BR122022000530B1 - ELECTROPORATION DEVICE AND MATRIX THEREOF CAPABLE OF PROVIDING AN INDIVIDUAL WITH A TOLERABLE ELECTRICAL POTENTIAL TO EPIDERMAL TISSUE RESULTING IN ELECTROPORATION OF CELLS IN SAID TISSUE - Google Patents

Abstract

A novel electroporation device for vaccine delivery that is effective in both generating a protective immune response and delivering tolerable (or nearly painless) delivery to an individual; and also methods of using the same device to vaccinate an individual against a variety of infectious diseases and types of cancer in an almost painless manner

Description

[001]Divided from BR 11 2012 021805 0, deposited on 03/01/2011.

[002] This work was partially supported by United States Army grant W23RYX-8141-N604:#08023003.

Field of invention

[003] The present invention relates, among other things, to electroporation devices and their use to facilitate the introduction of biomolecules into skin tissue cells in a mammal.

Fundamentals

[004] The magnitude of the immune response to a DNA vaccine generally depends on three main criteria: the optimized vector model, the use of a suitable adjuvant and the successful delivery and subsequent expression of the plasmid in the target tissue. In vivo electroporation has proven to be particularly effective in efficiently delivering DNA immunogens to muscle and skin, and in fact, several devices have already been part of human clinical trials.

[005] Drug delivery to dermal tissue (intradermal, ID) is an attractive method in a clinical setting for a number of reasons. The skin is the largest organ in the human body, the most accessible, the most easily monitored, as well as highly immunocompetent. However, the impenetrable barrier function of the skin is a major obstacle to efficient transdermal drug delivery.

[006] Human skin extends over approximately 2 square meters in area and is approximately 2.5 mm thick on average, making it the largest organ in the human body. Conventionally, the skin has two types of extensive tissue, the epidermis and the dermis. The epidermis is a continuously keratinized stratified epithelium. The outermost layer of the skin is the stratum corneum (SC) and serves as the skin's primary barrier function. The SC is a 15 to 30 cell thick layer of non-viable but biochemically active corneocytes. All three other strata of the epidermis (granulosum, spinosum and basal) contain keratinocytes in different stages of differentiation, as well as Langerhans immune cells and dermal dendritic cells.

[007] Physical and chemical methods for drug delivery and gene delivery transdermally have been detailed by groups around the world. Lipid supply and gene injection by iontophoresis are some examples. Another physical method to temporarily increase skin permeability is electroporation. Electroporation involves the application of brief electrical pulses that result in the creation of aqueous pathways within the lipid bilayer membranes of mammalian cells. This allows large molecules, including DNA, to pass through the cell membrane, which would otherwise be less permeable. Thus, electroporation increases absorption as well as the extent to which drugs and DNA are delivered to their target tissue. For electroporation to generate pore formation, a threshold energy must be reached and the movement produced by the electrophoretic effect depends on both the electric field and the pulse length.

[008] In the case of DNA vaccines, electroporation has been shown to quantitatively increase immune responses, increase the amplitude of these immune responses, as well as improve dose efficiency. Several advantages of delivery through the skin, in particular, presence of a variety of relevant immune cells, easy clinical accessibility as well as a target organ for immunization, and minimal depth of delivery (minimally invasive) are attractive, however , questions persist regarding the ability to achieve high transfection rates and subsequently resistant immune responses.

[009] Delivery of naked DNA via a standard intramuscular (IM) injection is notoriously inefficient outside of rodent models. This has led to an inability to achieve resistant immune responses in large mammals and humans. Various strategies have been developed to increase the expression of DNA-based vaccines, such as codon optimization, RNA optimization, leader sequence addition, and the development of optimized consensus sequences.

[0010] Despite improvements in the vector model and use of molecular adjuvants, there is still a need for an efficient method of delivering DNA vaccines that results in high-level expression of the plasmid in the desired cell strain of the desired tissue, most commonly , muscle, tumor or skin. Furthermore, there remains a need for an electroporation device and vaccine delivery method that is effective in both generating a protective and tolerable (or nearly painless) immune response.

Summary of the invention

[0011] In one aspect of the invention, there are electroporation devices capable of providing an individual with a tolerable electrical potential to layers of epidermal tissue between the stratum corneum and basal layers, which results in electroporation of cells in said tissue comprising: a voltage generator; and an array having a plurality of electrodes in electrical communication with the voltage generator. Each of the plurality of electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; the voltage generator, through the electrodes, provides an electrical potential of about 0.1 volt to about 50 volts to the epidermal tissue; and the electrodes have a tip at the distal end that is rounded and with a sharp tip, which allows the electrode to penetrate the layers of epidermis tissue between the stratum corneum and basal layers and provide the electrical potential of the voltage generator to the epidermis tissue.

[0012] In another aspect of the invention, there are electroporation devices capable of providing an individual with a tolerable electrical potential for epidermal tissue that results in electroporation of cells in said epidermal tissue comprising: a voltage generator; and an array having a plurality of electrodes in electrical communication with the voltage generator. Each of the plurality of electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; the voltage generator, through the electrodes, provides an electrical potential of about 0.1 volt to about 50 volts to the epidermal tissue; and the electrodes are adapted to penetrate epidermal tissue to depths of 0.1 mm or less.

[0013] In another aspect of the invention, there are electroporation devices capable of providing an individual with a tolerable electrical potential for epidermal tissue that results in electroporation of cells in said tissue comprising: a voltage generator; and an array having a plurality of electrodes in electrical communication with the voltage generator. Each of the plurality of electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; the voltage generator supplies the matrix with an electric field of about 0.1 to about 50 volts; and in which the electrodes deliver a tolerable electrical potential to the cells of the epidermal tissue, almost painlessly, as measured by a visual analogue scale.

[0014] In another aspect of the invention, there are electroporation devices capable of providing an individual with a tolerable electrical potential for epidermal tissue that results in electroporation of cells in said tissue comprising: a voltage generator; It is

[0015] a matrix having a plurality of electrodes in electrical communication with the voltage generator. Each of the plurality of electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; the voltage generator supplies the matrix with an electric field of about 0.1 to about 50 volts; and wherein the electrodes provide a tolerable electrical potential to the cells of the epidermal tissue, as evidenced by minimal tissue damage in said cells.

[0016] In another aspect of the invention, there are tolerable methods of delivering a biomolecule to epidermal tissue cells of an individual through electroporation-assisted delivery using the electroporation devices described herein, comprising: administering the biomolecule to the cells; contacting the electrodes with the epidermal tissue so that the electrodes penetrate through the stratum corneum and lodge in layers above the basal layers; and providing a tolerable electrical potential from the voltage generator to the cells of the epidermal layer through the electrodes.

Brief Description of Figures

[0017] Various objects and advantages of the present invention can be better understood by those skilled in the art with reference to the attached Figures, in which:

[0018] Figure 1 shows a technical drawing of an embodiment of a set of a minimally invasive EP device (“MIED”).

[0019] Figure 2 shows three-dimensional drawings of components of an embodiment of a MIED. “a” shows a sterile disposable matrix housing, “b” shows a durable housing, and “c” shows the electrode housing within the disposable matrix (2a).

[0020] Figure 3 shows three-dimensional drawings of an embodiment of a MIED which is a portable battery-charged device with detachable matrix (for sterilization). “a” shows the external view. “b” shows the internal view (including batteries).

[0021] Figure 4 shows images of a MIED modality.

[0022] Figure 5 shows fluorescent images of guinea pig skin tissue to detect GFP expression after administration of electroporation-facilitated delivery of GFP expression constructs using a MIED.

[0023] Figure 6 shows fluorescent images of skin tissue from various animals to detect GFP expression after administration of electroporation-facilitated delivery of GFP expression constructs using a MIED.

[0024] Figure 7 shows graphs that present immunogenicity results in protection against influenza in mice.

[0025] Figure 8 shows graphs that present immunogenicity results in protection against influenza in guinea pigs.

[0026] Figure 9 shows graphs that present immunogenicity results in protection against influenza in non-human primates.

Detailed description of the invention

[0027] The following abbreviated or reduced definitions are provided to aid understanding of the preferred embodiments of the present invention. The abbreviated definitions provided here are neither exhaustive nor contradictory to the definitions understood within the dictionary or field meaning. Abbreviated definitions are provided here to supplement, or more clearly, define definitions known in the art.

[0028] The term “constant current” is used here to define a current that is received or experienced by a tissue, or cells defining said tissue, over the duration of an electrical pulse supplied to the same tissue. The electrical pulse is provided by the electroporation devices described here. This current remains at a constant amperage in said tissue throughout the life of an electrical pulse, because the electroporation device provided herein has a feedback element, preferably having instantaneous feedback. The feedback element can measure tissue (or cell) resistance throughout the duration of the pulse and causes the electroporation device to change its electrical energy output (e.g., increase voltage) so that the current in it tissue remains constant throughout the electrical pulse (on the order of microseconds), and from pulse to pulse. In some embodiments, the feedback element comprises a controller. The current amperage values described as provided by the devices disclosed herein are preferably constant current amperage values.

[0029] The term “constant voltage” is used here to define an electrical voltage or potential that is received or experienced by a tissue, or cells defining said tissue, over the duration of an electrical pulse supplied to the same tissue. The electrical pulse is provided by the electroporation devices described here. This voltage remains at a constant voltage in said tissue throughout the life of an electrical pulse, because the electroporation device provided herein has a feedback element, preferably having instantaneous feedback. The feedback element can measure the resistance of the tissue (or cells) throughout the duration of the pulse and causes the electroporation device to change its electrical energy output so that the voltage in the same tissue remains constant throughout the electrical pulse. (in the order of microseconds) and, from pulse to pulse. In some embodiments, the feedback element comprises a controller. The voltage values described as provided by the devices disclosed herein are preferably constant voltage values.

[0030] The term “feedback” or “current feedback” is used interchangeably and means the active response of the provided skin EP devices, which comprises measuring the current in the tissue between electrodes and altering the energy output provided by the PE device accordingly, in order to maintain the current at a constant level. This constant level is pre-set by a user before starting a pulse sequence or electrical treatment. Preferably, feedback is performed by the electroporation component, e.g. controller, of the skin EP device, as the electrical circuit contained therein is capable of continuously monitoring the current in the tissue between electrodes and comparing the monitored current (or current within tissue) with a pre-defined current, and continually make power output adjustments to maintain the monitored current at pre-defined levels. In some embodiments, the feedback loop is instantaneous, just like an analog closed-loop feedback.

[0031] The term “biomolecule”, as used herein, refers to nucleic acid sequences, proteins, lipids, microbubbles (e.g., drug-filled vesicles) and pharmaceuticals. Preferably, the biomolecule is a vaccine, and more preferably, the biomolecule is a DNA vaccine, and even more preferably, a DNA plasmid vaccine.

[0032] The terms “electroporation”, “electropermeabilization” or “electrokinetic enhancement” (“EP”), as used interchangeably herein, refer to the use of a transmembrane electric field pulse to induce microscopic routes (po-ros) in a biomembrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions and water to pass from one side of the cell membrane to the other.

[0033] The term “decentralized current” is used herein to define the pattern of electrical currents delivered by various needle electrode arrays of the electroporation devices described herein, wherein the patterns minimize, or preferably eliminate, the occurrence of stress caused by electroporation-related heat in any area of tissue being electroporated.

[0034] The term “feedback mechanism”, as used herein, refers to a process performed by software or hardware (or firmware), which processes, receives and compares the impedance of the desired tissue (before, during and/or after of the energy pulse supply) with a present value, preferably current, and adjusts the supplied energy pulse to reach the pre-set value. The term “impedance” is used here when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus allowing comparisons with the pre-defined current. In a preferred embodiment, the “feedback mechanism” is realized by an analog closed-loop circuit.

[0035] The term “minimally invasive”, as used herein, refers to limited penetration by the needle electrodes of the provided electroporation device, and may include non-invasive electrodes (or non-penetrating needles). Preferably, the penetration is to such an extent that it passes through the stratum corneum and, preferably, enters the outermost living layer of tissue, the stratum granulosum, but does not penetrate the basal layer. The depth of penetration does not exceed 0.1 mm, and preferably depths ranging from about 0.010 mm to about 0.040 mm disrupt the stratum corneum. Preferably, this is accomplished using an electrode that has a trocar tip region to provide a sharp tip that allows penetration through the stratum corneum but prevents deep penetration.

[0036] The term “tolerable” or “almost painless” is used here interchangeably and, when referring to electroporation, means a substantially lower level of pain associated with electroporation, and not with commonly available electroporation devices. More specifically, tolerable (or nearly painless) electroporation is the result of combining the use of the MIED described here, avoiding muscle electroporation, along with delivering low electrical fields to the epidermal layers between the stratum corneum and the basal layers. Preferably, the electric fields will be composed of low voltage levels, i.e., for example, 0.01V to 70V, and preferably 1V to 15V. When assessed using a VAS, individuals experiencing MIED electroporation, according to the methods provided here, experience levels of pain that are within 20% (of the full scale) of their painless or no pain score, e.g., within 2 points, with a full scale of 0 to 10, and preferably within 10% of your painless score.

[0037] In one aspect of the invention, there are electroporation devices capable of providing an individual with a tolerable electrical potential to layers of epidermal tissue between the stratum corneum and basal layers that results in electroporation of cells in said tissue comprising: an voltage generator; and an array having a plurality of electrodes in electrical communication with the voltage generator. Each of the plurality of electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; the voltage generator, through the electrodes, provides an electrical potential of about 0.1 volt to about 50 volts to the epidermal tissue; and the electrodes have a tip at the distal end that is rounded and with a sharp tip, which allows the electrode to penetrate the layers of epidermis tissue between the stratum corneum and basal layers and provide the electrical potential of the voltage generator to the epidermis tissue. In some cases, electrodes have a tip at the distal end that is generally rounded but has a sharp tip, or in other words, a sharp tip followed by shallow or rounded angles. For example, the electrode may contain a distal end that is a 10° off-axis trocar tip, almost perpendicular to the needle axis.

[0038] In another aspect of the invention, there are electroporation devices capable of providing an individual with a tolerable electrical potential for epidermal tissue that results in electroporation of cells in said epidermal tissue comprising: a voltage generator; and an array having a plurality of electrodes in electrical communication with the voltage generator. Each of the plurality of electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; the voltage generator, through the electrodes, provides an electrical potential of about 0.1 volt to about 50 volts to the epidermal tissue; and the electrodes are adapted to penetrate epidermal tissue to depths of 0.1 mm or less.

[0039] In another aspect of the invention, there are electroporation devices capable of providing an individual with a tolerable electrical potential for epidermal tissue that results in electroporation of cells in said tissue comprising: a voltage generator; and an array having a plurality of electrodes in electrical communication with the voltage generator. Each of the plurality of electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; the voltage generator supplies the matrix with an electric field of about 0.1 to about 50 volts; and in which the electrodes deliver a tolerable electrical potential to the cells of the epidermal tissue, almost painlessly, as measured by a visual analogue scale.

[0040] In another aspect of the invention, there are electroporation devices capable of providing an individual with a tolerable electrical potential for epidermal tissue that results in electroporation of cells in said tissue comprising: a voltage generator; and an array having a plurality of electrodes in electrical communication with the voltage generator. Each of the plurality of electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; the voltage generator supplies the matrix with an electric field of about 0.1 to about 50 volts; and wherein the electrodes provide a tolerable electrical potential to the cells of the epidermal tissue, as evidenced by minimal tissue damage in said cells.

[0041] In some embodiments, the devices have electrodes that are adapted to penetrate epidermal tissue to depths of 0.1 mm or less and, preferably, to depths of about 0.01 mm to about 0.04 mm. Preferably, the electrodes are spaced from each adjacent electrode at a distance of about 1.5 mm. Furthermore, preferably, the voltage generator provides the epidermal tissue with an electrical potential of about 1 volt to about 15 volts, and more preferably about 15 volts.

[0042] In some embodiments, the devices provide tolerable electrical potential with a duration ranging from about 5 milliseconds to about 250 milliseconds, ranges and duration times therebetween.

[0043] In some embodiments, the devices provide a tolerable electrical potential that generates an assessment of pain by said individual that is almost zero, as measured by a visual analog scale. VAS is a 100 mm long horizontal line where 0 mm indicates no pain and 100 mm indicates worse pain. Near Painless is a score that uses the VAS methodology and produces a mean score of approximately <20 mm (within 95% confidence interval), and preferably <10 mm (within 95% confidence interval).

[0044] In some embodiments, the devices provide a tolerable electrical potential, which is an electrical potential that results in minimal tissue damage in said cells of the individual and, preferably, tissue damage not visualized by histopathological analysis of the tissue. Histological analysis can be used to assess visual damage.

[0045] Histological analysis demonstrates transfection in the upper layers of the epidermis

[0046] Histopathological analysis was performed on guinea pig skin tissue. Each panel showed tissue that had been electroporated by MID. All slides were stained with hematoxylin and eosin, as seen under a fluorescent microscope (20x objective) to visualize GFP positivity. Histopathology of skin tissue after animal sacrifice and 3 days after treatment was shown: analysis did not reveal any associated tissue damage after electroporation with a minimally invasive device. In other words, the histopathological analysis of the tissue with electroporation showed similarity to the tissue without electroporation.

[0047] In another aspect of the invention, there are tolerable methods of delivering a biomolecule to epidermal tissue cells of an individual through electroporation-assisted delivery using the electroporation devices described herein, comprising: administering the biomolecule to the cells; contacting the electrodes with the epidermal tissue so that the electrodes penetrate through the stratum corneum and lodge in layers above the basal layers; and providing a tolerable electrical potential from the voltage generator to the cells of the epidermal layer through the electrodes.

[0048] In some embodiments, the methods comprise manipulating the electrodes to penetrate the epidermal tissue to depths of 0.1 mm or less and, preferably, to depths of about 0.01 mm to about 0.04 mm. The matrix can be manually adjusted back and forth (or moved) at the injection site to ensure good contact and result in disruption of the stratum corneum layer by the tip of the electrodes. Preferably, the supply step comprises providing a tolerable electrical potential that generates an assessment of pain by said individual that is almost nil, as measured by a visual analogue scale. Furthermore, preferably, the supply step comprises providing a tolerable electrical potential that results in minimal tissue damage in said individual cells. The supply step preferably provides an electrical potential of about 0.1 volt to about 15 volts to the cells.

[0049] In some embodiments, the methods include the supply step comprising providing an electrical potential with a duration of about 5 milliseconds to about 250 milliseconds, and numbers and duration ranges therebetween and, preferably, 100 milliseconds.

General electroporation devices

[0050] There are electroporation device generators or controllers provided that can deliver a pulse of electrical energy to an epidermal tissue between the stratum corneum and the basal layers with low electrical fields to generate tolerable electroporation. Preferably, electroporation will occur in the stratum granulosum layer primarily or exclusively. The devices deliver the pulse of energy through the supplied MIED and through the needle electrodes that are capable of penetrating through the stratum corneum.

[0051] In some embodiments, receptivity of the present MIED, which preferably maintains a constant current in the treated tissue, is achieved through a feedback mechanism in the skin EP device, which prevents heating of a tissue and reduces damage of tissue, pain and contributes to the overall success of the electroporation technology provided. In some embodiments, the MIED may additionally comprise a controller, a waveform generator in electronic communication with the controller, a waveform recorder in electronic communication with the controller, and a battery electrically connected to the waveform generator. wave shape. The controller may receive data input from the user, instruct the waveform generator to deliver the energy pulse to the desired tissue in accordance with the data input, and transmit the data to the waveform recorder in accordance with the pulse. of energy supplied; and wherein the battery sends an electrical charge to the waveform generator, the battery being a lithium ion, nickel metal hydride, lead acid or nickel cadmium battery. Preferably, the MIED (Figure 3) is portable. The portable device can be operated via a battery pack and is suitable for mass vaccination, therapeutic or vaccination purposes.

[0052] The MIED may be a combination of the electrode array and applicator provided together with a variety of components or generators that generate an electric field (or generate an electric pulse). In some examples, the generators may be chosen from one of the known electroporation devices, including, but not limited to the following: the electroporation devices described in US Patent 7,245,963, entitled “Constant Current Electrode Assembly for Electroporation”, and US Patent 5,273,525, US Patent 6,110,161, US Patent 6,261,281, US Patent 6,958,060, and US Patent 6,939,862, among others. In more preferred examples, the generators are those used with the EP CELLECTRA® device and EP Elgen device (both from Inovio Pharmaceuticals, Inc., Blue Bell, PA). The patent references mentioned above are incorporated herein by reference in their entirety.

Electroporation Needles

[0053] A variety of known electroporation needles capable of delivering an electrical charge can be incorporated into the MIED. Electroporation needles are minimally invasive, including non-invasive needles. Preferably, the needles are sharpened, for example in some cases with a trocar sharpening, so that they can penetrate through the stratum corneum and reach the stratum granulosum. The trocar tip can be sharpened to leave a sharp point that allows penetration through the stratum corneum but prevents deep penetration. In some examples, the electrodes have a tip at the distal end that is generally rounded but has a sharp tip, or in other words, a sharp tip followed by shallow or rounded angles, for example, the electrode may contain a distal end which is a trocar tip with 10° off-axis sharpening, almost perpendicular to the needle axis.

Matrices

[0054] A large number of known needle matrix formations can be used with the present MIED. These contemplated needle array formations include any number of electrodes and any geometric pattern of electrodes in a general planar arrangement. Preferably, the electrodes are uniformly arranged and distributed along the electrode array (or along the base or substrate to which the electrodes are attached). More preferably, the needle electrodes are arranged in a square like array, with each adjacent needle electrode spaced at approximately the same distance (except for the electrodes on the edge of the square).

[0055] In some embodiments, the matrix consists of at least three equally spaced needles in a triangular pattern, at least four equally spaced needles in a circular pattern, or needles arranged in a 2 x 2, 3 x 3 quadrangular pattern, 4 x 4, 5 x 5, or larger. Alternatively, the pattern may be rectangular or rhomboid. Preferably, the needle electrodes are arranged in a 4 x 4 needle array arrangement. An example of the 4 x 4 needle array is shown in Figure 1. Figure 2 shows an example of the MIED housing, array housing and housing. of electrode. Each needle electrode may be spaced from each adjacent needle electrode at a distance of 150 mm or less, 100 mm to 1 mm, 50 mm to 1 mm, 40 mm to 1 mm, 30 mm to 1 mm, 20 mm to 1 mm, from 10 mm to 1 mm, from 5 mm to 1 mm, from 5 mm to 2 mm, from 5 mm to 2 mm and preferably 2 mm and more preferably 1.5 mm.

Electrical pulses (electrical potential supplied)

[0056] The provided devices operate at lower voltages and currents than typical EP methods to improve tolerability while maintaining successful transfection of the biomolecule (as evidenced by expression and subsequent immune response). The device is typically used with a pulse generator intended to provide a constant current, constant voltage, or a combination of both, as desired. The electrical pulses used by MIED to transfect cells in skin tissue are any known pulse patterns that provide the low electrical energy necessary to produce tolerable electroporation. In some embodiments, the MIED delivers an electrical pulse to the desired tissue at voltage levels of 0.01 V to 70 V, 0.01 V to 50 V, 0.01 V to 40 V, 0.01 V to 30 V, 0, 01V to 20V, 0.01V to 15V, 0.1V to 70V, 0.1V to 50V, 0.1V to 40V, 0.1V to 30V, 0.1V to 20 V, 0.1 V to 15 V, and preferably 1 V to 15 V. More preferably, the voltage levels are 15 V when adjacent electrodes of the MIED are spaced approximately 2 mm and preferably 1.5 mm.

[0057] In some embodiments, the MIED provides tolerable electrical energy that is characterized by an electrical pulse delivering the following current to the desired tissue: 0.2 mA to 100 mA, 0.1 mA to 100 mA, 0.5 mA to 100 mA, 1 mA to 100 mA, 1 mA to 80 mA, 1 mA to 60 mA, 1 mA to 50 mA, 1 mA to 40 mA, 1 mA to 30 mA and preferably 1 mA to 100 mA and, more preferably 1 mA to 30 mA and more preferably 10 mA.

[0058] The tolerable electrical pulses associated with MIED will all be characterized by short duration of each pulse, including pulse lengths of 5 milliseconds to 250 milliseconds, pulse lengths of 10 milliseconds to 250 milliseconds, 20 milliseconds to 250 milliseconds, 40 milliseconds to 250 milliseconds, 60 milliseconds to 250 milliseconds, 80 milliseconds to 250 milliseconds, 100 milliseconds to 250 milliseconds, 20 milliseconds to 200 milliseconds, 40 milliseconds to 200 milliseconds, 60 milliseconds to 200 milliseconds, 80 milliseconds to 200 milliseconds, 100 milliseconds to 200 milliseconds, 20 milliseconds to 150 milliseconds, 40 milliseconds to 150 milliseconds, 60 milliseconds to 150 milliseconds, 80 milliseconds to 150 milliseconds, 100 milliseconds to 150 milliseconds, 100 milliseconds to 140 milliseconds, 10 0 milliseconds to 130 milliseconds , 100 milliseconds to 120 milliseconds, 100 milliseconds to 110 milliseconds and, more preferably, 100 milliseconds.

[0059] The tolerable electrical pulses associated with MIED will also be characterized by low repeated pulses. The pulses provided by the MIED are repeated to provide the following number of pulses for each vaccination: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and, preferably, 1 to 6 pulses and , more preferably, 2 pulses. Fabric to administer vaccine + EP

[0060] The MIED is for supplying electroporation-stimulating electrical energy to skin tissue cells, helping to supply biomolecules to the cells present therein and, preferably, vaccination with DNA vaccines. Preferably, the electrodes penetrate only the skin tissue, which is the layer of living tissue, just below the stratum corneum and above the basal layer and, preferably, only in the stratum granulosum layers. The biomolecule is typically delivered to the target tissue using the Mantoux technique. Battery-charged version with portable electronics (see Figure 3)

[0061] The MIED can be a portable EP device. A portable stand-alone MIED device is provided, wherein the device is portable and wherein the source of electrical energy for electrical pulse is at least a battery having a voltage potential between 1.5 V and 12 V.

[0062] The portable MIED may comprise a housing within which said battery is located, a circuit board comprising an electrical circuit in electrical communication with and charged by said battery, and at least one capacitor capable of maintaining a capacitance between 1000 and 100000 uF (microFarads). In a related embodiment, the components (i.e., battery, circuit, and capacitor) are generally spatially arranged within said housing in a linear or side-by-side arrangement, such that the housing may generally contain a rectangular or cylindrical shape to which allows it to be held in the hand of a person operating the device. For example, the housing will have a proximal end and a distal end such that the cylinder or long portion of said rectangle is located in the middle position and wherein said battery is located within said proximal end of said housing. , and is accessible from that end through a removable cover. Electrical conduits may be located at the distal end of said housing, which are in electrical communication with said circuit and serve as connectors for connection to at least one cathode and at least one anode to be used in completing a circuit comprising an electroporative pulse. charged by said battery through said capacitor and circuit. The distal end of said housing may also comprise, in association with, or as part of, said housing, a mechanism for semi-permanently or permanently attaching to, and/or separating therefrom, a main assembly, said main assembly comprising any one of a reservoir for housing a fluid therapeutic agent, an orifice in fluid communication with said reservoir through which said fluid may be directed to a tissue of the body, a source of energy for moving said fluid from said reservoir through said hole, and at least one electrode comprising at least one anode and one cathode.

[0063] In some embodiments, the portable MIED is capable of delivering to the skin tissue of a mammal an electrical pulse capable of electroporating cells within said tissue, said electrical pulse having a voltage of generally between 0.1 V and 70 V, preferably 0.1V to 50 V, more preferably 1V to 15V. Furthermore, the device is capable of delivering an electroporation pulse of said field strengths for a period of time between 5 milliseconds and 250 milliseconds, and more typically between 10 and 100 milliseconds, even more commonly, between 30 and 70 milliseconds. liseconds, and more preferably, 50 milliseconds. Additionally, the device is capable, through its microprocessor, of delivering a bipolar pulse or a monopolar pulse of electroporation electrical energy, such bipolar or monopolar pulses may comprise a predetermined multiple pulse sequence and/or shaped pulses. of voltage, or even, exponentially, discharge pulses. Such variable pulse capability provides potential optimization of the electroporation process, including optimization of the voltage, pulse shape, duration, and polarity of the electroporation pulses.

Examples

[0064] The present invention is further illustrated in the following Examples. It should be understood that these Examples, although they indicate preferred embodiments of the invention, are provided by way of illustration only. From the above discussion and these Examples, a person skilled in the art can determine the essential features of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions. Thus, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the above description. Such modifications are also intended to be within the scope of the appended claims.

[0065] A trial can be conducted to evaluate the tolerability or reduced pain generated by the electroporation devices described here. The personnel responsible for the study can start a timer for each patient at the time of electroporation administration. Each patient will perform standard pain assessments under the supervision of the trained personnel responsible for the study, and they will record these assessments in a daily booklet at: 5, 10, 15, 20, 30 and 45 minutes, one hour and one and a half hours after electroporation , then hourly for 12 hours after electroporation, and at 16 hours and 24 hours after electroporation. Patients' pain intensity will be measured using both a categorical scale and a VAS. As described previously, the categorical scale included four categories: 0 = none, 1 = mild, 2 = moderate, and 3 = severe.

[0066] The VAS is a 100 mm long horizontal line where 0 mm indicates no pain and 100 mm indicates worse pain. Using this 100 mm scale, a nearly painless score would be a score that is within 20 mm of a score that indicates no pain (no pain felt). In some cases, almost painless will have scores within 10 mm of a pain-free score.

Methods

[0067] The following methods are used in each of the examples below, where appropriate by discussion.

[0068] Minimally Invasive Device Model - Electrode arrays consisting of a 0.0175 inch diameter 4x4 gold-colored trocar needle with a 1.5 mm spacing are designed to be used in conjunction with the Elgen 1000 pulse generator ( Inovio Pharmaceuticals, Inc., Blue Bell, PA) or a battery-charged low voltage circuit.

[0069] Plasmid preparation - The gWiz GFP plasmid was purchased from Al-devron (Fargo ND). The NP plasmid encodes full-length NP derived from Puerto Rico influenza type 8 (H1N1). The M2 plasmid encodes full-length M2 derived from influenza strain New Caledonia/99 (H1N1). The construct's central objective was to mutate signals, and was optimized and synthesized by Geneart (Germany). All plasmids were diluted in 1xPBS before injection. SYNCONTM (Synthetic DNA Construct) influenza vaccine and plasmid cocktail (100 μg/plasmid) containing pGX2005 (SYNCONTM vaccine construct encoding an H1HA consensus sequence) and pGX2009 (SYNCONTM vaccine construct encoding an H1HA consensus sequence) porcine H1HA) was diluted in 1xPBS before injection.

[0070] Animals - Female Hartley guinea pigs (strain code 051), female New Zealand rabbits and female Balb C mice were purchased from Charles River Laboratories. Female Wistar rats were purchased from Charles River Laboratories. Female Yorkshire pigs were purchased from S&S Farm’s (Ramona, CA). Guinea pigs, rats, rabbits, and mice were housed in BioQuant (San Diego, CA). (4) Male and (4) female monkeys (Macaca mulatto) were housed individually at BIOQUAL, Inc. (Rockville, MD), with free access to food and water. Monkeys were exposed to acclimatization for at least 30 days in quarantine before experimentation. All animals were housed and cared for in accordance with Institutional Animal Care and Use Committee (IACUC) standards.

[0071] Animal preparation - The GFP reporter results observed in the Hartley guinea pigs after hair removal were the same results observed in the IAF hairless guinea pigs in previous experiments. Since hair removal was shown to have no effect on the resulting transfection and due to cost considerations, it was decided to perform the remainder of the study in Hartley animals. Hartley guinea pigs were shaved and hair residues were removed with depilatory cream (Veet) 24 hours before treatment. Mice, rats, rabbits, pigs and monkeys were shaved before treatment.

[0072] DNA injections - Monkeys were sedated with injected ketamine. All other animals were sedated with inhaled isoflurane. All animals were injected intradermally (Mantoux method - 29 gauge insulin needle) with 50 μl of 1 x PBS containing the desired dose of plasmid. Mice were injected intramuscularly into the quadriceps with 50 μl of 1x PBS containing the desired dose of plasmid. Monkeys were injected intramuscularly into the quadriceps with 400 μl of 1x PBS containing the desired dose of plasmid. Empty vector was added to maintain equal amounts of DNA for each group.

[0073] Dermal device electroporation - Immediately after DNA injection, the dermal device was applied to the dermal injection site. The matrix was “shuffled” at the injection site to ensure good contact and electrotransfer achieved through pulse generation from the Elgen 1000 or a low voltage battery circuit. The parameters used were three 15 volt pulses of 100 milliseconds in duration.

[0074] Muscle electroporation - Immediately after IM injection, electroporation was performed by inserting a 27G needle array, 2 with an electrode spacing of 4 mm into the site of the injected muscle. Two pulses of 125 V/cm with a duration of 60 milliseconds each were delivered using the Elgen 1000.

[0075] Skin Imaging - Skin samples or biopsies were taken from autopsies of animals after their death and stored on ice until image capture by an OV 100 imaging microscope (Anti Cancer Inc., San Diego, CA) at 480 nm.

[0076] Histopathology - Skin samples or biopsies were removed from autopsies of animals after their death, immediately preserved in 10% neutral buffered formalin and sent to Pacific Pathology, San Diego, CA, for processing and histopathological analysis. Appropriate tissues were selected, processed, embedded in paraffin, sectioned at approximately 5 μm and labeled with hematoxylin and eosin. The resulting slides were examined by a board-certified pathologist. Sections were viewed using a Zeiss Axioplan microscope with a 10x objective at the Burnham Institute, San Diego, CA.

[0077] Detection of NP antibodies in immunized mouse sera - Antibody responses against NP were evaluated by ELISA using serum from immunized mice. The mice had blood samples taken from the retro-orbital region two weeks after the last immunization. Nunc MaxiSorp Immuno plates were coated with NP (5 μg/mL, Imgenex IMR-274) at 4 °C overnight. Unbound antigens were removed from the plate by an automated plate wash using PBS with 0.05% Tween-20. Plates were blocked for nonspecific binding by adding 200 μL of PBS with 0.5% BSA for one hour at 37°C. After washing as described above, serum was diluted 1:50 in PBS with 0.2% BSA and 0.05% Tween-20 and added to the first well. A serial dilution was made by diluting 1:3 to 1:5 for each well. The serum was incubated for two hours at 37°C before washing. Antimouse IgG-biotin (B9904-5ml; Sigma-Aldrich, St. Louis, MO, USA) was diluted 1:10000 and 50 μL was added to each well, incubating for one hour at 37°C before washing. Then, 50 μL of streptavidin-HRP (Southern Biotech, Birmingham, AL, USA) diluted 1:1000 was added to each well, incubating for one hour at 37 °C before washing. The final step was done by adding 50 μL of HRP substrate (P-9187, Sigma-Aldrich) and incubating at room temperature in the dark for 10 minutes before reading the optical density (OD) at 450 nm. A reading would be considered positive if the OD was three times greater than the OD of naïve mouse serum. The results were presented as endpoint titer, i.e., the last dilution at which the OD was greater than or equal to three times that of the naïve serum.

[0078] ELI Spot Assay - Two weeks after immunization, splenocytes from each group of mice were isolated. Single cell suspensions were cleaned by removing red blood cells using RBC lysis buffer (eBiosci-ence). The ELISPOT assay kit was purchased from R&D Systems. ELISPOT 96-well plates (Millipore) were coated with anti-mouse interferon (IFN-γ) monoclonal antibody. After overnight incubation at 4°C, wells were washed and blocked with blocking buffer according to the R&D Systems protocol. The pooled splenocytes from each group were added to the wells and incubated with 1 μg/mL NP147 (TYQRTRALV Biosynthesis Inc.) for 48 hours. The plates were then washed and examined following the protocol described by the manufacturer. The stains were counted and analyzed by Tecnologia Celular Ltd.

[0079] Intranasal Influenza Assay - Balb/c mice, in groups of 10, were immunized in weeks 0, 3 and 6. Mice were immunized: first with 30+30μg in week 0, addition of 30+30μg in week 3, addition of 100+100μg in week 10. In week 12, influenza assay experiments were performed using BSL IV protocols at The National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada, in accordance with ethical guidelines of its ethics committee. A dose of 100xLD50 of H5N1 type A/Vietnam/1203/04 was used for nasal challenge. The mice were then monitored for survival and body weight every day for 21 days following challenge.

[0080] HAI Assays - Animals had blood samples taken and serum samples were immediately stored on dry ice before being sent to BIOQUAL, Rockville, MD for processing. Sera were treated with killing receptor enzyme by diluting 1 part serum with 3 parts enzyme and were incubated overnight at 37°C in a water bath. The enzyme was inactivated by 30 minutes of incubation at 56°C, then 6 parts of phosphate-buffered saline were added for a final dilution of 1/10. HAI assays were performed in 96-well V-bottom microtiter plates using 4 hemagglutination units of virus and 1% red blood cells as previously described. Viruses used for the HAI assays were obtained from the CDC Influenza Branch.

Example 1

[0081] Electroporation using a minimally invasive device results in resistant reporter gene transfection

[0082] A minimally invasive electrode device (MIED) was designed for use with DNA vaccine delivery. This new applicator consists of gold-colored stainless steel needle electrodes with trocar sharpenings (the electrodes have a tip at the distal end that is rounded with a sharp tip) with 1.5 mm spacing in a matrix pattern 4 x 4 (Figures 4a and 4c). Alternating rows of four electrodes can be activated with reverse polarity: positive than negative. The electrodes are placed in electrical sockets within the plastic cable, allowing each electrode to be approached individually for alternative activation patterns. The device is designed to only make contact with the surface of the skin and not penetrate directly into the tissue. After adequate contact, sharpening the electrodes can allow the device array to break through the stratum corneum barrier layer, ensuring consistent impedance measurements and resulting in repeatable electroporation treatments. The prototype device was built with a connection cable for connection to the pulse generator (Figure 4b).

[0083] The lower limits of voltage parameters required for successful transfection and immune responses using this new minimally invasive needle electrode device (MIED) were studied. Separate skin sites on the flank of a guinea pig were injected using the Mantoux technique with 50 μl of 1 mg/ml GFP plasmid and immediately pulsed using the MIED set at a voltage as low as 15 V (Figure 5a). . Resistant and reproducible GFP transfection was visualized 8 hours after treatment and reached a maximum at 3 days. No or minimal GFP transfection was detectable after injection of isolated GFP plasmid (data not shown). Although a range of voltages and electrical parameters resulted in GFP transfection, pulses at 15 volts were more reproducible (data not shown). Reproducibility was quantified and assessed by positive pixel counting after fluorescence microscopy under standardized conditions. The most consistent pixel count numbers within a defined area were obtained at the 10 to 15 volt parameters. Actual GFP transfection patterns appeared as distinct “islands” around the point of contact with each electrode (Figure 5b). Effective EP is superficial and highly localized around the electrode tip at low voltage and consequently with no significant transfection below the stratum basale layers of the epidermis.

[0084] To ensure that resistant transfection was not a guinea pig-specific phenomenon, GFP localization was performed in a variety of species. Skin sites in mice, rabbits, rats, and pigs were injected with 50 μl (30 μl in mice) of 1 mg/ml GFP plasmid and immediately pulsed using the MIED. All species demonstrated resistant dermal GFP transfection after electroporation, with minimal or no transfection detected after injection of isolated GFP plasmid (Figure 6). In all species, strong GFP positivity was detected in all electroporated skin samples. However, only the rabbit samples demonstrated the distinct “island” pattern observed in the guinea pig samples (Figure 5). GFP localization in rat, mouse, and pig skin appeared more diffuse. The actual transfected area was similar between mouse, rabbit, and pig (approximately 4 square mm) as it was a function of injection bubble size. The transfected area on mouse skin was slightly smaller due to the smaller injection volume. In the mouse panel, the entire skin of the mouse is shown, so that the relative size can be appreciated. GFP positive cells were detected only on the skin surface in the rat, rabbit and pig samples. No GFP signal was observed at the bottom of these samples. The bottom of the mouse skins showed a positive GFP signal. However, this appears to be related to the thinness of the skin sample.

[0085] These data suggest that MIED can efficiently transfer plasmid to the skin across small and large animal models.

Example 2

[0086]Electroporation using a minimally invasive device - Histological analysis demonstrates transfection in the upper layers of the epidermis

[0087] To address the effect of electroporation by MIED at a cellular level, histological analysis was performed. Sites in a guinea pig were injected with 50 μl of 1 mg/ml GFP plasmid and immediately pulsed using the MIED. Biopsy samples were taken 3 days after treatment, selected, sectioned in paraffin, and stained with H&E (slide not shown). Pathological analysis revealed no associated tissue damage after electroporation with the minimally invasive device. High-power microscopy revealed that the majority of GFP transfection occurred in the stratum granulosum cell layer of the epidermis (slide not shown). GFP positive cells were also detected in the stratum basale. In contrast, only a few GFP-positive cells were detected in the biopsy that was injected with GFP plasmid alone. This was in direct contrast to the resistant signal observed in the tissue biopsy sample that received electroporation. A visual estimate of expression enhancement is more than 100- to 1000-fold based on the number of GFP-positive cells relative to the isolated DNA.

Example 3

[0088] Electroporation using a minimally invasive device results in resistant cellular responses and provides 100% lethal assay protection in mice

[0089] Mice were immunized with DNA coded for NP and M2 influenza antigens: first with 30+30μg in week 0, addition of 30+30μg in week 3, addition of 100+100μg in week 10. Combined Porto-type NP antigen Rico/39 and combined M2 antigen from strain New Caledonia/99 were optimized, synthesized, and then cloned into the backbone of a clinically approved mammalian expression vector, pMB76.5. The NP and M2e antigens were chosen to predominantly assess cellular immunity. These proteins are not capable of inducing neutralizing antibody responses.

[0090] Groups of mice were electroporated with the minimally invasive dermal device or the Elgen 1000 intramuscular device (Inovio Pharmaceuticals, Inc., Blue Bell, PA). Induction of potent cellular and humoral responses in the mouse model measured by antigen-specific T cell ELI Spot assays and antibody titers were observed in all mice. Antigen-specific CTL responses were 200 +/- 57.9 SFU/10A6 splenocytes versus NP for MIED and 85 +/- 36.3 SFU/10A6 splenocytes versus NP for IM (Figure 7a). These intradermally electroporated animals showed higher cellular responses and titers, although not statistically significant.

[0091] In an attempt to determine whether the induced immune responses were capable of impacting infection, mice were subjected to testing with a lethal dose of influenza A/H5N1/Vietnam/1203/04 via intranasal (i.n.) inoculation (Figure 7B). The influenza strain VN/1203/04 (H5N1) is known to cause rapid morbidity and mortality. While 100% of naive mice did not resist infection until the 11th day, 100% of mice immunized with MIED were protected against morbidity and mortality until the 15th day (end of the experiment). Likewise, until the 15th day, 90% of positive control animals immunized with IM survived.

Example 4

[0092] Electroporation using a minimally invasive device results in humoral immunogenicity and protective HI titers

Guinea pigs

[0093] It is difficult to reach the dermis in mice because of the extremely thin skin structure and the proximity of underlying muscle. For this reason, immune responses induced in mice are usually a combination of skin and muscle. Therefore, MIED was tested in a larger animal model, guinea pigs, which have a better defined skin structure.

[0094] Guinea pigs were immunized with a previously described consensus SYNCONTM influenza vaccine by intradermal electroporation through the MIED. Animals were vaccinated with a vaccine and plasmid cocktail (100 μg/plasmid) containing pGX2005 (SYNCONTM vaccine construct encoding an H1HA consensus sequence) and pGX2009 (SYNCONTM vaccine construct encoding a porcine consensus sequence). H1HA) in a volume of 50 μl diluted in 1 x PBS. Two weeks after two immunizations, each animal developed resistant HAI titers in 1:40 against the pandemic strain H1N1 Mexico/2009 and also to type A/H3N2 Brisbane and, in one dimension, to the strain A/H5N1 Vietnam/2005 ( Figure 8).

Monkeys

[0095] Monkeys were vaccinated with a previously described consensus influenza vaccine and plasmid cocktail (100 μg/plasmid) containing pGX2005 (SYNCONTM vaccine construct encoding an H1HA consensus sequence) and pGX2009 (vaccine construct SYNCONTM encoding a porcine H1HA consensus sequence) in a volume of 50 μl by intradermal electroporation (MIED) or intramuscular electroporation (Elgen 1000). After two immunizations, four of the four animals developed HAI titers of 1:40 against the H1N1/Mexico/2009 strain and against the A/H1N1/New Caledonia strain when immunized intradermally with MIED (Figure 9).

Example 5

[0096] Tolerability study with the minimally invasive device

[0097] To assess the level of EP pain with the minimally invasive EP device, it is applied to the epidermal layers between the stratum corneum and the basal layer. The visual response from tests with anesthetized animals does not produce the muscle contraction visualized with higher energy pulses or invasive electrode arrays, although it demonstrates effective transfection with reporter genes and immune response with antigenic plasmids. Pulse voltages of up to 15V and currents of up to approximately 30mA produce only a mild sensation during induction of protective immune responses in guinea pigs and rhesus monkeys against influenza.

[0098] In vivo EP applied using the invasive dermal device matrix showed marked reduction in pain compared to invasive intramuscular device matrices. MIED results are expected to further enhance pain reduction.

[0099] In vivo EP is applied using the MIED matrix after an ID injection of 0.9% saline into the stratum granulosum. Patients are injected with 0.15 mL of saline into the stratum granulosum layer and then EP is performed using CELLECTRA®2000 (Adaptable Constant Current Device or alternatively an Elgen 1000 device, Inovio Pharmaceuticals, Inc., Blue Bell, PA). EP parameters are 15 V, 100 millisecond pulse and 3 pulses in total.

[00100] For each individual, pain at the injection site will be assessed using a Visual Analogue Scale (VAS), which is determined immediately after PE.

Claims (28)

1. Electroporation device capable of providing an individual with a tolerable electrical potential to the epidermal tissue that results in electroporation of cells in said tissue, CHARACTERIZED by the fact that it comprises: a voltage generator; and an array having a plurality of electrodes in electrical communication with the voltage generator; wherein each of the plurality of electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; and the voltage generator delivers, through the electrodes, an electrical potential of about 0.1 volt to about 70 volts to the epidermal tissue; and further wherein the electrodes have a tip at the distal end that is rounded (blunt) with a sharp tip, which allows the electrode to penetrate the layers of epidermal tissue between the stratum corneum and basal layers and provide the electrical potential of the voltage generator to epidermal tissue, the electrodes being adapted to penetrate the epidermal tissue to depths of 0.1 mm or less; the electrodes providing an electrical potential to the cells of the epidermal tissue in order to cause minimal tissue damage to said cells. 2. Device according to claim 1, CHARACTERIZED by the fact that the electrodes are adapted to penetrate the epidermal tissue to depths of about 0.01 mm to about 0.04 mm. 3. Device according to claim 1, CHARACTERIZED by the fact that the electrodes are spaced from each adjacent electrode at a distance of about 1.5 mm. 4. Device according to claim 3, CHARACTERIZED by the fact that the voltage generator provides the epidermal tissue with an electrical potential of about 1 volt to about 15 volts. 5. Device according to claim 4, CHARACTERIZED by the fact that the voltage generator provides the epidermal tissue with an electrical potential at a current ranging from about 1 mA to about 50 mA. 6. Device according to claim 4, CHARACTERIZED by the fact that the voltage generator provides the epidermal tissue with an electrical potential for a duration ranging from about 5 milliseconds to about 250 milliseconds. 7. Electroporation device CHARACTERIZED by the fact that it comprises: an array having a plurality of needle electrodes, each configured to be individually in electrical communication with a voltage generator; wherein each of the plurality of needle electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; wherein the plurality of needle electrodes is configured to deliver an electrical potential of about 0.1 volts to about 70 volts to the epidermal tissue between the stratum corneum and the basal layers; and wherein each of the plurality of needle electrodes defines a central axis and a distal end, wherein the distal end defines a trocar tip having a sharp tip and a plurality of flat surfaces extending distally to the sharp tip, wherein the plurality of flat surfaces are oriented, respectively, at rounded angles between 10 degrees from the central axis and substantially perpendicular to the central axis, wherein the rounded angles and the sharp tip collectively allow the plurality of needle electrodes 1) to penetrate the layers of epidermal tissue between the stratum corneum and basal layers at depths of 0.1 mm or less, and 2) provide electrical potential to the epidermal tissue between the stratum corneum and basal layers so that at least a majority of cell electroporation occurs into cells of the stratum granulosum. 8. Device according to claim 7, CHARACTERIZED by the fact that the plurality of needle electrodes is adapted to penetrate the epidermal tissue at depths of about 0.01 mm to about 0.04 mm. 9. Device according to claim 7, CHARACTERIZED by the fact that the electrodes are spaced from each adjacent electrode at a distance of about 1.5 mm. 10. Device, according to claim 7, CHARACTERIZED by the fact that it comprises a voltage generator configured to provide to the cells of the epidermal tissue, through the plurality of needle electrodes, an electrical potential of about 0.1 volts to about 50 volts. 11. Device, according to claim 10, CHARACTERIZED by the fact that the electrical potential does not result in visible tissue damage in said cells of the individual according to a histopathological analysis of the tissue. 12. Device according to claim 11, CHARACTERIZED by the fact that the voltage generator provides the epidermal tissue with an electrical potential of about 1 volt to about 15 volts. 13. Device according to claim 12, CHARACTERIZED by the fact that the voltage generator provides the epidermal tissue with an electrical potential at a current ranging from about 1 mA to about 50 mA. 14. Device according to claim 12, CHARACTERIZED by the fact that the voltage generator provides the epidermal tissue with an electrical potential with a duration ranging from about 5 milliseconds to about 250 milliseconds. 15. Device, according to claim 10, CHARACTERIZED by the fact that the voltage generator comprises at least one battery. 16. Device according to claim 7, CHARACTERIZED by the fact that each of the plurality of needle electrodes comprises stainless steel and is gold plated. 17. Device according to claim 7, CHARACTERIZED by the fact that the plurality of flat surfaces consists of three flat surfaces. 18. Electroporation device capable of providing an individual with a tolerable electrical potential to layers of epidermal tissue between the stratum corneum and the basal layers that results in the electroporation of cells in said tissue, CHARACTERIZED by the fact that it comprises: a portable housing configured to manual operation; a voltage generator contained in the portable housing; an array housing carrying a plurality of needle electrodes; and an electrode housing coupled to the matrix housing, wherein the electrode housing has a plurality of electrical outlets, the plurality of needle electrodes is received within the plurality of electrical outlets, the plurality of electrical outlets places the plurality of electrical outlets of needle electrodes in electrical communication with the voltage generator, and the matrix housing is attachable and detachable from the portable housing; wherein each of the plurality of needle electrodes is spaced from each adjacent needle electrode at a spacing distance of from about 0.5 mm to about 2.5 mm; wherein the voltage generator provides through the plurality of needle electrodes an electrical potential of about 0.1 volts to about 70 volts to the epidermal tissue; and wherein the plurality of needle electrodes extends distally from the array housing and the plurality of needle electrodes each has a distal end defining a trocar tip having a sharp tip and a plurality of flat surfaces extending distally to the sharp tip, wherein the plurality of flat surfaces are oriented, respectively, at generally rounded angles (blunt), wherein the generally rounded angles and sharp tip of each needle electrode and spacing distance collectively allow the plurality of needle electrodes 1) penetrate the epidermal tissue layers between the stratum corneum and basal layers to depths of 0.1 mm or less, avoiding penetration into the basal layers, and 2) provide the electrical potential of the voltage generator to the layers of epidermal tissue between the stratum corneum and the basal layers, so that at least a majority of cell electroporation occurs in cells of the stratum granulosum. 19. Device, according to claim 18, CHARACTERIZED by the fact that the voltage generator comprises at least one battery. 20. Device according to claim 19, CHARACTERIZED by the fact that the portable housing further comprises a circuit board having an electrical circuit that is in electrical communication with the at least one battery and is powered by the at least one battery. 21. Device according to claim 20, CHARACTERIZED by the fact that the portable housing further comprises at least one capacitor in electrical communication with the electrical circuit, wherein the battery provides the electrical potential to the plurality of needle electrodes through of at least one capacitor and the electrical circuit. 22. Matrix for an electroporation device CHARACTERIZED by the fact that it comprises: a plurality of needle electrodes, each configured to be individually in electrical communication with a voltage generator; wherein each of the plurality of needle electrodes is spaced from each adjacent electrode at a distance of from about 0.5 mm to about 2.5 mm; wherein the plurality of needle electrodes is configured to deliver an electrical potential of about 0.1 volts to about 70 volts to the epidermal tissue between the stratum corneum and the basal layers; and wherein each of the plurality of needle electrodes defines a central axis and a distal end, wherein the distal end defines a trocar tip having a sharp tip and a plurality of flat surfaces extending distally to the sharp tip, wherein the plurality of flat surfaces are oriented, respectively, at rounded angles between 10 degrees from the central axis and substantially perpendicular to the central axis, wherein the rounded angles and the sharp tip collectively allow the plurality of needle electrodes 1) to penetrate the layers of epidermal tissue between the stratum corneum and basal layers at depths of 0.1 mm or less, and 2) provide electrical potential to the epidermal tissue between the stratum corneum and basal layers so that at least a majority of cell electroporation occurs into cells of the stratum granulosum. 23. Array according to claim 22, CHARACTERIZED by the fact that the plurality of needle electrodes is adapted to penetrate the epidermal tissue at depths of about 0.01 mm to about 0.04 mm. 24. Matrix, according to claim 22, CHARACTERIZED by the fact that the electrodes are spaced from each adjacent electrode at a distance of about 1.5 mm. 25. Matrix, according to claim 22, CHARACTERIZED by the fact that each of the plurality of needle electrodes comprises stainless steel and is gold plated. 26. Matrix, according to claim 22, CHARACTERIZED by the fact that the plurality of flat surfaces consists of three flat surfaces. 27. Matrix, according to claim 22, CHARACTERIZED by the fact that it comprises a matrix housing that carries the plurality of needle electrodes, wherein the plurality of needle electrodes extends distally from the matrix housing. 28. The array of claim 27, wherein the electrode housing has a plurality of electrical outlets, the plurality of needle electrodes is received within the plurality of electrical outlets, and the plurality of electrical outlets is configured to place the plurality of needle electrodes in electrical communication with a voltage generator.