissue. For instance, one or more the electrodes may have a blunt tip, or further, may have a flat shape, rounded shape, or the like, that simply presses against the tissue to be treated rather than piercing the tissue to be treated. In such instances, as the electrodes are atraumatic, the electrodes need not necessarily be actuatable, but instead can be positioned in a fixed location relative one another. Of course, in instances where the applicator is sized for passage through an access instrument, such as an endoscope, actuation of at least one of the electrodes may be necessary to allow for adequate spacing between the electrodes on the tissue to be treated. As such, at least one of the electrodes may be fixed while at least one of the other electrodes may be actuatable or, as discussed above, each of the electrodes may be independently or collectively actuatable. [00172] In this manner, as discussed previously, in certain embodiments, one or more of the electrodes may have the needle shape or some other projected shape suitable of pressing or piercing tissue to be treated, while the other electrode (e.g., the return or negative electrode) may be positioned on, or actually be, the distal tip of the applicator or endoscope which is positioned adjacent the tissue to be treated, and thus could be suitable for acting as an electrode. Furthermore, in this exemplary embodiment, the one or more positive electrodes need not be actuatable, but instead, can merely be positioned in a fixed location so as to project distally to a position sufficiently apart from the distal tip of the applicator (or to be positioned a suitable distance from the distal end of the endoscope or other access instrument) to allow for supply of an electrical pulse, as described herein.

[00173] In some embodiments, the actuation mechanism to control deployment of the electrodes may be passive (e.g., shape memory material for electrodes 400, spring 706 for electrodes 700). In some embodiments, the actuation mechanism to control deployment of the electrodes may be active (e.g., advancement of inner member 606, 610 through second actuator to cause electrodes 600 to move apart).

[00174] In some embodiments, an applicator may include a plurality of electrodes that are at an operative spacing for electroporation both before and after deployment from the applicator. In this manner, a spacing between the electrodes remains the same before and after deployment. The effect of deployment in this configuration is simply to axially advance the electrodes relative to the insertion tube of the applicator.

[00175] In some embodiments, applicators as described in the various embodiments of the application may include three electrodes, four electrodes, or more. Illustrative examples of these arrangements are provided elsewhere in the present disclosure. For each applicator, it is contemplated that the higher number of electrodes may be incorporated following the structural configuration of the existing design. Thus, for example, insertion tube 15 shown in FIG. 21 includes channels 204 at the tip that are angled outward from a centerline of the tube 15. In a variation of this embodiment with three electrodes, three channels 204 may be included, each equally spaced and extending away from the tube centerline toward an outer perimeter of the tube.

[00176] In some examples, an applicator may include four electrodes. The applicator may be rectangular in shape with electrodes spaced about 5 mm apart. In some examples, an applicator may include six or more electrodes positioned peripherally about a circumference with a diameter of about 5 mm. Details of the treatment performed and the results illustrative of the advantages of low voltage electroporation are found in Burkart et al., Improving therapeutic efficacy ofIL-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters , Gene Therapy, 25, 93-103 (9 March 2018), incorporated by reference herein in its entirety.

[00177] In any of the above-noted embodiments, the one or more electrodes may be deployed simultaneously and collectively with all electrodes or any portion of the total number of electrodes. Alternatively, each individual electrode may be actuated and deployed independently of the others.

[00178] In yet another embodiment, the electrodes may operate as a harpoon, whereby each electrode is inserted into the tissue such that each electrode separates from the applicator 14, tethered only by the wire or like structure which provides an electrical connection to the electrode. As such, each electrode can be positioned into the tissue at any location desired. For example, each electrode is deployed one at a time from the applicator at various locations in and around the target tissue. Each electrode remains tethered to the applicator and/or another electrode. Upon completion of the procedure, each electrode is drawn back to the applicator, whether by a spooling reel, a pulling of the wire, a magnetic attraction between the applicator and the electrode, or the like.

[00179] As discussed above, the electrodes are typically connected to a power source via a wire, though also present in most embodiments is a pusher member and an insertion tube. In some embodiments, the pusher member or the insertion tube could operate as the electrical connection to at least one of the electrodes, thereby eliminating the need for at least one of the wires. As one example, in an instance with two electrodes, the positive connection to one of the electrodes could be via the pusher member, while the negative or return connection to the other electrode could be the insertion tube body. Of course, adequate insulation of these structures would be required to avoid arcing of the electrodes and/or injury to the user.

[00180] In still another embodiment, the electrical connection between the electrical source and the at least one electrode could be wireless, for example, via the use of inductive power transfer via an electromagnetic field. Such a power connection could be completed transdermally, such that a wire would not be required to pass between the target tissue and the power source. Continuing with such an electrical connection, in certain embodiments, the harpoon-like electrode mentioned previously could be positioned in the target tissue, which would not be connected via wires to an electrical source. In this way, the drug delivery could occur by any desired procedure, and the electroporation could occur without being in a surgical setting. For example, once the electrodes are implanted into the target tissue, and whether or not treatment agents have been supplied to the patient and/or the target tissue, the patient could be removed from the operating room and the treatment could be supplied one or more times outside of the surgical setting using a drug delivery device such as a needle or the like, and a transdermal power delivery to the electrodes. The electrodes may then be removed at a later date or may be biodegradable, or if they are of a shape that is atraumatic (e.g., a disc-shaped electrode sutured to tissue) or is otherwise secured in the patient without fear of coming loose, the implant may remain inside the patient indefinitely.

Example Electrical Parameters

[00181] The nature of the electric field to be generated by the generator 12 is determined by the nature of the tissue, the size of the selected tissue and its location. It is desirable that the field be as homogenous as possible and of the correct amplitude. Excessive field strength results in lysing of cells, whereas a low field strength results in reduced efficacy. The electrodes may be mounted and manipulated in many ways including but not limited to those described herein. Using the system 10 described herein, the parameters of the electroporation (e.g., voltage, pulse duration, etc.) are all programmable and optimizable (e.g, via the one or more controllers described herein). In some embodiments, the parameters of the pulses are predetermined and employed in a consistent manner throughout the electroporation procedure. In some embodiments, the parameters of the pulses may be determined using a feedback mechanism while electricity is supplied to the applicator to continually adjust the parameters of the pulses during electroporation (e.g, EIS).

[00182] In some instances, electroporation uses high voltages and short pulse durations for treatment of tumors. The electrical field conditions of 1200-1300 V/cm and 100 ps have been used in vitro and in vivo with anticancer drugs like bleomycin, cisplatin, peplomycin, mitomycin c and carboplatin. These results refer to in vitro and in vivo work. Although such electrical conditions may be tolerated by patients in clinical situations, such treatments will typically produce muscle twitch and occasional discomfort to patients, and may produce worse results with certain treatment agents ( e.g ., larger molecules). Some of these problems could be considerably reduced by using low voltage high pulse durations for electrochemotherapy. Low voltage electroporation as contemplated by some embodiments of the present disclosure involves utilization of application of a voltage of about 600 V or lower, an electrical field of about 700 V/cm or lower, and a pulse length of between about 0.5 ms and about 1 s. In some examples, an electrical field of 400 V/cm or less may be utilized in a low-voltage generator configuration. In some embodiments, the generator 12 may apply a voltage of 300 V or less to the electrodes 100. In some embodiments, the generator 12 may apply a voltage of 60-300 V to the electrodes 100. In some embodiments, the generator 12 may apply a voltage of 150-200 V. In some embodiments, high voltages of greater than 1000V may cause irreversible electroporation (IRE). Thus, electroporation systems incorporating a low voltage generator are advantageous in that a risk of IRE is low compared with treatments employing a higher voltage.

[00183] The waveform of the electrical signal provided by the generator 12 can be an exponentially decaying pulse, a square pulse, a unipolar oscillating pulse train, a bipolar oscillating pulse train, or a combination of any of these forms. In some embodiments, the electrical parameters for the generator, encompassing a range for both low and high voltage generators, may encompass a nominal electric field strength from about 10 V/cm to about 20 kV/cm (the nominal electric field strength is determined by computing the voltage between electrode needles divided by the distance between the needles). In some embodiments encompassing a range for both low and high voltage generators, the pulse length can be about 10 ps to about 100 ms. In some embodiments encompassing a range for low voltage generators, the pulse length can be about 1 ms to about 1 s. In some embodiments, low voltage electroporation may be defined by a voltage and duration that causes reversible electroporation. There can be any desired number of pulses, typically one to 100 pulses per second. The wait between pulses sets can be any desired time, such as one second. The waveform, electric field strength and pulse duration may also depend upon the type of cells and the type of molecules that are to enter the cells via electroporation. The various parameters including electric field strengths required for the electroporation of any known cell is generally available from the many research papers reporting on the subject. An overview of the relationship between pulse strength and duration is described in Weaver et ah, A brief overview of electroporation pulse strength-duration space: A region where additional intracellular effects are expected , Bioelectrochemistry, 2012 October; 87: 236- 243. doi:10.1016/j.bioelechem.2012.02.007, which is incorporated by reference herein in its entirety. In some embodiments, any number of pulses may be used in a treatment. In some embodiments, 6 pulses are used. In some embodiments, 8 pulses are used. In some embodiments, 10 pulses are used.

[00184] Low voltage systems, in some embodiments, the generator may be a low- voltage generator. The electroporation therapy may be administered using the low-voltage generator producing an electric field of 700 V/cm or less, 600 V/cm or less, 500 V/cm or less, 400V/cm or less, 300V/cm or less, 200V/cm or less, or lOOV/cm or less. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 700 V/cm to 10 V/cm. The electroporation therapy may be administered using the low- voltage generator producing an electric field from 600 V/cm to 10 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 500 V/cm to 10 V/cm. The electroporation therapy may be administered using the low- voltage generator producing an electric field from 400 V/cm to 10 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 300 V/cm to 10 V/cm. The electroporation therapy may be administered using the low- voltage generator producing an electric field from 700 V/cm to 60 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 600 V/cm to 60 V/cm. The electroporation therapy may be administered using the low- voltage generator producing an electric field from 500 V/cm to 60 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 400 V/cm to 60 V/cm. The electroporation therapy may be administered using the low- voltage generator producing an electric field from 300 V/cm to 60 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 700 V/cm to 100 V/cm. The electroporation therapy may be administered using the low- voltage generator producing an electric field from 600 V/cm to 100 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 500 V/cm to 100 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 400 V/cm to 100 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 300 V/cm to 100 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 300 V/cm to 200 V/cm. The electroporation therapy may be administered using the low-voltage generator producing an electric field from 400 V/cm to 300 V/cm. In some embodiments, the pulse duration of the low-voltage generator may be from 1 millisecond (ms) to 1 second (s). [00185] Preferably, when low fields are used, the nominal electric field is from about 10 V/cm to 400 V/cm. In some embodiments, the nominal electric field may be from about 25 V/cm to 75 V/cm. In some embodiments, the low nominal electric field may be about 400 V/cm. In a particular embodiment, it is preferred that when the electric field is low, the pulse length is long relative to a high field pulse. For example, when the nominal electric field is in the “low” range discussed herein, it is preferred that the pulse length is about 10 msec.

[00186] With continuing reference to a system with a low voltage generator, in some embodiments, the low-voltage generator may produce a voltage ranging from 600V to 5 V. In some embodiments, the low-voltage generator may produce a voltage ranging from 500V to 5 V. In some embodiments, the low-voltage generator may produce a voltage ranging from 400V to 5 V. In some embodiments, the low-voltage generator may produce a voltage ranging from 300V to 5 V. In some embodiments, the low-voltage generator may produce a voltage ranging from 200V to 5 V. In some embodiments, the low-voltage generator may produce a voltage ranging from 100V to 5 V. In some embodiments, the low-voltage generator may produce a voltage ranging from 600V to 10V. In some embodiments, the low- voltage generator may produce a voltage ranging from 500V to 10V. In some embodiments, the low-voltage generator may produce a voltage ranging from 400V to 10V. In some embodiments, the low-voltage generator may produce a voltage ranging from 300V to 10V.

In some embodiments, the low-voltage generator may produce a voltage ranging from 200V to 10V. In some embodiments, the low-voltage generator may produce a voltage ranging from 100V to 10V. In some embodiments, the low-voltage generator may produce a voltage ranging from 600V to 50V. In some embodiments, the low-voltage generator may produce a voltage ranging from 500V to 50V. In some embodiments, the low-voltage generator may produce a voltage ranging from 400V to 50V. In some embodiments, the low-voltage generator may produce a voltage ranging from 300V to 50V. In some embodiments, the low- voltage generator may produce a voltage ranging from 200V to 50V. In some embodiments, the low-voltage generator may produce a voltage ranging from 100V to 50V. In some embodiments, the low-voltage generator may produce a voltage ranging from 600V to 100V. In some embodiments, the low-voltage generator may produce a voltage ranging from 500V to 100V. In some embodiments, the low-voltage generator may produce a voltage ranging from 400V to 100V. In some embodiments, the low-voltage generator may produce a voltage ranging from 300V to 1001 V. In some embodiments, the low-voltage generator may produce a voltage ranging from 200V to 100V. In some embodiments, the low-voltage generator may produce a voltage ranging from 600V to 200V. In some embodiments, the low-voltage generator may produce a voltage ranging from 500V to 200V. In some embodiments, the low-voltage generator may produce a voltage ranging from 400V to 200V. In some embodiments, the low-voltage generator may produce a voltage ranging from 300V to 200V. In some embodiments, the low-voltage generator may produce a voltage ranging from 600V to 300V. In some embodiments, the low-voltage generator may produce a voltage ranging from 500V to 300V. In some embodiments, the low-voltage generator may produce a voltage ranging from 400V to 300V. In some embodiments, the low-voltage generator may produce a voltage ranging from 600V to 400V. In some embodiments, the low-voltage generator may produce a voltage ranging from 500V to 400V. In some embodiments, the low-voltage generator may produce a voltage ranging from 600V to 500V.

[00187] Advantages of the low voltage generator may include an improved expression of therapeutic agents transfected over that of a high voltage generator. In some embodiments, the presence of a tissue sensing system as described elsewhere herein may further improve performance over that of another generator. Tissue sensing accomplished through the low voltage generator output may allow for characterization of the treatment site. In particular, the potential to gather feedback from therapy in order to determine unsafe treatment and potentially optimize therapy conditions may be highly comprehensive. Thus, following a series of pulses in a treatment, the expression of therapeutic agents may be significantly higher and more durable under the example embodiments described herein. Additionally, as noted elsewhere in the disclosure, production of a voltage below 600 V that produces an electric field below 700 V/cm with a low voltage generator mitigates the risk of irreversible electroporation which may cause damage to tissue in and around the target location for treatment. Moreover, electroporation with these parameters allows for an overall longer treatment duration, thereby increasing the likelihood of successful delivery of the treatment agent.

[00188] Preferably, the therapeutic method of the invention utilizes the systems described herein which may include an applicator, a plurality of electrodes configured to extend from the applicator, and a generator for applying an electric signal to the electrodes.

In some embodiments, the system may also include an insertion device as described elsewhere in the application, such as an endoscope. In some embodiments, the electric pulses from the generator may be proportionate to the distance between said electrodes for generating an electric field of a predetermined strength, such that field strength for a particular surgery is higher for systems that include an applicator with electrode tips at a greater distance from one another. In some embodiments, a system that includes a low voltage generator may include an applicator with electrodes that have tips spaced apart about 4 mm. In some embodiments, the above electrical parameters may be employed without using feedback from sensing circuitry to control and otherwise update the applied voltage during an electroporation procedure.

[00189] In some embodiments, the electrical pulses may be controlled via feedback from the sensing circuitry 31, which may measure the parameters of the electrodes 100 and target tissue continually during electroporation. In some embodiments, a sensing pulse may be transmitted between electroporation pulses, such that the generator quickly alternates between applying therapeutic electroporation and sensing the parameters of the electrodes and tissues. In some embodiments, an adaptive control method may be used to set the electroporation parameters in real time. One way in which the generator ( e.g ., via sensing circuitry 31, pulse circuitry 33, and controller 24) may measure the electroporation parameters and control the pulses of the generator is via Electrochemical Impedance Spectroscopy (EIS). In some embodiments, EIS may be used with a low-voltage generator. [00190] An adaptive control method for controlling electroporation pulse parameters during electroporation of cells or tissues using the electroporation system 10 includes providing a system (e.g., generator 12 and its corresponding circuitry) for adaptive control to optimize electroporation pulse parameters including electroporation pulse parameters, applying voltage and current excitation signals to the cells (e.g, via pulse circuitry 33), obtaining data from the current and voltage measurements (e.g, via sensing circuitry 31), and processing the data to separate the desirable data from the undesirable data (e.g, via controller 24 and processor 30), extracting relevant features from the desirable data (e.g, via controller 24 and processor 30), applying at least a portion of the relevant features to a trained diagnostic model, also referred to herein as “trained model” (e.g, via controller 24 and processor 30), estimating electroporation pulse parameters based on an outcome of the applied relevant features (e.g, via controller 24 and processor 30), where the initialized electroporation pulse parameters are based on the trained model and the relevant features, to optimize the electroporation pulse parameters, and applying, by the generator, a first electroporation pulse based on the first pulsing parameters.

[00191] To maximize the efficacy of electroporation, a quantifiable metric of membrane integrity that is measureable in real-time is desirable. As described herein, EIS is a method for the characterization of physiologic and chemical systems and can be performed with any of the standard electroporation, also referred to throughout the disclosure as ΈR”, electrodes described herein. This technique measures the electrical response of a system over a range of frequencies to reveal energy storage and dissipation properties. In biologic systems the extracellular and intracellular matrix resist current flow and therefore can be electrically represented as resistors. The lipids of intact cell membranes and organelles store energy and are represented as capacitors. Electrical impedance is the sum of these resistive and capacitive elements over a range of frequencies. To quantify each of these parameters, tissue impedance data can be fit to an equivalent circuit model. Real-time monitoring of electrical properties of tissues will enable feedback control over electroporation parameters and lead to optimum transfection in heterogeneous tumors. Using EIS feedback, will allow (1) delivery parameters to be adjusted in real-time, (2) delivery of only the pulses necessary to generate a therapeutic response, and (3) reduce the overall EP -mediated tissue damage as a result. [00192] In addition, in some embodiments, these EIS measurements can be used to determine ideal electroporation conditions described herein. In some embodiments, the method of the present invention may include contacting the tissue in the target site with a pair of electrodes 100. A low voltage power supply electrically connected to the electrodes 100 may be used to apply a low voltage excitation signal to the electrodes. Methods for sensing the impedance and/or capacitance may include but are not limited to waveforms such as phase locked loops, square wave pulses, high frequency pulses, and chirp pulses. A voltage sensor and a current sensor are used to sense a voltage drop and current flowing through the circuit, and these parameters may then be processed by the controller 24, as illustrated in FIG. 1, to determine an average impedance for all cells in the measured area. This detected impedance may then ( e.g ., via the trained model discussed above) determine any necessary changes to the electroporation parameters.

[00193] In some embodiments, the generator 12 (e.g., via sensing circuitry 31) is configured to measure dielectric and conductive properties of cells and tissues, and includes a voltage sensor to measure voltages across the tissue resulting from each of an excitation signal for sensing purposes and/or an electroporation pulse applied to the tissue, and a current sensor to measure current across the tissue resulting from each of the excitation signal for sensing purposes and/or the at least one applied electroporation pulse.

[00194] The pulsing circuitry 33 may include an initializing module configured to initialize electroporation pulsing parameters for performing electroporation in the cells or tissue, where initialized electroporation pulsing parameters are based at least in part on at least one trained model, such as the trained model described elsewhere in the present disclosure. In some embodiments, the controller 24 may direct the output of the pulsing circuitry 33. The generator 12 is configured to apply at least one of the excitation signals and/or the electroporation pulse to the tissue. The voltage sensor and current sensor of the sensing circuitry 31 may measure voltage and current across the cells of the tissue in response to the application of the excitation signals. The controller 24 may be configured to receive a signal relating to the measured sensor data from the sensing circuitry 31, corresponding to at least one of the excitation signal and the electroporation pulse, to fit the data to at least one trained model and to process the data into diagnostics and updated control parameters.

[00195] In the low voltage operation, the generator may output any of the parameters described herein, including, for example, a minimum of 10 V and maximum of 300 V with pulse durations ranging from 100 to 10 ms. EIS may be data captured before and between pulses and obtained by the generator 12 over a range of 100 Hz to 10 kHz with 10 data points acquired per decade. Acquisition of EIS data over this spectra is accomplished in 250 ms, which is rapid enough to: (1) execute routines to determine a time constant for the next pulse; (2) store EIS data for post analysis; and (3) not interrupt clinically used electroporation conditions. The generator may be capable of a minimum output load impedance of 20 ohms and a maximum load impedance of an open circuit. To allow hands-free operation of the generator a foot pedal ( e.g ., foot pedal 58) may be added to trigger, pause, or abort the electroporation process.

[00196] The controller 24 may include a pre-processing module to receive the signal relating to the data from the current and voltage measurements, and process the data to separate desirable data from undesirable data, a feature extraction module to extract relevant features from the desirable data, a diagnostic module to apply at least a portion of the relevant features of the desirable data to at least one trained diagnostic model, and a pulse parameter estimation module to estimate at least one of initialized pulsing parameters and subsequent pulsing parameters based on an outcome of at least one of the measured data, the diagnostic module and the feature extraction module. The memory 36 stores the desirable and undesirable data, sensor data and the trained models for feature extraction by the controller.

Methods of Operation

[00197] Various methods associated with the electroporation system 10 will now be described. In any of the embodiments described herein, such methods can be used for treatment of one or more cancers, and more specifically, can be used to treat a tumor or other visceral lesion, particularly those found within a patient and which are not superficial or in the dermal layers. Such tumors or other lesions may be either primary or metastatic malignancies. Each of the methods described herein may be used in connection with the other treatments and therapies disclosed herein ( e.g ., with treatment using a hemostatic agent) unless otherwise noted.

[00198] With reference to FIG. 18, an example method of using the electroporation system 10 described herein is shown. In some embodiments, the method of FIG. 18 is used for treatment of one or more cancers. In some embodiments, the method of FIG. 18 is used to treat a tumor or other visceral lesion. At depicted step 150, the method may include inserting an insertion device into a patient until a distal end of the insertion device is positioned adjacent to a target site. The insertion device may be advanced through an internal passage in a variety of ways as described, for instance, in the specific examples below. In some embodiments, the insertion device may be an endoscope, including flexible endoscopes or rigid endoscopes, such as a trocar. In some embodiments, the applicator may be inserted itself with no insertion device. At depicted step 152, the method may include inserting a portion of a drug delivery device into a working channel of the insertion device, such that the portion of the drug delivery device is positioned adjacent to the target site. At depicted step 154, the method may include administering a treatment agent to the target site from the drug delivery device. At depicted step 156, the method may include removing the portion of the drug delivery device from the insertion device. At depicted step 158, the method may include inserting an insertion tube of an applicator into the working channel of the insertion device, such that a distal end of the insertion tube, including a plurality of electrodes, is positioned adjacent to the target site. At depicted step 160, the method may include delivering one or more electrical pulses to the electrodes to electroporate the tissue at the target site. At step 162, the method may include removing the applicator and insertion device from the patient. In some embodiments, the applicator may include a piercing tip 130 such that the method may further include piercing one or more tissues of the patient prior to delivering the electrical impulse and/or treatment agents. In some embodiments, as described above, the drug and/or plasmid may be administered through any of a number of means, including IM, IT, and IV delivery. In embodiments in which the drug delivery device operates through the applicator, steps 152-156 may be combined with steps 158-162. In the above described method, a low voltage generator may be used, including the particular configurations described herein. The method may be performed with or without EIS. In one example of the method performed with a low voltage generator and without EIS, the voltage applied may be the same for each pulse of the treatment, irrespective of the characteristics of the tissue encountered (e.g., the variable impedance of the tissue that may be encountered through performance of the method) and a result should be obtained that is not affected by the characteristics of the tissue. Further, as noted elsewhere in the disclosure, treatment using this approach has been shown to be successful and to possess advantages relative to treatment that employs a high voltage generator.

[00199] Advantages of performing the method using a low voltage generator and the applicator as described herein include that less heat stress is applied to the cells at the target site during electroporation, thereby increasing the likelihood that the cells will survive throughout and after the treatment. Additionally, with a lower voltage, electrical pulses may be delivered over a longer period of time compared to a high voltage electroporation procedure. With a longer duration treatment, the cells are kept open for a longer period and a greater amount of the treatment agent may be absorbed by the cells, increasing the likelihood of successful treatment.

[00200] With reference to FIG. 67, another example method of using the electroporation system 10 described herein having an insertion device is shown, for example in an embodiment where the applicator is flexible, and/or in an embodiment where an endoscope, trocar, or the like is used. In some embodiments, the method of FIG. 67 is used for treatment of one or more cancers. In some embodiments, the method of FIG. 67 is used to treat a tumor or other visceral lesion. At depicted step 6700, the method may include inserting the insertion device into a patient until a distal end of the insertion device is positioned adjacent to a target site. In some embodiments, the insertion device may be an endoscope, including flexible endoscopes or rigid endoscopes, such as a trocar. Alternatively, the applicator may be inserted itself with no insertion device. At depicted step 6705, the method may include inserting an insertion tube of an applicator into the working channel of the insertion device, such that a distal end of the insertion tube, including a plurality of electrodes and a drug delivery channel, are positioned adjacent to the target site. At depicted step 6710, the method may include administering a treatment agent to the target site from a drug delivery device connected to the drug delivery channel. At depicted step 6715, the method may include delivering one or more electrical pulses to the electrodes to electroporate the tissue at the target site. At step 6720, the method may include administering a hemostatic agent to the target site from a drug delivery device connected to the drug delivery channel, which may use any of the drug delivery techniques described herein. At step 6725, the method may include removing the applicator and insertion device from the patient. In some embodiments, the applicator may include a piercing tip 130 such that the method may further include piercing one or more tissues of the patient prior to delivering the electrical impulse and/or treatment agents. In some embodiments, as described above, the drug and/or plasmid may be administered through any of a number of means, including IM, IT, and IV delivery.

In some embodiments, the hemostatic agent may be delivered locally or systemically according to any of the embodiments disclosed herein. In the above described method, a low voltage generator may be used, including the particular configurations described herein. The method may be performed with or without EIS.

[00201] With reference to FIG. 68, another example method of using embodiments of the electroporation system 10 described herein, which may or may not include an insertion device, is shown. In some embodiments, the method of FIG. 68 is used for treatment of one or more cancers. In some embodiments, the method of FIG. 68 is used to treat a tumor or other visceral lesion. At depicted step 6800, the method includes inserting an insertion tube of an applicator into the patient, such that a distal end of the insertion tube, including a plurality of electrodes and a drug delivery channel, are positioned adjacent to a target site. At depicted step 6805, the method includes administering a treatment agent to the target site from a drug delivery device connected to the drug delivery channel. At depicted step 6810, the method includes delivering one or more electrical pulses to the electrodes to electroporate the tissue at the target site. At step 6815, the method may include administering a hemostatic agent to the target site from a drug delivery device connected to the drug delivery channel, which may use any of the drug delivery techniques described herein. At step 6820, the method includes removing the applicator from the patient at step 6820. Steps 6805 and 6810 may occur simultaneously, or step 6805 may occur prior to step 6810. In some embodiments, the applicator may include a piercing tip 130 such that the method may further include piercing one or more tissues of the patient prior to delivering the electrical impulse and/or treatment agents. In some embodiments, as described above, the drug and/or plasmid may be administered through any of a number of means, including IM, IT, and IV delivery. In some embodiments, the hemostatic agent may be delivered locally or systemically according to any of the embodiments disclosed herein.

[00202] The methods, systems, and apparatus described herein may be used with a number of endoscopic procedures, including but not limited to procedures in the respiratory tract ( e.g ., rhinoscopy or bronchoscopy), the abdominal cavity, general soft tissue and/or bone, the gastrointestinal tract (e.g., enteroscopy, rectoscopy, colonoscopy, anoscopy, sigmoidoscopy, or esophagogastroduodenoscopy), the urinary system and in the cerebrum. Examples of the application of the method in these procedures is provided in greater detail below. It should be appreciated that in these and other procedures described throughout the disclosure, references to diseased tissue includes, but is not limited to, tumors, cancerous cells, and other lesions in general. Cancers treated may include soft tissue sarcomas. Tumors contemplated for treatment through the methods of the present disclosure include, for example, primary tumors, metastatic tumors, or both.

[00203] In some embodiments, the present disclosure relates to a method of treating diseased tissue (e.g., primary and/or metastatic tumors) in the respiratory tract. In some embodiments of the method, the lung may be accessed using bronchoscopy. In some embodiments, prior to performance of surgery, pre-operative planning may be performed to confirm the specific location of the diseased tissue and to perform applicator advancement path or endoscopic path planning. Pre-operative surgical planning may involve capturing images using cone beam computed tomography (CBCT) and using such images to generate a 3D model of the patient’s lungs. Other techniques may also be used to capture images, including computed tomography, magnetic resonance, positron emission tomography, fluoroscopy and x-rays. The image data taken from any number of the above modalities may be extrapolated to create the 3D model of the patient anatomy. An analysis of the 3D model is then performed to identify the location of the diseased tissue. Once identified, a surgical plan may be developed for access to the diseased tissue. Based on an identified target site, details of an approach to the site may be established. In some embodiments, pre-operative planning may involve other known approaches to identifying diseased tissue. For example, where the diseased tissue is closer to an orifice, a surgical plan may be established without the creation of a 3D model. In other examples, it may be sufficient to use one or more of the modalities for capturing images of the patient without analysis and extrapolation to identify a location of diseased tissue and to establish a path of access.

[00204] Turning to the performance of the bronchoscopy, in some embodiments, the patient is adjusted to a sitting or supine position. Then, the applicator is inserted into an endoscope or bronchoscope in preparation for advancement into the patient. In particular, the insertion tube of the applicator is inserted into the endoscope. The endoscope may be flexible or rigid. Using the established pre-operative surgical plan, the endoscope is inserted through the nose or mouth into and through the upper airway, trachea, and into the bronchial system, and then into, in some examples, the lungs. Visualization tools included with the endoscope are used to aid in reaching the diseased tissue at the target site. The endoscope is advanced until its distal tip is proximal to or contacts the target site. In some embodiments, the advancement of the endoscope may be monitored with a connected navigation system.

Where pre-operative planning includes the generation of a 3D model, additional images may be taken during the advancement steps at the discretion of the surgeon to make any adjustments based on actual conditions if evidence suggests that conditions have changed since the original images were taken to create the 3D model. In some embodiments, the visualization tools described herein may be used with embodiments of a separate drug delivery applicator ( e.g ., the separate drug delivery applicator 19 discussed herein) to facilitate identification of the injection site and alignment of the applicators (e.g., applicator 14 and separate applicator 19) for collocating delivery of the drug and electroporation.

[00205] With the distal end of the applicator located at the target site, electroporation and/or drug delivery may commence in a manner as described in any of the embodiments set forth herein. In some embodiments, electroporation and delivery of the treatment agent(s) may be simultaneous or otherwise occur at about the same time. In some embodiments, electroporation may commence prior to delivery of the treatment agent(s). In some embodiments, delivery of the treatment agent(s) is followed by electroporation.

[00206] In some examples, the bronchoscopy procedure described may be similarly employed in a rhinoscopy procedure or other procedure in the respiratory tract.

[00207] In some examples, the method of treating diseased tissue in the respiratory tract may be performed with the aid of robotics. For instance, the applicator may be used with a robotic system to perform the bronchoscopy. In particular, the applicator may be advanced through the body of the patient and/or the electrodes of the applicator may be deployed through control of the robotic device of the robotic system. To perform these functions, for example, an arm of the robotic device may be manipulated to rotate and position the applicator during the procedure. Similarly, the arm of the robotic device may be manipulated to control electricity flow into the applicator. In some examples, other steps of the method may also be aided by the use of the robotic system.

[00208] In some embodiments, the present disclosure relates to a method of treating diseased tissue in the abdominal cavity. In some embodiments, the method may commence with pre-operative surgical planning as described in detail above. With a location of the diseased tissue and a path to access the diseased tissue identified, access to the target site and treatment may commence. In preparation for entry, the applicator may be inserted into an endoscope, though the endoscope may be positioned at least partially into the patient prior to inserting the applicator therethrough.

[00209] In some embodiments, the applicator used includes a sharp tip, such as tip 130 on applicator 110, for example. Initially, the endoscope is positioned through a mouth of the patient, through the esophagus and into the stomach. From within the stomach, the applicator is advanced to a stomach wall to create a gastric opening using tip 130, thereby advancing the endoscope with applicator therein into the peritoneal cavity. Alternatively, a standard trocar or other instrument may be used to pierce the stomach wall. Visualization aids accompanying the endoscope, in conjunction with optional navigation system and imaging information may then be used to direct the endoscope and applicator to the target site on a wall of the peritoneal cavity under guided imagery.

[00210] With the distal end of the endoscope located at the target site, electroporation and/or drug delivery may commence in a manner as described in any of the embodiments set forth herein. In some embodiments, electroporation and delivery of the treatment agent(s) may be simultaneous or otherwise occur at about the same time. In some embodiments, electroporation may commence prior to delivery of the treatment agent(s). In some embodiments, delivery of the treatment agent(s) is followed by electroporation.

[00211] In another embodiment, a method for treating diseased tissue in the abdomen may be performed using a laparoscope, whereby one or more keyhole cuts may be formed in the patient through which a laparoscope and the applicator are passed and navigated to the target tissue. As discussed above, drug delivery can be performed using the applicator, or alternatively, a separate instrument can be used to deliver the treatment agent(s) to the target tissue. At least one additional cannula may be used to provide a passageway for the applicator and/or drug delivery device to the target tissue. Typically, rigid cannula(e) are used, and thus, an applicator with a rigid insertion tube may also be used.

[00212] In some examples, the method of treating diseased tissue in the abdomen may be performed with the aid of robotics. For instance, the applicator may be used with a robotic system to perform the procedure. In particular, the applicator may be advanced through the body of the patient and/or the electrodes of the applicator may be deployed through control of the robotic device of the robotic system. To perform these functions, for example, an arm of the robotic device may be manipulated to rotate and position the applicator during the procedure. Similarly, the arm of the robotic device may be manipulated to control electricity flow into the applicator. In some examples, other steps of the method may also be aided by the use of the robotic system.

[00213] In some embodiments, the present disclosure relates to a method of treating diseased tissue in the gastrointestinal tract, such as in the pancreas. In some embodiments of this method, an ultrasound endoscope is used with the applicator inserted therein. The ultrasound endoscope uses high frequency sound waves to produce detailed images of anatomy, including lining and walls of the stomach and pancreas. As described above, in some embodiments, pre-operative surgical planning may be performed to identify a specific location of the diseased tissue and to evaluate the intended insertion path for the applicator and/or endoscope. Once ready for surgery, the applicator is inserted into the ultrasound endoscope, though the endoscope may be positioned at least partially into the patient prior to inserting the applicator therethrough. Note than an ultrasound endoscope may also be utilized in the other methods described herein in which an endoscope or other endoscopic- type instruments, such as bronchoscopes and laparoscopes, are used.

[00214] To access the diseased tissue target site, the ultrasound endoscope is inserted through the mouth and into the stomach. Using the images generated through the ultrasound as well as the information harnessed through pre-surgical planning, if used, the endoscope is manipulated within the stomach so that its distal tip faces a stomach wall abutting the portion of the pancreas having the diseased tissue. Then, the applicator is advanced from the endoscope so that a pointed tip on the applicator may penetrate the stomach wall and thereby reach a location abutting the target site on the pancreas. Alternatively, a standard trocar or other instrument may be used to pierce the stomach wall. In circumstances where the target site on the pancreas does not abut the stomach, the endoscope may be guided further once in the peritoneal cavity to direct the applicator to the target site. Additionally, visualization aids may accompany the endoscope, along with an optional navigation system and imaging information from pre-operative planning, to aid in the direction of the applicator to the target site.

[00215] In some examples, and as described elsewhere in the disclosure, an endoscope can be positioned through the mouth into the stomach/small intestine, where the applicator, with a flexible body, can be guided into pancreatic lesions, for sequential plasmid injection and electroporation. The flexible body ( e.g ., insertion tube 15) may have a length of approximately 100 cm to allow for navigation toward the target lesions via an endoscope or laparoscope, depending on the specific application and/or tumor indication.

[00216] With the distal end of the endoscope located at the target site, electroporation and/or drug delivery may commence in a manner as described in any of the embodiments set forth herein. In some embodiments, electroporation and delivery of the treatment agent(s) may be simultaneous or otherwise occur at about the same time. In some embodiments, electroporation may commence prior to delivery of the treatment agent. In some embodiments, delivery of the treatment agent is followed by electroporation. Upon completion of the electroporation, the applicator, and as applicable guiding device such as an endoscope, are removed and, when applicable, the stomach incision is closed as appropriate. [00217] It is further contemplated that the procedure described above for the pancreas may also be similarly performed for a colonoscopy.

[00218] In some examples, the method of treating diseased tissue in the gastrointestinal tract may be performed with the aid of robotics. For instance, the applicator may be used with a robotic system to perform a procedure to reach the pancreas with an ultrasound endoscope or the like. In particular, the applicator may be advanced through the body of the patient and/or the electrodes of the applicator may be deployed through control of the robotic device of the robotic system. To perform these functions, for example, an arm of the robotic device may be manipulated to rotate and position the applicator during the procedure. Similarly, the arm of the robotic device may be manipulated to control electricity flow into the applicator. In some examples, other steps of the method may also be aided by the use of the robotic system.

[00219] In some embodiments, the present disclosure relates to a method of treating diseased tissue in the urinary system, such as in the urethra or the bladder. In some embodiments an endoscope is used with an applicator inserted therein. In some embodiments, the endoscope is rigid, while in others, it is flexible. In some embodiments, a urethral catheter is used with an applicator. In some embodiments, an applicator is used by itself without any guiding device. As described above, in some embodiments, pre-operative surgical planning may be performed to identify a specific location of the diseased tissue and to evaluate the intended insertion path for the applicator and/or endoscope. Once ready for surgery, the applicator is inserted into the endoscope or urethral catheter, or if the applicator is being used on its own, it is ready for use on its own. As with the other exemplary methods discussed above, the applicator need not be positioned in the endoscope or urethral catheter prior to insertion of either access instrument into the patient (assuming an access instrument of some type is being used at all).

[00220] In some embodiments, the endoscope (or urethral catheter) is advanced directly into the urethra from outside the patient and the tip of the endoscope is directed to the diseased tissue. In some embodiments, the endoscope is advanced into the urethra from outside the patient and from the urethra into the bladder. From within the bladder, the endoscope tip is directed to a diseased tissue on the bladder. Whether in the urethra or bladder, the applicator is advanced from within the endoscope so that the applicator is in position for the electroporation procedure. Additionally, visualization aids may accompany the endoscope, along with an optional navigation system and imaging information from pre operative planning, to aid in the advancement of the applicator to the diseased tissue. [00221] With the distal end of the endoscope located at the target site, electroporation and/or drug delivery may commence in a manner as described in any of the embodiments set forth herein. In some embodiments, electroporation and delivery of the treatment agent(s) may be simultaneous or otherwise occur at about the same time. In some embodiments, electroporation may commence prior to delivery of the treatment agent(s). In some embodiments, delivery of the treatment agent(s) is followed by electroporation.

[00222] In some examples, the method of treating diseased tissue in the urinary system may be performed with the aid of robotics. For instance, the applicator may be used with a robotic system to perform the procedure. In particular, the applicator may be advanced through the body of the patient and/or the electrodes of the applicator may be deployed through control of the robotic device of the robotic system. To perform these functions, for example, an arm of the robotic device may be manipulated to rotate and position the applicator during the procedure. Similarly, the arm of the robotic device may be manipulated to control electricity flow into the applicator. In some examples, other steps of the method may also be aided by the use of the robotic system.

[00223] In some embodiments, the present disclosure relates to a method of treating diseased tissue in the brain through a neurosurgical procedure. In some examples, the procedure may be used to treat various types of tumors in the brain or in the neurological system more generally. In some embodiments an endoscope is used with an applicator inserted therethrough. In some embodiments, a catheter is used with an applicator. In some embodiments, an applicator is used by itself without any access device. As described above, in some embodiments, pre-operative surgical planning may be performed to identify a specific location of the diseased tissue and to evaluate the intended insertion path for the applicator and/or endoscope.

[00224] In some embodiments, an endovascular approach to the diseased tissue in the brain is used. This approach may be used to treat a glioblastoma, glioblastoma multiforme, or the like, for instance. In one example, the applicator, disposed in a catheter or an endoscope, is introduced percutaneously into the body of the patient through the femoral artery, then steered superiorly through the aorta, vena cava, carotid or vertebral artery. Other access points are also suitable for an approach into the cerebrum. Alternatively, the catheter or endoscope is positioned in the patient’s vasculature first, prior to positioning the applicator therein. To determine where to steer the applicator from the carotid or vertebral artery, the location of the diseased tissue is compared with the location of the applicator. The applicator is then advanced through the appropriate blood vessels of the brain. In some unique circumstances, it may be possible to further steer the applicator through intra-cranial blood vessels if necessary. However, prior to doing so, the surgeon will assess whether such access is feasible by comparing an outer diameter of the endoscope or catheter compared with the intra-cranial blood vessels to be traversed. In some examples, the applicator may be configured to be advanceable relative to the endoscope or catheter, thereby reducing the minimum diameter necessary for access of the device for electroporation. Additionally, visualization aids may accompany the endoscope, along with an optional navigation system and imaging information from pre-operative planning, to aid in the advancement of the applicator to the diseased tissue. Once advancement of the applicator to the diseased tissue at the target site is complete, electroporation may be performed.

[00225] In some embodiments, areas around the brain may be accessed through the nose through a transsphenoidal procedure. This may be desirable when the diseased tissue is on or near the pituitary gland or when the diseased tissue is a tumor that grows from the dura (membrane surrounding the brain). Thus, the procedure may be used to treat, for example, pituitary adenoma, craniopharyngioma, rathke’s cleft cyst, meningioma and chordoma. In some examples, the applicator is disposed in an endoscope or a catheter and then advanced through the nose and the sphenoid sinus to reach the diseased tissue for the performance of electroporation. In some embodiments, a small incision may be made in one or more of the nasal septum, sphenoid sinus and the sella to reach the diseased tissue. A similar approach involving the creation of small holes in the nasal area may also be used to access the diseased tissue through the mouth. In some examples of the above embodiments, a microscope may also be used to complement the applicator in a procedure.

[00226] In each of the described methods of accessing tissue in and around the cerebrum, once the distal end of the applicator is positioned at the target site, electroporation and/or drug delivery may commence in a manner as described in any of the embodiments set forth herein. In some embodiments, electroporation and delivery of the treatment agent(s) may be simultaneous or otherwise occur at about the same time. In some embodiments, electroporation may commence prior to delivery of the treatment agent(s). In some embodiments, delivery of the treatment agent(s) is followed by electroporation.

[00227] In some examples, the method of treating diseased tissue in the cerebrum may be performed with the aid of robotics. For instance, the applicator may be used with a robotic system to perform the procedure. In particular, the applicator may be advanced through the body of the patient and/or the electrodes of the applicator may be deployed through control of the robotic device of the robotic system. To perform these functions, for example, an arm of the robotic device may be manipulated to rotate and position the applicator during the procedure. Similarly, the arm of the robotic device may be manipulated to control electricity flow into the applicator. In some examples, other steps of the method may also be aided by the use of the robotic system.

[00228] The above described methods demonstrate that the electroporation technology and systems described herein may be employed in a wide variety of surgical applications.

The specific examples outlined are intended to demonstrate how the system may be employed in specific applications, and in no way are intended to be limiting in any way. To be clear, further to use of the system to access diseased tissue with the applicator alone, with an endoscope, or with a catheter, it is further contemplated that a trocar may be used to access a target site to perform electroporation. A trocar may be advantageous to provide direct access into bone malignancies, for example, such as primary or secondary sarcomas.

[00229] In some embodiments, the methods described herein may be used in combination with tissue imaging procedures in addition to those described elsewhere in the application. For example, procedures including fluorescence imaging, white light imaging, or a combination thereof may be used. In some examples, fluorescence imaging may employ the use of an agent or a dye. Well known examples of such agents include indocyanine green. Such fluorescence imaging agent and visualization capabilities may be used to direct the electroporation applicator to the target tissue. In some instances, the blood flow through a tumor may cause an incidence of dye in the tumor, illuminating the tumor under visualization. Such a process may increase the effectiveness of electroporation as the operator can see and thus treat areas of the tumor which may have not been seen under normal white light visualization.

[00230] In some embodiments, the methods, systems, and apparatus described herein may be used with other surgical procedures, including laparoscopies. The methods, systems, and apparatus described herein described herein may also be used with a number of treatments including but not limited to gene therapies ( e.g ., plasmid therapies) or drug treatments for any of a number of cancers and other diseases.

[00231] Referring back to FIG. 1, in some embodiments, the electrodes 100 may be used to detect an impedance of the body tissue between the electrodes at the electroporation site. In particular, the electrical responses of a tissue may be measured over a range of interrogation frequencies transmitted through the electrodes via electrochemical impedance spectroscopy. The collected data may then be fit to equivalent circuit models to determine the electrical properties of the tissue. In some embodiments, the electrical pulses of any of the methods and apparatus disclosed herein may be supplied by a low-voltage generator. [00232] The controller 24 that controls the electroporation process may interface with the generator 12 to provide a feedback loop that fine tunes the generator output to a desired level based on the impedance detected at the electrodes. This process may be implemented for any of the electrode and electroporation systems, methods, and apparatus discussed herein.

[00233] Accordingly, blocks of the flowcharts support combinations of means for performing the specified functions and combinations of operations for performing the specified functions. It will also be understood that one or more blocks of the flowcharts, and combinations of blocks in the flowcharts, can be implemented by special purpose hardware- based computer systems which perform the specified functions, or combinations of special purpose hardware and computer instructions.

Methods of Treatment

[00234] The electroporation devices described herein may be used in therapeutic treatments and in the delivery of treatment agents. In some embodiments, therapeutic treatments include electrotherapy, also referred to herein as electroporation therapy (EPT), using the described apparatuses for the delivery of one or more treatment agents ( e.g ., molecules) to a cell, group of cells, or tissue, for performing electroporation on the cell, group of cells, or tissue, and delivering a hemostatic agent to the cell, group of cells, or tissue. In some embodiments, the molecule or treatment agent is a drug (i.e., active pharmaceutical ingredient). Combining any of the treatment agent(s) discussed herein or otherwise generally known in the art with EPT, as discussed herein, may provide an effective treatment even in patients who did not respond to the treatment agent(s) on their own. In some embodiments, the drug is a small molecule. In some embodiments, the drug is a macromolecule. A drug can be, but is not limited to, a chemotherapeutic agent. A macromolecule can be, but is not limited to, a chemotherapeutic agent, nucleic acid (such as, but not limited to, polynucleotide, oligonucleotide, DNA, cDNA, RNA, peptide nucleic acid, antisense oligonucleotides, siRNA, miRNA, ribozyme, plasmid, and expression vector), and polypeptide (such as, but not limited to, peptide, antibody, and protein). In some embodiments, therapeutic treatments include delivery of a therapeutic electric pulse to a cell, group of cells, or tissue using any of the described electroporation devices. The cell, group of cells, or tissue may be, but is not limited to, a tumor cell or tumor tissue.

[00235] Drugs or treatment agents contemplated for use with the methods include chemotherapeutic agents having an antitumor or cytotoxic effect. A drug can be an exogenous agent or an endogenous agent. In some embodiments, the drug is a small molecule exogenous agent. Small molecule exogenous agent agents include, but are not limited to, bleomycin, neocarcinostatin, suramin, doxorubicin, carboplatin, taxol, mitomycin C and cisplatin. Other chemotherapeutic agents will be known to those of skill in the art (see, for example, The Merck Index). In some embodiments, the drug is a membrane-acting agents. "Membrane acting" agents act primarily by damaging the cell membrane. Non-limiting examples of membrane-acting agents include, N-alkylmelamide and para-chloro mercury benzoate. In some embodiments, the drug is a cytokine, chemokine, lymphokine, or hormone. In some embodiments, the drug is a nucleic acid. In some embodiments, the nucleic acid encodes one or more cytokines, chemokines, lymphokines, therapeutic polypeptide, adjuvant, or a combination thereof.

[00236] The molecule or treatment agent can be administered to a subj ect before, during, or after administration of the electric pulse. The molecule can be administered at or near the cell, group of cells or tissue in a patient. In some embodiments, the molecule can be co-localized with the electric pulse using an applicator having electrodes and a drug delivery channel extending therethrough ( e.g ., applicator 110; electrodes 100, 200, 400, 500, 600; and drug delivery channel 18 shown in FIGS. 47-66). The chemical composition of the treatment agent will dictate the most appropriate time to administer the agent in relation to the administration of the electric pulse. For example, while not wanting to be bound by a particular theory, it is believed that a drug having a low isoelectric point (e.g., neocarcinostatin, IEP=3.78), would likely be more effective if administered post electroporation in order to avoid electrostatic interaction of the highly charged drug within the field. Further, such drugs as bleomycin, which have a very negative log P, (P being the partition coefficient between octanol and water), are very large in size (MW=1400), and are hydrophilic, thereby associating closely with the lipid membrane, diffuse very slowly into a tumor cell and are typically administered prior to or substantially simultaneous with the electric pulse. In addition, certain treatment agents may require modification in order to allow more efficient entry into the cell. For example, an agent such as taxol can be modified to increase solubility in water which would allow more efficient entry into the cell. In some embodiments, electroporation facilitates entry of the molecule into a cell by creating pores in the cell membrane.

[00237] In some embodiments, the molecule or treatment agent is delivered to modulate expression of a gene. The term "modulate" envisions the decrease (suppression) or increase (stimulation) of expression of a gene. Where a cell proliferative disorder is associated with the expression of a gene, nucleic acid sequences that interfere with the gene's expression at the translational level can be used. In some embodiments, one or more antisense nucleic acids, ribozymes, siRNAs, miRNA, triplex agents, or the like are delivered via electroporation to block transcription or translation of a specific mRNA. In some embodiments, a nucleic acid is delivered to express an RNA or polypeptide. The nucleic acid can be recombinant, single stranded or double stranded, DNA or RNA or a combination of DNA and RNA, circular or linear, and/or supercoiled or relaxed. The nucleic acid can also be associated with one or more of proteins, lipids, virus, viral vector, chimeric virus, or viral particle. The nucleic acid can also be naked. A virus can be, but is not limited, adenovirus, herpes virus, vaccinia, DNA virus, RNA virus, retrovirus, murine retrovirus, avian retrovirus, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), Rous Sarcoma Virus (RSV), gibbon ape leukemia virus (GaLV) can be utilized. Similarly a viral vector, chimeric virus, and/or viral particle can be derived from any of the above described viruses.

[00238] In embodiments, an effective dose of a hemostatic agent is administered to the subject after administration of the electric pulse. In embodiments, an effective dose of a hemostatic agent is administered to the subject after the effective dose of the at least one treatment agent. In embodiments, an effective dose of a hemostatic agent is administered to the subject at the same time as the effective dose of the at least one treatment agent. The hemostatic agent may be delivered via any of the drug delivery mechanisms disclosed herein, including via a drug delivery device 16 through a drug delivery channel 18 of an applicator or via a separate drug delivery applicator 19, or systemically via IV, injection or the like. For example, in some embodiments, any of a number of means, including IM, IT, and IV delivery may be used for the hemostatic agent to be delivered to the target site. In some embodiments, multiple delivery devices may be used depending upon the number of treatment agents, hemostatic agents, and other therapies being delivered.

Therapeutic polypeptides [00239] Therapeutic polypeptides (one type of treatment agent listed above) include, but are not limited to, immunomodulatory agents, biological response modifiers, co stimulatory molecule, metabolic enzymes and proteins, antibodies, checkpoint inhibitors, and adjuvants.

[00240] The term "immunomodulatory agents" is meant to encompass substances which are involved in modifying an immune response. Examples of immune response modifiers include, but are not limited to, cytokines, chemokines, lymphokines, and antigen binding polypeptides. Lymphokines can be, but not limited to, tumor necrosis factor, interleukins (IL, such as, but not limited to IL-1, IL-2, IL-3, IL-12, IL-15), lymphotoxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, and alpha-interferon, beta-interferon, gamma-interferon, and their subtypes. In some embodiments, the immune response modifier comprises a nucleic acid encoding one or more cytokines, chemokines, lymphokines or subunits of cytokines, chemokines, and lymphokines. In some embodiments, the immunomodulatory agent is an immune stimulator. Non-limiting examples of immune stimulators include, IL-33, flagellin, IL-10 receptor, sting receptor,

IRF3. The term "cytokine" is used as a generic name for a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. As used herein an "immunostimulatory cytokine" includes cytokines that mediate or enhance the immune response to a foreign antigen, including viral, bacterial, or tumor antigens. Immunostimulatory cytokines include, but are not limited to, TNFa, IL-1, IL-10, IL-12, IL-12 p35, IL-12 p40, IL-15, IL-15Ra, IL-23, IL-27, IFNa, IFNp, IFNy, IL-2, IL-4, IL-5, IL-7, IL-9, IL-21, and TGFp. In some embodiments, the immunostimulatory cytokine is a nucleic acid encoding one or more of TNFa, IL-1, IL-10, IL-12, IL-12 p35, IL-12 p40, IL-15, IL-15Ra, IL-23, IL-27, IFNa, IFNp, IFNy, IL-2, IL-4, IL-5, IL-7, IL-9, IL-21, and TGFp.

[00241] Another treatment agent, a "co-stimulator," refers to any of a group of immune cell surface receptor/ligands which engage between T cells and antigen presenting cells and generate a stimulatory signal in T cells which combines with the stimulatory signal (i.e., "co stimulation") in T cells that results from T cell receptor ("TCR") recognition of antigen on antigen presenting cells. Co-stimulatory activation can be measured for T cells by the production of cytokines. As used herein the term "co-stimulatory molecules" includes a soluble co-stimulator or agonists of co-stimulators. Co-stimulatory molecules include, but are not limited to, agonists of GITR, CD137, CD134, CD40L, CD27, and the like. Co-stimulator agonists include, but are not limited to, agonistic antibodies, co-stimulator ligands, including multimeric soluble and transmembrane co-stimulator ligands, co-stimulator ligand peptides, co-stimulator ligand mimetics, and other molecules that engage and induce biological activity of a co-stimulator. In some embodiments, a soluble co-stimulatory molecules derived from an antigen presenting cell may be, but is not limited to, GITR-L, CD137-L, CD134-L (a.k.a. OX40-L), CD40, CD28. Agonists of co-stimulatory molecules may be soluble molecules such as soluble GITR-L, which comprises at least the extracellular domain (ECD) of GITR- L. The soluble form of a co-stimulatory molecule derived from an antigen presenting cell retains the ability of the native co-stimulatory molecule to bind to its cognate receptor/ligand on T cells and stimulate T cell activation. Other co-stimulatory molecules will similarly lack transmembrane and intracellular domains, but are capable of binding to their binding partners and eliciting a biological effect. In some embodiments, for intratumoral delivery by electroporation, the co-stimulator molecule is encoded in an expression vector that is expressed in a tumor cell. In some embodiments, the co-stimulatory molecule is a nucleic acid encoding one or more of GITR, GITR-L, CD137, CD137-L, CD134, CD134-L, CD40, CD40L, CD27, and D28, and the like or a functional fragment thereof. A co-stimulatory molecule includes a molecule that has biological function as co-stimulatory molecule and shares at least 80% amino acid sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity GITR, GITR-L, CD137, CD137-L,

CD 134, CD134-L, CD40, CD40L, CD27, or D28 or a functional fragment thereof. In some embodiments, a co-stimulatory agonist can be in the form of antibodies or antibody fragments, both of which can be encoded in a plasmid and delivered to the tumor by electroporation.

[00242] Other treatment agents, such as metabolic enzymes and proteins, include, but are not limited to, antiangiogenesis compounds. Antiangiogenesis compounds include, but are not limited to, Factor VIII and Factor IX. In some embodiments, the metabolic enzyme or protein comprises a nucleic acid encoding one or more metabolic enzyme or protein comprises or functional fragments thereof.

[00243] The term "antibody" as used herein is another treatment agent including immunoglobulins, which are the product of B cells and variants thereof as well as the T cell receptor (TcR), which is the product of T cells, and variants thereof. An immunoglobulin is a protein comprising one or more polypeptides substantially encoded by the immunoglobulin kappa and lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Also subclasses of the heavy chain are known. For example, IgG heavy chains in humans can be any of IgGl, IgG2, IgG3, and IgG4 subclass. Antibodies exist as full-length intact antibodies or as a number of well-characterized fragments thereof. Antibody fragments can be produced by the modification of whole antibodies or synthesized de novo or antibodies and fragments obtained by using recombinant DNA methodologies. Antibody fragments include, but are not limited to, F(ab')2, and Fab', scFv, and ByTE fragments. In some embodiments, antibody comprises a nucleic acid encoding one or more antibodies or antibody fragments.

[00244] An "adjuvant," yet another treatment agent, is a substance that enhances an immune response to an antigen. In some embodiments, adjuvants include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral salts such as aluminum hydroxide or aluminum phosphate, various cytokines, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. In some embodiments, an adjuvant is or comprised keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, cholera toxin or functional fragments thereof. In some embodiments, an adjuvant is or comprises Granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand. LAMP1, calreticulin, human heat shock protein 96, CSF Receptor 1 or a functional fragment thereof. In some embodiments, an adjuvant comprises a nucleic acid encoding one or more adjuvants or adjuvant fragments (i.e., genetic adjuvants). In some embodiments, a genetic adjuvant is fused to an antigen. An antigen can be, but is not limited to, a tumor antigen, shared tumor antigen or viral antigen. Non-limiting examples of antigens include, NY-ESO-1 or a fragment thereof, MAGE-A1, MAGE-A2, MAGE- A3, MAGE- A 10, SSX-2, MART-1, Tyrosinase, GplOO, Survivin, hTERT, PRS pan-DR, B7-H6, HPV-7, HPV16 E6/E7, HPV11 E6, HPV6b/l 1 E7, HCV-NS3, Influenza HA, Influenza NA, and polyomavirus. In some embodiments, a genetic adjuvant is fused to a cytokine, or co-stimulatory molecule.

[00245] Another treatment agent, an immune checkpoint molecule, refers to any of a group of immune cell surface receptor/ligands which induce T cell dysfunction or apoptosis. These immune inhibitory targets attenuate excessive immune reactions and ensure self tolerance. As used herein "checkpoint inhibitor" comprises a molecules that prevent immune suppression by blocking the effects of an immune checkpoint molecule. Checkpoint inhibitors include, but are not limited to, antibodies and antibody fragments, nanobodies, diabodies, soluble binding partners of checkpoint molecules, small molecule therapeutics, peptide antagonists, etc. In some embodiments, a checkpoint inhibitor can be, but is not limited to, CTLA-4 antagonist, PD-1 antagonist, PD-L1 antagonist, LAG-3 antagonist, TIM3 antagonist, KIR antagonist, BTLA antagonist, A2aR antagonist, HVEM antagonist. In some embodiments the checkpoint inhibitor is selected from the group comprising: nivolumab (ONO-4538/BMS-936558, MDX1 106, OPDIVO), pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011), and MPDL3280A (ROCHE). In some embodiments, a checkpoint inhibitor polypeptide can be encoded by a nucleic acid that is delivery to a tumor.

Expression vectors

[00246] Any of the described polypeptides may be encoded on nucleic acid, to form yet another treatment agent. The nucleic acid can be, but is not limited to, an expression vector or plasmid. The term “plasmid” or “vector” includes any known delivery vector including a bacterial delivery vector, a viral vector delivery vector, an episomal plasmid, an integrative plasmid, or a phage vector. The term “vector” refers to a construct which is capable of expressing one or more polypeptides in a cell.

[00247] An encoded polypeptide may be linked, in an expression vector to a sequence encoding a second polypeptide. In some embodiments, an expression vector encodes a fusion protein. The term “fusion protein” refers to a protein comprising two or more polypeptides linked together by peptide bonds or other chemical bonds. In some embodiments, a fusion protein is be recombinantly expressed as a single-chain polypeptide containing the two polypeptides. The two or more polypeptides can be linked directly or via a linker comprising one or more amino acids.

[00248] In some embodiments, the nucleic acid (i.e., expression vector) encodes two polypeptides expressed from a single promoter, with an intervening exon skipping motif that allows both polypeptides to be expressed from a single polycistronic message. In some embodiments, the expression vector comprises:

P-A-T-C, P-C-T-A, or P-A-T-B wherein P is a promoter, A, B, and C are nucleic acid sequences encoding therapeutic polypeptides, and T is a translation modification element. A translation modification element can be, but is not limited to, an internal ribosome entry site (IRES) and a ribosomal skipping modulators, such as, but not limited to P2A, T2A, E2A or F2A. In some embodiments, A and B comprise nucleic acid sequences encoding immunomodulatory molecules. In some embodiments, A and B encode cytokines or cytokine subunits, such as, but not limited to, IL- 12 p35 and IL-12 p40.

[00249] In some embodiments, the nucleic acid (i.e., expression vector) encodes three polypeptides expressed from a single promoter, with intervening ribosome skipping motifs to allow all three proteins to be expressed from a single polycistronic message. In some embodiments, the expression vector comprises:

P-A-T-B-T-C or P-C-T-A-T-B wherein P is a promoter, A, B, and C are nucleic acid sequences encoding therapeutic polypeptides, and T is a translation modification element. A translation modification element includes, but is not limited to, an internal ribosome entry site (IRES) and a ribosomal skipping modulators, such as, but not limited to P2A, T2A, E2A or F2A. In some embodiments, A and B comprise nucleic acid sequences encoding immunomodulatory molecules and/or co-stimulatory molecules, or subunits thereof. In some embodiments, A and B encode chains of a heterodimeric cytokine. In some embodiments, C comprises a nucleic acid sequence encoding a costimulatory molecule, genetic adjuvant, antigen, a genetic adjuvant-antigen fusion polypeptide, chemokine, or antigen binding polypeptide.

Chemokines include, but are not limited to CXCL9. An antigen binding polypeptide can be, but is not limited to, a scFv. A scFv can be, but is not limited to, an anti-CD3 scFv and an anti-CTLA-4 scFv.

[00250] The promoter can be, but is not limited to, human CMV promoter, simian CMV promoter, SV-40 promoter, mPGK promoter, and b-Actin promoter.

[00251] In some embodiments, A encodes an IL-12 p35, IL-23pl9, EBI3, or IL-15, and B encodes an IL-12 p40, IL-27p28, or IL-15Ra.

[00252] In some embodiments, the genetic adjuvant comprises Flt3 ligand; LAMP-1; Calreticulin; Human heat shock protein 96; GM-CSF; and CSF Receptor 1.

[00253] In some embodiments, the antigen comprises: NYESO-1, OVA, RNEU, MAGE-A1, MAGE-A2, Mage-AlO, SSX-2, Melan-A, MART-1, Tyr, GplOO, LAGE-1, Survivin, PRS pan-DR, CEA peptide CAP-1, OVA, HCV-NS3, and an HPV vaccine peptide. [00254] The IL-12 p35 and IL-12 p40 polypeptide may be mouse or human IL-12 p35 and IL-12 p40.

[00255] In some embodiments P is a CMV promoter, A encodes an IL-12 p35 polypeptide, T is an IRES and B encodes an IL-12 p40 polypeptide.

[00256] In some embodiments P is a CMV promoter, A encodes an IL-12 p35 polypeptide, T is P2A element, and B encodes an IL-12 p40 polypeptide. [00257] In some embodiments P is a CMV promoter, A encodes a human IL-12 p35 (h IL-12 p35) polypeptide, T is an IRES and B encodes a human IL-12 p40 (hIL-12 p40) polypeptide.

[00258] In some embodiments P is a CMV promoter, A encodes a human IL-12 p35 polypeptide, T is P2A element, and B encodes a human IL-12 p40 polypeptide.

[00259] In some embodiments, A encodes an IL-12 p35, B encodes an IL-12 p40 polypeptide and C encodes a co-stimulatory polypeptide.

[00260] In some embodiments, A encodes an IL-12 p35, B encodes an IL-12 p40 polypeptide and C encodes aNY-ES01-Flt3L or Flt3L-NY-ES01 fusion polypeptide.

[00261] In some embodiments, A encodes a hIL-12 p35 polypeptide, T is a P2A element, B encodes a hIL-12 p40 polypeptide and C encodes a FLT3L-NYES01 fusion polypeptide.

[00262] In some embodiments, A encodes a hIL-12 p35 polypeptide, T is an IRES element, B encodes a hIL-12 p40 polypeptide and C encodes a FLT3L-NYES01 fusion polypeptide.

[00263] In some embodiments, P is a CMV promoter, A encodes a hIL-12 p35 polypeptide, T is a P2A element, B encodes a hIL-12 p40 polypeptide and C encodes a FLT3L-NYES01 fusion polypeptide.

[00264] In some embodiments, P is a CMV promoter, A encodes a hIL-12 p35 polypeptide, T is an IRES element, B encodes a hIL-12 p40 polypeptide and C encodes a FLT3L-NYES01 fusion polypeptide.

[00265] In some embodiments, A encodes an IL-12 p35, B encodes an IL-12 p40 polypeptide and C encodes a polypeptide comprising an anti-CD3 scFv. In some embodiments, A encodes a hIL-12 p35 polypeptide, T is a P2A element, B encodes a hIL-12 p40 polypeptide and C encodes a polypeptide comprising an anti-CD3 scFv. In some embodiments, A encodes a hIL-12 p35 polypeptide, T is an IRES element, B encodes a hlL- 12 p40 polypeptide and C encodes a polypeptide comprising an anti-CD3 scFv. In some embodiments, P is a CMV promoter, A encodes a hIL-12 p35 polypeptide, T is a P2A element, B encodes a hIL-12 p40 polypeptide and C encodes a polypeptide comprising an anti-CD3 scFv. In some embodiments, P is a CMV promoter, A encodes a hIL-12 p35 polypeptide, T is an IRES element, B encodes a hIL-12 p40 polypeptide and C encodes a polypeptide comprising an anti-CD3 scFv.

[00266] In some embodiments, A encodes an IL-12 p35, B encodes an IL-12 p40 polypeptide and C encodes a CXCL9. In some embodiments, A encodes a hIL-12 p35 polypeptide, T is a P2A element, B encodes a hIL-12 p40 polypeptide and C encodes a CXCL9. In some embodiments, A encodes a hIL-12 p35 polypeptide, T is an IRES element,

B encodes a hIL-12 p40 polypeptide and C encodes a CXCL9. In some embodiments, P is a CMV promoter, A encodes a hIL-12 p35 polypeptide, T is a P2A element, B encodes a hlL- 12 p40 polypeptide and C encodes a CXCL9. In some embodiments, P is a CMV promoter, A encodes a hIL-12 p35 polypeptide, T is an IRES element, B encodes a hIL-12 p40 polypeptide and C encodes a CXCL9.

[00267] In some embodiments, A encodes an IL-12 p35, B encodes an IL-12 p40 polypeptide and C encodes a CTLA-4 scFv. In some embodiments, A encodes a hIL-12 p35 polypeptide, T is a P2A element, B encodes a hIL-12 p40 polypeptide and C encodes a CTLA-4 scFv. In some embodiments, A encodes a hIL-12 p35 polypeptide, T is an IRES element, B encodes a hIL-12 p40 polypeptide and C encodes a CTLA-4 scFv. In some embodiments, P is a CMV promoter, A encodes a hIL-12 p35 polypeptide, T is a P2A element, B encodes a hIL-12 p40 polypeptide and C encodes a CTLA-4 scFv. In some embodiments, P is a CMV promoter, A encodes a hIL-12 p35 polypeptide, T is an IRES element, B encodes a hIL-12 p40 polypeptide and C encodes a CTLA-4 scFv.

[00268] Described are methods for the treatment of malignancies, wherein the administration of a plasmid or expression vector encoding one or more therapeutic polypeptides, in combination with electroporation has a therapeutic effect on lesions ( e.g ., primary or secondary tumors). Also described are methods for the treatment of malignancies, wherein the administration of a plasmid or expression vector encoding one or more therapeutic polypeptides, in combination with electroporation has a therapeutic effect on primary tumors as well as distant tumors and metastases. In some embodiments, the plasmid or expression vector encodes one or more of immunomodulatory agents, biological response modifiers, co-stimulatory molecule, metabolic enzymes and proteins, antibodies, checkpoint inhibitors, and/or adjuvants.

[00269] In some embodiments, the plasmid or expression vector encodes at least one immunostimulatory cytokine, chosen from IL-12, IL-15, and a combination of IL-12 and IL- 15.

[00270] In some embodiments, the plasmid or expression vector encodes a co stimulatory molecule. The co-stimulatory molecule can be, but is not limited to, GITR, CD137, CD134, CD40L, and CD27 agonists. Co-stimulatory agonists may be in the form of antibodies or antibody fragments, both of which can be encoded in a plasmid or expression vector and delivered to the tumor by electroporation. [00271] In some embodiments, the plasmid or expression vector encodes CXCL9, anti- CD3 scFv, or anti-CTLA-4 scFv.

[00272] Described are methods of treating a cancer comprising administering to a subject, by electroporation using the described electroporation systems and applicators, a therapeutically effective amount one or more of the described expression vectors. The one or more expression vectors are injected into a tumor, tumor microenvironment, tumor margin tissue, peritumoral region, lymph node, intradermal region, and/or muscle, and electroporation therapy is applied to the tumor, tumor microenvironment, tumor margin tissue, peritumoral region, lymph node, intradermal region, and/or muscle. The electroporation therapy may be applied by the described electroporation systems and/or applicators. The described expression vectors, when delivered using the described electroporation systems and applicators, result in local expression of the encoded proteins, leading to T cell recruitment and anti-tumor activity. In some embodiments, the methods also result in abscopal effects, i.e., regression of one or more untreated tumors. In some embodiments, regression includes debulking of a solid tumor.

[00273] In some embodiments, therapy is achieved by intratumoral delivery of plasmids or expression vectors encoding therapeutic polypeptides using electroporation.

Combination Therapy

[00274] In some embodiments, a therapeutic method includes a combination therapy.

A combination therapy comprises a combination of therapeutic molecules or treatments. Therapeutic treatments include, but are not limited to, electric pulse (i.e., electroporation), radiation, antibody therapy, and chemotherapy alone or in combination with the hemostatic agent treatments described herein. In some embodiments, administration of a combination therapy is achieved by electroporation alone or with electroporation and hemostatic agent. In some embodiments, administration of a combination therapy is achieved by a combination of electroporation and systemic delivery of the treatment agent and/or hemostatic agent. In some embodiments, a plasmid expressing one or more immunomodulatory peptides is administered by intratumoral electroporation and a checkpoint inhibitor is administered systemically. In some embodiments, the immunomodulatory peptide is IL-12, CD3 half-BiTE, CXCL9, or CTLA-4 scFv. In some embodiments, the one or more immunomodulatory peptides included IL-12 and CD3 half-BiTE, CXCL9, or CTLA-4 scFv. In some embodiments, administration of a combination therapy is achieved by a combination of electroporation and radiation. Therapeutic electroporation can be combined with, or administered with, one or more additional therapeutic treatments. The one or more additional therapeutics can be delivered by systemic delivery, intratumoral delivery, and/or radiation. The one or more additional therapeutics can be administered prior to, concurrent with, or subsequent to the electroporation therapy. In some embodiments, the therapeutics (i.e., a treatment agent) can be administered co-locally with the electric pulse or other treatment using an applicator having both electrodes and a drug delivery channel extending therethrough ( e.g ., applicator 110; electrodes 100, 200, 400, 500, 600; and drug delivery channel 18 shown in FIGS. 47- 66). In such embodiments, the generator may deliver an electrical pulse to the electrodes to electroporate target tissue to allow the treatment agent administered via the drug delivery channel to permeate and treat the target tissue.

[00275] In some embodiments, intratumoral electroporation of an expression vector encoding a co-stimulatory agonist can be administered with other therapeutic entities, all of which can be treatment agents. In some embodiments, the co-stimulatory molecule is combined with one or more of: CTLA4, cytokines (i.e. IL-12 or IL-2), tumor vaccine, small molecule drug, small molecule inhibitor, targeted radiation, anti -PD 1 antagonist, and anti- PDL1 antagonist Ab. A small molecule drug can be, but is not limited to, bleomycin, gemzar, cytozan, 5-fluoro-uracil, adriamycin, and other chemotherapeutic drug agent. A small molecule inhibitor can be, but is not limited to: Sunitinib, Imatinib, Vemurafenib, Bevacizumab, Cetuximb, rapamycin, Bortezomib, PI3K-AKT inhibitors, and IAP inhibitors. In some embodiments, the co-stimulatory molecule can is combined with one or more of: TLR agonists (e.g., Flagellin, CpG); IL-10 antagonists (e.g., anti-IL-10 or anti-IL-lOR antibodies); TGFp antagonists (e.g., anti-TGFp antibodies); PGE2 inhibitors; Cbl-b (E3 ligase) inhibitors; CD3 agonists; telomerase antagonists, and the like. In particular, various combinations of IL-12, IL-15/IL-15Ra, and/or GITR-L are contemplated. IL-12 and IL-15 have been shown to have synergistic anti-tumor effects. In some embodiments, two or more therapeutic polypeptides are delivered by intratumoral electroporation therapy. The therapeutic polypeptides can be expressed from a single expression vector or plasmid or multiple expression vectors or plasmids.

[00276] In some embodiments, combination therapy comprises administration of treatment agents including a checkpoint inhibitor and an immunostimulatory cytokine. In some embodiments, the checkpoint inhibitor is encoded on an expression vector and delivered to a tumor by electroporation therapy. In some embodiments, the immunostimulatory cytokine is encoded on an expression vector and delivered to a tumor by electroporation therapy. In some embodiments, the checkpoint inhibitor and the immunostimulatory cytokine are encoded on an expression vector, wherein expression is driven by a single promoter, and delivered to the cancerous tumor by electroporation therapy. In some embodiments, the checkpoint inhibitor is a systemically administered polypeptide and the immunostimulatory cytokine is administered by intratumoral electroporation of an expression vector encoding the immunostimulatory cytokine. In some embodiments, the expression vector encoding the immunostimulatory cytokine further encodes a CD3 half- BiTE, CXCL9 or CTLA-4 scFv.

[00277] Checkpoint inhibitor therapy may occur before, during, or after intratumoral delivery by electroporation of an immunostimulatory cytokine. A checkpoint inhibitor may be in the form of antibodies or antibody fragments, both of which can be encoded in a plasmid and delivered to the tumor by electroporation, or delivered as proteins/peptides systemically. In some embodiments, the checkpoint inhibitor is encoded on an expression vector and delivered to the tumor by electroporation therapy. In some embodiments, the checkpoint inhibitor is administered after electroporation of the immunostimulatory cytokine, whereby administration of certain treatment agents are staggered and administered at different times relative to the electroporation step. In some embodiments, a hemostatic agent may be delivered before, after, or simultaneous with a checkpoint inhibitor.

Hemostatic Agents

[00278] A hemostatic agent (also known as antihemorrhagic agent) is a substance that promote hemostasis or the ability to stop excessive bleeding. These agents can work systemically, by promoting coagulation, or locally by incorporating fibrinogen, coagulation factors, platelet aggregation, or vasoconstriction. When a blood vessel is injured, it sets off a coagulation cascade involving vessel constriction to reduce blood flow, adherence of circulating platelets to the vessel wall at the site of the trauma, and platelet activation and aggregation. Additionally, a series of enzymatic reactions involving coagulation proteins produce fibrin to form a stable hemostatic plug. Therefore, some hemostatic agents mimic, promote, or bypassing these events.

[00279] Some examples of hemostatic agents that can be used in this invention are oxidized regenerated cellulose, microfibrillar collagen, thrombin, gelatin matrix. Additional examples of hemostatic agents are aprotinin, desmopressin, and rFVIIa. The effective dose of the hemostatic agent will vary based on the hemostatic agent used. The effective dose of the hemostatic agent is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result, e.g, hemostasis. For example, the recommended dose for desmopressin is 0.3 pg per kg. An exemplary high-dose of aprotinin is 2.2 +/- 0.4 U and an exemplary low-dose of aprotinin is 3.4 +/- 0.9 U. rFVIIa may be administered, for example, from about 30pg to about 90pg

[00280] The use of hemostatic agents is beneficial in situations that call for emergency blood control including minor or major surgical procedures. In embodiments, administering a hemostatic agent after an electric pulse decreases wound healing time and improves recovery in post treatment of surface or visceral lesions. Even in situations in which substantial damage is not caused to the target tissue, the use of a hemostatic agent may improve the efficacy of the therapies disclosed herein and delivery of a treatment agent by increasing the efficacy of the delivered therapy. For example, the hemostatic agent may be used in combination with reversible electroporation to deliver the additional benefits described herein. In some embodiments, the hemostatic may additionally improve wound healing to the extent minor damage is caused during reversible electroporation or more major damage is caused during irreversible electroporation. In embodiments, the combined administration of the therapeutic agent and hemostatic agent induces an immune response along with wound healing factors which increased safety and efficacy. Without wishing to be bound to a particular theory, it is believed that a therapy of electroporation and treatment with a treatment agent may be improved by a hemostatic agent even during reversible electroporation by increasing the efficacy of the treatment agent via platelet aggregation caused by the hemostatic agent at the target site.

[00281] In embodiments, an example method of treatment may include: (1) inserting applicator, navigating to target site (e.g, via an insertion device, such as a trocar or flexible endoscope or via the applicator itself without an additional device), and piercing the visceral lesion, (2) beginning injection of a therapeutic agent, (3) delivering electroporation therapy, (4) replacing therapeutic agent with a hemostatic agent and using the same injection port, delivering the hemostatic drug thereby inducing hemostasis, (5) removing applicator. In embodiments, the electroporation therapy comprises low-voltage electric pulses. In embodiments, the electric pulses have a field strength of 700V/cm or less and a pulse length of between about 0.5 ms and about 1 s using approximately 6 to 8 pulses, or in any other variation of low-voltage electroporation described herein. Other variations may be used according to any of the embodiments described herein, such as variations of treatment agent, therapy type, applicator, delivery method, electrode configuration, or any other treatment method or device described herein. In some other embodiments, delivery of a hemostatic agent may be beneficial in connection with a high-voltage, irreversible electroporation. [00282] In embodiments, the hemostatic agent is administered after the electroporation therapy. In embodiments, the hemostatic agent is administered after the administration of the effective dose of the at least one treatment agent and after electroporation therapy. In some embodiments, a localized treatment with hemostatic agent is sufficient to cause significantly reduced healing time. In embodiments, the treatment agent is an antitumor agent.

[00283] In embodiments, a hemostatic agent and an antitumor agent are formulated to be administrable to a cancerous tumor by intratumoral electroporation. In embodiments, a pharmaceutical composition comprising a hemostatic agent and an antitumor agent. In embodiments, the pharmaceutical composition is formulated to be administrable by intratumoral electroporation. In embodiments, a composition in the form of a medicament for cancer comprising a hemostatic agent and an antitumor agent. In embodiments, the composition is formulated to be administrable by intratumoral electroporation.

Treatment

[00284] The term "treatment" includes, but is not limited to, inhibition or reduction of proliferation of cancer cells, destruction of cancer cells, prevention of proliferation of cancer cells or prevention of initiation of malignant cells or arrest or reversal of the progression of transformed premalignant cells to malignant disease, or amelioration of the disease.

[00285] The term “effective dose” of a treatment agent or therapeutic molecule, e.g., a pharmaceutical composition, as used herein, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. In some embodiments, an effective dose refers to an amount of a compound of the present invention that (i) treats the particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition or disorder described herein.

[00286] In some embodiments, methods are provided for reducing the size of a tumor or inhibiting the growth of cancer cells in a subject, or reducing or inhibiting the development of metastatic cancer in a subject suffering from cancer.

[00287] In some embodiments, one or more of the methods comprises, treating a subject having a cancerous tumor comprising: injecting the cancerous tumor with an effective dose of a therapeutic molecule or treatment agent; and administering electroporation therapy to the tumor. In some embodiments, one or more of the methods comprises, treating a subject having a cancerous tumor comprising: injecting the cancerous tumor with an effective dose of an expression plasmid encoding a therapeutic polypeptide; and administering electroporation therapy to the tumor.

[00288] In some embodiments, the described devices can be used for the therapeutic application of an electric pulse to a cell, groups of cells, or tissue of a subject for damaging or killing cells therein. In some embodiment the cell is a cancer cell. In some embodiments, the cancer cell is malignant.

[00289] In some embodiments, the described devices can be used for the therapeutic application of an electric pulse to a cell, groups of cells, or tissue of a subject thereby facilitating entry of a therapeutic molecule into the cell, groups of cells, or tissue. In some embodiments, the described devices can administer the therapeutic molecule to the cell, groups of cells, or tissue. In some embodiments, the described devices may be used both for the therapeutic application of an electrical pulse and for administration of the therapeutic molecules, such that the electrical pulse and the therapeutic molecules are co-localized at the same cell, groups of cells, or tissue without having to reposition the applicator or change the treatment apparatus. In some embodiments the cell is a cancer cell. In some embodiments, the cancer cell is malignant.

[00290] In some embodiments, the therapeutic molecule or expression vector is administered substantially contemporaneously with the electroporation treatment. The term "substantially contemporaneously" means that the molecule and the electroporation treatment are administered reasonably close together with respect to time, i.e., before the effect of the electrical pulses on the cells diminishes. The administration of the molecule or therapeutic agent depends upon such factors as, for example, the nature of the tumor, the condition of the patient, the size and chemical characteristics of the molecule and half-life of the molecule. [00291] In some embodiments of the treatment agent, the molecule is combined with one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than an active pharmaceutical ingredient (API, therapeutic product) that are intentionally included with the API (molecule). Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the API during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance. Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents. [00292] The described electroporation devices and methods can be used to treat a cell, group of cells, or tissue. In some embodiments, the described electroporation devices and methods can be used to treat one or more lesions. In some embodiments, the described electroporation devices and methods can be used to treat tumor cells. The tumor cells can be, but are not limited to cancer cells. The term "cancer" includes a myriad of diseases generally characterized by inappropriate cellular proliferation, abnormal or excessive cellular proliferation. The cancer can be, but is not limited to, solid cancer, sarcoma, carcinoma, and lymphoma. The cancer can also be, but is not limited to, pancreas, skin, brain, liver, gall bladder, stomach, lymph node, breast, lung, head and neck, larynx, pharynx, lip, throat, heart, kidney, muscle, colon, prostate, thymus, testis, uterine, ovary, cutaneous and subcutaneous cancers. Skin cancer can be, but is not limited to, melanoma and basal cell carcinoma. Melanoma can be, but is not limited to, cutaneous and subcutaneous melanoma. Breast cancer can be, but is not limited to, ER positive breast cancer, ER negative breast cancer, and triple negative breast cancer. In some embodiments the tumor cells may include glioblastoma. The cancer can be, but is not limited to, a cutaneous lesion or subcutaneous lesion. In some embodiments, the described devices and methods can be used to treat are used to treat cell proliferative disorders. The term "cell proliferative disorder" denotes malignant as well as non-malignant cell populations which often appear to differ from the surrounding tissue both morphologically and genotypically. In some embodiments, the described devices and methods can be used to treat a human. In some embodiments, the described devices and methods can be used to treat non-human animals or mammals. A non-human mammal can be, but is not limited to, mouse, rat, rabbit, dog, cat, pig, cow, sheep and horse. The administration of the molecule or therapeutic agent and electroporation can occur at any interval, depending upon such factors, for example, as the nature of the tumor, the condition of the patient, the size and chemical characteristics of the molecule and half-life of the molecule. [00293] The described electroporation devices and methods are contemplated for use in patients afflicted with cancer or other non-cancerous (benign) growths. These growths may manifest themselves as any of a lesion, polyp, neoplasm (e.g. papillary urothelial neoplasm), papilloma, malignancy, tumor (e.g. Klatskin tumor, hilar tumor, noninvasive papillary urothelial tumor, germ cell tumor, Ewing's tumor, Askin's tumor, primitive neuroectodermal tumor, Leydig cell tumor, Wilms' tumor, Sertoli cell tumor), sarcoma, carcinoma (e.g. squamous cell carcinoma, cloacogenic carcinoma, adenocarcinoma, adenosquamous carcinoma, cholangiocarcinoma, hepatocellular carcinoma, invasive papillary urothelial carcinoma, flat urothelial carcinoma), lump, or any other type of cancerous or non-cancerous growth. Tumors treated with the devices and methods of the present embodiment may be any of noninvasive, invasive, superficial, papillary, flat, metastatic, localized, unicentric, multicentric, low grade, and high grade.

[00294] The described electroporation devices and methods are contemplated for use in numerous types of malignant tumors (i.e. cancer) and benign tumors. For example, the devices and methods described herein are contemplated for use in adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer) bladder cancer, benign and cancerous bone cancer (e.g. osteoma, osteoid osteoma, osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinoma, clear cell) esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer, both melanoma and non-melanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma). As described herein, a lesion may be described in relation to the organ or region on or in which it resides. For example, a lesion may be considered “at a lung” if it is attached to, disposed on, or disposed within any portion of the lungs and/or lung tissue or would otherwise be associated with the lung by a person of skill in the art in light of this disclosure.

[00295] In some embodiments, an electric pulse of electric energy is applied to tissue near or surrounding the target site (e.g. tumor margin tissue). The electric pulse can be applied to tissue near or surrounding the tumor site either before or after excision of the tumor. The electric pulse and optionally a therapeutic molecule can be applied to tissue near or surrounding the tumor site to kill or damage cancerous cells or to deliver one or more therapeutic molecules. The therapeutic molecule can be administered to a subject or tissue intravenously or by injecting directly onto and around the tumor. The electric pulse and optionally a therapeutic molecule can be delivered to a tumor margin tissue to reduce relapse of growth of tumor cells, tumor branches, and/or microscopic metastases in a mammalian tissue at or adjacent to a localization for a tumor excised from a subject. The therapeutic molecule can be administered to the margin tissue before or simultaneously with administration of an electroporating electrical pulse. The electric pulse and optionally the therapeutic molecule can be administered prior to or after surgical resection or ablation of a tumor. In some embodiments, surgical resection or ablation of the tumor is performed with 24 hours of electroporative electric pulse administration. The tumor margin tissue comprises tissue within 0 5 2.0 cm around the tumor. In some embodiments, the tumor margin tissue comprises an open surgical wound margin.

[00296] In some embodiments, methods of treating a subject having a cancerous tumor comprise: a) injecting the cancerous tumor with an effective dose of a therapeutic molecule (e.g., treatment agent), and b) administering an electric pulse to the tumor using a described electroporation device. In some embodiments, therapeutic molecule comprises a nucleic acid. In some embodiments, the therapeutic molecule encodes one or more co-stimulatory molecules, metabolic enzymes, antibodies, checkpoint inhibitors, or adjuvants.

[00297] In some embodiments, methods of treating a subject having a cancerous tumor comprise: a) injecting the cancerous tumor with an effective dose of at least one expression vector coding for at least one immunostimulatory cytokine(s) and at least one co-stimulatory molecule; b) administering electroporation therapy to the tumor use a described electroporation device.

[00298] In some embodiments, the methods further comprise administering an effective dose of one or more checkpoint inhibitors to the subject. In some embodiments, methods of treating a subject having a cancerous tumor comprise: a) injecting the cancerous tumor with an effective dose of at least one plasmid coding for at least one immunostimulatory cytokine(s); b) administering electroporation therapy to the tumor use a described electroporation device; and c) administering an effective dose of one or more checkpoint inhibitors to the subject.

[00299] In some embodiments, the electroporation therapy may be any of the therapies detailed herein. In some embodiments, the electroporation therapy may comprise a low- voltage therapy without the performance of EIS. In some embodiments, the controller of the system may cause the generator to perform EIS between pulses of the low-voltage therapy to determine and optimize the parameters of the generator based on the operating conditions and treatment agents used. For example, the parameters ( e.g ., voltage, pulse duration, etc.) of the generator may be controlled by the controller to cause optimum permeation of the treatment agent.

[00300] In some embodiments, the electroporation therapy comprises the administration of one or more voltage pulses having a duration of approximately 0.1ms each. In another embodiment, the checkpoint inhibitor is administered systemically. In some embodiments, low voltage may be used with the treatment therapies and apparatus disclosed herein.

[00301] Specific embodiments described herein include:

1. A method of treating a lesion or tumor in a subject, the method comprising: administering to the lesion or tumor an effective dose of a hemostatic agent; administering to the lesion or tumor an effective dose of at least one treatment agent; administering electroporation therapy to the lesion or tumor; and wherein administering the electroporation therapy comprises administering an electric pulse to the lesion or tumor using an electroporation system comprising: an applicator comprising: a plurality of electrodes comprising a first electrode having a first tip and a second electrode having a second tip; and a generator electrically connected to the plurality of electrodes, wherein administering the electric pulse to the lesion or tumor comprises disposing the first electrode and the second electrode into or adjacent to the lesion or tumor, and delivering the electric pulse via the first electrode and the second electrode.

2. The method of embodiment 1, wherein the electroporation therapy is administered between administering the hemostatic agent and administering the effective dose of the at least one treatment agent.

3. The method of embodiment 1, wherein the generator is configured to output low- voltage electric pulses.

4. The method of embodiment 3, wherein the electric pulses have a field strength of 700V/cm or less.

5. The method of embodiment 1, wherein the electroporation therapy is reversible electroporation therapy.

6. The method of embodiment 1, wherein the treatment agent is a plasmid coding for a cytokine, a checkpoint inhibitor, a plasmid encoding an immunomodulatory polypeptide wherein the immunomodulatory polypeptide comprises: a cytokine, a costimulatory molecule, a genetic adjuvant, an antigen, a genetic adjuvant-antigen fusion polypeptide, a chemokine, or an antigen binding polypeptide, or a combination thereof.

7. The method of embodiment 6, wherein the plasmid coding for a cytokine is a plasmid coding for IL-12.

8. The method of embodiment 7, wherein the plasmid comprises tavokinogene telseplasmid.

9. The method of embodiment 1, wherein the applicator further comprises a drug delivery channel configured to deliver at least one of the at least one treatment agent and the hemostatic agent co-locally with the electroporation therapy. 10. The method of embodiment 9, wherein each of the at least one treatment agent and the hemostatic agent delivered co-locally with the electroporation therapy are delivered via the drug delivery channel of the applicator.

11. The method of embodiment 1, wherein the plurality electrodes are configured to move between a retracted position and a deployed position.

12. The method of embodiment 1, wherein a distance between the first tip of the first electrode and the second tip of the second electrode is greater in the deployed position than in the retracted position.

[00302] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Moreover, although the foregoing descriptions and the associated drawings describe example embodiments in the context of certain example combinations of elements and/or functions, it should be appreciated that different combinations of elements and/or functions may be provided by alternative embodiments without departing from the scope of the appended claims. In this regard, for example, different combinations of elements and/or functions than those explicitly described above are also contemplated as may be set forth in some of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

2. The method of claim 1, wherein the electroporation therapy is administered between administering the hemostatic agent and administering the effective dose of the at least one treatment agent.

3. The method of any one of claims 1-2, wherein the generator is configured to output low-voltage electric pulses.

4. The method of claim 3, wherein the electric pulses have a field strength of 700V/cm or less.

5. The method of any one of claims 1-4, wherein the electroporation therapy is reversible electroporation therapy.

6. The method of any one of claims 1-5, wherein the treatment agent is a plasmid coding for a cytokine, a checkpoint inhibitor, a plasmid encoding an immunomodulatory polypeptide wherein the immunomodulatory polypeptide comprises: a cytokine, a costimulatory molecule, a genetic adjuvant, an antigen, a genetic adjuvant-antigen fusion polypeptide, a chemokine, or an antigen binding polypeptide, or a combination thereof.

7. The method of claim 6, wherein the plasmid coding for a cytokine is a plasmid coding for IL-12.

8. The method of claim 7, wherein the plasmid comprises tavokinogene telseplasmid.

9. The method of any one of claims 1-8, wherein the applicator further comprises a drug delivery channel configured to deliver at least one of the at least one treatment agent and the hemostatic agent co-locally with the electroporation therapy.

10. The method of any one of claims 1-9, wherein each of the at least one treatment agent and the hemostatic agent delivered co-locally with the electroporation therapy are delivered via the drug delivery channel of the applicator.

11. The method of any one of claims 1-10, wherein the plurality electrodes are configured to move between a retracted position and a deployed position.

12. The method of any one of claims 1-11, wherein a distance between the first tip of the first electrode and the second tip of the second electrode is greater in the deployed position than in the retracted position.

13. A method of using an electroporation and drug delivery system, comprising: dispensing a dose of a hemostatic agent; dispensing a dose of at least one treatment agent; and operating an electroporation applicator using an electroporation system comprising: an applicator comprising: a plurality of electrodes comprising a first electrode having a first tip and a second electrode having a second tip; and a generator electrically connected to the plurality of electrodes, wherein operating the electroporation applicator comprises disposing the first electrode and the second electrode into or adjacent to a location of the dose of the hemostatic agent and the dose of the at least one treatment agent, and delivering the electric pulse via the first electrode and the second electrode.

14. A hemostatic agent and an treatment agent formulated to be administrable to a cancerous tumor by intratumoral electroporation, wherein the electroporation is administrable by an applicator comprising: a plurality of electrodes comprising a first electrode having a first tip and a second electrode having a second tip; and a generator electrically connected to the plurality of electrodes.

15. A pharmaceutical composition comprising a hemostatic agent and an treatment agent.

16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is formulated to be administrable by intratumoral electroporation.

17. A composition in the form of a medicament for cancer comprising a hemostatic agent and an treatment agent.

18. The composition of claim 17, wherein the composition is formulated to be administrable by intratumoral electroporation.

19 . The pharmaceutical composition of claim 15 or the composition of claim 17, wherein the treatment agent is a plasmid coding for IL-12.

20. The pharmaceutical composition or composition of claim 19, wherein the plasmid comprises tavokinogene telseplasmid.