English:
[0005] In vivo plasmid transfer technology, particularly as it relates to the transfer of plasmids to intradermic ("ID") or subcutaneous ("SQ") tissue or cells, has traditionally been limited in scope because in vivo expression levels resulting from the naked DNA (plasmid) transfer have been low, with only fractions of those achieved by viral gene transfer. The doses of plasmid that can be delivered in a volume adapted for the ID or SQ is small compared to doses that can be used in intramuscular injections ("IM"). Numerous investigators have outlined the safety and toxicological concerns with injecting viruses as DNA vectors into animals and humans (Frederickson, et al., MoI. Ther. 8:8-10 (2003)). Consequently, direct injection of plasmid DNA has become a desired delivery technique, however, efficient delivery into cells and protein expression levels have been a challenge. Skeletal muscle cells have provided a main target for direct plasmid transfer for DNA vaccines and other applications (Prud'homme, et al., Curr. Gene Ther. 6:243-273 (2006)).
[0006] A recent advance for plasmid delivery in vivo has been that of electroporation ("EP"). EP is used for delivery of a large variety of molecules: from ions to drugs, dyes, tracers, antibodies, and oligonucleotides, to RNA and DNA (Gehl, Acta Physiol Scand. 177:437-447 (2003)). This process exposes the target tissue to a brief electric field pulse that induces temporary and reversible pores in the cell membrane. During the period of membrane destabilization, molecules such as plasmids may gain intracellular access. Previously, reports have shown that improvements in the EP process increases the efficacy of transfer and lowers the amount of plasmid needed to generate targeted levels of antigens in vivo by using a constant-current device (Draghia-Akli and Smith, Page 245 in Gene Therapy - Therapeutic Mechanisms and Strategies, N. S. Templeton and D. D. Lasic, eds. Marcel Dekker, Inc., New York. (2003)). Enhancement of plasmid delivery using EP allows the injected tissue to be used as a bioreactor for the high production and secretion of proteins into the blood stream, and/or antigen presentation. The expression levels are increased by as much as two to three orders of magnitude over plasmid injection alone, to levels comparable to those of adenoviral-mediated gene delivery and may in some cases reach physiological ranges.
[0008] One of the challenges of skin EP in large mammals and humans is the individual variation of skin thickness, as well as variation in skin thickness between different anatomical regions. For instance, skin thickness at the deltoid level in humans is approximately 2 millimeters ("mm"), while at the suprascapular level it is approximately 2.6 mm, at the waist it is approximately 1.7-1.9 mm and at the thigh it is approximately 1.6-1.7 mm. Skin is sensitive, thus electrodes have to also be adapted in gauge and configuration to create the least discomfort. While for therapeutic purposes a certain degree of discomfort is sometimes acceptable to patients during therapy, it has been suggested that only a relatively painless method can be used for prophylactic vaccination. Electrode array configuration and characteristics, and device pulse patterns, have to be adapted to this challenge. It is known that the maximum uniform electric field is generated during electroporation from approximately 2 mm beyond the tip of an electrode to a point one-third the electrode from its tip.
[0009] U.S. Patent No. 7,245,963 by Draghia-Akli, et al. describes modular electrode systems and their use for facilitating the introduction of a biomolecule into cells of a selected tissue in a body or plant. The modular electrode systems comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant. The biomolecules are then delivered via the hypodermic needle into the selected tissue. The programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes. The applied constant-current electrical pulse facilitates the introduction of the biomolecule into the cell between the plurality of electrodes. The entire content of U.S. Patent No.7,245,963 is hereby incorporated by reference.
[0010] U.S. Patent Pub. 2005/0052630 submitted by Smith, et al. describes an electroporation device which may be used to effectively facilitate the introduction of a biomolecule into cells of a selected tissue in a body or plant. The electroporation device comprises an electro-kinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device produces a series of programmable constant-current pulse patterns between electrodes in an array based on user control and input of the pulse parameters, and allows the storage and acquisition of current waveform data. The electroporation device also comprises a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire content of U.S. Patent Pub. 2005/0052630 is hereby incorporated by reference.
[0015] When tissues have a small proportion of conducting ions, resistance is increased, heat is generated and cells are killed. Ohm's law expresses the relationship between current ( "I"), voltage ( "V"), and resistance ( "R "):
[0019] When electricity is delivered to the cells of a subject, the dose of electricity can be accurately described in terms of charge ( "Q "), which is the current ( "/") and the time ( "t"), according to the formula:
[0021] The effectiveness of electroporation is limited by the fact that there is a threshold value for the pulse intensity below which electroporation does not occur, and an upper limit above which the cells are destroyed. Experimental evidence shows that the difference between the upper and lower limits is so small that it is very difficult to design effective pulsing protocols without undue experimentation. This makes use of the technique difficult, mostly when one targets tissues that inherently have a non-homogeneous cell composition, such as skin - cells in epidermis and dermis of different thickness and number of strata, fat, fascia, blood vessels of different dimensions from capillaries to small vessels; only a true software driven device capable of instantaneously analyzing the instant conditions variable from individual to individual and between locations on the skin surface of each individual and adapting the output to the specific conditions would fulfill these requirements. Also, the electrode should be adapted to the skin morphology and prevent tissue damage and bleeding.
[0024] Previous investigators have utilized electroporation devices for plasmid DNA transfer that are conceptually based on constant voltage systems, utilizing a predetermined voltage between the electrodes. Because the impedance between electrodes that are embedded in a tissue can vary from case-to-case, or tissue-to-tissue, a predetermined voltage does not produce a predetermined current. A predetermined voltage pulse causes an unregulated increase in the current flowing through a muscle tissue during the duration of the pulse in addition to the loss of the perfect square wave function. By contrast, a constant- current source actually maintains a square wave function constant current through muscle tissue. However, the existing commercial electroporation devices do not have the firmware designed to enable measurement of the exact amount of current to which the cells are exposed. The unregulated current generated with conventional electroporation devices may generate amounts of heat in tissues that can easily kill cells. For example, a typical electronic 50 milliseconds (ms) pulse with an average current of 5 Amperes ("A," or "Amp") across a typical load impedance of 25 ohms ("ω") can theoretically raise the temperature in tissue 7.5°C, which is enough to kill cells. The physics of tissue injury caused by electrical shock is reviewed by Lee, et al. (Lee, et al., Annu. Rev. Biomed. Eng 2:477-509.:477-509 (2000)). Thus, there is a need to avoid the technological problems associated with constant voltage electroporation by providing a means to control effectively the amount of electricity delivered to the cells and thereby achieve proficient electroporation while limiting the destruction of cells.
[0025] The difficulties present in many electrodes stem from the fact that the pulse energy is concentrated in the center of the array, the point where the material to be transfected is deposited. As a result, the spatial distribution of energy delivery assumes a very nonuniform character. Therefore, only a fraction of the cells in the volume encompassed by the electrode assembly is electroporated. Thus, there is also a need for some applications to provide a means to effectively control the dosage of electricity delivered to the cells in the inter-electrode space by precisely controlling the ionic flux that impinges on the conduits in the cell membranes. Also, commercially existing electrodes for large animal applications and humans generally do not allow for ID or SQ targeting of dendritic cells, as they are typically too long, of an inappropriate gauge and improper bevel orientation. Thus, there is a need for certain applications for a skin electroporator, facilitating the in vivo delivery of a biomolecule, such as a plasmid, into skin tissue, e.g., ID or SQ spaces in an animal.
[0027] In addition, electroporation devices which use skin and muscle invasive replaceable needle arrays as electrodes to deliver the electric current require maintenance of sterile conditions when the needle array replacement occurs. This is necessary from both a medical practice and regulatory compliance viewpoint. In the same time, a disposable skin electrode is needed to allow for lower production costs and usage in mass vaccination of both therapeutic and prophylactic purposes. Thus, there is also a need to provide a skin electrode disk that allows easy replacement of the needle skin array.
[0033] Figure 1 shows a diagram of the structure of the skin. The most superficial region is the epidermis (1.1), which in turn can be divided into 5 sub-layers: stratum comeum (1.1.1), stratum spinosum (1.1.2), stratum lucidium (1.1.3), stratum granulosum (1.1.4), and stratum basale (1.1.5). The dermis (1.2) is situated just under the epidermis, and it is a very well vascularized region; also the hair roots and sweet glands are located in this layer. The subcutaneous layer is situated just underneath the dermis;
[0034] Figure 2 A shows a system diagram of a preferred embodiment of the EP devices described herein.
[0035] Figure 2B shows an example of a controller with may be a part of the EP devices described herein.
[0036] Figure 2C shows an example of an actual pulse sequence recording downloaded at the end of the pulse sequence, using the skin EP device as a pulse generator, and the described skin electrode array to deliver the electric pulses after plasmid formulation injection into the ID and SQ layers of skin in a 25 kg pig: ("2.1") shows the file name that has been downloaded via the infrared port onto a personal computer or hand-held device; ("2.2") shows the subject number, as entered directly into the skin EP device through the numerical pad, as a prerequisite condition before the EP process could start; ("2.3") shows the characteristics of the electric pulses selected by the operator, including the number of pulses in the pulse sequence, wait time before and between pulses (in seconds, "5"), the pulse width (in milliseconds, "ms"), the pulse current amplitude (in amperes, "A"); ("2.4") shows the electrical charge of each electrode during each pulse - for each of the skin electrodes 1, 2, 3 any of the following positions is possible: positive, "pos"; negative, "neg"; or off "off - the "off electrode(s) are importing back information into the device before, during and after each pulse ("feed-back"); ("2.5") shows the actual amperage ("A"), voltage ("V") and skin tissue resistance, Z, at the injection area ("Ohms") during each pulse of the pulse sequence; as the feed-back arises every 0.1-0.2 ms (and in this case the pulses were 52 ms in length), only a small proportion of the feed-back file has been included, for example purposes;
[0037] Figure 3 shows an example of the graphical display of downloaded data when using a skin skin electrode delivery of electric pulses by the skin EP device device. The graph depicts both the voltage ("3.1.1") and current ("3.1.2") as measured by the skin EP device in Pulse 1 ("3.1") as well as the voltage ("3.2.1") and current ("3.2.2") as measured by the skin EP device in Pulse 2 ("3.2"). The tissue resistance (as measured during the electric pulses) is also displayed for Pulse 1 ("3.3") and Pulse 2 ("3.4");
[0038] Figure 4 shows an example of the graphical display of downloaded data when using a large electrode array delivery of electric pulses by the skin EP device device to the skin. The graph depicts both the voltage ("4.1.1") and current ("4.1.2") as measured by the skin EP device in Pulse 1 ("4.1"), the voltage ("4.2.1") and current ("4.2.2") as measured by the skin EP device in Pulse 2 ("4.2"), and the voltage ("4.3.1") and current ("4.3.2") as measured by the skin EP device in Pulse 3 ("4.3"). The tissue resistance (as measured during the electric pulses) is also displayed for Pulse 1 ("4.4"), Pulse 2 ("4.5") and Pulse 3 ("4.6");
[0039] Figure 5 shows a schematic representation of a skin electrode array, including detailed characteristics: ("5.1") side view depicting length and angle of insertion end of a single needle-electrode; ("5.2") front view depicting angles and degrees for revere- trochar point; ("5.3") notes indicating needle-electrode specifications;
[0040] Figure 6 shows a visual representation ("6.1") and schematics ("6.2") of the skin electrode handle: ("6.1.1") trigger used to activate the impedance check and then electroporation sequence; ("6.1.2") "LED" displays green or red when the skin EP device is ready to treat or during an error, respectively; ("6.1.3") skin electrode array locked into place; ("6.1.4") cord containing individual wires and signals between the skin electroporation handle and skin EP device unit; ("6.1.5") individual wires whose detail is depicted in electrical wiring diagram in Figure 8; ("6.2.1") trigger schematic; ("6.2.2") LED schematic; ("6.2.3") schematic depicting the raised ridge on the skin electrode array used to facilitate ejection from the skin electrode handle following completion of electroporation pulse sequence; ("6.2.4") schematic of needle-electrodes of skin electrode array; ("6.2.5") side view schematic of skin electrode array locked into place on handle; ("6.2.6") schematic of locking arm and detent (on handle); ("6.2.7") schematic depicting sockets inside skin electrode handle used to receive and make contact for circuit connection; ("6.2.8") schematic depicting cord housing individual wires whose detail is shown in Figure 8;
[0041] Figure 7 shows the skin electrode array mounted on the handle: ("7.1") side view schematic of individual sockets for receiving and making electrical connection between the individual needle-electrodes in skin electrode array and skin electrode handle;
[0042] Figure 8 shows electrical wiring diagram for array and handle assembly;
[0043] Figure 9 shows the schematic representation of the multi skin electrode array clamshell folding packaging container that allows for sterile loading of the handle between treatment subjects: ("9.1") lid; ("9.2") base, including individual wells custom- designed to house arrays; ("9.3") side-view of wells; and
[0044] Figure 10 shows the average expression levels of GFP in animals which were injected with different amounts oftheplasmidpSP5-12-GFP and electroporated with the skin EP device using the skin electrodes array and skin specific EP conditions (MA), compared to electrodes and conditions usually employed for plasmid delivery to the skeletal muscle (LA).
[0045] Figure 11 shows a three-electrode needle array where distance L = kxn, where n represents the number of electrodes and k represents a proportionality constant.
[0046] Figure 12 shows a graph of SEAP expression levels (pg/mL/kg) over 1 1 days from the time of electroporation using the respective SEAP expressing plasmids.
[0047] Figure 13 shows a graph of the SEAP expression levels at day 11 for various samples along with related ELISA results (in average antibody dilution).
[0048] Figure 14 shows a graph of IGF-I expression levels (mg/mL) over 11 days from the time of electroporation using the respective IGF-I expressing plasmids.
[0049] Figure 15 displays fluorescent images that represent green fluorescent protein expression after ID/SQ plasmid administration followed by electroporation (EP) in pig skin. Plasmids were delivered at different doses and volumes with either the larger EP array customary used for IM + EP (A), or the skin EP (B).
[0050] Figure 16 displays a graph showing levels of secreted embryonic alkaline phosphatase (SEAP) detected in pig serum at 4-11 days after pSEAP administration. Animals received the same plasmid dose (1 mg), in either a concentrated formulation (at 10 mg/mL) or a regular formulation (at 2mg/mL) (n=5/group). The data are shown as group mean responses ± SEM.
[0052] Figure 18 displays a graph showing enhanced cellular immune responses to HIV-I immunogens with ED/SQ co-injection of plasmid encoded IL-12 followed by EP. IFNγ ELISpots were performed two weeks after each immunization. Responses to env are depicted as white bars and gag are depicted as black bars with the data shown as stacked group mean responses ± SEM.
[0053] Figu re 19 displays a graph showing enhanced memory responses to HIV- 1 immunogens with ID/SQ EP. Ten weeks after the last immunization, ELISpot assays were performed to determine antigen-specific memory responses to gag and env in the ID/SQ and ID/SQ+EP groups. The data are shown as group mean responses ± SEM.
[0054] Figure 20 displays a graph showing induction of T H 1 and not T H 2 mediated cellular response following ID/SQ immunization of HIV-I gag, env and plasmid rhIL-12. An IL-4 ELISpot was performed at week 10 to assess the induction of a gag or env- specific T H 2 response. Responses to env are depicted as white bars and gag are depicted as black bars with the data shown as stacked group mean responses ± SEM.
[0055] Figure 21 displays a graph showing induction of p24 (21A) and gplόO (21B) antibodies. Serum was collected at weeks 0, 4, 8, 12, and 18. Gag and env antibody titers were determined by p24 and gplόO ELISA, respectively, in the ID/SQ and ID/SQ+EP immunized groups. The data are shown as group mean responses ± SEM.
[0056] Figure 22 displays bar graphs showing SEAP expression measured in different muscles in mice. Mice were injected with 10 μg C5- 12-SEAP plasmid in the tibialis anterior (TA) muscle or gastrocnemius (G) muscle and EP after either 4s or 80s delay. Mice were also injected with 10 μg pf C5-12-SEAP plasmid without EP (no EP). Serum SEAP levels were higher in TA muscle and G muscle injected mice compared to control mice that received the plasmid without EP (*P < 1.3 E-21). Mice injected with the expression cassette without the plasmid backbone (NB) and then electroporated had higher SEAP levels than NB group without EP. However, the NB groups were significantly lower than mice administered C5-12-SEAP in the identical muscle (NB 4s compared to TA 4s **P < 0.008; NB 80s compared to TA 80s ***P < 0.004). Mice injected into the TA muscle yield higher expression than animals injected into the G muscle and lowering the delay between injection and EP in the TA muscle from 80s to 4s did not affect expression.
[0057] Figure 23 displays bar graphs showing average GFP expression quantified in mice after intramuscular (IM) or intradermal (ID) injection of 50 μg of CMV-EGFP plasmid at concentrations of 2 mg/mL or 10 mg/mL in a total volume of 25 or 5 μL, respectively. GFP expression was highest in groups administered 10 mg/mL concentration as compared to animals administered 2 mg/mL concentration (ED and IM, *P = 0.01), although ED and IM injection sites yielded similar overall scores when compared to each other.
[0058] Figure 24 displays bar graphs showing SEAP expression, which was driven by the ubiquitous CMV promoter, measured in different muscles in mice under various current settings. The plasmid formulation was tested: saline, saline + LGS, water, or water + LGS. Saline + LGS formulation at 0.1 A in the TA muscle yielded the highest expression. Animals receiving plasmid formulated into water and electroporation at 0.2A yielded significantly lower SEAP levels than those receiving 0. IA for the same muscle (*P < 0.05 for TA and **P < 0.001 For G). When water + LGS was used as the plasmid formulation, the differences in serum SEAP levels were not significant for the TA muscle, but were for the G muscle (***P < 0.04).
[0059] Figure 25 displays titers for anti-SEAP antibodies measured 14 days post- treatment, which were highest in mice treated with 0.1A of current in the G muscle when plasmid was formulated in (A) saline + LGS and in the TA muscle when formulated in (B) water + LGS.
[0060] Figure 26 displays a schematic of one embodiment of a relay contact matrix.
[0061] Figure 27A displays a first part of a flowchart of the electroporation functional flow.
[0062] Figure 27B displays a second part of the flowchart of the electroporation functional flow.
[0065] 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 tissue between electrodes and altering the energy output delivered by the EP device accordingly in order to maintain the current at a constant level. This constant level is preset by a user prior to initiation of a pulse sequence or electrical treatment. Preferably, the feedback is accomplished by the electroporation component, e.g., controller, of the skin EP device, as the electrical circuit therein is able to continuously monitor the current in tissue between electrodes and compare that monitored current (or current within tissue) to a preset current and continuously make energy-output adjustments to maintain the monitored current at preset levels. In some embodiments, the feedback loop is instantaneous as it is an analog closed-loop feedback.
[0066] The term "biomolecule" as used herein refers to nucleic acid sequences, proteins, lipids, microbubbles (e.g. drug-loaded vesicles), and pharmaceuticals.
[0067] The terms "electroporation," "electro-permeabilization," or "electro-kinetic enhancement" ("EP") as used interchangeably herein refer to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cellular membrane to the other.
[0069] The term "skin region" (or "skin tissue") as used herein refers to skin tissue, dermis, subdermis, and intradermic ("ID"), intracutaneous, subcutaneous ("SQ") layers or spaces. The skin region does not include muscle tissue.
[0070] The term "without substantially penetrating" as used herein refers to penetration of no more than about 1 mm to 2 mm by an object (e.g., needle) of interest, and preferably no more than about 5 mm.
[0074] Figure 2A shows a system diagram of one preferred embodiment of the EP devices provided herein. Major functional elements of the EP devices are shown in the diagram, but are for illustrative purposes only as not all functional elements are necessary for all embodiments. Each element is described in terms of the hardware functionality of each element. The sequences of events that are enabled by the hardware can be controlled by software, as described herein. The central element of the device is the controller in this example. One example of a controller can be seen in the schematic shown in Figure 2B, which is a microcontroller, which in some embodiments is a Texas Instruments msp430F149 or Motorola 68HC908AZ60A. Other alternative controllers are known to those skilled in the art of electrical engineering and can readily replace the illustrative examples, above.
[0075] hi some embodiments, the skin electrodes are needle electrodes, conductive rods, or conductive film areas, and preferably needle electrodes. Preferably, the needle electrodes are capable of contacting an intradermic or a subcutaneous tissue without substantially penetrating a muscle tissue. In some embodiments the skin electrodes can be mounted on a base, which can of plastic material, forming a replaceable, or exchangeable, skin electrode disk having an array of skin electrodes (or skin array or skin needle array) which can be used in association with the skin EP devices described herein. In some embodiments, the skin electrodes can be from about 1 mm to 20 mm; 1 mm to 12 mm; 2 mm to 10 mm; 2 mm to 7 mm in length; or about 5 mm in length. In some embodiments the skin electrode can be 22 to 28 gauge; 23 to 27 gauge; 24 to 27 gauge, or about 26 gauge. Preferably, the skin electrode is approximately 5 mm in length and 26 gauge. In some embodiments the skin electrode is the only part of the skin EP device that actually touches the skin of the patient (to prevent cross-contamination). A vaccine formulation can be delivered to the selected area, the area surrounded by the skin array, and the skin electrodes inserted into the skin. The skin array preferably creates a uniform pressure around the skin electrodes inserted into the skin, and thereby helps to generate a uniform electric field during the EP process in the target area.
[0076] In some embodiments, the skin electrode assembly further comprises: an activator switch, and a status indicator for reporting activation of the skin electrode assembly. In some embodiments, the electrode array comprises at least three skin electrodes, and preferably the three skin electrodes have a spatial arrangement that is a triangle. Preferably, the triangle is an isosceles triangle. In some embodiments, the electrode array comprises three skin electrodes spatially arranged as an isosceles triangle having sides of 5 mm in length and a base of 3 mm in length. In some embodiments, the electrode array is disposable and removably connected to the skin electrode assembly. Preferably, the disposable electrode array is a skin electrode disk, and more preferably, the skin electrode disk is sterilizable.
[0078] In some embodiments, the feedback mechanism is performed by either hardware or software. Preferably, the feedback mechanism is performed by an analog closed- loop circuit. Preferably, this feedback occurs every 50 μs, 20 μs, 10 μs or 1 μs, but is preferably a real-time feedback or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). In some embodiments, the neutral electrode measures the impedance in the desired tissue and communicates the impedance to the feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the pulse of energy to maintain the constant current at a value similar to the preset current. In some embodiments, the feedback mechanism maintains the constant current continuously and instantaneously during the delivery of the pulse of energy.
[0082] In an example of operation of the skin EP devices described herein, the controller can deliver pulses of energy according to a programmed sequence by at least setting the desired constant current to that of the preset current. In one embodiment, an analog closed-loop feedback circuit continuously maintains the desired constant current by adjusting the voltage applied across the electrodes as the target tissue resistance changes, preferably skin. The controller can end the pulse when the pulse time length has been met by setting the desired output current to zero. When the sequence of pulses is over, the controller can review the waveform data and alert the user if the current in the desired tissue was outside a window of error around the preset current during the electroporation sequence.
[0083] The controller can be a single-chip microcontroller, preferably a processor that has one or more of the following properties: abundant I/O, on-board peripherals, low power consumption, and extensive memory capability. In some embodiments, the controller comprises a Texas Instruments processor msp430F149. Software can direct the steps of the electroporation procedure, and can direct the steps in conjunction with the hardware, and is preferably firmware because it resides permanently within and runs from the single-chip microcontroller. In some embodiments, the software performs an algorithm that includes receiving a device user's input, receiving information from the needle electrodes on resistance in pulse receiving tissue, or current therein, and adjusting electrical output based on received inputs within the duration of each electrical pulse.
[0084] In some embodiments, the skin electroporation device also includes a current waveform generator in communication with the electroporation component and in electrical communication with the skin electrode assembly; the current waveform generator generating a current pulse train waveform for delivery through the skin electrode assembly. In some embodiments, the user inputs a programmed sequence to the electroporation component, which communicates the programmed sequence to the current waveform generator; wherein the current waveform generator is capable of generating the current pulse train waveform according to the provided programmed sequence. Preferably, the current waveform generator is a power-transistor analog circuit. The current waveform generator can generate a current pulse train waveform that passes through the electrodes of the electrode array in accordance with the programmed sequence. The pulse train is preferably square in shape and the number of pulses is limited by the software or firmware, and preferably from 1 to 10 pulses, and more preferably from 2 to 5 pulses, and more preferably 2 to 3 pulses. One pulse is applied to each electrode set. In some embodiments, a pulse is from 20 ms to 52 ms in duration and occurs at a rate of 1 Hz. The amplitude of the pulse train is programmable by the operator and ranges from 0.1 Amperes (A or Amp) to 1.5 A in increments of 0.1 A. In one embodiment for the present invention, the pulse amplitude is below 0.4 A, and preferably between 0.1 and 0.2 A (see Figures 2 and 3). The amplitude of the pulse train can be adapted as a function of the individual tissue resistance, both between individuals and in targeting different areas of the body, preferably skin tissue, of the same individual. The current waveform generator can comprise a general power-transistor analog circuit, which can function under the direction of the controller.
[0085] In some embodiments, the skin electroporation device also includes an impedance tester capable of testing for establishment of an electrical connection between the skin electrodes and the desired tissue. The impedance tester can be used to determine whether the skin EP devices' circuitry has established proper electrical connection to the tissue. This test verifies that the inserted skin electrodes have established a good connection to the handle, and that the skin electrodes also have a good connection to the tissue. EP treatment can be preceded by an impedance test. If any of the impedance measurements fall outside a programmable range of resistance (measured in Ohms, ω), the impedance test fails and the electroporation sequence is not initiated. See the electroporation functional flow in Figure 27A, particularly the decision box 27A.10, which can lead to a delay of firing if the impedance is not enabled (in other words, an electrical contact is not made between the electrodes and the desired tissue).
[0087] The impedance tester can function as a safety feature in the skin EP device. It can indicate, for example, whether all of the electrodes have penetrated the same tissue and a circuit can be established. Electrodes in contact with air, especially dry air, have an extremely high resistance. If electroporation starts and one or more electrodes have not penetrated the tissue (a real possibility when dealing with skin electrodes and skin EP procedures), the resulting electrode voltages can be thousands of volts, which might have lethal or damaging consequences for the subject and also damage the skin EP device. For this reason, overload voltage protection may be implemented to prevent excessive voltages on the electrodes. In some embodiments, the electroporation device includes a safety feature, the safety feature being a voltage cap that prevents the device from delivering the pulse of energy to the tissue when adjustment to the pulse of energy would yield a voltage above the voltage cap. Regardless of the electrical load (e.g. air, skin or muscle tissue), the over-voltage γ protection may be engaged (or safety cap may be triggered) if v exceeds 200V for a period of γ no more than 1 ms. tJ is the voltage across electrode i and j where i, j = 1 to 5. If the over- γ voltage protection engages (or safety cap is triggered), IJ goes to approximately 0 V until the next electroporation pulse is fired. While the skin EP device is in the off state, the voltage across any electrode pair preferably does not exceed 10V.
[0088] In some embodiments, the skin electroporation device also includes a waveform logger in communication with the electroporation component. Preferably, the waveform logger is capable of recording electroporation voltage and current waveforms continuously during the delivery of the pulse of energy. More preferably, the waveform logger is capable of recording electroporation voltage and current waveforms at a rate of 2000 samples per second. The waveform logger, by sampling and monitoring the parameters of the electroporation procedure, enables the user to receive and process possible problems and adjust the settings in the event that an electroporation procedure fails or does not achieve desirable results. An exemplary sample rate is 2000 samples per second, about 104 samples for each of the 2-3 current pulses of 52 milliseconds (ms). An exemplary total sample period is 2208 ms with sampling starting approximately 50 ms before the first pulse is fired and stopping about 50 ms following the last pulse in a 2-pulse pattern. The voltage and current waveforms may be quantified into a 12-bit digital representation with ±1 least significant bit ("LSB") linearity. The current waveform resolution should preferably be at least 10 milliamperes (mA) and the voltage waveform resolution should preferably be at least 1.0 V. The waveform logger may be composed of general operational amplifier analog circuits and an analog to digital ("A/D") converter suitable for use with the controller. An example of the waveform logger characteristic for skin delivery of DNA formulations is tabulated in Figure 2C (please refer to "2.5").
[0089] In some embodiments, the skin electroporation device also includes an input device in direct communication with the user and the electroporation component, the input device capable of receiving input commands and communicating the input commands to the electroporation component. Preferably, the input device is a numeric keypad or a touch screen. In one example, the skin EP devices operating parameters may be entered by an operator via a numeric keypad (such as, Grayhill 88AB2). In a preferred embodiment, the numbers input into the keypad are displayed on a liquid crystal display ("LCD"). Typical parameters that can be programmed are the electroporation pulse current, impedance test enable/disable, and electroporation firing delay. The features related to the keypad can also be directed by the controller. An example of the information that is useful for electroporation using one of the described skin EP devices includes: subject identification number, number of pulse in sequence, pre-wait time (seconds, s, sec), pulse width (ms), pulse current (Amp), and the switching pattern for the needle electrodes of the skin electrode array (subject identification number, as well as all the electroporation conditions are automatically recorded and available for analysis). One example of the information that is useful for electroporation using one of the described skin EP devices is given in Figure 2C (note that subject identification number, as well as all the electroporation conditions are automatically recorded and available for analysis).
[0090] In some embodiments, the skin EP devices can further include a status reporting element for displaying or otherwise notifying the operator as to the status of the system. In some embodiments, the skin electroporation device also includes a status reporting element in communication with the electroporation component. Preferably, the status reporting element is an information display panel, an audible notification, a light-emitting diode, or a combination thereof. The status reporting element can report confirmation of the generation of the pulse of energy and delivery of the constant current. These status reporting elements may include an information display panel, such as a liquid crystal display ("LCD") (such as, Lumex LCM-S02004DSF). The LCD is preferably of the character display type and is preferably capable of displaying 4 lines of 20 characters each. The LCD is also preferably equipped with a back-light that can be switched on and off by means of a toggle switch. Status information may also be provided by audible notification, such as a buzzer (such as, CUI CEP-2202 AS) sounding at various pitches. Each pitch preferably has a different meaning, as controlled by the software or firmware. For example, the volume of the buzzer may have 3 programmable settings and range roughly from 60 to 80 dB at a distance of 1 meter from the buzzer. The sound pressure level range is only given as reference. The sound level is deemed acceptable if it is audible in a noisy environment (e.g. a farm, or a military compound) if set to its highest level and it is not too loud in a quite environment (e.g. an office) if set to its lowest level. In addition, the SKIN EP DEVICE may be equipped with a light emitting diode ("LED") (such as, Lumex SSI-LXR1612ID, or any panel-mount red LED) to designate whether the unit is turned on or off.
[0092] In some embodiments, the skin electroporation device also includes a communication port in communication with the electroporation component. In some embodiments, the communications port that can be used to upload electroporation waveform data to an external electronic device, such as a handheld personal computer (e.g., "Pocket PC"), personal digital assistant ("PDA") or personal computer ("PC"), for viewing purposes. Preferably, the communications port is an optical serial communications port, such as an infrared ("IR") port (such as, Transceiver: ACTiSYS ACT-IR220LN115, orZilogZHX1201; Encoder: Microchip MCP2120, or TI TIRlOOO).
[0093] In some embodiments, the skin electroporation device also includes a memory component in communication with the electroporation component. The memory component can store electroporation waveform data and operating parameters. Preferably, the memory component (such as, Atmel AT45DB321B or AT45DB32 IC-TU) is nonvolatile, meaning it retains its data even if the skin EP device is off. To conserve memory, electroporation waveform data only can be saved to memory during the active periods of the electroporation pulse train. During the inactive periods, sampled data only can be stored to memory if either one of the waveforms exceeds a specified threshold. For example, these thresholds may be a voltage threshold of 2 V and a current threshold of 10 mA. Data stored to memory during the inactive periods of the current pulse train may be time stamped so that the time index of the data is known once the waveforms are reconstructed. Provision may be made for the storage of up to 40 samples (20 ms) of data that occur during the inactive periods of the pulse train. Storage can be limited to 20 ms because the software can specify that the remainder of the electroporation sequence will be aborted if anyone of the thresholds is exceeded for a cumulative period of more than 20 ms. An electroporation waveform data set requires about 2 kB of memory when the above compression technique is implemented. The memory component of the skin EP devices preferably contains sufficient memory to save at least 8000 pulses (each pulse is stored separately).
[0094] In some embodiments, the skin electroporation device also includes a power source in communication with the electroporation component. Preferably, the power source is a battery, and more preferably, a battery such as a lithium ion, nickel metal hydride, lead acid, or nickel cadmium battery. In some embodiments, the power source is preferably a battery (such as, two 6V Panasonic LC-R064R5P 4.5Ah, two 6V genesis NP7-6 6V 7.0 Ah) and is responsible for providing power to each of the skin EP devices' circuits. These circuits include a low voltage/low power capacity power supply for the controller and its peripherals, a low voltage and low power capacity power supply for the impedance tester, and a high power capacity power supply for the current waveform generator. The power switch (such as, E-Switch R5 CB LKBLKEFO, or any DPDT 1OA panel-mount rocker switch) controls power to the skin EP devices and can be either on or off. In the off position, all electrical connections to the electrode assembly are electrically neutral within 5 seconds after power is turned off.
[0096] hi some embodiments, the skin EP devices also include a skin electrode handle assembly. Preferably, the handle assembly includes three elements: a skin electrode array (preferably, a skin needle electrode array), a status indicator for reporting the status of the skin EP device, and an activator switch. The array can include any number of needle electrodes in one or more different spatial arrangements, and preferably include odd number of electrodes and also preferably a lower number of electrodes, preferably 7 electrodes or less. In a preferred embodiment, the needle skin electrode array is circular and comprises three needle skin electrodes placed in an isosceles formation, with the large sides of 5 mm and the small base of 3 mm; this placement is believed to be important for the pulse pattern and electric field generation, and consequently quality of the skin EP. The status of the skin EP device is preferably indicated on the handle assembly through the use of status indicators, such as one or more LED's, which can be in varying colors and programmed to flash intermittently to signify various steps of the electrode firing sequence. The handle assembly activator switch is preferably used to initiate various steps of the electrode firing sequence ("6.1.1").
[0097] In some embodiments, the handle assembly is wireless and can be operated remotely from the electroporation component (or controller). The wireless handle assembly can receive information from the electroporation component (or controller), including the programmed sequence and pulse parameters, and deliver a pulse of energy to the desired tissue and maintain the constant current in same tissue. The wireless handle assembly can include an internal battery or a capacitor that holds an electrical charge for its operation. Preferably, the wireless handle assembly will include the feedback mechanism, preferably the analog closed-loop circuit. The wireless handle assembly can dock with the electroporation component and exchange information and provide one or more of the functionalities of the skin EP devices described herein.
[0098] In some embodiments, the electrode array is disposable and removably connected to the skin electrode assembly. Preferably, the disposable electrode array is a skin electrode disk, and more preferably, the skin electrode disk is sterilizable.
[0099] In one embodiment of the present invention, the skin EP devices include a replaceable skin electrode disk which can be removably mounted in the handle assembly. In a preferred embodiment, the replaceable skin electrode disk is mounted on the handle assembly of the skin EP device. Figures 6.1 and 6.2 depict the side view of the electrode array ("6.2.3" and "6.2.5"). In Figures 6.1 and 6.2, the electrode disk has a plurality of needle skin electrodes mounted on a support structure in a spatial arrangement for penetrating the selected tissue, in particular skin. In a preferred embodiment, the spatial arrangement is a circular skin array. Individual skin electrodes in the needle array on the handle side of the skin electrode disk are blunt-ended and deburred for insertion into the complementary electrical contact fittings in the handle. The handle preferably houses an electrical connector from the needle skin electrodes to the pulse generator or skin EP device.
[00100] In a preferred embodiment, the needle skin electrodes in the skin EP device skin electrode assembly as well as in the replaceable skin electrode disk are in a circular array. In a further preferred embodiment, the plurality of needle skin electrodes consists of three needle skin electrodes. In an additional preferred embodiment, the center of the three needle skin electrodes fall in a circular array in the shape of a formation inscribed by 2 sides of 5 mm and a small base of 3 mm.
[00101 ] In a preferred embodiment, the needle skin electrodes are stored sterile in a specific packaging system (Figure 9) that allows for rapid change of the skin electrode array between treated subjects, while maintaining each skin electrode array sterile.
[00102] In some embodiments, a programmed sequence is provided for the operation of the skin EP devices, which controls whether each electrode of the skin needle array can function as a positive, negative, or neutral electrode. For example, as shown in Figure 2C, in a programmed sequence each electrode can function as a positive, negative or off position. The entire sequence in Figure 2C is depicted in which electrodes 1 through 3 become the positive electrode successively, with two negatively charge electrodes at opposite vertices of the skin array ("2.4" - second column), or one electrode off, one positive and one negative ("2.4" - forth column).
[00104] In some embodiments of the present invention, provided are methods of using the skin electroporation devices described herein to deliver the pulse of energy to the desired skin tissue to produce the constant current similar to a preset current input by a user. The methods include: inserting a plurality of needle skin electrodes into skin tissue without substantially penetrating a muscle tissue; and applying the pulse of energy to the plurality of needle skin electrodes to deliver a current equal to the preset current in the skin tissue; and measuring impedance of the skin tissue with a neutral one of the plurality of needle skin electrodes and using a feedback mechanism in the electroporation device to adjust the pulse of energy applied in response to the measured impedance to maintain the current delivered to the skin tissue constant. In one example of a preferred embodiment, the methods of using the skin EP devices include progressing through an electroporation functional sequence such as that depicted in the flow chart provided in Figures 27A and 27B. In particular, 27B.10 in Fig. 27B shows a blow-up of the "produce current pulse" step and depicts the feedback mechanism, which preferably is performed by an analog closed-loop circuit and operates in real-time (or instantaneously).
[00105] In some embodiments, the methods further include measuring impedance prior to applying the pulse of energy to determine whether electrical contact is made between the desired skin tissue and needle skin electrodes. In some embodiments, the methods of the invention are used to place at least one DNA vaccine in contact with said intradermic or subcutaneous tissue.
[00107] In some embodiments of the present invention, provided are methods comprising the steps of: providing an skin electrode assembly having a plurality of needle skin electrodes, the skin electrode assembly in electrical communication with a current waveform generator; contacting skin tissue of a mammal with the plurality of needle skin electrodes without substantially penetrating a muscle tissue of the mammal; and applying an electrical pulse of energy from the current waveform generator to the plurality of needle skin electrodes for a time and under conditions effective to expose the contacted skin tissue to a substantially constant current.
[00110] U.S. Patent No. 7,245,963 and U.S. Patent Pub. 2005/0052630 discloses system diagrams of certain electroporation components that can be used in accordance with the present invention, where they are adaptable into or as part of the skin EP devices. Each element is described therein in terms of the hardware functionality of such element. The entire content of each of these publications is hereby incorporated by reference in their entirety. The sequences of events that are enabled by the hardware are controlled by software or firmware or combination of both, as described herein.
[00111] hi some embodiments, the electroporation component of the skin EP device comprises an electrode-connect relay matrix, which can facilitate the programmed sequence and operate the firing of the electrodes in the skin electrode assembly. The relay matrix can control each of the electrodes, preferably needle electrodes, so that each electrode is in one of five states: off, positive low voltage, impedance-measuring input, positive high voltage, and negative (regulating) high voltage. In one embodiment, the relay matrix includes 1 1 double pole, double throw (DPDT) relays that create a switch matrix. 10 of those relays are driven using a set of shift registers connected to the serial peripheral interface (SPI) port as an SPI- to-parallel port. The SPI slave in, master out (SIMO) is connected to the Data-in pin of both 74VHC595 (or approved equivalent) shift registers. The 74VHC595 has 2 clock inputs. The universal serial synchronous/asynchronous communication interface claock (UCLK) signal is connected to the shift register clock input (SCK) pins of the registers used to shift data into the register. Separate general purpose input/output (GPIO) pins go to the storage register clock (RCK) clock inputs used to clock data from the shift register, in parallel, into the output register. The result is the same serial data is shifted into both shift registers, but then the data is only clocked into the output register of the one to change.
[00114] One example of a current pulse produced by the skin EP devices is shown in Figure 3.1 and 3.2. Figure 3.1 and 3.2 shows the waveform of each current pulse. The waveform parameters are:
[00117] One aspect of the present invention pertains to skin EP devices for introducing biomolecules into one or more cells of an animal, in particular skin, for ID or SQ vaccination purposes. Preferably, the skin EP devices introduce the biomolecules to skin tissue, such as SQ or ID tissue, by delivering and maintaining a constant current in same tissue throughout the duration of the electrical pulse delivered. In some embodiments, the handle of the skin electrode handle assembly is non-conductive and designed to provide a user an easy means for implanting the needle skin electrode assembly into a selected tissue, in particular skin. The utilization of disposable needle skin electrode disks with snap-on mounts on the handle allows a user to quickly attach and detach the needle skin electrode disks, and allowing for rapid (and sterile) change between treated subjects. The power source of the skin EP devices can utilize battery packs for use in the field where access and use of an electrical outlet or other power source is dangerous or inconvenient, as for instance in a mass vaccination during a bioterrorism attack.
[00121] Examples of drugs that can be used as biomolecules for delivery to the skin tissue by skin EP include chemotherapeutic agents having an antitumor or cytotoxic effect, including bleomycin, neocarcinostatin, suramin, doxorubicin, carboplatin, taxol, mitomycin C and cisplatin. Other chemo-therapeutic agents are known to those skilled in the art, and can be found in references including The Merck Index. In addition, agents that assist transmembrane delivery ("delivery agents") are also contemplated as biomolecules for delivery and can include: N-alkylmelamide and para-chloro mercury benzoate. In embodiments where the biomolecules are DNA plasmids or DNA vaccines, a further biomolecule for delivery along with, prior to, or subsequent to the DNA plasmids or DNA vaccines, is an additional one or more DNA plasmids or DNA vaccines. In embodiments where the biomolecules are DNA plasmids or DNA vaccines that are intended to express target proteins that elicit an immune response, a further biomolecule for delivery along with, prior to, or subsequent to the DNA plasmids or DNA vaccines, is one or more genes for proteins which further enhance the immune response against such target proteins. Examples of such genes are those which encode other cytokines and lymphokines such as alpha- interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-I, IL-2, IL-4, IL-5, IL-6, IL-IO, IL-12, IL- 18, MHC, CD80,CD86 and IL- 15 including IL- 15 having the signal sequence deleted and optionally including the signal peptide from IgE. Other genes which may be useful include those encoding: MCP-I, MIP-ID, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-I, VLA-I, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL- 18, CD40, CD40L, vascular growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, DcB, Inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPl, TAP2 and functional fragments thereof.
[00122] One of ordinary skill understands that numerous changes and modifications of the skin EP devices can be made without departing from the spirit and the scope of the present invention. The skin EP devices can include any number of electrodes, such as a three- electrode array arranged in a different pattern, which can be seen in Figure 11. The distance L is chosen so that the energy intensity at point B is one third of that at point A. After three pulses, (1 to 2, 2 to 3 and 3 to 1), point B has received a cumulative dose equal to that of point A. As the number of electrodes in the array is increased, the distance L necessary to yield a uniform energy distribution becomes proportionately longer. L = k x n where n is the number of electrodes, and k is a proportionality constant. Thus, by selecting a greater number of electrodes a greater volume of tissue can be encompassed. The optimal number of skin electrodes chosen may depend on the volume of the material to be transfected and how far it is dispersed between injection and electroporation.
[00124] Preferably the DNA formulations for use with the skin EP devices described herein have high DNA concentrations, preferably concentrations that include gram quantities of DNA in small volumes that are optimal for delivery to the skin, preferably small injection volume, ideally 25-200 microliters (μL). In some embodiments, the DNA formulations have high DNA concentrations, such as 1 mg/ml or greater (mg DNA/volume of formulation). More preferably, the DNA formulation has a DNA concentration that provides for gram quantities of DNA in 200 μL of formula, and more preferably gram quantities of DNA in 100 μL of formula.
[00125] The DNA plasmids for use with the skin EP devices of the present invention can be formulated or manufactured using a combination of known devices and techniques, but preferably they are manufactured using an optimized plasmid manufacturing technique that is described in a commonly owned, copending U.S. provisional application U.S. Serial No. 60/939,792, which was filed on May 23, 2007. In some examples, the DNA plasmids used in these studies can be formulated at concentrations greater than or equal to 10 mg/mL. The manufacturing techniques also include or incorporate various devices and protocols that are commonly known to those of ordinary skill in the art, in addition to those described in U.S. Serial No. 60/939792, including those described in a commonly owned patent, US Patent No. 7,238,522, which issued on July 3, 2007. The high concentrations of plasmids used with the skin EP devices and delivery techniques described herein allow for administration of plasmids into the ID/SC space in a reasonably low volume and aids in enhancing expression and immunization effects. The commonly owned application and patent, US Serial No. 60/939,792 and US Patent No. 7,238,522, respectively, are hereby incorporated in their entirety.
[00126] First, the power to the skin EP device is turned on. The firmware remains in the idle state until input is received from the user. To start an electroporation sequence, a password is entered to obtain an introductory prompt on the LCD. At this point, the handle assembly activator switch is pressed. The user then enters a number, preferably a subject identification number, which is logged with the data of every pulse stored for later download. The number is preferably entered using a numeric keypad. The biomolecule formulation is than administered as an ID or SQ injection in a reasonably small injection volume, ideally 25- 200 skin liters and the skin electrode array is inserted into the skin to completely surround the injection area which is easily visualized. The user is then prompted, via a "beep" from the buzzer, to press the activation switch to continue the electroporation sequence. After the activation switch is pressed, the firmware establishes whether or not the impedance tester is enabled. If the impedance tester is enabled, the software immediately performs a series of impedance measurements. The firmware tests the impedance between electrodes with a low DC voltage.
[00127] After the electrodes have been inserted into the target tissue, and during the impedance check, the processor checks the conductivity of the tissue at the electrode level. This is done to ensure that electrodes are making electrical contact with the target tissue in the patient. These measurements are performed in a matter of seconds with enough time to achieve accurate readings, often less than 2 seconds. During the impedance testing, a red LED on the handle assembly is lit. If any of the impedance measurements fail, a long error "beep" will sound, the handle LED will stay red, the LCD will display the error, and the firmware will return to the idle state.
[00130] When the EP sequence is completed successfully, the skin EP device returns to the idle state. There is further visual confirmation of the successful generation of an electric field on the LCD following the completion of the electric pulse sequence. Following each sequence, the LCD will exhibit a list of three or five numerals, either "1" or "0", one for each of the pulses of the electroporation sequence. A "0" indicates that the pulse was normal and a "1 " indicates that the pulse was abnormal. More specifically, each pulse must achieve at least 90% of the set current to be described as normal or a "0" will be displayed in the LCD. If a certain pulse achieved less than 90% of the set current, the pulse is abnormal and a "1" will be displayed. Furthermore, the lowest and average current of the abnormal pulses are displayed. For example, for a five pulse electroporation sequence set at current of 0.5 Amps, if pulses 3 and 5 achieve only 0.4 Amps and 0.3 Amps, respectively, " 0 0 1 0 1" will be displayed as well as "Low: 60% Average 80%". This control mechanism is essential when delivering vaccines for therapeutic or prophylactic applications. Should the vaccine delivery be inadequate, there is a high probability that the antibody or cellular responses needed for treatment or protection will not be achieved, and the subject at risk to developing the disease (Roth et al., 2005).
[00131 ] The skin EP device software or firmware enables real time data acquisition and storage in non- volatile memory. Figure 2C illustrates a first portion of data that may be collected during the electroporation process. The first section of the file header contains the file name ("2.1") and the animal number ("2.2"). The columnar data ("2.3") describes the pulse in sequence, the wait time before pulsing, the pulse width, and the pulse current for each of the three electrodes. Figure 2.4 illustrates a second portion of data, which identifies the configuration of each electrode during a given pulse sequence. Figure 2.5 illustrates a formatted version of a third portion of raw data for the same electroporation. The file is downloaded from the skin EP device as a Microsoft Excel CSV file. The data are copied and pasted into a template Microsoft Excel Spreadsheet file. Columns in the spreadsheet represent measured voltage (V) and current (Amps) during the electroporation pulse as well as the calculated tissue resistance (Ohms). Data downloaded after smaller electrodes were used in pigs' skin yielded almost double the skin resistance than after using larger electrodes (700 ω to 2200 ω).
[00132] Plasmid construction. pEGFP-Nl (Clontech, Mountain View, CA) used in the experiments described herein, encodes a red-shifted variant of wild-type GFP which has been optimized for brighter fluorescence and higher expression in mammalian cells (excitation maximum = 488 nm; emission maximum = 507 nm.) pEGFP-Nl encodes the GFPmutl variant which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences. Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site to further increase the translation efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T- antigen. A neomycin-resistance cassette (neor), consisting of the S V40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex thymidine kinase gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pEGFP-Nl backbone also provides a pUC 19 origin of replication for propagation in E. coli and an fl origin for single-stranded DNA production.
[00141] SEAP used in this study is a human protein and, thus, it elicits an immune response in pigs. The measurement of the antibody response using an ELISA was performed, and the results can be seen in the graph shown in Figure 13. The antibody response in serum samples at Day 11 was graphed with the serum SEAP results at Day 11. The concentrated plasmid samples yielded among the highest mean antibody titers.
[00144] IGF-I expression was higher in animals injected IM and electroporated with 0.5 Amps current intensity. Lower current intensities gave lower expression levels, but the small number of animals made the difference non-statistically different.
[00146] pEGFP-Nl or pSEAP-2 Basic Vector (Clontech Laboratories, Inc., Palo Alto, CA) were used in experiments 1 and 2, respectively. pEGFP-Nl, used in experiment 1 (Exp.1 ), encodes a red-shifted variant of wild-type green fluorescent protein (GFP) which has been optimized for brighter fluorescence and higher expression in mammalian cells (excitation maximum = 488 nm; emission maximum = 507 nm). A ubiquitous cytomegalovirus promoter (CMV) was driving the expression of a secreted embryonic alkaline phosphatase (SEAP) in the pSEAP-2 Basic Vector; this plasmid was used in pig experiment 2 (Exp.2). Plasmids were obtained using a commercially available kit (Qiagen Inc., Chatsworth, CA, USA). Endotoxin levels were at less than 0.01 EU/μg, as measured by Kinetic Chromagenic LAL (Endosafe, Charleston, SC). Plasmid preparations were diluted in sterile water and formulated 1 % weight/weight with poly-L-glutamate sodium salt (MW=I 0.5 kDa average) (Sigma, St. Louis, MO), further HPLC purified at VGX Pharmaceuticals, Immune Therapeutics Division (The Woodlands, TX).
[00147] At Day 0 of Exp. 1, animals were weighed and anesthetized with isoflurane (5% induction, 2-3% maintenance). Two 3" x 3" sites on each flank were shaved and cleaned carefully. One inch blocks were marked off using tattoo ink for later identification. Each plasmid injection was delivered in the center of the block. Animals received BD/SQ injections; at various EP conditions (see below), injection volumes and doses: plasmid dose, injection volume (50μg/50μL, 50μg/100μL, lOOμg/lOOμL, and 200μg/100μL), current amplitude and pulse length were varied and biopsies of the treated area were scored by an independent observer blind to the identities of the treatment groups. Photographs of dissected skin sections were taken and fluorescence was measured 5 days post-injection. A numerical score was calculated based on the area of distribution and fluorescence intensity, as compared to the sample with the highest expression (0 = no fluorescence, no distribution; 5 = brightest fluorescence, greatest distribution). Square wave pulses were used in all experiments, and administered using a skin EP device, the CELLECTRATM device (VGX Pharmaceuticals, Immune Therapeutics Division, The Woodlands, TX), which can deliver an adaptive constant current (constant current as experience by the underlying tissue). In all cases, adaptive constant-current parameters were between 0.1 and 0.4 Amps, with 2 or 3 pulses, for 20 milliseconds/pulse or 52 milliseconds/pulse, and with one second between pulses.
[00148] Two types of arrays were used in Exp. 1 : the electrode array previously used for intramuscular (IM) EP which is a circular array (1 cm diameter) of five equally spaced 21 -gauge solid stainless steel needle electrodes, mounted on a non-conductive material; the ID/SC skin needle electrodes consisting of three 26-gauge solid stainless steel needle electrodes, 3 mm in length, placed in an isosceles triangle formation (two long sides are 5 mm in length, and the short side is 3 mm in length), mounted on non-conductive material (see also Figure 17B).
[00149] In Exp. 2, the animals were injected ID/SQ and skin EP with 1 mg pSEAP, as either a standard plasmid preparation at 2 mg/mL (500 μL, similar to an TM injection), or as a concentrated plasmid preparation at 10 mg/mL (100 μL), and SEAP expression measured up to 11 days after plasmid administration.
[00150] On days 0, 4, 7, and 11 of Exp. 2, animals were weighed at 8:30 AM and blood was collected by jugular vein puncture into MICROT AINER serum separator tubes. Blood was allowed to clot for 10 to 15 min at room temperature and subsequently centrifuged at 3000 x g for 10 min and the serum stored at -80 0 C until further analysis.
[00151] Serum samples were thawed and 50μL was assayed for SEAP activity using the Phospha-Light Chemiluminescent Reporter Assay Kit (Applied Biosystems, Bedford, MA), per manufacturer instructions. The lower limit of detection for the assay is 3pg/mL. More concentrated serum samples were diluted 1 : 10 in mouse serum before assaying for SEAP activity. All samples were read using LUMIstar Galaxy luminometer (BMG Labtechnologies, Offenburg, Germany).
[00154] Rhesus macaques were immunized twice with a low dose of DNA (0.2mg/antigen) and then immunized three times with a high dose of DNA (1.Omg/antigen) by ID/SQ injection followed by skin EP. The two low dose immunizations resulted in weak cellular immune responses as determined by IFNγ ELISpot in both ED/SQ alone and ED/SQ injection followed by skin EP (DD/SQ+EP) groups. However, an increase in DNA dose boosted responses to 1,000 SFU/10 6 PBMCs in the ID/SQ group after 3 immunizations. The KHEP group had 50% more IFNγ-producing cells than the ID/SQ group. The enhancement of immunogenicity in the ID/SQ+EP group was also seen in memory T cell responses with the ID/SQ+EP group having twice as many antigen-specific IFNγ-producing cells than the ID/SQ group. ID/SQ immunization alone did not result in a significant production of gag or env antibody titers (<50 endpoint titer). However, plasmid delivery with EP resulted in peak gag endpoint titers of 8800 and env endpoint titers of 1600. Finally, the co-immunization of plasmid-encoded rhIL-12 also resulted in a T H 1 polarized immune response as determined by an IL-4 ELISpot assay.
[00160] Animals were bled every two weeks for the duration of the study. 10 mL of blood were collected in EDTA tubes. PBMCs were isolated by standard Ficoll-hypaque centrifugation and then resuspended in complete culture medium (RPMI 1640 with 2mM/L L- glutamine supplemented with 10% heat-inactivated fetal bovine serum, 100 FU/mL penicillin, lOOμg/mL streptomycin, and 55μM/L β-mercaptoethanol.) RBCs were lysed with ACK lysis buffer (Cambrex Bio Science, East Rutherford, NJ).
[00164] Data are analyzed using Prism Graphpad software, and expressed as means ± SEM.
[00166] The induction of the cellular immune response after each immunization was determined by IFNγ ELISpot (Figure 18). After one low dose immunization of 0.2mg per antigen, both the ID/SQ and the ED/SQ+EP groups had very weak responses (72 ± 11 SFU/10 6 PBMCs and 85 ± 34 SFU/10 6 PBMCs, respectively). A second low dose immunization doubled the number of IFNγ-producing cells in the ID/SQ group (173 ± 77 SFU/10 6 PBMCs) and tripled the response in the ID/SQ+EP group (287 ± 34 SFU/10 6 PBMCs). Having observed a very weak cellular response following the two low dose immunizations we used a higher dose (lmg per antigen) of DNA for the next three immunizations. The immune response did not increase significantly with the third immunization in the ID/SQ group (176 ± 72 SFU/10 6 PBMCs). However the ID/SQ+EP group did boost with the higher dose to double the amount of antigen-specific IFNγ-producing cells (383 ± 162 SFU/10 6 PBMCs). The animals were immunized for a fourth time with the lmg dose of DNA. A three-fold increase in IFNγ response was observed in the ID/SQ group compared to the previous immunization (376 ± 210 SFU/10 6 PBMCs). As with the third immunization, delivery of the high dose of DNA with skin EP resulted in a doubling of the IFNγ response (1466 ± 762 SFU/10 6 PBMCs). These high levels of antigen-specific responses were maintained through the final immunization (1453 ± 873 SFU/10 6 PBMCs). The last immunization further doubled the amount of IFNγ-producing cells in the ID/SQ group (927 ± 191 SFU/10 6 PBMCs).
[00167] The induction of a robust memory T cell response is an important aspect of a successful vaccine. To evaluate the memory T cell population induced by the ID/SQ DNA immunizations, ELISpot analysis was performed 10 weeks after the last DNA vaccination (Figure 19). The ID/SQ+EP group had a memory IFNγ response that was twice the magnitude of the ID/SQ group (998 ± 290 and 449 ± 108 SFU/10 6 PBMCs, respectively).
[00168] Studies using gene gun for skin immunizations have demonstrated the induction of T H 2 biased T cell responses. However it has been shown that this bias could be reversed to a T H 1 response if IL- 12 protein was also delivered. The co-delivery of rhesus IL- 12 in an ID DNA immunization was performed to determine whether its co-delivery would lead to an induction of T H I biased response (Figure 20). The induction of antigen-specific T H 2 responses was determined by an IL-4 ELISpot assay 10 weeks after the final immunization. All animals in the ID/SQ only group had a negative IL-4 response (>50
[00169] A weakness of DNA immunizations lies in its inability to induce antibody responses in non-human primates and in human clinical studies. Each group's ability to induce both HIV-I gag and env specific antibody titers to recombinant p24 and gpl60 antigens was determined by an ELISA assay (Figure 21). For both antigens, the ID/SQ group did not show significant antibody titers (<50 endpoint titer). The DD/SQ+EP group developed a low level of antibodies after the second low dose immunization (endpoint titer: 100±50). However, a greater induction of antibodies was seen after the dose of DNA delivered was increased in the third immunization with the high dose of DNA (2220±l 000). Antibody titers were boosted through the fourth immunization (8800 ± 4000). The env antibody responses also reflected the results we observed with the gag antigen with the ID/SQ group having low titers (<50 endpoint titer) and the ID/SQ+EP group reaching a maximum endpoint titer of 1600 ± 800.
[00170] Rhesus macaques were immunized three times with the same HIV gag, env and rhIL-12 constructs (as discussed, above) at l.Omg/mL by IM immunization. Comparing the induction of cellular immune responses, both the IM and the ID/SQ routes had similar levels of EFNγ-producing cells after the first two immunizations. In contrast, immunization by ID/SQ+EP resulted in high levels of HIV gag antibody titers after one high dose immunization compared to EvI+EP. After two immunizations, the ID/SQ+EP group had HIV gag endpoint titers that were twice that of the EVI+EP group.
[00171 ] In these experiments, we used skin needle electrodes for delivery into the ID/SQ compartment, and compare the resulting immune response to those achieved after IM+EP (see Table 1).
[00173] This study tested the delivery and expression of SEAP or EGFP-expressing plasmids to determine which electroporation parameters (lowest effective current), lag time (time between injection and onset of electroporation), formulation and target muscle impact expression and antibody levels. Animals were electroporated using the CELLECTRATM skin EP device (including use of 3 x 26 gauge electrodes) with varying current and lag time for either IM or ID injection.
[00174] In a first experiment (Exp. 1), groups of C57/B16 mice (n=10/group) were injected with 10 μg of a plasmid expressing SEAP under the control of a muscle-specific synthetic promoter (pSPc5-12-SEAP) into the tibialis anterior (TA) or gastrocnemius (G) muscle at a lag time of 4 or 80 seconds and 0.1 A constant current. A control group received the plasmid in the TA without EP (No EP). Additional groups of animals received in equimolar concentration an expression cassette with no plasmid backbone (NB), but identical promoter, transgene and 3 ' polyadenylation signal, in TA and G muscles with and without EP.
[00175] In the second experiment (Exp.2), groups were mice (n=4) were injected with 50 μg of pCMV-EGFP, in a total volume of 5 or 25 μL, respectively, for a final plasmid concentration of 10 mg/mL or 2 mg/mL, either IM or ED. Photographs of dissected skin and muscle sections were taken and fluorescence was measured 5 days post-injection. Treated area was scored by an independent observer blind to the identities of the treatment groups. A numerical score was calculated based on the area of distribution and fluorescence intensity, as compared to the sample with the highest expression (0 = no fluorescence, no distribution; 5 = brightest fluorescence, greatest distribution).
[00176] In the third mouse experiment (Exp. 3), 16 groups x 5 mice per group for a total of 80 mice were used (TABLE 2). Mice (n=5/group) were injected with 50 μg of CMV-SEAP (at 10 mg/mL) at varying formulations (saline, saline + 1% LGS, water, water + 1% LGS) and current intensities. Animals were electroporated with 4 seconds lag time with varying current (0.1 A to 0.2A) for IM injection in the TA or G muscle.
[00177] In all cases, animals were acclimated for 3 days, weighed and ear-tagged. Food and water were available αd libitum for the length of the experiment. On Study Day 0 animals were weighed, bled and anesthetized using the combination anesthetic for rodents - xylazine (37.5 mg/mL; 0.05 mL/30 grams body weight), ketamine (1.9 mg/mL; 0.016 mL/10 grams body weight), acepromazine (0.37 mg/mL; 0.025 mL/15 grams body weight), plasmid was administered per design and electroporated. For Exp. 1 and 3, blood was collected via retro- orbital bleed and allowed to clot. All collected blood was centrifuged to isolate serum and plasma and then aliquoted into tubes on ice for SEAP assays and anti-SEAP ELISA, as performed previously with minor modifications. After Day 14, all animals were exsanguinated under surgical plane of anesthesia.
[00179] Serum samples were thawed and 50 μL was assayed for SEAP activity as previously described using the Phospha-Light Chemiluminescent Reporter Assay Kit (Applied Biosystems, Bedford, MA), per manufacturers' instructions. The lower limit of detection for the assay is 3 pg/mL. More concentrated serum samples were diluted 1:10 in control species-specific serum before assaying for SEAP activity. All samples were read using LUMIstar Galaxy luminometer (BMG Labtechnologies, Offenburg, Germany).
[00180] Immunogenicity to the human SEAP protein was measured in mice using the following modified procedure. Nunc Maxisorb (Rochester, NY) plates were coated with purified human placental alkaline phosphatase in PBS (100 μl containing 100 nanograms/well of human placental alkaline phosphatase (Sigma)) overnight at 4°C. Plates were decanted and washed three times. Plates blocked using 1% BSA (Sigma) in 0.05% Tween 20 in PBS (blocking solution) and incubated for 2 hours room temperature. Serum samples were diluted 1 : 100 followed by serial dilutions of 1 :4 in 1% BSA (Sigma) in 0.05% Tween 20 in PBS in separate dilution plates. The block solution was decanted from plates. 100 μl of diluted test serum was added to each well and incubated at room temperature for 2 h. Serum dilutions were decanted and washed three times. Secondary antibody was diluted (rabbit anti-mouse conjugated with horseradish peroxidase) 1 : 1000 in 1% BSA in 0.05% Tween 20 in PBS and incubated 1 h at room temperature. Plates were decanted and washed. 100 μl of o- phenylenediamine (OPD) substrate per well was added (0.67mg/mL) in 0.1 M citric acid buffer and incubated 8 min; 100 μl of 1 M H 2 SO 4 was added to stop the reaction. Absorbance was measured at 490 nm on Spectramax Plus 384 plate reader (Molecular Devices, Sunnyvale, CA).
[00181] Expression was increased if the SEAP-expressing plasmid injection is administered into the TA (Figure 22) versus the G muscle, at both 80s and 4s lag time between injection and EP using the skin EP device. When the SPc5-12-SEAP plasmid was injected into the TA muscle, serum SEAP levels were 285 fold higher than in control animals that received the plasmid in the absence of EP (P < 1.3 E-21); no difference was observed between the 80s and 4s lag time for the TA muscle (357±6 vs. 357±6.2 pg/mL/g); the injection under identical conditions into the G muscle resulted in SEAP levels of 90 to 182 fold higher than controls (G 80s vs. no EP controls, P < 0.003; G 4s vs. no EP controls, P < 7.7 E-06). When plasmid fragments containing the expression cassettes only were injected in the TA and G (no backbone (NB), but identical promoter, transgene and 3' polyadenylation signal) in an equimolar formulation, the expression levels were 210-250 fold higher than controls that did not receive IM+EP (80s vs. no EP, P < 1.8 E-08; 4s vs. no EP, P < 3.8 E-05). SEAP expression in the TA 80s was 24% higher than the NB 80s group (P < 0.008), while TA 4s was 37% higher than the NB 4s group (P < 0.004). Both groups administered C5-12-SEAP and NB without EP (No EP) demonstrated negligible SEAP expression.
[00183] The differences in SEAP levels were assessed when SEAP transgene is under control of a ubiquitous promoter (versus a muscle-specific promoter as used in Exp. 1). Consistent with the previous experiments, expression increased if the plasmid injection-EP procedure is performed in the TA (Figure 24) versus the G muscle (P = 0.05). In this particular experiment, saline + LGS formulation resulted in higher serum SEAP levels as compared to saline formulation (41.1±7.9 pg/mL/g vs. 31.0±5.9 pg/mL/g, respectively), although this did not attain statistical significance due to high intra-group variability. In general, animals that were electroporated at 0.1 A current setting yielded slightly higher SEAP expression than animals that received identical LGS plasmid formulation, delivered at 0.2A constant current. Nevertheless, the animals that received 0.2A plasmid formulated into water yielded significant lower SEAP levels than those receiving 0.1 A for the same muscle (P < 0.05 for TA and P < 0.001 For G). When LGS was added to the water formulation, the differences were not significant for the TA muscle, but were still maintained for the G muscle (P < 0.04).
[00184] Formulation of SEAP plasmid with saline + LGS yielded higher protein expression, however, the titers of anti-SEAP antibodies were lower when compared with animals injected with SEAP plasmid formulated in water + LGS (Figure 25), although this did not attain statistical significance due to high intra-group variability.

中文:
[0005]体内质粒转移技术,尤其是当其涉及转移质粒至真皮内(“ID”)或皮下(“SQ”)组织或细胞,已传统地受到局限,因为裸露DNA(质粒)转移引起的体内表达水平一直较低,仅有部分是通过病毒基因转移完成。与可用于肌肉内注射(“IM”)的剂量相比以适用于ID或SQ的体积给予的质粒剂量较小。许多研究均已表明对向动物和人注射病毒作为DNA载体的安全性和毒理学的关注(Frederickson等,Mol.Ther.8:8-10(2003))。因此,直接注射质粒DNA已成为期望的递送技术,然而,有效传送至细胞和蛋白表达水平是一大难题。骨骼肌细胞提供了直接质粒转移DNA疫苗和其他应用的主要靶标(Prud′homme等,Curr.Gene Ther.6:243-273(2006))。
[0006]质粒体内递送的最近进展为电穿孔(“EP”)。EP可用于递送各种分子:从离子到药物、染料、示踪剂、抗体和寡聚核苷酸至RNA和DNA(Gehl,Acta PhysiolScand.177:437-447(2003))。该方法将目标组织暴露于简单的电场脉冲下,其可诱导在细胞膜中形成瞬时和可逆的孔。在膜去稳定化过程中,分子例如质粒可进入细胞内。之前,报导已显示对EP方法的改良可增加转移效率并降低通过使用恒流装置体内产生目标水平抗原所需的质粒量(Draghia-Akli和Smith,GeneTherapy第245页-Therapeutic Mechanisms and Strategies,N.S.Templeton和D.D.Lasic编辑,MarcelDekker,Inc.,New Yor k.(2003))。使用EP增强质粒传送使得被注射组织可用做大量生产和向血流中分泌蛋白质的生物反应器和/或用于抗原递呈。与单独质粒注射相比表达水平增加了二至三个数量级,增加至可与腺病毒介导的基因传送可比的水平并且在一些情况下可达到生理学范围。
[0008]大型哺乳动物和人的皮肤EP的一大难题是皮肤厚度的个体差异,以及不同解剖学区域皮肤厚度的差异。例如,人三角肌皮肤厚度约为2毫米(“mm”),而肩胛水平约2.6mm,腰部约1.7-1.9mm,大腿为约1.6-1.7mm。皮肤是敏感的,因此也需要调节电极规格(gauge)和配置(configuration)将不适降至最低。当用于治疗目的时,在治疗中一定程度的不适对于患者而言有时是可接受的,已建议只有相对无痛的方法可用于预防接种。针对这一难题已调整电极阵列结构和特性以及装置脉冲模式。已知在电穿孔中从电极尖端约2mm至距其尖端三分之一电极处的位点可产生最大均一电场。
[0009]Draghia-Akli等的美国专利7245963号描述了模块电极系统和它们在辅助向身体或植物所选组织的细胞中引入生物分子的应用。模块电极系统包含多个针状电极;皮下针;可提供程序控制型恒流脉冲控制器与多个针状电极之间传导性连接的电连接器;和一个能量源。操纵器可抓起这些安装在支撑结构上的针状电极并将它们插入所选身体组织或植物。接着通过皮下针将生物分子递送至所选组织。程序控制型恒流脉冲控制器被激活并将恒流电脉冲施加至多个针状电极。所施加的恒流电脉冲辅助将生物分子引入多个电极之间的组织中。美国专利7245963的完整内容以参考形式并于本文。
[0010]Smith等提交的美国专利出版物2005/0052630描述了可用于有效辅助将生物分子引入所选身体组织或植物细胞的电穿孔装置。该电穿孔装置包含电动(electro-kinetic)装置(“EKD”装置),其操作由软件或固件(firmware)设定。EKD装置基于使用者控制和脉冲参数输入在任何阵列的电极之间产生一系列的程序可控型恒流脉冲模式并允许存储和采集电流波形数据。该电穿孔装置也包含具有针状电极阵列的可替换电极盘、注射针的中心注射管道以及可移除的导盘。美国专利出版物2005/0052630的完整内容以参考形式并于本文。
[0015]当组织具有小比例的导电离子时电阻增加,产生热量并杀死细胞。欧姆(Ohm′s)定律表达了电流(“I”)、电压(“V”)和电阻(“R”)之间的关系:
[0019]当电流递送至受试者细胞时,电流的剂量可根据下式由电荷(“Q”)精确描述,其为电流(“I”)和时间(“t”):
[0021]电穿孔的有效性受限于这一事实:脉冲强度存在阈值和上限,阈值之下不发生电穿孔而高于上限会破坏细胞。实验数据显示上限和下限之间的差异很小以致于很难设计有效的脉冲方案而不存在不适当的实验。这使得该技术使用困难,主要是当目标组织天然具有非均一的细胞组分时,例如不同厚度的表皮和真皮中的皮肤细胞以及层(strata)、脂肪、筋膜(fascia)的数量,不同大小的血管从毛细血管至小血管;只有可以即时(instantaneously)分析个体与个体之间以及个体皮肤表面位点之间的即时条件变化的精确(true)软件驱动装置可以满足这些要求。而且,电极应该适应皮肤形态学并防止皮肤损伤和出血。
[0024]之前的研究者已使用电穿孔装置用于质粒DNA转移,其概念性的基于恒定电压系统,使用电极间的预设定电压。因为包埋在组织中的电极之间的阻抗在不同情况下和不同组织中存在差异,预设定电压不能产生预定电流。除了丧失完美的方波功能,预定电压脉冲引起在脉冲持续中流经肌肉组织的电流未经调节的增长。相反,恒流电源事实上可以维持流经肌肉组织的方波功能恒定电流。但是,现有的市售电穿孔装置不具有经设计可测定细胞所暴露的精确电流量的固件。传统电穿孔装置产生的未调节电流可能在在组织中生成很容易杀死细胞的大量热量。例如,流经典型的25欧姆(“Ω”)负载阻抗的平均电流为5安培(“A”或“Amp”)的典型的50毫秒(ms)电脉冲理论上可将组织温度升高7.5°C,这足以杀死细胞。Lee等人综述了电休克引起的组织损伤物理学(Lee等,Annu.Rev.Biomed.Eng2:477-509.:477-509(2000))。因此,需要通过提供有效控制递送至细胞的电量避免与恒定电压电穿孔相关的技术问题并藉此达到熟练的电穿孔同时限制对细胞的破坏。
[0025]许多电极存在的问题根源于这一事实:脉冲能量浓缩在阵列中央,即放置被转染材料的位点。结果,能量传送的空间分布具有非常不均一的特性。因此,只有电极组件所包容体积中的一部分细胞被电穿孔。因此也需要一些应用以通过精确控制与细胞膜中的管道碰撞的离子流提供有效控制递送至电极间空间的细胞的电流量的方法。而且,用于大型动物应用和人类的现有市售电极都不允许ID或SQ靶向树突状细胞,因为它们通常太长并且具有不适当的规格和不适宜的斜面方向。因此,需要皮肤电穿孔仪的某些应用,可以辅助内体递送生物分子(例如质粒)至皮肤组织,例如动物的ID或SQ空间。
[0027]此外,使用皮肤和肌肉侵入性可替换针阵列作为电极递送电流的电穿孔装置在发生针阵列替换时需要维持无菌条件。这从医疗实践和调控顺应性角度而言都是必要的。同时,需要一次性(disposable)皮肤电极以允许更低的生产成本和在治疗和预防目的的大规模接种中使用。因此,需要提供允许简易替换针皮肤阵列(needle skin array)的皮肤电极盘。
[0033]图1显示了皮肤结构示意图。最表面的区域为表皮(1.1),其可进一步分为5层:角质层(1.1.1)、棘层(1.1.2)、透明层(1.1.3)、颗粒层(1.1.4)和基底层(1.1.5)。真皮(1.2)位于表皮之下,是血管化非常丰富的区域;毛根和汗腺均位于这一层。皮下层位于真皮之下。
[0034]图2A显示了本文所述EP装置优选实施方案的图解。
[0035]图2B显示了可为本文所述EP装置一部分的控制器的实例。
[0036]图2C显示了在脉冲序列末端下载的实际脉冲序列记录,使用皮肤EP作为脉冲发生器并且使用所述皮肤电极阵列在质粒制剂注射至25kg猪的ID和SQ皮肤层之后递送电脉冲;(“2.1”)显示通过红外线端口下载至个人电脑上或便携装置上的文件名;(“2.2”)显示了受试者数量,通过数字键盘直接输入皮肤EP装置,作为EP过程可以开始之前的前提条件;(“2.3”)显示了操作人员选择的电脉冲特性,包括脉冲序列中的脉冲数量,脉冲之前和之间的等待时间(以秒计算,“s”),脉冲宽度(以毫秒计,“ms”),脉冲电流振幅(以安培计,“A”);(“2.4”)显示了在各次脉冲中各电极的电荷-对于各皮肤电极1、2、3任何下列位置都是可能的:“正极”,“pos”;负极,“neg”;或关闭“off”-“关闭”电极在各次脉冲之前、过程中和之后向装置输入信息(“反馈”);(“2.5”)显示了在脉冲序列的各次脉冲中的实际安培(“A”)、电压(“V”)和注射区域的皮肤电阻,Z(“欧姆”);由于每0.1-0.2ms(并且这种情况下脉冲长度为52ms)产生反馈,所以为了举例目的仅包括了一小部分反馈文件。
[0037]图3显示了通过皮肤EP装置使用皮肤电极递送电脉冲时所下载数据的图解显示实例。该图描述了脉冲1(“3.1”)中皮肤EP装置测定的电压(“3.1.1”)和电流(“3.1.2”)以及脉冲2(“3.2”)中通过皮肤EP装置测定的电压(“3.2.1”)和电流(“3.2.2”)。也显示了脉冲1(“3.1”)和脉冲2(“3.2”)的组织电阻(在电脉冲过程中测定)。
[0038]图4显示了通过皮肤EP装置使用大电极阵列向皮肤递送电脉冲时所下载数据的图解显示实例。该图描述了脉冲1(“4.1”)中皮肤EP装置测定的电压(“4.1.1”)和电流(“4.1.2”),脉冲2(“4.2”)中通过皮肤EP装置测定的电压(“4.2.1”)和电流(“4.2.2”)以及脉冲3(“4.3”)中通过皮肤EP装置测定的电压(“4.3.1”)和电流(“4.3.2”)。也显示了脉冲1(“4.1”)、脉冲2(“4.2”)和脉冲3(“4.3”)的组织电阻(在电脉冲过程中测定)。
[0039]图5显示了皮肤电极阵列的图解表示,包括详细的特性:(“5.1”)侧视图描述了单个针状电极的长度和插入末端的角度;(“5.2”)前视图描述了反套针点(revere-trochar point)的角度和程度;(“5.3”)表明了针状电极的规格。
[0040]图6显示了皮肤电极手柄的视觉展示(“6.1”)和图解:(“6.1.1”)用于活化阻抗检查和电穿孔序列的触发器;(“6.1.2”)当皮肤EP装置准备治疗或出现错误时“LED”分别显示绿色或红色。(“6.1.3”)固定于位置的皮肤电极阵列;(“6.1.4”)包含单股电线的电缆以及皮肤电穿孔手柄和皮肤EP装置单位之间的信号;(“6.1.5”)单股电线,其细节描述于图8的电线图解中;(“6.2.1”)触发器图解;(“6.2.2”)LED图解;(“6.2.3”)描述了用于在完成电穿孔脉冲序列后辅助从皮肤电极手柄弹出的皮肤电极阵列的凸起的脊(raised ridge)的图解;(“6.2.4”)皮肤电极阵列的针状电极图解;(“6.2.5”)固定于手柄上的皮肤电极阵列的侧视图图解;(“6.2.6”)锁臂和锁销(手柄上)的图解;(“6.2.7”)描述用于接收和进行回路连接接触的皮肤电极内部插座的图解;(“6.2.8”)描述包含单股电线的电缆的图解,该电线的细节显示于图8。
[0041]图7显示了安装于手柄上的皮肤电极阵列:(“7.1”)用于接收和产生皮肤电极阵列中单个针状电极和皮肤电极手柄之间的电连接的单个插座的侧视图图解。
[0042]图8显示阵列和手柄组件的电线图解。
[0043]图9显示了允许治疗受试者之间无菌加载手柄的多个皮肤电极阵列蛤壳式折叠包装容器:(“9.1”)盖子;(“9.2”)基底,包括用户设计的用于放置阵列的单个的孔;(“9.3”)孔的侧视图;以及
[0044]图10显示了GEP在注射了不同量的质粒pSP5-12-GFP并使用皮肤电极阵列和皮肤特异性EP条件(MA)用EP装置电穿孔的动物中的平均表达水平,与通常用于向骨骼肌(LA)递送质粒的电极和条件相比。
[0045]图11显示了三电极针阵列,其中距离L=kxn,其中n表示电极数量而k表示比例常数。
[0046]图12显示了使用各自的SEAP表达质粒从电穿孔的时间开始11天中SEAP表达水平(pg/mL/kg)的图片。
[0047]图13显示了多个样品第11天的SEAP表达水平以及相关ELISA结果(平均抗体稀释)一起的图片。
[0048]图14显示了使用各自的表达IGF-1质粒从电穿孔的时间开始11天中IGF-1表达水平(mg/mL)的图片。
[0049]图15展示了在猪的皮肤中给予ID/SQ质粒接着进行电穿孔(EP)之后的绿色荧光蛋白的荧光图像。以不同剂量和体积使用通常用于IM+EP(A)的较大EP阵列或皮肤EP(B)递送质粒。
[0050]图16展示的图片显示了给予pSEAP之后的4-11天猪血清中检测到的分泌型胚胎碱性磷酸酶(SEAP)的水平。动物以浓缩制剂(10mg/mL)或常规制剂(2mg/mL)(n=5/组)接受相同的质粒剂量(1mg)。数据以小组平均应答±SEM表示。
[0052]图18展示的图片显示了ID/SQ共注射质粒编码的IL-12之后进行EP对HIV-1免疫原的细胞免疫应答增强。各次免疫后两周进行IFNγELISpots。对env的应答用白色柱形表示而gag表示为黑色柱形,数据以叠加小组平均应答±SEM表示。
[0053]图19的图片显示了使用ID/SQEP对HIV-1免疫原的记忆应答增强。最后一次免疫后10周进行ELISpost测定法以检测ID/SQ和ID/SQ+EP组中对gag和env的抗原特异性记忆应答。数据以小组平均应答±SEM。
[0054]图20的图片显示了ID/SQ免疫HIV-1gag、env和质粒rhIL-12之后对TH1而不是TH2介导的细胞应答的诱导。10周时进行IL-4ELISpot以检测对gag或evn-特异TH2应答的诱导。对env的应答以白色柱形表示,gag以黑色柱形表示,数据以叠加小组平均应答±SEM。
[0055]图21的图片展示了p24(21A)和gp160(21B)抗体的诱导。在第0、4、8、12和18周收集血清。分别通过p24和gp160ELISA测定ID/SQ和ID/SQ+EP免疫组中的gag和env抗体滴度。数据以小组平均应答±SEM表示。
[0056]图22的柱形图显示了在不同小鼠肌肉中测定的SEAP表达。将10μg C5-12-SEAP质粒注射入小鼠的胫骨前肌(TA)和腓肠肌(G)并在4s或80s延迟后进行EP。也给小鼠注射10μg C5-12-SEAP而不进行EP(未EP)。与接受质粒而未EP的对照小鼠相比TA肌和G肌注射小鼠的血清SEAP水平更高(*P<1.3E-21)。注射了不含质粒骨架(NB)的表达盒并接着进行电穿孔的小鼠比未EP的NB组具有更高的SEAP水平。但是,NB组显著低于在相同肌肉给予C5-12-SEAP的小鼠(NB4s与TA4s相比**P<0.008;NB80s与TA80s相比***P<0.004)。进行TA肌注射的小鼠比进行G肌注射的小鼠产生更高的表达并且将TA肌注射与EP之间的延迟从80s降低至4s不影响表达。
[0057]图23的条形图显示了分别以2mg/mL或10mg/mL以总体积25或5μL肌肉(IM)或皮下(ID)注射50μg CMV-EGFP质粒之后在小鼠中定量的平均GFP表达。与给予2mg/mL浓度((ID和IM,*P=0.01)的动物相比给予10mg/mL浓度的小组中GFP表达最高,尽管相互比较时ID和IM注射位点得到相似的总体评分。
[0058]图24的柱形图显示了由普遍存在的CMV启动子驱动在各种电流设置下在小鼠不同肌肉中测定的SEAP表达。检测质粒制剂:盐水、盐水+LGS或水+LGS。TA肌肉中于0.1A的盐水+LGS制剂产生最高表达。接受配制于水中的质粒并以0.2A电穿孔的动物产生与相同肌肉接受0.1A的动物相比显著较低的SEAP水平(TA,*P<0.05;G,**P<0.001)。当水+LGS用作质粒制剂时,TA肌肉的血清SEAP水平的差异不显著,而G肌肉的差异显著(***P<0.04)。
[0059]图25显示了治疗后14天测定的抗SEAP抗体的滴度,其在用0.1A电流在G肌肉(当质粒配制于(A)盐水+LGS中时)和在TA肌肉(当配置于水+LGS中时)处理的小鼠中最高。
[0060]图26显示了继电接触矩阵实施方案的图解。
[0061]图27A显示了电穿孔功能性电流流程图的第一部分。
[0062]图27B显示了电穿孔功能性电流流程图的第二部分。
[0065]术语“反馈”或“电流反馈”可替换使用并意指所提供皮肤EP装置的活性应答,其包括测定电极之间组织的电流并据此改变由EP装置递送的能量输出以将电流维持在恒定水平。恒定水平由使用者在起始脉冲序列或电子治疗之前预先设定。优选通过皮肤EP装置的电穿孔组件例如控制器完成反馈,因为其中的电流回路可以持续监控电极间组织的电流并将所监测电流(或组织内的电流)与预设定电流对比并持续进行能量输出调节以维持所检测电流在预设定水平。在一些实施方案中,反馈环为即时的因为其为类比(analog)闭环反馈。
[0066]本文术语“生物分子”用于指核酸序列、蛋白质、脂类、微泡(例如载药囊泡)和药物。
[0067]术语“电穿孔”、“电透化作用”或“电动(electro-kinetic)增强”(“EP”)在本文中可替换使用并指使用跨膜电场脉冲诱导生物膜中的显微路径(孔);它们的存在允许生物分子例如质粒、寡聚核苷酸、siRNA、药物、离子和水从细胞膜的一边通过至另一边。
[0069]术语“皮肤区域”(或“皮肤组织”)在本文中用于指皮肤组织、真皮、真皮下和真皮内(“ID”)、皮内,皮下(“SQ”)层。皮肤区域不包括肌肉组织。
[0070]术语“基本不刺入”在本文中指所关注物体(例如针)刺入不超过约1mm至2mm,并优选不超过约5mm。
[0074]图2A显示了本文提供的EP装置的优选实施方案的体系图解。该图解显示了EP装置的主要功能元件,但是仅用做说明目的因为并不是所有功能元件对于所有的实施方案都是必需的。以各元件的硬件功能描述各元件。由硬件启用的事件序列可由软件控制,如本文所述。该装置的中心元件为该实施例中的控制器。控制器的一个实例图解显示于图2B,其为微控制器,其在一些实施方案中为TexasInstruments msp43OF149或Motorola 68HC908AZ60A。其它可选择的控制器为电子工程领域技术人员已知并可轻易取代上述例证性实例。
[0075]在一些实施方案中,皮肤电极为针状电极、导电杆或导电薄膜区域并优选针状电极。优选地,针状电极可与皮内或皮下组织接触并基本不刺入肌肉组织。在一些实施方案中,该组织电极可安装于基座上,其可为塑料材料,形成具有皮肤电极阵列(或皮肤阵列或皮肤针阵列)的可替代或可替换皮肤电极,其可与本文所述的皮肤EP装置相连使用。在一些实施方案中,该皮肤电极的长度可为约1mm至20mm;1mm至12mm;2mm至10mm;2mm至7mm;或长约5mm。在一些实施方案中该皮肤电极可为规格22或28;规格23至27;规格24至27,或约规格26。优选地,该皮肤电极长约5mm并为规格26。在一些实施方案中该皮肤电极仅为真正与患者皮肤接触的皮肤EP装置的一部分(以防止交叉污染)。可向所选区域递送疫苗制剂,该区域被皮肤阵列包围并且皮肤电极插入皮肤中。该皮肤阵列优选在插入皮肤的皮肤电极四周产生均一压力,并藉此辅助在目标区域的EP过程中生成均一的电场。
[0076]在一些实施方案中,该皮肤电极组件进一步包含:活化器开关以及可报告皮肤电极组件活化的状态指示器。在一些实施方案中,该电极阵列包含至少三个皮肤电极并优选具有三角形空间排布的三个电极。优选地,该三角形为等腰三角形。在一些实施方案中,电极阵列包含空间排列成等腰三角形的三个皮肤电极,其腰长为5mm并且底边长3mm。在一些实施方案中,该电极阵列为一次性的并与皮肤电极组件可移除性地相连。优选地,该一次性电极阵列为皮肤电极盘并更优选该皮肤电极盘为无菌的。
[0078]在一些实施方案中,反馈机制由硬件或软件执行。优选地,该反馈装置由类比闭环回路执行。优选地,该反馈每50μs、20μs、10μs或1μs发生但是优选实时反馈或即时(即由测定响应时间的可用技术测定为基本即时)。在一些实施方案中,中性电极测量期望组织中的阻抗并将阻抗传递至反馈机制,反馈机制响应该阻抗并调节能量脉冲以将恒定电流维持在与现有电流相似的数值。在一些实施方案中,该反馈机制可在能量脉冲的传递中持续和即时维持恒定电流。
[0082]在本文描述的EP皮肤装置操作的实例中,该控制器可根据程序化序列通过至少将期望恒定电流设置成现有电流递送能量脉冲。在一个实施方案中,类比闭环反馈回路通过调节施加至电极的电压(由于目标组织电阻变化,优选皮肤)维持期望的恒定电流。当通过设定期望输出电流至0满足脉冲时长时控制器可结束脉冲。当脉冲序列结束时,控制器可复查波形数据并在期望组织的电流超出电穿孔序列中预定电流周围的误差时提醒使用者。
[0083]控制器可为单芯片微控制器,优选具有一个或更多以下性质的处理器:充足的I/O,板上外设(on-board peripherals)、低耗电和大记忆容量。在一些实施方案中,该控制器包含Texas Instrumentsprocessor msp430F149。软件可指导电穿孔过程的步骤并指导与硬件的连接步骤,优选固件因为其永存其中并从单芯片微控制器运行。在一些实施方案中,该软件执行一种算法,其包括接受装置使用者的输入,接受脉冲接受组织中电阻上的来自针状电极的信息或其中的电流并基于在各电脉冲中的输入调节电流输出。
[0084]在一些实施方案中,皮肤电穿孔装置也包括与电穿孔组件连接并与皮肤电极组件电子连接的电流波形发生器;该电流波形发生器生成经皮肤电极组件递送的电流脉冲串波形的电流波形发生器。在一些实施方案中,使用者输入程序化序列至电穿孔组件,其将程序化序列传递至电流波形发生器;其中该电流波形发生器可根据所提供的程序化序列生成电脉冲串波形。优选地,该电流波形生成器为功率晶体管类比回路。该电流波形生成器可根据程序化序列生成通过电极阵列电极的电脉冲串波形。该脉冲串优选方形并且脉冲的数量受限于软件或固件并优选1至10个脉冲,更优选2至5个脉冲,更优选2至3个脉冲。向各电极组合施加一个脉冲。在一些实施方案中,脉冲持续时间为20ms至52ms并且以1Hz的速率发生。该脉冲串的振幅可由操作者程序设计并且范围从0.1安培(A或Amp)至1.5A增量为0.1A。在本发明的一个实施方案中,脉冲振幅低于0.4A并优选0.1至0.2A之间(见图2和3)。可以个体组织电阻(个体之间以及相同个体身体不同区域,优选皮肤组织)调节脉冲串的振幅。电流波形发生器可包含一般功率晶体管类比回路,其可在控制器的指导下发挥功能。
[0085]在一些实施方案中,皮肤电穿孔装置也包括可检测皮肤电极和期望组织之间电子连接建立的阻抗检测器。阻抗检测器可用于测定皮肤EP装置的回路是否与组织之间建立了适当的电子连接。这一检测验证所插入的皮肤电极已与手柄建立了良好的连接以及皮肤电极与组织之间具有良好连接。EP治疗之前可进行阻抗检测。如果任何阻抗测量落在电阻程序化范围之外(以欧姆测量,Ω),阻抗检测失败并且不启动电穿孔序列。参见图27A的电穿孔功能流程图,尤其是决策箱27A.10,如果阻抗被启用(即电极和期望组织之间的电子接触未建立)其可导致点火延迟。
[0087]阻抗检测器可在皮肤EP装置中作为安全装置发挥功能。它可以指示,例如,是否所有的电极都已插入相同的组织并且可建立一个回路。与空气接触尤其是干燥空气接触的电极具有极高的电阻。如果开始电穿孔并且一个或多个电极未刺入皮肤(使用皮肤电极和进行皮肤EP操作时真正存在的可能性),所得电极电压可为上万伏特,其对受试者可能具有致死或损伤后果并且也损伤皮肤EP装置。由于这一原因,可实施过载电压保护以防止电极上的过量电压。在一些实施方案中,电穿孔装置包括安全特征(safety feature),当调整能量脉冲将得到高于电压限(voltage cap)的电压时该安全特征为防止装置向组织递送能量脉冲的电压限。不考虑电负荷(例如,空气、皮肤或肌肉组织),如果Vij在多于1ms的时间内超过200V可使用过电压保护(或触发安全限(safety cap))。Vij为电极i和j之间的电压,其中i,j=1至5。如果使用过电压保护(或触发安全限),Vij接近0V直至点火下一个电穿孔脉冲。当皮肤EP装置为关闭状态时任何电极对之间的电压优选不超过10V。
[0088]在一些实施方案中,皮肤电穿孔装置也包括与电穿孔组件连接的波形记录器。优选地,该波形记录器可在能量脉冲过程中持续记录电穿孔电压和电流波形。更优选地,该波形记录器可以2000个样品每秒的速度记录电穿孔电压和电流波形。通过取样和监控电穿孔操作的参数,波形记录器使得使用者能够接受并处理可能的问题并调节电穿孔操作失败或不能达到预期结果这些事件中的设置。示例性取样速度为2000个样品每秒,52毫秒(ms)的2-3个电脉冲约104个样品。示例性总取样时间为2208ms,取样起始于点火第一次脉冲前约50ms并以2-脉冲模式在最后一次脉冲后50ms结束。电压和电流波形以具有±1个最低有效位(′′LSB′′)线性的12位数字表示法定量。电流波形分辨率优选至少10毫安(mA)并且电压波形分辨率应当优选至少1.0V。波形记录器可由一般的运算放放大器类比回路和适用于控制器的模拟-数字(“A/D”)转换器组成。皮肤转输DNA制剂特性的波形记录器的实例列于表2C(请参考“2.5”)。
[0089]在一些实施方案中,皮肤电穿孔装置也包括与使用者和电穿孔组分直接通信的输入装置该输入装置可接受输入指令并将该输入指令传递至电穿孔组件。优选地,该输入装置为数字键盘或触屏。在一个实例中,可由操作者通过数字键盘(例如Grayhill 88AB2)输入EP装置操作参数。在优选的实施方案中,输入键盘的数字显示在液晶显示器(“LCD”)上。可程序控制的典型参数为电穿孔脉冲电流、阻抗检测启用/禁用和电穿孔点火延迟。与键盘相关的装置也可由控制器指导。可用于使用所述皮肤EP装置之一进行电穿孔的信息实例包括:受试者识别号码、顺序中的脉冲数量、预等待时间(秒,s,sec)、脉冲宽度(ms)、脉冲电流(Amp)以及皮肤电极阵列针状电极的转换类型(受试者识别号码以及所有电穿孔条件为自动记录并可用于分析)。可用于使用所述皮肤EP装置之一进行电穿孔的信息实例见图2C(注意受试者识别号码以及所有电穿孔条件为自动记录并可用于分析)。
[0090]在一些实施方案中,皮肤EP装置可进一步包括用于显示或提醒操作者系统状态的状态报告元件。在一些实施方案中,皮肤电穿孔装置也包括与电穿孔组件通信的状态报告元件。优选地,状态报告元件为信息显示板、语音提示、发光二极管或其组合。该状态报告元件可报告能量脉冲生成的确认和恒定电流的递送。这些状态报告元件可包括信息显示板,例如液晶显示器(“LCD”)(例如,LumexLCM-S02004DSF)。LCD优选为特征显示类型并优选可以显示4行20个字。LCD也优选安装有可由肘节开关打开和关闭的背景光。状态信息也由语音提示器提供,例如以各种音调发声的蜂鸣器(例如,CUICEP-2202AS)。各音调优选具有不同的含义,由软件或固件控制。例如,蜂鸣器的音量可具有3个程序可控的设置并且距蜂鸣器1米处的范围大略为60-80dB。提供声压水平范围仅作为参考。如果设定至其最高水平在嘈杂环境中(例如农场或军事围地(military compound))可以听见并且当设定至其最低水平在安静环境中(例如办公室)声音不是很高那么该声音水平是可接受的。此外,皮肤EP装置可安装发光二极管(“LED”)(例如,Lumex SSI-LXR1612ID或任何安装于面板的红色LED)以指示元件是打开还是关闭。
[0092]在一些实施方案中,皮肤电穿孔装置也包括与电穿孔组件通信的通信端口。在一些实施方案中该通信端口可用于上传电穿孔波形数据至外部电子装置,例如便携个人电脑(例如,“Pocket PC”)、个人数据助手(“PDA”)或个人电脑(“PC”)用于复查。优选地,该通信端口为串行通信端口,例如红外(“IR”)端口(例如,收发器(Transceiver):ACTiSYS ACT-IR220LNl15,或Zilog ZH1201;编码器(Encoder):Microchip MCP2120或TI TIR1000)。
[0093]在一些实施方案中,皮肤电穿孔装置也包括与电穿孔组件通信的存储器组件。存储器组件可储存电穿孔波形数据和操作参数。优选地,该存储器组件(例如,Atmel AT45DB321B或AT45DB321C-TU)为非易失的,意味着即使关闭皮肤EP装置仍然保留这些数据。为了保存存储器,仅可在电穿孔脉冲序列的活化阶段将电穿孔波形数据保存至存储器。在非活化阶段,只有任一波形超过特定阈值时才可将取样数据存储在存储器中。举例而言,这些阈值可为2V的电压阈值和10mA的电流阈值。在电脉冲序列的非活化阶段存储至存储器的数据可能具有时戳(time stamped)这样一旦重构波形即可知数据的时间指数。存储量可存储脉冲序列的非活化阶段发生的多至40个样品(20ms)的数据。存储可限制于20ms因为软件可设定如果超过任一阈值的累积时间多于20ms将停止之后的电穿孔序列。当实施上述压缩技术时电穿孔波形数据设置需要约2kB的内存。皮肤EP装置的存储器组件优选包含足够的内存保存至少8000个脉冲(各脉冲单独存储)。
[0094]在一些实施方案中,皮肤电穿孔装置也包括与电穿孔组件相连的能量源。优选地,该能量源为电池,更优选为例如锂离子、镍金属氢化物、铅酸或镍镉电池这样的电池。在一些实施方案中,该能量源优选为电池(例如,两个6V Panasonic LC-R064R5P 4.5Ah,两个6V genesis NP7-66V 7.0Ah)并用于向皮肤装置的各回路提供电源。这些回路包括用于控制器及其外围的低电压/低功率容量电源,阻抗检测器的低电压和低功率容量电源以及电流波形发生器的高功率容量电源。电源开关(例如,E-Switch R5CBLKBLKEFO,或任何DPDT10A安装于面板的翘板开关)控制皮肤EP装置的电源并可打开或关闭。在关闭位置,与电极组件的所有电连接在关闭电源5秒内为电中性。
[0096]在一些实施方案中,皮肤EP装置也包括皮肤电极手柄组件。优选地,该手柄组件包括三个元件:皮肤电极阵列(优选皮肤针状电极阵列)、用于报告皮肤EP装置状态的状态指示器和活化器开关。该阵列可包括任何数量的具有一种或更多种不同空间排列的针状电极并优选包括奇数个电极并且优选较低数量的电极,优选7个电极或更少。在优选的实施方案中,针状皮肤电极阵列为环形并包含以等腰形式放置的三个针状皮肤电极,长边为5mm,底为3mm;这一放置形式被认为对于脉冲模式和电场生成以及最终皮肤EP的质量是重要的。优选在手柄组件上通过使用状态指示器指示皮肤EP装置的状态,例如一个或多个LED,其可具有多种颜色并经程序化间歇闪烁以指明电极点火序列的多个步骤。手柄组件活化器开关优选用于起始电极点火序列的多个步骤(“6.1.1”)。
[0097]在一些实施方案中,手柄组件是无线的并可由电穿孔组件(或控制器)远程操作。该无线手柄组件可接受来自电穿孔组件(或控制器)的信息,包括程序化序列和脉冲参数并向期望组织递送能量脉冲并在相同组织内维持恒定电流。无线手柄组件可包括维持其操作电荷的内置电池或电容器。优选地,该无线手柄组件将包括反馈机制,优选类比闭环回路。无线手柄组件可与电穿孔组件对接并交换信息以提供本文描述的皮肤EP装置的一个或多个功能。
[0098]在一些实施方案中,电极阵列为一次性的并与皮肤电极组件可移除式连接。优选地,该一次性电极阵列为皮肤电极盘并且更优选地,该皮肤电极盘为无菌的。
[0099]在本发明的一个实施方案中,皮肤EP装置包括可替换皮肤电极盘,其可移除地安装于手柄组件中。在优选的实施方案中,该可替换的皮肤电极盘安装在皮肤EP装置的手柄组件上。图6.1和6.2描述了电极阵列的侧视图(“6.2.3”和“6.2.5”)。在图6.1和6.2中,该电极盘具有以空间排列安装于支架结构上的针状皮肤电极用于刺入所选组织尤其是皮肤。在优选的实施方案中,该空间排列为环形皮肤阵列。皮肤电极盘上手柄一侧上的针状阵列中的单个皮肤电极为钝头并经去毛刺用于插入手柄中的互补电接触配件中。手柄优选安装从针状皮肤电极至脉冲发生器或皮肤装置的电连接。
[00100]在优选的实施方案中,皮肤EP装置皮肤电极组件以及可替换皮肤电极盘中的针状皮肤电极为环形阵列。在优选的实施方案中,该多个针状皮肤电极由三个针状皮肤电极组成。在另一优选的实施方案中,三个针状皮肤电极的中心落于由2条5mm的边和一条3mm的小的底形成的形状中的环形阵列中。
[00101]在优选的实施方案中,针状皮肤电极在特定的包装系统(图9)中无菌保存,该系统允许在接受治疗的受试者之间快速改变皮肤电极阵列并同时维持各皮肤电极阵列无菌。
[00102]在一些实施方案中,提供了用于操作皮肤EP装置的程序化序列,其控制各皮肤针状电极阵列是否可作为正极、负极或中性电极发挥功能。例如,如图2C所示,在程序化序列中各电极可以正极、负极或关闭状态发挥作用。描述了图2C中的完整序列,其中电极1至3相继成为正极电极,皮肤阵列对角具有两个负电荷电极(“2.4”-第二列),或者一个电极关闭,一个正极,一个负极(“2.4”-第四列)。
[00104]在本发明的一些实施方案中提供了使用本文描述的皮肤电穿孔装置向期望皮肤组织递送能量脉冲以产生与使用者输入的预设定电流相似的恒定电流的方法。这些方法包括:将多个针状皮肤电极插入皮肤组织而基本不刺入肌肉组织;向多个针状皮肤电极施加能量脉冲以递送与皮肤组织中的预设定电流相等的电流;用多个针状皮肤电极中的一个中性电极测量皮肤组织的阻抗并响应所测定的阻抗使用电穿孔装置中的反馈机制调节所施加的能量脉冲以维持传送至皮肤组织的电流恒定。在一个优选的实施方案中,使用皮肤EP装置的方法包括按照例如图27A和27B提供的流程图描述的电穿孔功能序列进行。具体而言,图27B中的27B.10显示了“产生电脉冲”步骤的放大(blow-up)并描述了反馈机制,其优选由类比闭环回路执行并实时(或即时)操作。
[00105]在一些实施方案中,这些方法进一步包括在施加能量脉冲之前测定阻抗以确定是否在期望皮肤组织和针状皮肤电极之间建立了电连接。在一些实施方案中,本发明方法用于将至少一种DNA疫苗与所述皮内或皮下组织接触。
[00107]在本发明的一些实施方案中提供了包含以下步骤的方法:提供具有多个针状皮肤电极的皮肤电极组件,与电流波形发生器电子相连的皮肤电极组件;将哺乳动物的皮肤与多个针状皮肤电极接触而基本不刺入哺乳动物的肌肉组织;并将来自电流波形发生器的能量电脉冲施加至多个针状皮肤电极一段时间并在可将皮肤组织有效暴露于基本恒定电流的条件下。
[00110]美国专利7245963号和美国专利出版物2005/0052630公开了根据本发明可使用的某些电穿孔组件的系统图解,其中它们可适用于皮肤EP装置中或作为其一部分。本文以该元件的硬件功能描述各元件。这些出版物的完整内容以参考形式并于本文。由硬件启用的事件序列由软件或固件或二者的组合控制,如本文所述。
[00111]在一些实施方案中,皮肤EP装置的电穿孔组件包括电极-连接继电器(relay)矩阵,其可辅助程序化序列并可操作皮肤电极组件中电极的点火。继电器矩阵可控制各电极,优选针状电极,这样各电极处于五种状态之一:关闭、正极低电压、阻抗测量输入、正极高电压和负极(调控)高电压。在一个实施方案中,继电器矩阵包括11个双极、可产生切换矩阵的双掷(DPDT)继电器。这些继电器中的10个由一组与串行外设接口(SPI)端口连接作为SPI至并行端口的移位寄存器驱动。SPI从入主出(SIMO)与两个74VHC595(或经认可的等同芯片)的移位寄存器的数据输入引脚连接。74VHC595具有两个时钟输入。通用串行同步/异步通信接口时钟(UCLK)信号与用于转移数据至寄存器的寄存器移位寄存器时钟输入(SCK)引脚连接。单独的通用输入输出(GPIO)引脚通向存储寄存器时钟(RCK)的时钟输入,用于将移位寄存器数据并行时钟(clock)至输出寄存器。结果是相同的串行数据移动(shifted)到两个移位寄存器中,但随后数据仅被时钟(clocked into)到待变的输出寄存器中。
[00114]由皮肤EP装置产生的电脉冲的实例显示于图3.1和3.2。图3.1和3.2显示了各电脉冲的波形。波形参数为:
[00117]本发明一方面涉及向动物的一个或多个细胞尤其是皮肤中引入生物分子的皮肤EP装置,用于ID或SQ接种目的。优选地,该皮肤EP装置向皮肤组织例如ID或SQ组织中引入生物分子,其方式为在整个电脉冲传送过程中在相同的组织中递送和维持恒定电流。在一些实施方案中,皮肤电极手柄组件的手柄为非传导性的并经设计向使用者提供向所选组织尤其是皮肤组织植入针状皮肤电极组件的简便方法。使用在手柄上具有搭锁式固定装置(snap-on mounts)的一次性针状皮肤电极盘使得使用者可以快速连接和分离针状皮肤电极盘并允许在接受治疗的受试者之间的快速(并且无菌)转换。皮肤EP装置的电源可使用在电源插座和其他电源的获得和使用是危险的或不方便的情况下(例如在生物恐怖袭击中的大规模接种)使用的电池组。
[00121]可作为生物分子通过皮肤EP递送至皮肤组织的药物的实例包括具有抗肿瘤或细胞毒性作用的化疗药物,包括博来霉素、新制癌菌素、苏拉灭、多柔比星、卡铂、紫杉醇、丝裂霉素C和顺铂。其它化疗药物为本领域技术人员已知并可在包括默克索引的参考文献中找到。此外,有助于跨膜递送的试剂(“递送剂”)也可作为生物分子考虑进行递送并包括:N-烷基醇酰胺和对氯苯甲酸汞。在生物分子为DNA质粒或DNA疫苗的实施方案中,与DNA质粒或DNA疫苗同时递送,在其之前或之后递送的另一生物分子可为附加的一个或多个DNA质粒或DNA疫苗。在其中的生物分子为预期表达诱导免疫应答的靶蛋白的DNA质粒或DNA疫苗的实施方案中,与DNA质粒或DNA疫苗同时、在其之前或之后递送的另一生物分子可为进一步增强对该靶蛋白的免疫应答的一个或多个蛋白的基因。该类基因的实例为编码其它细胞因子和淋巴因子者,例如α干扰素、γ干扰素、血小板衍生的生长因子(PDGF)、TNFa、TNFP、GM-CSF、内皮生长因子(EGF)、IL-1、IL-2、IL-4、IL-5、IL-6、IL-10、IL-12、IL-18、MHC、CD80、CD86和IL-15,包括信号序列缺失的IL-5并任选包括来自IgE的信号肽。可使用的其他基因包括编码以下者:MCP-1、MIP-1、MIP-1p、IL-8、RANTES、L-选择蛋白、P-选择蛋白、E-选择蛋白、CD34、GlyCAM-1、MadCAM-1、LFA-1、VLA-1、Mac-1、p150.95、PECAM、ICAM-1、ICAM-2、ICAM-3、CD2、LFA-3、M-CSF、G-CSF、IL-4、IL-18的突变形式、CD40、CD40L、血管生长因子、IL-7、神经生长因子、血管内皮生长因子、Fas、TNF受体、Flt、Apo-1、p55、WSL-1、DR3、TRAMP、Apo-3、AIR、LARD、NGRF、DR4、DR5、KILLER、TRAIL-R2、TRICK2、DR6、Caspase ICE、Fos、c-jun、Sp-1、Ap-1、Ap-2、p38、p65Re1、MyD88、IRAK、TRAF6、IkB、Inactive NIK、SAP K、SAP-1、JNK、干扰素应答基因、NFkB、Bax、TRAIL、TRAILrec、TRAILrecDRC5、TRAIL-R3、TRAIL-R4、RANK、RANK LIGAND、0x40、0x40 LIGAND、NKG2D、MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、TAP1、TAP2及其功能片段。
[00122]普通技术人员理解可对皮肤EP装置进行很多改变和修改而不偏离本发明精神和范畴。皮肤EP装置可包括任何数量的电极例如不同形式的三电极阵列,其可参见图11.选择距离L这样点B的能量强度是点A的三分之一。三次脉冲之后(1至2,2至3,3至1),点B接受了与点A相等的累积剂量。由于阵列中电极数量增加,产生均一能量分布所必须的距离L也成比例增长。L=kxn,其中n为电极数量,k为比例常数。因此,通过选择更大数量的电极可包围更大体积的组织。皮肤电极最佳数量的选择取决于被转染材料的体积和其在注射和电穿孔之间分散的远近。
[00124]优选地,与本文所述皮肤EP装置一起使用的DNA制剂应具有高DNA浓度,优选最佳用于递送至皮肤的小体积内包括毫克量DNA的浓度,优选小注射体积,理想为25-200微升(μL)。在一些实施方案中,该DNA制剂具有高DNA浓度。例如1mg/mL或更高(mg DNA/制剂体积)。更优选地,DNA制剂具有可在200μL制剂中提供克量DNA的浓度并且更优选在100μL制剂中提供克量DNA。
[00125]与本发明皮肤EP装置一起使用的DNA质粒可使用已知装置和技术的组合配制或生产,但是优选使用描述于共同拥有的未决美国临时申请美国序列60/939792号(2007年5月23日申请)的经优化的质粒生产技术生产。在一些实施例中,可以大于或等于10mg/mL的浓度配制用于这些研究中的DNA质粒。这些生产技术也包括或并入本领域技术人员熟知的各种装置和方案,除美国序列60/939792号所描述之外还包括共同拥有的美国专利7238522号(2007年7月3日颁布)所描述者。与本发明皮肤EP装置和递送技术一起使用的高浓度质粒可以适当的低容量给予质粒至ID/SC空间并辅助增强表达和免疫作用。共同拥有申请和专利即美国序列60/939792号和美国专利7238522号均以其完整内容并于本文。
[00126]首先将皮肤EP装置的电源打开。固件维持空闲状态直至接收到使用者的输入。为了起始电穿孔顺序,输入密码在LCD上获得介绍性提示以起始电穿孔序列。这时,按压手柄组件活化器开关。接着使用者输入数字,优选受试者识别号码,其与各次脉冲一起被记录并保存用于之后的下载。优选使用数字键盘输入号码。然后以适当的小注射体积以ID或SQ注射给予生物分子制剂,理想为25-200皮肤升(skinliters)并且将皮肤电极阵列插入皮肤以完全包围轻易可见的注射面积。接着使用者在蜂鸣器“蜂鸣声(beep)”的提示下按压活化器开关以继续电穿孔序列。当按压活化器开关之后,固件确立阻抗检测器是否被启用。如果阻抗检测器被启用,软件立即执行一系列的阻抗测定。固件检测低DC电压电极之间的阻抗。
[00127]将电极插入目标组织后并且在阻抗检查阶段,处理器在电极水平检查组织的传导性。这样做以确保电极与患者的目标组织发生了电子接触。这些测定在数秒左右内进行并具有足够时间完成准确读数,通常小于2秒。在阻抗检测过程中,手柄组件上的红色LED发光。如果任何阻抗测定失败,表示错误的长蜂鸣声响起,手柄LED仍为红色,LCD将显示错误并且固件返回至空闲状态。
[00130]当成功完成EP序列时,皮肤EP装置回复至空闲状态。在完成电脉冲序列之后LCD上会进一步可视确认成功生成电场。每次序列之后,LCD将显示一列三或五个数字,其为“0”或“1”,各数字表述电穿孔序列的各次脉冲。“0”指示脉冲正常而“1”指示脉冲不正常。更具体而言,各次脉冲必须达到设定电流的至少90%才称之为正常或在LCD上显示“0”。如果某一脉冲达到小于设定电流的90%,该脉冲为不正常并且显示“1”。而且,显示不正常脉冲的最低和平均电流。例如,对于设定于0.5安培电流的五次脉冲电穿孔序列,如果脉冲3和5分别仅达到0.4安培和0.3安培,将显示“00101”以及“低:60%平均80%”。当递送治疗或预防应用的疫苗时这一控制机制是必须的。如果疫苗递送不足,很有可能达不到治疗或保护需要的抗体或细胞应答并且受试者可能生病(Roth等,2005)。
[00131]皮肤EP装置软件或硬件可实现实时数据采集并存储至非易失性存储器中。图2C显示了在电穿孔过程中采集的第一部分数据。文件标题的第一部分包含文件名称(“2.1”)和动物编号(“2.2”)。分栏(columnar)数据(“2.3”)描述了脉冲序列,脉冲前的等待时间,脉冲宽度和三个电极各自的脉冲电流。图2.4显示了数据的第二部分,其鉴定在给定脉冲序列中各电极的结构。图2.5显示了相同电穿孔的原始数据第三部分的格式化版本。该文件以Microsoft Excel CSV文件下载自皮肤EP装置。拷贝数据并粘贴至模板Microsoft Excel Spreadsheet文件。Spreassheet的列显示电脉冲过程中测定的电压(V)和电流(Amps)以及计算的组织电阻(Ohms)。将较小电极用于猪皮肤之后下载的数据比使用较大电极(700Ω至2200Ω)之后得到的皮肤电阻几乎增加一倍。
[00132]质粒构建。用于本文所述实验中的pEGFP-N1编码野生型GFP的红移变异体,其经优化具有更亮的荧光和在哺乳动物细胞中更高的表达(最大激发光=488nm;最大发射光=507nm)。EGFP-N1编码包含Phe-64至Leu和Ser-65至Thr两个氨基酸替换的GFPmut1。EGFP基因的编码序列包含多于190个沉默碱基变化,其与人密码子使用偏好一致。侧接EGFP的序列被转化成Kozak一致序列翻译起始位点以进一步增加在真核生物细胞中的翻译效率。EGFP基因下游的SV40多腺苷酸化信号指导EGFP mRNA 3′端的适当加工。载体骨架也包含用于在哺乳动物细胞中复制的SV40复制起点并表达SV40T抗原。新霉素耐药盒(neor,由SV40早期启动子、Tn5的新霉素/卡那霉素抗性基因和疱疹单纯胸苷激酶基因的多腺苷酸化信号组成)使得可使用G418筛选稳定转化的真核生物细胞。这一盒上游的细菌启动子在大肠杆菌中表达卡那霉素抗性。pEGFP-N1骨架也提供在大肠杆菌中增殖的pUC19复制起点和用于单链DNA生产的f1起点。
[00141]用于本实验的SEAP为人蛋白质并且因此其在猪中诱导免疫应答。使用ELISA进行抗体应答的检测,结果见图13所示的图中。第11天血清样品中的抗体应答与11天的血清SEAP结果一起作图。浓缩质粒样品得到最高的平均抗体滴度。
[00144]IM注射并以0.5Amps电流强度电穿孔的动物中IGF-I表达更高。更低的电流强度得到更低的表达水平,但是小数量的动物使得该差异为非统计学差异。
[00146]pEGFP-N1或pSEAP-2基础载体(Clontech Laboratories,Inc.,PaloAlto,CA)分别用于实验1和2。用于实验1(Exp.1)的pEGFP-N1编码野生型绿色荧光蛋白(GFP)的红移变异体,其经优化在哺乳动物细胞中具有更亮的荧光和更高的表达(最大激发光=488nm;最大发射光=507nm)。普遍存在的细胞巨化病毒启动子(CMV)驱动pSEAP-2基本载体中分泌型胚胎碱性磷酸酶(SEAP)的表达;该质粒用于猪实验2(Exp.2)。使用可商业购买的试剂盒(Qiagen Inc.,Chatsworth,CA,USA)获取质粒。内毒素水平经Kinetic Chromagenic LAL(Endosafe,Charleston,SC)测定小于0.01EU/μg。在无菌水中稀释质粒制剂并用多聚L谷氨酸钠盐(MW=10.5kDa平均)配制成1%重量/重量(Sigma,St.Louis,MO),在VGX Pharmaceuticals,Immune Therapeutics Division(The Woodlands,TX)进一步HPLV纯化。
[00147]在实验1的第0天,将动物称重并用异氟烷(5%诱导,2-3%维持)麻醉。将两侧肋上的两个3′′×3′′位点剃毛并仔细清理。用纹身墨水标记一英寸的区域用于之后识别。在该区域的中央进行各质粒注射。动物接受ID/SQ注射;在不同EP条件下(见下文)、注射体积和剂量:质粒剂量、注射体积(50μg/50μL、50μg/100μL、100μg/100μL和200μg/100μL)、电流振幅和脉冲长度均变化并且由不了解处理组身份的独立观察者对经处理区域的活组织检查评分。对剖离的皮肤区域照相并在注射后5天测量荧光。基于分布面积和荧光强度计算数字分数,与具有最高表达的样品相比(0=无荧光,无分布;5=最亮荧光,最大分布)。在所有实验中使用方波脉冲并使用皮肤EP装置CELLECTRATM装置(VGX Pharmaceuticals,Immune TherapeuticsDivision,The Woodlands,TX)给予。其可递送适当的恒定电流(如下层组织经受的恒定电流)。在所有情况下,适当的恒定电流参数为0.1-0.4Amps之间,具有2或3次脉冲,持续20毫秒/脉冲或52毫秒/脉冲,并且脉冲间相隔一秒。
[00148]在实验1中使用两种阵列类型:之前用于肌肉内(IM)EP的电极阵列,其为5个等空间排列的规格21固体不锈钢针状电极的环形阵列(直径1cm),安装于非传导性材料上;ID/SC皮肤针状电极由三个长3mm以等腰三角形(两个长边长5mm,短边长3mm)排列的安装于非传导性材料上的规格26固体不锈钢针状电极组成(见图17B)。
[00149]在实验2中,ID/SQ注射动物并用1mg pSEAP(或者为2mg/mL的标准质粒制剂(500μL,与IM注射相似)或者为10mg/mL的浓缩质粒制剂)进行皮肤EP,测量SEAP表达直至给予质粒后11天。
[00150]在实验2的第0、4、7和11天,于8:30AM称重动物并通过颈静脉穿刺收集血液至MICROTAINER血清分离管中。使血液在室温下凝结10或15分钟,接着3000xg离心10分钟并将血清-80°C保存用于进一步分析。
[00151]将血清样品冻融并使用Phospha-Light ChemiluminescentReporter Assay Kit(Applied Biosystems,Bedford,MA)根据操作说明对50μL进行分析。该测定法的检测下限为3pg/mL。测定SEAP活性之前在小鼠血清中1∶10稀释更浓缩的血清样品。使用LUMIstar Galaxy光度计(BMG Labtechnologies,Offenburg,Germany)读数所有样品。
[00154]用低剂量DNA(0.2mg/抗原)免疫两次猕猴,然后用高剂量DNA(0.1mg/抗原)通过ID/SQ注射免疫三次,之后进行皮肤EP。经IFNγELISpot测定在单独ID/SQ组和ID/SQ注射接着进行皮肤EP(ID/SQ+EP)的组中两次低剂量免疫得到弱细胞免疫应答。但是,三次免疫后在ID/SQ组中DNA剂量的增加将应答提高至1000SFU/106PBMCs。ID+EP组比ID/SQ组的IFNγ产生细胞多50%。ID/SQ+EP组免疫原性的增强也表现于记忆T细胞应答,其中ID/SQ+EP组比ID/SQ组的抗原特异性IFNγ产生细胞多50%。单独ID/SQ免疫未得到gag或env抗体滴度的显著产生(<终点滴度)。但是用EP进行质粒递送得到8800的峰gag终点滴度和1600的env终点滴度。最后,同时给予质粒编码的rhIL-12也可得到经IL-4ELISpot测定法测定的TH 1特异性免疫应答。
[00160]在实验过程中每两周采集动物血液。将10mL血液收集至EDTA试管中。通过标准Ficoll-hypaque离心分离PBMCs并接着重悬于完全培养基(含有2mM/L L谷氨酸并添加了10%加热灭活的胎牛血清、100IU/mL青霉素、100μg/mL链霉素和55μM/L β巯基乙醇的RPMI1640)中。用ACK裂解缓冲液(Cambrex Bio Science,EastRutherford,NJ)裂解RBC。
[00164]使用Prism Graphpad软件分析数据并表示为平均值±SEM。
[00166]通过IFNγELISpot测定每次免疫之后细胞免疫应答的诱导(图18)。在0.2mg每抗原的一次低剂量免疫后,ID/SQ和ID/SQ+EP组均具有弱应答(分别为72±11SFU/106PBMCs和85±34SFU/106PBMCs)。第二次低剂量免疫使ID/SQ组中IFNγ产生细胞的数量加倍(173±77SFU/106PBMCs)并且使ID/SQ+EP组的应答增加至三倍(287±34SFU/106PBMCs)。已在两次低剂量免疫后观察到较弱的细胞应答,我们使用高剂量DNA进行接下来的三次免疫。ID/SQ组第三次免疫的免疫应答并未显著增加(176±72SFU/106PBMCs)。但是ID/SQ+EP组确实在较高剂量将IFNγ产生细胞的数量提高至两倍(383±162SFU/106PBMCs)。用1mg剂量的DNA第四次免疫动物。与之前免疫相比在ID/SQ组中观察到IFNγ应答的三倍增长(376±210SFU/106PBMCs)。在第三次接种时,使用皮肤EP递送高剂量DNA得到双倍的IFNγ应答(1466±762SFU/106PBMCs)。在最后的免疫中维持了这些高水平的抗原特异应答(1453±873SFU/106PBMCs)。最后一次免疫进一步加倍了ID/SQ组中IFNγ产生细胞的数量(927±191SFU/106PBMCs)。
[00167]诱导强效的T细胞应答是成功免疫的一个重要方面。为了评价ID/SQ DNA免疫诱导的记忆T细胞群,在最后一次DNA接种后10周进行ELISpot分析(图19)。ID/SQ+EP组的记忆IFNγ应答是ID/SQ组强度的两倍(分别为998±290和449±108SFU/106PBMCs)。
[00168]使用基因枪用于皮肤免疫的研究已例证对TH2偏倚(biased)T细胞应答的诱导。但是已显示如果也递送IL-12可将这一偏倚逆转至TH1应答。在ID DNA免疫中一起递送给猕猴IL-12以确定其共递送是否可引起对TH1偏倚应答的诱导(图20)。在最后一次免疫后10周通过IL-4ELISpot测定法测定对抗原特异TH2应答的诱导。仅进行ID/SQ的组中所有动物具有阴性IL-4应答(>50SFU/106PBMCs)并且ID/SQ+EP组中仅有一只动物具有阳性IL-4应答(136SFU/106PBMCs),表明显著的TH1应答。
[00169]DNA免疫的一个弱点在于其不能在非人灵长类和人类临床实验中诱导抗体应答。通过ELISA测定法测定各组诱导对重组p24和gb160抗原的HIV-gag和evn特异抗体滴度的能力(图21)。对两种抗原而言,ID/SQ组未显示显著的抗体滴度(<50终点滴度)。ID/SQ+EP组在第二次低剂量免疫后具有低水平的抗体(终点滴度:100±50)。但是在使用高剂量DNA的第三次免疫中增加所递送DNA的剂量之后观察到更大的抗体诱导(2220±1000)。第四次免疫中提高了抗体滴度(8800±4000)。env抗体应答也反映了我们在有低滴度(<50终点滴度)的ID/SQ组观察到的gag抗原的结果并且ID/SQ组达到最大终点滴度1600±800。
[00170]用相同的HIV gag、env和rhIL-12构建体(如上所述)以1.0mg/mL通过IM免疫将猕猴免疫三次。与细胞免疫应答诱导相比,前两次免疫之后IM和ID/SQ途径二者具有相似水平的IFNγ产生细胞。相反的,与IM+EP相比在第一次高剂量免疫之后ID/SQ+EP免疫得到高水平的HIV gag抗体滴度。在两次免疫之后,ID/SQ+EP组的HIV gag终点滴度是IM+EP组的两倍。
[00171]在这些实验中,我们使用皮肤针状电极递送至ID/SD区室,并比较所得免疫应答与IM+EP之后得到者(见表1)。
[00173]这一实验检测SEAP或EGFP表达质粒的递送和表达以确定哪些电穿孔参数(最低有效电流)、延迟时间(注射和开始电穿孔之间的时间)、制剂和目标肌肉影响表达和抗体水平。使用CELLECTRATM皮肤EP装置(包括使用3×规格26电极)用各种电流和用于IM或ID注射的延迟时间电穿孔动物。
[00174]在第一个实验(Exp.1)中,将10μg在肌肉特异性合成启动子(pSPc5-12-SEAP)控制下表达SEAP的质粒注射至C57/B16小鼠组的胫骨前肌(TA)或腓肠肌(G)肌肉中,延迟时间为4或80秒,恒定电流为0.1A。对照组TA注射质粒而不进行EP(无EP)。其它组的动物TA或G肌肉接受等摩尔的无质粒骨架(NB)但是具有相同的启动子、转基因和3′多腺苷酸化信号的表达盒,不进行EP。
[00175]在第二个实验(Exp.2)中,给小鼠组(n=4)IM或ID注射总体积分别为5或25μL的50μg pCMV-EGFP,终质粒浓度为10mg/mL或2mg/mL。对剖离组织和肌肉部分照相并在注射后5天测量荧光。对经处理组的身份不了解的独立观察着对经处理区域评分。与具有最高表达的样品相比基于分布面积和荧光强度计算数字评分(0=无荧光,无分布;5=最亮荧光、最大分布)。
[00176]在第三个小鼠实验(Exp.3)中,总共使用80只小鼠,分为16组,每组5只(表2)。以不同制剂(盐水、盐水+1%LGS、水、水+1%LGS)和电流强度给小鼠(n=5/组)注射50μg CMV-SEAP(10mg/mL)。以4秒延迟时间和不同电流(0.1A至0.2A)对TA或G肌肉IM注射的动物进行电穿孔。
[00177]在所有情况下,使动物适应三天,称重并在耳朵进行标记。在整个实验过程中可随意获取食物和水。在研究一开始称重动物、采血并使用啮齿类组合麻醉剂麻醉,即甲苯噻嗪(37.5mg/mL;0.05mL/30克体重)、氯胺酮(1.9mg/mL;0.016mL/10克体重)、乙酰丙嗪(0.37mg/mL;0.025mL/15克体重),根据设计给予质粒并进行电穿孔。对于实验1和实验3,眼眶后采血并使其凝结。离心所有收集的血液以分离血清和血浆,接着分装至置于冰上的试管中用于SEAP测定法和抗-SEAP ELISA,如之前所进行并存在较小的修改。第14天后,在手术麻醉计划下将动物放血。
[00179]将血清样品冻融并使用Phospha-Light ChemiluminescentReporter Assay Kit(Applied Biosystems,Bedford,MA)根据操作说明对50μL进行分析。该测定法的检测下限为3pg/mL。测定SEAP活性之前在小鼠血清中1∶10稀释更浓缩的血清样品。使用LUMIstar Galaxy光度计(BMG Labtechnologies,Offenburg,Germany)读数所有样品。
[00180]在小鼠中使用以下经修改的操作测定对人SEAP蛋白的免疫原性。用经纯化的PBS中的人胎盘碱性磷酸酶(每100μL包含100纳克/孔的人胎盘碱性磷酸酶(Sigma))在4°C包被Nunc Maxisorb(Rochester.NY)板过夜。倾倒板子并洗涤三遍。用含有1%BSA(Sigma)和0.05%吐温20的PBS封闭板子并于室温下温育2小时。将样品1∶100稀释并接着在单独的稀释板中用含有1%BSA(Sigma)和0.05%吐温20的PBS以1∶4序列稀释。将封闭液从板中倾倒出。向各孔中加入100μL经稀释的待测血清并于室温下温育2小时。倾去血清稀释液并洗涤三次。在含有1%BSA(Sigma)和0.05%吐温20的PBS中以1∶1000稀释第二抗体(与辣根过氧化酶结合的兔抗小鼠抗体)并于试问下温育1小时。倾倒板子并洗涤。将每孔100μL的邻苯二胺(OPD)底物加入(0.67mg/mL)0.1M柠檬酸缓冲液中并温育8分钟;加入100μL 1MH2SO4终止反应。在Spectramax Plus 384读板仪(Molecular Devices,Sunnyvale,CA)上于490nm测量吸光度。
[00181]与G肌肉相比如果将SEAP表达质粒注射剂给予TA(图22),注射和使用皮肤EP装置进行EP之间的时间为80s和4s,则表达增加。当将SPc5-12-SEAP质粒注射至TA肌肉时,血清SEAP水平比在不进行EP条件下接受质粒的对照动物高285倍(P<1.3E-21);未发现80s和4s延迟时间对于TA肌肉的差异(357±6vs.357±6.2pg/mL/g);在相同条件下注射至G肌肉得到比对照高90至182倍的SEAP水平(G 80s vs.无EP对照,P<0.003;G4s vs.无EP对照,P<7.7E-0.6)。当以等摩尔制剂将仅包含表达盒的质粒片段注射至TA和G(无骨架(NB),但是具有相同的启动子、转基因和3′多腺苷酸化信号),表达水平比未接受IM+EP的对照组高210-250倍((80s vs.无EP,P<1.8E-08;4s vs.无EP,P<3.8E-05)。TA80s中的SEAP表达比NB80s组高24%(P<0.004)。给予C5-12-SEAP和NB而不进行EP(无EP)的两组均显示可忽略不计的SEAP表达。
[00183]当SEAP转基因由普遍存在的启动子(相对于实验1中使用的肌肉特异性启动子)控制时评价SEAP水平的差异。与之前的实验一致,与G肌肉(P=0.05)相比如果质粒注射-EP操作在TA中进行(图24)则表达增加。在这一具体实验中,与盐水制剂相比盐水+LGS制剂得到更高的血清SEAP水平(分别为41.1±7.9pg/mL/g vs.31.0±5.9pg/mL/g),尽管由于组内差异性并未达到统计学显著性。总体而言,与接收相同LGS质粒制剂并以0.2A恒定电流递送的动物相比以0.1A电流设置进行电穿孔的动物得到更高的SEAP表达。此外,接受0.2A以水配制的质粒的动物与相同肌肉接受0.1A的动物相比得到显著较低的SEAP水平(对TA,P<0.05;对G,P<0.001)。当将LGS加入水制剂时对TA肌肉的差异并不显著但是对G肌肉仍然保持(P<0.04)。
[00184]SEAP质粒与盐水+LGS的制剂得到更高的蛋白表达,但是与注射用水+LGS配制的SEAP质粒的动物相比抗SEAP抗体的滴度更低(图25),尽管由于组内差异性并未达到统计学显著性。