CA2337652C - Skin and muscle-targeted gene therapy by pulsed electrical field - Google Patents

Skin and muscle-targeted gene therapy by pulsed electrical field Download PDF

Info

Publication number
CA2337652C
CA2337652C CA2337652A CA2337652A CA2337652C CA 2337652 C CA2337652 C CA 2337652C CA 2337652 A CA2337652 A CA 2337652A CA 2337652 A CA2337652 A CA 2337652A CA 2337652 C CA2337652 C CA 2337652C
Authority
CA
Canada
Prior art keywords
skin
electrode
dna
needle
pulse
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
CA2337652A
Other languages
French (fr)
Other versions
CA2337652A1 (en
Inventor
Nagendu B. Dev
Gunter A. Hofmann
Edward Nolan
Dietmar Rabussay
Arnt Tonnessen
Georg Widera
Lei Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genetronics Inc
Original Assignee
Genetronics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genetronics Inc filed Critical Genetronics Inc
Publication of CA2337652A1 publication Critical patent/CA2337652A1/en
Application granted granted Critical
Publication of CA2337652C publication Critical patent/CA2337652C/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00Electrotherapy; Circuits therefor
    • A61N1/18Applying electric currents by contact electrodes
    • A61N1/32Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/327Applying electric currents by contact electrodes alternating or intermittent currents for enhancing the absorption properties of tissue, e.g. by electroporation
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00Electrotherapy; Circuits therefor
    • A61N1/02Details
    • A61N1/04Electrodes
    • A61N1/0404Electrodes for external use
    • A61N1/0408Use-related aspects
    • A61N1/0412Specially adapted for transcutaneous electroporation, e.g. including drug reservoirs
    • A61N1/0416Anode and cathode
    • A61N1/0424Shape of the electrode
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00Electrotherapy; Circuits therefor
    • A61N1/18Applying electric currents by contact electrodes
    • A61N1/32Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/325Applying electric currents by contact electrodes alternating or intermittent currents for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00Electrotherapy; Circuits therefor
    • A61N1/02Details
    • A61N1/04Electrodes
    • A61N1/0404Electrodes for external use
    • A61N1/0408Use-related aspects
    • A61N1/0452Specially adapted for transcutaneous muscle stimulation [TMS]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00Electrotherapy; Circuits therefor
    • A61N1/18Applying electric currents by contact electrodes
    • A61N1/20Applying electric currents by contact electrodes continuous direct currents
    • A61N1/30Apparatus for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body, or cataphoresis
    • A61N1/303Constructional details
    • A61N1/306Arrangements where at least part of the apparatus is introduced into the body

Abstract

The present invention describes an in vivo method, using pulsed electric field to deliver therapeutic agents into cells of the skin and muscle for local and systemic treatments. In particular, therapeutic agents include naked or formulated nucleic acid, polypeptides and chemotherapeutic agents.

Description

we ooroZ6zycrNS~ns~ss SKIN AND MUSCL&TARGETED GENE THERAPY
BY IrIJhSBD ELBCT1:ICAL FIELD
The prasedt inv~tion relates geatrnaUy to the use of elatric pulses to incteasa the permeability of cells, and more spxifically to a method and apparatus for the application of controlled electric fields for in viva delivery of ptrumaoartical compounds and genes into cells by electroporation therapy (EP1~, also known as cell poration therapy (CP'I~ and electrochemotheTapY (ECT').
The skin is an especially at~vo target for gene therapy. In particular, the ability to target genes to the epidermis of the skin could be used to correct skin-specific disorders as well as for the production of proteins secreted into the skin to correct certain systemic diseases. For example, genes expressing cytokines, intarferons or other biologically active I S molecules could be used to treat skin tumors or other lesions. In addition, keratinocytes and tibroblasts in the skin caa secrete protein factors which circulate to treat systemic conditions such as hemophilia. Despite the clear pote~ial in using skin as a target for gene therapy, the major technical problem of an in viva method of gorse delivery remains largely unresolvexl.
Since the sNarurre aorneurre (SC) acts as a sig~nificaat physical barrier to gene transfer rote the akin, the technical problem of how to deliver genes through this layer persists.
Similarly, muscle cells ate also useful tars for gene therapy duo to their ubiquity.
Nonetheless, as with skin, there exists a need for a medrod to reliably introduce exogenous therapeutic material into muscle cells.
Gene therapy does not include only intrinsically therapeutic genetic mat~iai (i.e., gases that encode a missing or ssed gars product), but also those which elicit an immune r~pocrsc. One of the oldest and most effective forms of preventative care against infectious diseases is vaccination. Safe and effective vaccines are available to protect against a variety of bacterial and viral dise~s. These vaccines consist of inactivated patlrogcOS, recombinant of aatural subunits, and live attenuated or live recombinant microorganisms.

we oorozm hcrnrs~n sass DNA immunization, a novel method to induce protective immune responses, was neeently introduced into the scientific community and proven to be very effective in animal models. This technology is currently in first safety and efficacy trials in human volunteers.
DNA immunization entails the direct, in vivo administration of plasmid-based DNA vectors that encode the production of defined microbial antigens. The de novo production of these antigens in the host's own. ceps results in the elicitation of antibody (i.e., humoral) and cellular immune responses that provide protection against live virus challenge and persist for extended periods in the absence of farther immunizations. The unique advantage of this technology is its ability to mimic the effects of live attenuated vaccines without the safety and stability concerns associated with the parenteral administration of live infectious agents, Because of these advarnages, considerable research efforts have focused on refining in vivo delivery systems for naked DNA that result in maximal antigen production and resultant immune responses.
The most widely used administration of vaccine DNA is direct injection of the DNA
into muscle or skin by needle and syringe. This method is effective in inducing immune responses in small animals, as mice, but even here it requires the administration of relatively large amounts of DNA, ca. 50 to 100 ug per mouse. To obtain immune responses in larger animals, as rabbits, non-human primates, and humans, very large amounts of DNA
have to be injected. It has to be seen whether this requirement for very large amounts of vaccine DNA
turns out to be practical, for safety and commercial reasons, in huiman applications.
A cell has a nature) r~esiatance to the passage of molecules through its membranes into the cell cytoplasm. Sciein the 1990's first discovered "el~p~ion", where electrical fields arc used to create pores in cells without causing permanent damage to the cells. Electroporation made possible the insertion of large molecules directly iMo call cytoplasm by temporarily creating pores in the cells through which the molecules pass irno the cell.
Blectroporation techniques (both fn vlero and in vivo) function by applying a fief high voltage pulse to elodrodes positiamd around the trestatent region. The electric field generated between the electrodes ~s die cell membranes to tmnporarily become porous, wheroupon molecules of the imp4nt agent enter the cells.
Electropotation has bean used to implant materials into many different types of cells.
Such cells, for example, include eggs, platelets, human cells, red blood cells, mammalian cells, plant protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, and sponm. Further-more, electroporation has been used to implant a variety of different materials, including nucleic acids, polypaptides, and various chemical agents.
E3a~mporation has bxn used in both irr vitro and in vivo procedures to introduce foreign material into living cells. With lrr vhro appiicahons, a sample of live cells is fast mixed with the implant agent and placed between electrodes such as parallel plates. Then, the electrodes apply an electrical field to the cell/implant mixture.
With in vivo applications of electmporation, electrodes arc provided in various configWatioas such as, for example, a caliper that grips the epidermis overlying a region of cells to be treated. Alternatively, nesadle-shaped electrodes may be inserted into the patient, to access more deeply located cells. In either case, sitar the therapeutic agent is injected into the treatment region, the electrodes apply an electrical fteki to the region.
In some electroporMion applications, this electric field comprises a single square wave pulse on the order of 100 to S00 V/cm, of about 10 to 60 ms duration. Such a pulse may be generated, for example, in known applications of the Elactro Square Porator T820, made by the BTX
Division of G~tronics, Inc.
Elxtropocation has baa~ recently :ms an alienate approach to the troatment of certain diseases such as cancer by introducing a chemotherapy. drug directly into the coil.
For example, in the tromtrnent of certain types of cancer with chemotherapy it is necessary to use a high enough dose of a drug to kill the ceocer cells withwrt killing an uascoepbble high number of normal cells. If the chemotherapy drug could be inserted directly inside the cancer cells, this objective could be achieved. Some of the best anti-cancer drugs, for example, bleom~in, normally cannot penetrate the mmnbranes of certain cancer cells effectively. However, olectroporation makes it possible to insert the bleomycin into the cells.
Despite the suitability of the epidermis as a target tissue for gene tbetapy, there are significant barriers to safe, easy, efficient, and economical gone delivery.
In particular, the lipid-rich SC, which is composed of dead kecatinocytes surrounded by multiple, parallel bilayer m~nbrsaes, represents a formidable physical barrier to epidermal gene transfer. To we oo~ozszi rc~rnrs~ns~ss overcome this barrier a novel, non-vial approach, involving the basic concept of elec>xoporation to introduce genes into the epidermis is provided by the prcsatt invention.
As described above, the technique of eloctroporation is now a wo!!-established physical method for transf~ion of cello that allows introduction of marker molecules, drugs, genes, antisense oligonucleotides and proteins intracellularly. However, thorn still exists a need to introduce therapeutic ages directly into skin cells or through the skin and into muscle tolls without direct injection.
The present invention describes an in viva method, using pulsed electric field to deliver therapeutic agents into colts of the skin and muscle for local and systemic treatments.
In particular, therapeutic agents include caked or formulated nucleic acid, polypeptides and chemotherapeutic agents.
l~erapeutic agents can be employed directly as palliative agents (i.e., those which directly exert a therapeutic effects ~ as agents with a less direct effect (e.g., g~os encoding polypeptides that elicit an immune response).
The advantages offered by electeoporation for skin and muscle-directed gene therapy irmlude: (1) elimination of the risk of gen~ing covet diseasr,-causing agents, (2) delivery of DNA molecules much larger than can be packaged into a virus, (3) no immune responses or . , ~cvlde offods by non-DNA material, eg., viral proteins or cationic lipids, (~l) DNA .esters the cell through a non-endasomal pathway, diminishing the rate of DNA
degradation, and (5) the method is simple, highly reproducible and cost effective.
In accordance with one anbodimeM of the present invention, there is provided an ire viva method for introducing a thorapautic agent into skin cells of a subject, comprising applying a pulsed electric field to the skin calls substantially contemporaneously with the applicatian of therapeutic agent to the akin calls, such that the therapeutic agent is introduced into the skin cells.
In accordance with another embodiment of tlu presets invention, there is provided a meshed fa inducing an immune response in a subject, comprising applying a pulsed electric we ooroz~i Pcrms~n~ss field to skin andlor muscle cells of the s~joct subsrandaiiy contampo~raneously with the application of an immune responso-inducing aged to the skin andlor muscle cells, such that the immune response-inducing agent is introduced into the skin and/or muscle cells thereby inducing in the subject an immune response.
In accordance with stilt another embodiment of the present invention, there is provided a method for the therapeutic application of eloGdoparnion to skin and/or muscle cells of a subject for introducing topically applied molecules into said tails, comprising providing an array of electrodes, at least one of the electrodes having a needle configuration for penetrating tissue; inserting the needle electrode into selected tissue;
positioning a second electrode of the stray of electrodes in conductive relation to the selected tissue; and applying pulses of high amplitude electric signals to the elaarodes, proportionate to the distance between the electrodes, for electroporation of said tissue; such that said topically applied molecules are introduced into said skin andlor muscle cells.
In another embodiment of the present invention, there is providaf a micropatch electrode for use with an electroporation apparatus, said micropatch electrode having a substantially planar array of patch elmnants, each patch element comprising two sets of electrodes, wherein each set of elecxrodes comprises a fast electrode and a second electrode electrically insulated from one another, such that when different electric potentials are applied to said first and second electrodes, a voltage is produced therobetvveen.
2p.;. . . . An electrode kit..for use.in.coayjunction with ele~n therapy, said.kit having a miaopatch eleChode as described herein, and an injection needle, optionally comprising one or more holes disposed along itq length and proximal to the needle tip, wherein said holes are in fluid communicsttion with the hollow interior of said iqiection needle.
In accordanx with another embodiment of the present invention there is provided an electrode for use with an electroporation apparatus, said electrode having a ring-shaped electrode having an electrically iaawhting shield, wherein the electrically insulating shield provides support to the ring-shaped electrode and electrically insulates a tissue under treatment anploying the electrode, end an electrically oooducdng injection needle, optionally comprising one or more holes disposed along its bngth and proximal to the needle tip, wherein the holes are in fluid communication with the hollow interior of the injection needle, we no~om rcrms~ns~rss wherein a potential difference applied to the ring shaped electrode and the needle electrode aestes a voltage therebetween.
In accordance with yet another mnbodiment of the present invention there is provided an electrode for use with an electroporation apparatus, said electrode having an array of a plurality of paired electrode noodles and, at Ieast one injection needle, wherein a potential difference applied said paired electrode needles creates a voltage therobetween.
In acxordance with another embodiment of the present invention there is provided an electrode for use with an electroporation apparaAts, said electrode having a suction g ~~ ~mprising a ring epode dabout an injection needle electrode, such that when said ring electrode is contacted with skin of a subject and suction is generated by the suction generating devicx, the skin is pulled up around the injection noodle electrode, causing the injection needle eltectxtrode to pierce the skin, and wherein a potential difference applied to the ring-shaped electrode and said injection needle electrode creates a voltage therebetween.
In accordance with anotl~ anbodiment of the present invention them is provided an electrode for use with an electroporation apperadis, said electrode having an injection needle and paired electrically conducting wires disposed within the hollow come of the injection needle and protruding from the tip thereof, the elxtrieelly conducting wires having an eiec~ricaUy insulated portioa and an expoeod electrically conducting portion, the exposed ically conducting portion being at the end of said wires protruding from the tip of said injection needle.
In accordance with another embodiment of the present invention there is provided a needle electrode for use with an electropotation apparatus, said needle electrode having disposed around a portion of its length a substance-releasing material.
In accordance with another embodiment of the present invention there is provided a needle electrode for use with an elocbroporation ~paratus, said needle electrode having a hollow central core, at least four holes along its leagth, wherein the four holes cmnprise two paired holes, each of said two paired holes comprising a hole proximal to the tip of the needle and a hole distal to the tip of said needle, and s pair of electrically conducting wiros having an electrically insulated portiwt ~d an exposed electrically conducting portion, wherein the pair of electrically conducting wines are located in said hollow core, except for the exposod electrically conducting portion which cans outside of the needle, ceding outward from the distal paired hole to the proximal paired hole where it re-enters the hollow core.
In accordance w'tth another embodiment of the present invention there is provided a needle electrode for use with an electroporation apparatus, said electrode having a plurality of electrically conducting needles disposod within a depth guide, wherein the tip of the needles extend far a predetermined distance boyond the depth guide.
BIZ1EF DES!~IZ1PTION nF TH_ . FI ' 1RRS
FIG. 1 shows a micropatch etectroporation applicator that has a micropatch member 110 sect an injection noodle 1Z0.
FIG. 2 shows an electroporation applicator that replaces the micropatch member in FIG. 1 with a ring-shaped eloarode Z10.
FIG. 3 shows a device having three iajection needles 11.0A,1?~OB, and 120C
ccmnected to an electrical control circuit 300 to function as a pair of electrodes.
F1G. 4A shows an ekctroporation device based ~ the device shown in FIG. 2. A
s~rtinge 410'is implemented to hold an injection needle electrode 42.0 and a ryng electrode . , 430.
FIGS. 5A and SB show a ta;edle applicator having a hollow needle 510 and two electrical wins 520A and 5208 that aro held in the hollow portion of the needle 310.
FIG. 6 shows a pair of needle electrodes 6i0 and 6Z0 that have substance-releasing sections 612 and 622 near the needle tips.
FIG. T shows a single needle applicator having a hollow center portion and at least four holes 91?,A, T1~B, ~14A, and ~14B on the side walls near the needle tip.

mus99ns~ss FIG. 8 shows an applicator having an array of 6 needle electrodes. Each needle electrode is used to apply both electrical pulses and a therapeutic agent.
FIG. 9 shows and applicator having an array of noodle electrodes {9Z0A, 920B, ate.). A substance-releasing needle 910 is located in the center of the array and has holes near the needle tip for releasing a DNA.
FIG. 10A depicts an example of a caliper type electrode (PIN 384, available from Gcnetronics, Inc.) comprising of two brass electrodes, each measuring about 1 cm X lcm, mounted on a caliper scale which allows for accurate measuramatt of the inter-alactrode distance.
FIG. l OB depicts a meander electrode, comprising an array of interweaving electrode forgers. In this embodiment, the electrode finer width is about 2mm and the electrode gap is about 0.2 mm.
FIG. 11 depicts a method for introducing naked DNA into the skin comprising walk aro~md iotradermal injection of DNA into citoa A-E, followed by doctmpulsiog a thin fold of skin using a caliper electrode.
FIG. 12 depicts a microneedle electrode array with integrated container for therapaltic agent and therapeutic agent injector needle.
FIG.13, p~enels a-d, depict photomicrographs of akin samples stained with X-gal 3 days pit application of IacZ DNA and: {a) no pulse (caliper pre~ue ouly); (b) pulsing (i.e., three exponential decay pulses of amplitude 120 V and pulse length of 10 ms administered from a BTX 13CM 600 palae Generator within about 1 minute) and caliper pressure treatment for 1 min.; (c) and (d) after the same treatment as panel (b) but with pulse length and post-pulso pressure times of 10 ms with 10 min.. pressure and 20 ms with 10 min.
pressure, respectively.
FIG. 14A graphically depicts the of IacZ gene expression under the excel conditions described for FIG. 13 (a-d).

WO t10r026Z1 PCTlUS99/15156 FIG. 14A graphically dapiots the trensfeetion effciency of lacZ gene undo the experimental condirions described for FIG. 13 (a-d).
FIG. 15, panols 1 A/8 - 4 AB show photomicrographs comparing integation and expression of GFP following a~li~ion of ptmanid DNA combining the GFP gene, followed S by electroporation using meander or calipor type electrodes.
FIG. 16A and 16B depict pbOtomicrogriphs using confocat fluorescent optics aad FITC filtration of SF-295 cells 24 hoots post electroporation with 30 ltg of unlabeled and 10 ug of TO'fO-t labeled plasmid, each encoding GFP. Fig. 16A shows GFP
expressing cells are visible as well as small gin fluorescent dots that indicate TOTO-1 labeled plasmid within cells that are not ex~sing the plesmid (100X magnif'4cation, fluorescxnt light only).
Fig. 1 tiB shows white light from a tung~en source added to the fluorescent light to simultaneously illuminate the GFP positive and negative coils (460X
magnifcati~).
FIG. 17 graphically depicts the results of DNA immunization using 5 pg, 20 ltg, and 50 pg in tibialis muscle of mice.
FIG. 18 depicts the dramatic increase in SE,AP expression in blood obtained by combining electroporation with i.d. injection of plasmid encoding SF.AP.
FIG. 19 graphically depicts the dramatic incnraxs in SF.AP eacp~sion in blood by using different configuration of shallow needle arrays when combining electroponttion with i.d. injection of plasmid encoding SF.AP.
FIG. 20 depicts an alternative embodiment of a shallow needle electrode atxay.
FIG. 21 shows the relative expression of lucife~ase (RLt~ 24 hours after gee delivery to human skin xenogaRed onto nude mice, plus and minus electroporation.
FIG. 22 depicts results obtains from the comparison of caliper vs. meander elxtrodes in delivering CMV-lucifuase plaemid DNA to hairless mouse skin.

WO U0I~2621 PCTNS99I15755 In accordance with the present invention, there are provided in vivo methods for introducing a therapeutic agent iMo skin or muscle cells of a subject, said method comprising applying a pulsed electric fteld to said skin or muscle cells substantially contemporaneously 5 with the application of said therapeutic agent to said skin or muscle cells, such that said therapeutic agent is introduced into said skin or muscle cells respectively.
The term "substantially c~teanporanea~sly" means that the molecule and the olectroporation treatment are administered reas~ably close together with respect to time.
'The administration of the molecule or therapeutic agent can at any interval, depending upon 10 such factors, for example, as the nature of tlu tumor, the condition of the patient, the size and chemical characteristics of the molecule and half fife of the molecule.
As used heroin, the terms "impulse," "pulse," "electrical impulse,"
"electrical pulse,"
"electric pulse," "electropulse" and grammatical variations thereof are interchangeable and all refer to an electrical stimulus. Although the various terms are frequently used heroin in the singular, the singular forms of the terms include multiple pulses.
Preferred electrical impulses are pulsed electric fields applied via elochoporation. The pulse can be unipolar, bipolar, exponential or square wave form. Electric pulses contemplated for use in the practice of the present invention include those pulses of sufficient voltage and duration to cause elo~opo<ation.
As used herein, the term "therapeutic agait" as used heroin refers to drugs (e.g., chemot>xrapattic agents), nucleic acids (e.g., polynueleotides), peptides and polypeptides, including antibodies. The tenor "polynucleotides" inctudo DNA, cDNA and RNA
sequences, as further elaborated herein.
Therefore, in accordance with anMher dnbodiment, the present invention provides a method for the introduction of nucleic acid into the colts of the skin and/or muscle, profaably human, by contacting the skin with nnckic acid and applying an electrical pulse to the targeted region. The electrical pulse is of sufficient voltage and duration to cause elechopomtion to happen so that the nucleic acid can penetrate into the cells of the skin and/ muscle and be expressed as a transg~tic molecule. The biological expression of the WO 00~026ZI PGTNS9911S1SS

nucleic acid component results in the transcription and translation of the delivered gene so that the targeted cells synthesize gang product de reeve. Therapeutic applications include, for example, the augmentation of missing or under-axpnsssed gangs; the expression of gas that have a then4peutic value (e.g., inhibiting the action of harmful gene products by expressing a S rocxptor to bind the product of an over-acpr~d gene); the expression of genes, the product of which elicits a desired immune response; std the like.
As will be understood by those of skill in the art, efficient expression of a nucleic acid ~g a thaapaur~ polypeptida gena~ily ~equiras that the nucleic mid sequence be operably associated with a regulatory sequencx. Regulatory sequences coatamplated for use in the practice of the present invention inaluda promoters, aihancers, and die like. As those of skill in the art will also approciata, even when a promoter sequemce is op~ably associated with the therapeutic nucleic acid, expression may be further augmented by operably associating an enhancer element or the like.
Promoters contemplated for use in the practice of the present invention include the CMV, RSV LTR, MPSV LTR, SV40, the group of keratin specific premolars (e.g., the keeatin and involucrin group of ~snoters. Preaenttyr, it is preferred that the promoters employed in the practice of the present invention are specifically active in skin cells. The >xan~xiptional promoters of a number of genes expressed in the epidermis have boon charactaiud. Furthermore, such promoters fond to restrict axpr~ession to wither the basal compartrnent or the suprabasal comparlme~. Keratin 14, for example, is expressed by basal kaatiotocytes, v~hereas invohrcrin is expressed by suprabasal keratinocytes.
In addition, the keratin 14 and involucrin genes are highly expt~ssed in keratinocytes, thus use of their promoters to drive transgene transcription yields not only target specificity, but also high levels of eacpression. The promoters for both gas have boon sueo~sfully used to direct compartment-specific expression to the epidermis of transgenic mice.
Ch~od~a~apeutic agents o~tampia~ed for use in the method of the invention typically have an antitumor or cytotoxic effect. Such drugs or ages iuchuk bleomycin, neocarcinostatin, suramin, doxoiubicin, carboplatin, taxol, mitomycin C, cisplatin, and the like. Other chematherapeutic agams w01 he known to those of skill in the art (see, f~
example The Merck Index). In addition, chamothaapeutio agents that arc "membranes we oom p~~~

acting" agenu are also included is invention methods. Thex ag~ts may have palliative effxu as those listed above, or alternafrvely, they may be agents wbich act primarily by damaging the cell membrane. Examples of membrane-acting agents inchtda N-atkyimelamide and para-chloro mercury benaoate. '!he chemical composition of the agant wilt dictate the most appropriate time to administer the agent in rotation to the administration of the elxtric pulse. For example, while n~ wanting to he bound by a particular theory, it is believed that a drug having a low isoelectric point (e.g., neocatcinostatin, IEP=3.78), would likely be more effective if administered post.electroprnration in order to avoid electrostatic interaction of the highly charged drag within the fmld. Further, such drugs as bloomycin, which have a very negative log P, (P being the partition coefficiait between octanol and water), are very large in size (MW=1400), and are hydrophilic, thereby associating closely with the lipid membrane, diffux vay slowly i~o a tumor cell and are typically administered prior to or substantially simultaneous with the electric pulse. In addition, certain agents may require modification in order to allow more efficient e~y into the cell. For example, an agent such as taxol can be modifitd to increase solubility in water which would allow mare effcieM entry into the cell. Eloctroporation facilitates entry of bleomycin or other similar drugs into the tumor cell by creating pores in the cell membrane.
In one embodiment of the presant invention, t6cre is provided a method for the therapeutic application of ele~pot'abon to skin andla muscle of a sabject for introducing applied molecules into cells therein, comprising providing an array of electrodes, at least one of the electrodes having a needle cor~gtusxiotv for penatraximg tissue;
inserting the needle electrode into xleGtod tissue; positioning a aeeo»d eleet~ode of the array of electrodes in conductive rotation to the xlected tissue; applying pulxa of high amplitude electric signals to the electrodes, proportionate to the distance between the electrodes, for electroporation of the tissue, such that said applied molecules an introduced into said akin or muscle cells. In one aspect of the present invention, the molecules to be introduced arc topically applied. In another aspect of the present inventi~, the molecules to be introduced sre applied by other means such as injection, of the lint. It should be understood that the electroporation of tissue can be performed itr vitro, in viva, or ex vivo. Eleetropotation can ahsa be performed utilizing single calls, eg., single cell suspensions or ire vitro or ax vivo in cell culture.

wo oo~osszi pcrnls99ns~ss It may be desirable to mo~lates the expression of a gene in a cell by the introduction of a molecule by the method of the invention. 'Ihe ternt "modulate" envisions the suppression of lion of a gene when it is ov~x-fed, or augmertation of expression whoa it is under-expressed. Where a cell pmlifemtive disorder is associated with the expression of a gene, for example, nucleic acid aequon~ that with expression of the gene at the translational level can be used. This approach utilizes, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or ttx~tslation of a spxific mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme.
Nucleic acids contemplated for use in the practice of the present invontian include asked DNA, naked RNA, asked plasmid DNA, either wpercoiled or linear, and encapsulated DNA or RNA (e.g., in liposomes, microspheres, or the Like). As will be understood by those of skill in the art, paeticles mixed with phumid so as to "condense" the DNA
molecule may also be employed.
Amisenae nucleic acids arse DNA or RNA molecules that are complementary roe at least a portion of a specific mRNA molecule (see, e.g., Weintraub, Scientific Arhericarr, 262:40, 1990). In the cell, the aatiaenae nucleic acids hybridise to the oorrespoadiag mRNA, forming a doublo-stranded molecule. The antisensc nucleic acids interfere with the tcanslati~ of the mRNA, since the cell will not translate a mRNA that is doubfo-stranded.
Aatisonsa oligomers of about 15 nucleotides are preferred, since they are easily synthesized a~ are lesslikely to cause deloterioas effeCb than larger molecules when introduced into the target cell. The use of antisense methods to inhibit the i~ vitro translation of genes is well known in the art (see, e.g., Marcus-Saknra, Anal. Biochan., 172:289,1988).
Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around doublalmlical DNA, fonaing a threo-strand helix.
Theref~e, these triplex compounds can be designed to neeognize a unique site on a chosen gene (Maher, et al.. Mtisenae Res. art Dev., 1(3):227,1991; Heleae, C.,.lrtticanctr lkug Design, 6(6):569, 1991 ). Accordingly, electrvp~on of nucleic acids useful for triplex formation is also coatanplated as within the scope of the paaaa~t invention.

WO OOI~OZ6Z1 PCT/US99/15755 Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a mam~er analogy to DNA restriction endonucleaaes.
Through the modification of nuchtide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide aoquences in an RNA molecule and cleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences art inactivated.
There art two basic types of ribozymes namely, tctrahyrn~rea-type (Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type. Tetrafrynrena-type ribozymes recognize sequences which are four bases in length, while hammerhead-type ribozymes recognize base sequences in the range of 11-18 bases in length. The longs the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species.
Consequently, hammerhead-type ribozymes are peeferablt to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
The present invention also provides methods of gene therapy for the treatment of cell proliferative or immunologic disorders mediated by a particular gene or absence thereof. The tam "cefi proliferative disorder" denotes malignant as well as non-malignant cell populations which often appear to differ from the surrounding tissue both morphologically and genotypically. Such therapy would achieve its therapeutic effect by introduction of a specific sense or antisense polynuclootide into cells having the disorder.
Delivery of polynucleotides can bb achieved using a recombinant expression vector such as a chimeric virus, or the polynucleotide can be delivered as "naked" DNA for example.
The polynucleotide sequcnas of the invention are DNA or RNA sequences having a therapeutic effect after being taken up by a cell. Nucleic acids cont~nplated for use in the practice of the present invention can be double stranded DNA (e.g., plasmid, cosmid, phage, viral, YACS, BACS, other artficial chromsomos, and the like), single stranded DNA or RNA.
The nucleic acids may be uneomplexed (i.e., "naked") or compiexed (e.g., with chemical agents tech as lipids (e.g., cationic), dendrimers, or other polyplexes that facilitate DNA
penetration into tissues snd through cell membranes, and the like). The DNA
may also be encapsulated or formulated with protein complexes.

we ooroz6il pcrros~ns~ss Exampks of polynucleotides that aro themselves therapeutic are anti-sense DNA
and RNA; DNA coding for an anti-sense RNA; or DNA coding for tRNA or rRNA to replace defective or deficient endogenous molecules, and the like. The polynucleotides of the invention can also code for therapeutic polype~ides. As used herein, "polypeptido" is 5 undetsWod to be any translation product of a polyweleotl~ rogerdkss of size, and whether gfycosylated or not. Therapeutic polypeptides cant~nplated for use in the practice of the present invention include, as a primary example, those polypeptides that can compensate for r defective or deficient species in an animal, or those that act thmugh toxic effects to limit or remove harmful cells from the body.
10 Also included are polynuclootides which encode metabolic enzymes and proteins, such as blood coagulation compounds (e.g., Factor VIII or Factor IX), and the ldce.
In accordance with another embodiment of the present invention, there are provided methods for inducing an immune response in a subjecx. invention methods of this embodiment comprise applying a pulsed electric field to akin or muscle cells of the subject 15 substantially contemporaneously with the application of an immune response-inducing agent to ~e akin or musck cells, such that the immune responso-ind~ing agent is i~oduced into the akin or muscle cells thereby inducing in the subject an immune response.
As used herein, "immune response-ind~ing agent" means any agent, which upon introduction into the skin or muscle cells of a subject, results in an immune response, whether the response be a cellular response, a humoral roaponse, or both. Immune response-inducing agents 'contemplated for use in the practice of the present invention include expressible nucleic aids, and polypepbdes.
Expressible DNA and mRNA can be delivaod to ills to form therein a polypeptide trap product. If the n~leic acids aro opeeatively associated with the proem regulatory sequences, enhanced synthesis of the encoded protein is achievable. DNA or RNA
encoded polypeptides contemplated for use in the practice of the present invention include immunizing potypaptiaes, pathogen-derived prnbeiaa, blood coagulation factors, pe~ide hormones, sad the IdCe. Peptide hormones include, for exasaple, calcit~in (C7), parathyroid hormone (P1'lEi), erythmpoietin (Epo), insulin, cybakinea, growth hormone, growth factors, and the like). Lymphokines contemplated for use in the paactice of the present invention wo o~ro~sZ~ ~cr/us99/is~55 include tumor necrosis factor, intuieukins 1, 2, and 3, lymphotoxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, alpha-interferon, beta-int~erferon, gamma-interferon and subtypes t6eroof. Blood c~guladon factors contemplated for use in the practicx of the present invention include Factor VIII or Factor IX.
When the DNA or mIZNA delivered to the cells codes for an immunizing peptide, invention methods can be applied to achieve improved and more et~ective immunity against infectious agents, including bacteria, intracellular viruses, tumor cells, and the like.
Therapeutic polynucleotides provided by the invention can also code for immunity-conferring polypeptides, which can act as endogenous immunogens (i.e., aniigen-containing polypeptides) to pmvoke a humoral immune response, a cellular immune response-inducing agent response, or both. Methods for inducing such responses and targeting specific calls for specific rosponsas are described, for example, in U.S.
Patent No. 5,589,466.
The polynucleotides employed in accordance with the present invention can also code for an antibody. In this regard, the term "antibody" encompasses whole immunoglobulin of any class, chimeric antibodies and hybrid antibodies with dual or multiple antigen or epitope specificities, and fragments, such as F(ab~, Fab', Fab, and the like, including hybrid fisgments thereof. Also included within the moaning of "antibody" are conjugates of such fragments, and so-called antigen binding proteins (single chain antibodies) as described, for example, in U.S. Pat. No. 4,704,692, hereby incorporated by reference herein in its entirety.
Thus, an isolated polynueleotide eodlng for variable regions of an antibody can be introduced, in accordance with tlm pint invention, to enable the treated subject to produce antibody in sfru. For illustrative methodology relating to obtaining antibody-encoding polynucleotides, see Ward et al. Nanve, 341:544-546 (1989); Gillies et al., Bfoteclsnol.
7:799-804 (1989). The antibody in turn exerts a therapeutic effect, for example, by binding a surface antigai associated with a pathogen. Alternatively, the encoded antibodies can be anti-idiotypic antibodies (antibodies that bind other antibodies) as described, for example, in U.S.
Pat. No. 4,699,880. Such anti-idiotypic antibodies could bind endogenous or foreign antibodies in a treated individual, thereby ameliorating or preventing pathological conditions associated with an immune response, (e.g., in the context of an autoimmune disease such as lupus and the like).

we oorom Pcrn~nsrss 1~
It is presently preferred that polynucleotide sequences used in the practice of the preset invention code for therapeutic or immunogeaic polypeptides. These polynuclootide sequences may be used in association with other polynucleotide sequences coding for regulatory proteins that control the expression of the thoraperrtic or immunogenic polypeptides. The regulatory proteins) so employed can act in any number of regulatory warmers known to those of skill in the art, such as by binding to DNA so as to regulate its transcription, by binding to messenger RNA to incxease or decrease its stability or translation efficiency, and the like.
The polynucleotide material delivered to the cells in viva can take any number of forms, and the pmesent invention is not limited to any particular polynucleotide coding for any particular polypeptide. Plaamids containing yes coding for a large number of physiologically active peptides and antigens or immunogens are contemplated for use in the practice of the present invention and can be readily obtained by those of skill in the art.
Various viral vectors can also be utilized in the practice of the present invention and include adenovims, herpes virus, vaccinia, RNA virus, and the like. It is presently preferred that the virus be an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a marine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be include, but are not limilvd to: Moloney marine leukemia virus (MoMuLV), Harvey mur'me sarcoma virus (HaMuSV), marine mammary tumor virus (MuMTV~ and Roux Sarcoma Virus (RSV). When the subject is a human, a vector such as the gibbon ape leukemia virus (GaLV), or the like csm be utilized. A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a geese for a selectable marker so that transduced cells can be identified and generated.
Therapeutic peptides or polypeptides may also be included in the therapeutic method of the invention. For example, immunomodulatory agents and other biological response modifiers can be administered for incorporation by a cell. As used heroin, the form "biological respoosc modifmrs" encompasses ~bs~s which aro involved in modifying the immune response. Examples of immune response modifiers include such compounds as lymphOltiaes, and the It~ce. Lymphokines include, for example, tumor necrosis factor, wo aor~o~s~l Pcrnrs~nsrss i~erleukins 1, 2, and 3, lymphotoxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, alpha-interferon, beta-interferon, gamma-interferon and their subtypes.
Administration of a cbomofeutic agent, polynuclootide or polypeptde, is the practice of invention methods will typically be by topical application.
Accordingly, a "parneaxion esths»eer" also can be included with eloctropulsing oo increase imroduction of a composition. As used herein, the term "permeation enhsncer" refers to any action (e.g., mechanical, physical, chemical) or any composition that can increase or "augment"
introduction of a composition into skin andlor mnscle cells. The term "augment," when used hmein as a modifier of introduction, means that the rate (over time) or amount of composition introduced into skin andlor muscle cells via eiectropulsing is gteaur than that produced by electropulsing in the absence of the peraneation enhancer. Thus, administering a parmeation enhancer prior to, substantially contemporaneously with or aftm topical application of a therapeutic agent serves to "augment" electrically induced introduction of the composition into skin and/or muscle culls. Ahernatively, a permeation enhancer can be mixed with the composition in the pharma~tical formulation to be iotroducod.
Permeation aihancer compositions that increase the permeability of skin andlor muscle cells include, for exaosple, alcohols (e.g., methanol), alkyl methyl sulfoxides (e.g., DMSO~
pyrrotidona (e.g., 2-pyrrolidane), surfactants, urea, glycerol monolaurata, polyethylene glycol monoisurate, glycerol moaolavram, docainehyde~ochlor~e, hydrooostisone, m~thol, methyl salicylate, and the like. Permeation enhancers fi~rr)xr inchrde mechanical or physical actions that function is asrociation with an electrical impulse (cg., eibeaaloo, vibration, ultrasound, and the like).
Depending on the nature of the therapeutic agent, the desired depth of penetration, the target tissue type, and the like, 'rt may be desirable to conduct electroporation in combination with other electrically-based tnmtment modalities. Electropulsing conducted substantially contemporaneously with iontophotesis (IPH), can produce a greater merapeutic effect than either applying the pulse or iontophoteais alone. Furthermore, elcctroincorporation (E1) (see, e.g., US Patent Na. 5,464,386, which is hereby incorporated by referwce herein in its entirety), or eleciropulaing in combination with IPH
and liposomal formulation can enhance delivery significantly. (sea, e.g., Badkar, et al., Doug Delivery 6 (1999) 111-115). Accordingly, in another embodiment of the present invetrtion, P~r~' el~pulsing is used in conjuncrion with one or moro of iontophoresis and ekctroincorporation.
As used herein, the term "tranadermally introducing" and grammatical variations thereof, refers to tho delivery of a composition into dre skin, through/across the skin, or a combination thereof.
Targeting the cells of the skin for gene therapy has several advantages. First of all, the epidernris is an accessible tisane, which simplifies approaches for introduction of a transgone. Keratinocytes, the predomiaant cell type in the epidermis and hence the cellular target for gene transfer, form the outer-most barriers of the skin, making them amenable to in vivo manipulation. The accassibilily of the epidermis raises the potential for use of non-invasive, topical methods for game transfer. The epidermis is a srrriified squamous epithelium consisting of a basal proliferating compartrnent and a suprabasal, differentiating compartment. By ta>g~ing gene transfer to the basal compertrnmrt, gene can be iat<odrxxd into epidermal stem cells. Various treatraent regimens aro thus malt possible.
For example, t 5 single gene recessive disord~crs such as lamellar ichthyosis (I I) or X-linked ichthyosis (XLI) could be treated using the gore transglutaminax 1, or the gee for the steroid sulfatase arylsulfatase C, reapectivoly. Epidermal stem cells give rise to basal, transiently amplifying cells, which have a limited capacity to divide. In turn, these cells give rise to the differentiating keratinocytes of the suprabasal epidermis. Thus, by achieving transgene in prog~itar basal keratiQOCytes, method: for Imr~tam, srrs<ained genre therapy are provided.
Keratirrocytes fimedaa wail as synd~tic and secxetory cells. ICexatirrocytes have been shown to synthesize and secrete In-vhro the products of transfected genes. Circulating transgeno-derived proteins such as growth hormone (GH)(22 kD), ApoE (34 kD), and FIX
(57 kD) have boon detected in athymic mice bearing grafts of kenrtinocytes.
This dema~strateS that traosgene products e~xpr~d in the epidermis can penetrate the basement membrane zone and rich the systemic circulation.
I . In one embodiment, the electrode is s wire electrode (useful for in vitro studies, and the like). In another embodlmart, the eiocaode is a plurality of electrodes (e.g., a micropatch electrode as deaaribed in US Patent A~lication Serial No.
09/134,245, filed we oorom rc~rms~nsrss on August 14, 1998, which is heroby iueotporated herein in its entirety by referonce). In still yet anatha~ embodimwri, the electrode comprises a meander electrode (e.g., an array of interweaving electrode fingers, with a typical elodrode width in the range of about 0.2 up to about 1 mm, and an electrode gap of about 0.2 mm, wherein the gap can be filled 5 with an electrically insulating substance). In an additional embodiment, the electrode is a porous eleetrbde. The various electrodes used herein are preferably insulated to protect against excess boat or burning, ctut~ent leakage, shock, etc. Appropriate electric pulsing parameters are set forth herein or can be de~ennined using the teachings heroin and, in view of these parameters, the skilled artisan can select among various suitable electrode 10 types (e.g:, ceramic, metal, etc.) and contlgurations (single wire, multiple wire, ate.) available in the art. In one embodiment of the present invention, a sqnarc wavy pulse is applied, whm~ein the pulse is at least 50 V for about 10 up to 20 ms. Of course other pulse types, voltages and times may be appropriate, as wlll be understood by those of skill in the art.
15 In accordance with additional embod~ants of the present invention, these are provided a number of improved eledroporation devices that may be advantageously used in different applications. Examples of these additional embodiments are described below, with rrrferrrttce to attached figures.
FIG. l shows a micropatch electropotatiort applicator 100 that has a micropatch 20 member 110 and as injection needle IZO. The micropatch 100 includes c plurality of patch elemetrts 110 that era positioned relative ~ orra another to form a substantially planar array.
Each patch elemart 110 includes two pairs of eiedrades, a first pair having etxtrodes liOA
and 1108 and a second pair having electrodes IZOA and 1Z0B. The two electrodes I 10A and i lOB (or II2A and 11ZB) in each pair era aleatrioally insulated from each other and are applied with different electrical poterttisls to peodrrCe a voltage th~ebdweera~. Far example, the electrode ilOA may be at a positive vohage and the electrode I lOB may be at a negative voltage. An electrical control circuit (not shown) may be connected to the microlmtch member 100 to produce desired electrical palaes at each patch element 110. One .
anbodiment of the micropatch m~tber 100 is disclosed in pending U.S.
Applicadan No.
08/905,240, "Method and Apparatus for Using Eiodroporation Mediated Delivery of Drags and Genes," filed on August 1,199'7, which es incorporated herein by reference in its we ooiozazt PcrnJS~ns~ss entirety. The micropatch member 180 can be placed upon a tissue surface 102 such as a person's skin to apply electrical pulses to the tissue surface 102.
The micropatch member 100 also includes a plurality of gaps 114 between adjacent patch elements 110. Such gaps 114 are formed of an electrically insulating material and can be penetrated by sharp objects such as the injection needle 120. The injection needle 120 is configured to include a conduit 121 for transporting an injection substance to the needle tip 122. One or more holes 124 are optionally formed on the side walls of the needle 120 near the needle tip I22 so that the injection substarxe can be released not only from the needle tip 122 but also from these holes 124. The injection needle 120 may be formed of a metal but may also be formed of a suitably rigid insulating malarial. In operation, the injection needle 120 supplies injection substance to a target tissue area and the micropatch member 100 operates in conjunction with a power supply to cause electroporation of the target tissue.
FIG. 2 shows an electroporation applicator wherein the mieropatch member 100 depicted in FIG. 1 is replaced with a ring-shaped electrode 210. Unlike the applicator shown in FiG. I, the injection needle 120 in this embodiment also functions as an electrode.
Hence, at least a part of the injection needle 120 must be fotmed of an electrical conductor. In the ~bodiment illustrated in F1G. 2, the needle 120 is formed of a metal. The ring-shaped electrode 220 may be in any shape, including but not limited to, a circular ring as shown, a square ring, or the like. An electrical insulating shield 220 is implemented to provide a support to the ring-shaped electrode ?AO and to electrically insulate a tissue under treatment from the electrode 200: A through bola 222 is formed on shield 220 within the center opening of the ring-shaped electrode 200 for positioning the itljection needle 120. The needle electrode may be modified to provide for radial delivery of an injection substance, for example, by providing one or more holes disposed along its length and proximal to the needle tip, wherein said holes are in fluid communication with the hollow interior of the injection needle.
An electrical control circuit (tat alzown) is connected to both the injection needle 120 and the ring-shaped electrode 210 to produce desired electrical pulses, causing el~roporation and augments delivery of the mjecti~ substance to the target tissue.

FIG. 3 shows a device having a plurality of paired electrode needles and at least one injection needle. In the embodiment depicted in Fig. 3, the electrode has throe needles l2pA,1?,OB, and 120C. Needles 120A and 120B are connected to an eketrical control circuit 300 to function as a pair of electrodes. Additional paired electrode needles may be employed. The needle 1ZOC is not used to apply electrical pulses to the tissue under trea~nont but is used as an injection port to supply injection substance to the alectroporated tissue area. Substantially contemporaneously with the injection of an injection anrbstance, voltage difference is applied between the needle electrodes IZO to produce desired electrical pulses, causing alectroporation and augmented ~livexy of the injection substance to the target tissue. The injection needle may be modified to provide for rr<dial delivery of an injection substance, for example, by providing one or more holes disposed along its length and proximal to the needle tip, wherein said holes are in fluid communication with the hollow interior of the injection needle.
FIG. 4A shows an olectroporation device based am the device shown in FIG. 2. A
srmtion generating device such as a syringe 4I0 is implemented to hold an injection noodle electrode 420 and a ring electrode 430. In the depicted embodiment, the suction device is a syringe 410 having a piston 412 which is slidably with, and seatingly disposed about the injection needle elactrnde 420. As used herein, "sealingly disposed about"
means that the piston is fitted around the needle closely enough that a partial vacuum can be created upon withdrawal of the piston. The close ftt may be facili~, for exstnple, by providing a gasket, or the like. In this marmar, when the piston 41Z is retracted, or suction is otherwise generated, a partial vacuum is created in the syringe 410 and skin to which the device is applied is partially fitted ap by action of the vactmm (FIG. 4B)~ This facilitates the penetration of the skin by the injection needle electrode 4Z0. Substantially contemporaneously with the injection of an injection substance, voltage difference is applied between the needle electrode 4Z0 and the ring electrode 430 so that electrical pulses can be generated, causing electnoporation and augmented delivery of the injection substance to the target tissue.
FIGS. 5A and SB show a treedk applicator having a hollow needle SIO and two electrical wires 520A and SZOB that are held in the hollow portion of the needle 518. Each wire (eg, SZOA) has an insnlsted portion (eg., 522A) snd an exposed tip portion (eg., we ooroasm Pcrms~ns~ss SZ2B). In operation, the needle 510 penetrates a tissue and then is withdrawn to "plain" the exposed tip portions of the two wires 520A and SZOB in the tissue (Fig. 5B).
Substantially contemporaneously with the injection of an injection substance, voltage dityerence is applied between the wires SZOA and 5208 such that eketrical pulses can be generated, causing electroporakion and augmented delivery of the injection substance to the target Hssue.
FIG. 6 shows a pair of needle eloctzodes 610 and 620 that have substance-releasing sections 611 and 62Z near the needle tips. The substance-releasing sections 61Z and 622 are configured to have an injection substance (e.g., a selected DNA) associated therewith. The substance-releasing sections may be fornted of any biocompatible suitably absorbent material or material to which injection substance will adhere. Materials contemplated for use as substance-releasing sections include lipids, cationic lipids, heparin, hyaluronic acid, and the like. When the needles are penetrated within a tissue, electrical pulses are applied to the needle electrodes 610 and 620 so that injection substance applied substantially contemporaneously therewith can be electroporated into the cells between the electrodes.
As used herein, "biocompatible" or "biocompatible material" means a material that is suitable for introduction into the human body for therapeutic purposes.
FIG. 7 shows a single needle applicator. A needle ?1O has a hollow center portion and at least four holes ?12A, ?1Z8, ?14A, and ?148 on the side walls proximal to the needle tip. Paired holes (e.g., ?12A and ?1Z8) are preferably arranged relatively proximal and distal 24 to the needle tip along the same side of the needle body. Holes ?14A and ?148 are similarly arranged along the needle body but are radially spaced from holes ?12A and ?128. The noodle ?10 is either formed of an insulator or coated with an insulating layer. Two electrical wires ?20 and ?30 located in the hollow center portion of the noodle respectively run through the holes ?14A, 7148 and ?12A, 7128 to so that wire sections ?20A and ?208 are exposed on the exterior of the needle body ?10. Each wire is coated with an insulator layer except for the exposed section. Different electrical potentials are applied to the wires ?20 and ?30 to produce electrical pulses between the axposed sections ?ZOA and ?208.
Additional holes may be formed near the holes ?12A, ?128, ?14A, ?148 for delivering an injection substance.
FIG. 8 shows an applicator having as array of 6 needle electrodes. Each needle electrode is used to apply both electrical pulses and an injection substance.
As illustrated, we oorozszl pc~rms99ns~ss holes are formed on the side walls near the needle tip for releasing an injection substance into the tissue.
FIG. 9 shows anothesr applicator having an array of needle elextrodes (920A, 9ZOB, arc.). A substance-releasing needle 910 is located in the center of the array and has holes near the needle tip for releasing an injection substance. Substantially contemporaneously with the injextion of an injection subsraace, voltage dit>f'ereace is applied bexvreen pairs of needles such that electrical pulses can be generated, causing elextroporation and augmented delivexy of the injection substance to the tsrgtt tissue.
The needle 910 may be either electrically active to be pulsed against the sing nexdk elextrodes or is electrically passive.
As used herein, "injection substance" means any therapeutic agent to be delivered W
the target tissue. As described heroin, therapeutic agents contemplated for use in the practice of the present invention include nucleic acids, polypeptidex, ~notherapaitic agents and the like.
In accordance with another embodiment of the present invention, there arc provided electrode kits for use in conjanction with elextroporatioo thmapy, each kit containing the components of the elxtrodes described hereon. For example, in one aspect, there is provided an electrode kit comprising a micropatch e;lexdnde, and an inje~erion needle, optionally comprising one or more holes disposes along 'rts length and ptnximal to the needle tip, wherein the holes are in fluid communiextion with the hollow interior of the injextion needle.
With all of the above ela~roporation devices, a brief time of iontophoresis may be applied to distribute the DNA between the electrodes before pulsing for elextroporation. The ~edles can be used for ionto~or~s between two or among three; or more ntodks.
Iontophoresis can also be performed between any needle and other electrodes such as baween the injection needle I20 and the ring electrode 210 in FIG. 2.
Therefore;, in aexordaoce with another embodiment of the present inve~ion, thesre are provided methods for introducing therapaitic agents into skin cells of a subject, said method comprising employa~g an electroporation method as eieaaibed herein in conjunction with ibis and or elextroincorporation.

~0 1 l~~s~~~
Additional functional components that can 6e added to the invartion electrical appa~us include, for example, an io~ophoretia unit (IPH), which can be used in combination with an electrical impulse be transdermally introduce a grater amount of the composition into andlor acres the skin than rntlsing alone, or that can drive the composition 5 deeper into the skin and/or muscle, if desired. A controlling means (e.g., a switching unit), such as an automated switch, optionally programmable, may be used to control any of the parameters of invention apparatus, including, fa example, application of pulse aloe, tire time between applying the impulse and applying IPH, as well as optionally controlling the time during which IPH is applied. Each parameter will be determined by the connpositi~
10 introduced, the desired effect, the concentration etc. In one aspect of this embodiment, electrodes can be operated by an electroporation unit and an IPH unit respectively in a sequential m. More specifically, two caliper electrodes can be disposed on either side of a skin fold (e.g., electrodes 1 and 2 on the left side and electrodes 3 and 4 on the right side). In elxtrc~otation mode electrode 1 is pulsed against eiectrodc 2, likewise electrode 3 15 is pulsed against electrode 4. In IPH mode, electrode 1 is connected to electrode 2 (with either positive or negative polarity) while electrode 3 is connected to electrode 4 with an opposite polarity', then electrodes 1 and 2 will be pulsed ag~iast electrodes 3 and 4. Of course, these operation parameters can be set or programmed into the mini-generator.
A vibration unit also can optionally be included in the apparatus, which can be used 20 in combination with an electrical impulse to transdermaliy introduce a composition into and/or across die skin, if desired. A phonophoresis rmit, which can transdermally introduce a composition into the skin by means of ultrasound, also can optionally be included in the apparatus, if desired. Thus, by applying vibration or ultrasound before, after or during puisieg and/or io~pharesis, the composition can be driven deeper into die largest tissue or a 25 greater amount of the composition can be driven into the target tissue than pulsing alone. As above, a switching unit, such as an automated switch, optionally programmable, could be used to control the time between applying the iatpulse sod applying vibration or ultrasound, as well as optionally controlling the time during which impulse, vibration or ultrasound is applied A me~ms for administering a composition can optionally be included in the electrical appsr~us, which can be used to admiaiatcr the caruposition to the target tissue prior to, wo ea~o~2i rCrnrs~ns~ss substantially contemporaneously with, or after applying an electric pulse, iontophoresis, vibration or ultrasound, in their various embodiments. Depending on the specific formulation, a composition can be incorporated into a patch reservoir (e.g., as a nicotine patch), which is then aibched both to the electrode and the skin. Formulations employed for IPH are advantagovusly used in this manner.
As used in the above e~text, tha term "~rbstantially c~ntanporaoeously" moans that the electric pulse and the composition are applied to the skin reasonably close together in time. Preferably, the composition is administered prior to or concurrontly with electropulsing. When applying muhiple electrical impulses, the composition can be administered before or after each of the pulses, or at any tirae between the electrical pulses.
When applying any auxiliary electrically-basod therapy (i.e., IPH, EI, and the like), vibration err ultrasound, the composition can be adminisbxed before or after each, and at any time Although electrodes of the pent invention are designed to work with coramercially available electroporation power supplies, the invaition apparatus can have a variety of other functionalities in addikion to the optional controlling means for applying an electric pulse, indicating means and fastening means. For example, the apparatus can have an indi means for indicating apparatus rally, the various pulse parameter settings (eg , voltage, capacitance, pulse duration, tune delay between pulses, pulse wave type), pulses) applied, parameters of the applied pulses) (e.g., voltage, capacitance, pulse duration, pulse wave type, number of pulses) or a combination theroof. Such indicating means can be visual, audible, or a combination thereof. For example, a single audible "boep" can indicate that the "apparatus is ready," two audible "beeps" can indicate that a pulse has been correctly applied and three audible "beeps" can indicato a malfunction or that the pulse was not or was imply applied. Visual india~ng means include analog or digital alpha-numeric displays (e.g., LCD, LED and the liked as in watches, and further can include illuminating means for low light visualization, for example, by white light, electroluminescent backlighting for LCD or electroluminescent lamps (t.e., INDIGLO'"'), or by various fliaresceat or radioactive illuminating cotnpositi~s, and the like.

we ouroz6zi Pcrms~ns~ss Additional "user friendly" fiu~ions include the aforementioned controlling moans for applying an electric pulse (e.g., pushbutton, knob, lever switch, dial and the like) as well as means for adjusting parameters (e.g., pushbutton, knob, lever switch, dial and the like) including, for example, pulse duration, voltage, capacitance, field strength, number, wave type, and the like. Means for adjust~g, setting, storing or retrieving one or more pulse parameters also are included herein. Such means include traditional mechanical elxtronic controls (e.g., a selector switch controlling each parameter in which the switch has a plurality of settings; exemplary pulse length settings, 5 cosec, 10 cosec, 25 cosec, 35 cosec, 50 cosec, for example) as well as a chip control (e.g., silicon wafer types commonly used in the computer industry) which is controlled, for example, by a pushbutton interface, as in watches for example. A chip, optionally removable from the apparatus or, user and/or manufacturer programmable for control of the various pulse parameters set forth herein also is contemplated. Storage capacity of such a chip is sutlacient to provide virtually unlimited fine control of the various parameters, as will as storing diffemnt pulse parameter settings for different compositions, users and the like. As each of the various electronic functionalities of the invention apparatus described herein can be controlled or managed by a computer chip, a chip affords the option of additionally incorporating software, if desired, said software optionally user programmable.
In addition to efficacy, both sensation and user softly art important. Thus, in another ombodim~t, the invention further provides an apporatus having means for preventing applying excess pulse voltage, duration, field strength and/or number. Any moans which passively or actively interrupts or disrupts the electric circuit, including fuses, circuit breaker switches, sad the like, or devices that actively monitor the various pulse parameters and internipt or disrupt the electric circuit to prevent pulse voltage, duration, field strength, pulse number from being applied can be incorporates into the circuit path. Those skilled in the art of electrical devices will know of other eve elements that prevent applying excess pulse voltage, duration, field strength or ewmber.
The electric pulse can be provided by any electrauric device that provides an appropriate electric pulse or tlectric source sufOcieot for transdermally introducing a composition into skin andlor muscle. The nature of the electric field to be generated is determined by the nature of the tissue, the size of the xlected tissue and its location. It is WO liOlOZb21 PCTNS99/15755 desitable~that the field be as homogenous as possible and of the corrext amplitude. Excessive field strength results in lysing of cells, arhoress a low field strength results in reductd efficacy. The electrodes may be mounted and manipulated in many ways including but not limited to those in the parent application. The electrodes may be conveniently manipulated on and by forceps to internal position.
fhe waveform of the electrical signal provided by the pulse generator can be an exponentially decaying pulse, a square pulse, a unipolar oscillating pulse train, a bipolar oscillating pulse train, or a combination of any of these forms. The nominal electric field strength can be from about 10 V/cm to about 20kVlem (the nominal electric field strength is determined by computing the voltage b~vve~ electrode needles divided by the distance between the needles). The pulse length cau be about 10 ps to about 100 ms.
There can be any desired number of pulses, typically one to 100 pulses per second. The wait between pulses sets can be any desired time, such as orse second. The waveform, electric field strength and pulse duration may also depend up<m the type of cells and the type of molecules that are to enter the cells via electroporation. Each pulse wave form has particular advantages; square wave form pulses provide increased effrcioncies in transporting compounds imo the cells in comparison to exponential decay wave form pulses, and the ease of optimization over a broad range of voltages, for example (Saunders, "Guide to Electroporation and Elecuofusion," 1991, pp. 227-47). Preferably, the waveform used is an exponential or a square wave pulse.
The electric fields needed for is vivo cell eleetroporation are generally similar in magnitude to the fields required for cells frr vftro. Presently preferred magaiiudes are in the range of firm 10 Vlcm to about 1300 Vlcm. The higher end of this range, over about 600 V/cm, has been verified by in vivo experiments of others reported in scientific publications.
The nominal electric field can be designated either "high" or "low". It is presently preferred that, when high fields are used, the nominal electric field is from about 700 V/cm to 1300 V/cm and more preferably fmm about 1000 Vlcm to 1300 Vlcm. It is presently preferred that, when low fields are used, the nominal electric field is from about 10 V/cm to 100 Vlem, and more preferably from about 25 V/em to 75 Vlcm. In a particular embodiment of the present invention, it is pr~ntly prefemd that when the electric field is low, the pulse we ooroz62~ rcTnrs~ns~ss length is long. For example, what the nominal electric fteld is about 25-75 V/cm, it is preferred that the pulse length is about 10 cosec.
It is presently prefenrod that practice of the therapeutic method of the invention otiliaes the appstatos of the invention which provides an elet~nodc apparatus for the application of clectroponititon to a portion of the body of a patient and comprises a support member, a phtrality of needle electrodes mounted on said support member for insertion into tissue at sets positions and distar~s from one another, and means including a signs) generator responsive to said distance signal for applying an electric signal to the electrodes ptvpottionate to the distance bctwoert said ela~rodes for g~terating an electric field of a predetermined strength.
Altattatively, it is undussood that other systems could be utilized in the therapeutic method of the invention (e.g., for tow voltage, long pulse treatment), for example, a square wave pulse electroporation system. Exemplary pulse generators capable of generating a pulsed electric field include, for example, the F.CM600, which can generate an exponential wave form, and the ElectroSquarePorator (T820), which cea generate a square wave form, both of which are available from BTX, a division of Genetronics, Inc. (San Diego, CA).
Square wave electroporation systems deliver controlled electric pulses that rise quickly to a set voltage, stay at that level for a set length of time (pulx lengthy and than quickly drop to zero. This type of system yields better transformation efficiency for the electroporation of plant prcrtoplast and mammalim cell litres than as expon~iai daxy system.
The ElectroSqttsnPorator (T820) is the first commercially available square wave ei~oporation system capable of gaoecating up to 3000 Volts. The pulse lettgdt can be adjusted from 5 Itsec to 99 cosec. The square wave elactroporation pulses have a gentler effect on the cells which results in higher cell viability.
The T820 EloctroSquarePorstor is active in both the High Voltage Mode (HVM) (100-3000 Volta) and the Low Voltage Mode (LVM) (10-500 Volts). The pulse length for LVM is about 0.3 to 99 cosec and for HVM, 5 to 99 psec. The T820 has multiple pulsing capability firm abotrt 1 to 99 pulses.

WO OOI!0?621 PCTNS99/15755 Additional elxtroporation type apparatus are commercially available and can be used to generate the pulse for the invention s and in practicing the invention m~Ods.
The invention will now be described in greater detail by reference to the following, non-limiting examples.

we ooro~sZ~ rcrms~ns~ss Ezamplea Ezample 1. Electroporatbe of lacZ DNA
LacZ DNA (40 Itg DNA in 20 pt Tris-EDTA), whemin the lacZ gene is under control of the CMV promoter, was topically applied to the skin of six to seven-week-old SICHI
hairless mice (female) (Charles River Laboratories. Wilmington, MA). A caliper electrode (Fig. 10A) was then placed on both sides of a skin-fold at the dorsal region of the mouse and electrical pulses ~ delivered. Unpulsed mice vie used as pressu~only and blank controls. The skin was harvested for X-gal staining 3 days after ~pliof the lacZ DNd.
Pulse application and electrical measurements: Three exponential decay pulses of amplitude 120 V and pulse length of 10 ms or 20 ms were administered 5om a BTX
ECM
600 pulse Generator within about 1 minute. Pressure was maintained with the caliper for up to 10 min. following pulsing. A caliper type electrode (I em= each electrode) was applied on a mouse skin-fold of about 1 mm in thickness. Resistance of the skin was measured before, during and after pulsing. Expression of IacZ DNA was assayed by X-gal staining with 0.1%
nuclear fast red. Standard histologlcal analysis was then carried out.
$; Typical results of gene expression after 3 days are shown in Fig. 13 (a-d).
A cell that expresses the lacZ gage ua~xgoes blue rtaming of its cytoplasm when exposed to X-gal. Efficient gene transfer aad expression were found in the dermis by pulsing and pressure treatment for 1 min. (Fig. 13b, Fig. 14A and H). In the case of control (pressure only by caliper electrodes gene Vision appeared only arowrd hair follicles in the vesry upper layers of the skin with light blue staining (Fig. 13a, Fig. 14A end B).
This indicates that the electrical pulse creates new pathways to permit passage of DNA
through the epidermis. Pressure maintained at~r the pulses, increases the depth as well as the efficiency of gene expression in the dermis. See, for example Fig. 13c, wherein a pulse of 10 ms delivered over about 1 min. was followed by caliper pressure being maintained for about 10 min. The third bar on graphs 14 A and B show depth of penetration and efficiency of transfection, respectively, for the name parameters. A groater number of transfected cells and more intense IocZ gene expreSSion were found with a 20 ass pulse length (Fig.13d and Figs.
14 A and B, fourth bar, compared to t0 ms. 'The maximum depth of lac2 gene expression we ooro~Zl Pc~rms~ns~ss below the epidermis with pressure maintained for 10 minutes after pulsing was more than twofold that of the control in the hair follicles only.
The In vtvo resistance measurements have demonstrated that the high resistance of the SC decreased dramatically during electropon~tion, and the recovery time (i.e., recovery of original resistance) depends on the electrical parameters. No ~-gal activity was found in the control groups such as: no lcx2; pulse (i.e., eleetmporation); no pulse, IacZ;
no lacZ or poise. There was no evidence of tissue injury in the pulsed skin by visual observation as well as in histological studies.
Example 2. ElectroporaHon o! GFP
Is vtvo sk'~ GFP gene delivery fFiQ. 15): A procedure similar to that used in Example 1 was used for investigation the etyectiveness of GFP expression in skin.
Hairless mice were anesthetized with isoflurane inhalation and their dorsal hindquarter skin was swabbed with alcohol and air-dried. Fifty micrograms of a plasmid containing GFP and associated regulatory sequences (CMV promoter) was either applied topically or directly injected into the skin followed by electroporation with the caliper electrode or meander electrode. The animals were sacrificed three days later and skin of the treated area was excised and frozen. Frozen cross sections (12 pm) were immediately prepared.
Viewing under a microscope with confocal fluorescent optics and FITC fihration, indicated GFP
expression in the epidermal and dermal layers of the rehitively thin murine skin (Fig. 15).
The results of several different treatment regimens are depicted in Fig. 15.
Panels 1A and 1B
of Fig. 15 show photomicrographs of direct injection into abdominal muscle of plasmid.
Panels 2 AB and 3 A/B of Fig. 15 show the results of topical application of plasmid followed by three 100 V, 20 ms pulses applied using the BTX ECM 600 generator, using a caliper electrode (2 AIH) or a meander electrode (3 A/B). Panels 4 AB of Fig.15 show the results of subcutsnaous injection of plasmid followed by du~ee 120 V, 20 ms pulses applied using the BTX ECM 600 generator, using a caliper or meander electrode. Both the topical applicatiat and dermal injection of DNA gave detectable GFP expression with little apparent difference in the level of activity.

we oorom Pcrnrs99ns~ss Eiample 3. In vitro electroporation of Kaman gliobiaatoma cell line SF-29s To evaluate and track the delivery of plasmid DNA to target cells, a fluoresca~tly labeled vector using TOTO-l (Moieculu Probes, Inc.) was developed. The TOTO-1 molecule is attached to the green fluore~x~at protein (GFP)-expressing vector pEC3FP-C 1 (Clonetech). Both the plasmid, and the plasmid product (GFP) fluoresce ands the same FITC excitation and filtration, and are thus bob observable simultaneously ands a microscope with confocal fluorescent optics and FITC filtration. (Fig. 16). 30 pig of unlabeled and 10 ug of TOTO-1 labeled piasmid was electroporated into the SF-295 cells and 24 hours later. GFP expression was assessed through an inverted fluorescervt microscope. In Fig. 16A, ( 100X magnification, fluorescent light only) GFP expressing cells are visible as well as small green fluorescent dots that indicate TOTO-i labeled plasmid within cells that are not expressing the plasmid. In Fig. 16B, (460X magnification) white light from a tungsten source was added to the fluorescent light to simultaneously illuminate the GFP
positive and negative cells. This image shows the TOTO-1 labeled plasmid is aggregated into subceliular vesicles but the ceslls have not yet expressai GFP. Thus, the intracellular delivery sad expression of a plasmid vector can be evaluated by this technique for in vivo skin applications.
Ezample 4: DNA aptaka is the skin DNA encoding the firefly lucifmasc gone was injected intraderrnally into skin of hairless mice (no shaving) or rats (after shaving of fur). DNA ampunts.shown in. Table 1 were injaxed in 50 p1 PBS as shallow as possible into the skin, using a 30 gauge needle.
Electroporation was done with a ealipm electrode (1 cm2) or with a Micro-patchII flat electrode. Where indicated, three 120V, 20 mace. pulses were given, using a instrument (BTX, lne., San Diego, CA). The site of DNA injection was marked and the skin was removed 24 hours after treatment. Skin flaps were minced by scissor in lysis buffer provided in the commercial luciferase assay kit and luciferase aet'rvity was measured following the kit protocol. The results shown in Table I indicate that eleetroporation dramatically 'sncreases gene exit after infra-dermal DNA delivery into mouse and rat skin.

wo ooroz6m rcrnJS99ns~ss Table 1 EP Enhancement of Luccrifase Activity after Intra-Derma! DNA Delivery (RLU) 20 pg in 50 ltl i.d. i.d. + pulse Mice 38 156,995 (caliper) 7229 211,636 (caliper) 1831 116,725 (micro-patchII) 415 1,704,558 (micro-patchIl) Rat 57 194,398 (micro-patchII) Etg in 50 ~t i.d. i.d. + pulse Mice 160 49,247 (caliper) 9,021 57,134 (caliper) " . 879 46.264"(micro-patchII) 116 36,679 (micro-patchII) background: <50, with or without pulse WO OOIiO'1621 PCT/US99/15?55 Example 5: DNA uptake i~ the muscle DNA encoding the firefly lucifersse gene (20 pg in 50 pl PBS) was injected into the hind-limb muscle of hairless mice using a two noodle electroporation array, where the 5 injection needle also serves as negative electrode. The distance between the two electrodes was 2 mm. Where indicated, six pulses were given (20V per mm electrode distance, 50 msec., reversed polarity after 3 pulses; one pulse every 15 seconds) using the instrument. After 24 hours, the treated muscle was removed and minced by scissor for determination of luciferase activity using a commercial kit.
10 Genes encoding human growth hormone (hfiH) or ~creted alkaline phosphatase (SF.AP) were injected into the hind-limb muscle of rats using the same device as used in mice and (where indicated) eloetroporation was done as described for mouse muscle.
Growth hormone was measured in muscle tissue and serum using a commercial kit, SFrAP
activity was measured in serum using a commercial kit 24 hours after treatment. The results 15 shown in Table 2 indicate that electroporation dramatically increases gene expression after infra-muscular DNA delivery in mice and rats.

WO 00/02621 PCf/US99/15755 Table 2 EP Enhancement of Gene Expression in Muscle hGH in rats (20 ~g in 50 1i1) (RLU) i.m. i.m. + pulse 3,341 41,465 3,462 21,540 Serum 1,027 1,449 Background: muscle:3,242 3,397 serum: 952 Luciferase in mice (20 ~g in 50 Eal) (RLU) i.m. i.m. + pulse 576 27,441 140 74,637 Background: 50,62 SEAP in rats (20 ~g in 50 E,il) (RLU in serum) i.m. i.m. + pulse 5,009 191,398 Background: 2143 we oorom rcrnrs~!rnsrss Eiamp~le 6: Immrae respopsa her isl~annsc~lnr DNA delivery An example of a model antigen is the Hepatitis B Virus surface Antigen (I~sAg).
The gene encoding this antigen was clone into an eukaryotic exprossion vector so that expression of the sAg gene is driven by the human elongation factor 1 promoter. Expression was verified by transient expression in B16 cells.
DNA was injected into the gracialis muscle of the hind limbs of BalbJc mice:
SO Etg DNA in 50 p1 PBS, both hind limbs were injected. One cohort of four animals was injected only (animals # 1-4 in Table 3~ o~ other cohort was treaned with a two needle electrode array and pulsed: 20V per mm noalle distance, 2x3 pulses at 50 msec: with polarity reversion after the first set of three pulses (animals #5-8 in Table 3). Two weeks after DNA
delivery, l out of 4 mice was earl-HBSAG positive in the non EP treated cohort. All mice in the treated group were positive, pointing towards a positive e!~'ect of EP on the generation of an immune response. All mice were boosted four wedcs post prime and tested again for antibody titers. The geometric mean titer (GMT) in the treated group was 193 mIU/ml, with 3/4 mice having reached protaxive antibody levels. The GMT in the untc~eated group was 3.2 mILT/ml, with only 1/4 mice reaching protective antibody fifes. DNA
immunization iMo the tibialis anterior muscle confirmed the above results: mice #1-4 in Table 4, untreated, GMT:
2.6 mIU/ml; mice #13-1 to 13-4, Table 3, EP tro~ed, GMT: 13,547. In additioa to showiag the significant improvement of EP treatment on an immune response after DNA
immunization, these data also show the supa3mity of the tibialis muscle for DNA
immunization in mice.
Eumple 7: DNA Titration To test whether electroporation could iNCroase the efliciancy of DNA
vaccination after i.m. injection by allowing the use of lower DNA doses, DNA titration studies were performed. The need for extremely large doses of DNA is a major obstacle which has yet to be overcome in the DNA vaccination of larger animals than mice, and elatroporati~ helps to overcome this problem. Except as otherwise noted, the experimental portions of this Example are as in Example 6.

wo oom rcrrtrs~n sass DNA immunizations with 5 lag, 20 Etg, a 50 pg gave str~g and consistent antibody responses two weeks after prime, which showod strongly boosted secondary responses after a booster immuni~stion. These responses wero achieved only with electroporation.
(Mice #5-14, Table 4, #11, 12, Table 5, #13-1-13-4, Tsble 3). Down titnnion of the DNA
amamt usai to immunize showed the consistent primary rosponses could be achieved with 3 a of DNA. With this amount, a booster immunizai~ was necessary to give better then protxtive levtls in all animals of this cohort. Mice #1-5, Table 6). One microgram or less DNA was found to be not sufficient to induce antibody responses, even after electroporation. (Mice #6-20, Table 6). Untreated cohorts receiving 20 ~g or 5 pg of DNA showed GMTs of <10 mlU/ml (mica #1-10, Table 5). The results of DNA immunization using 5 ltg, 20 itg, and 50 Etg in tibialis muscle are summarized in Fig. 17.

wo ooroZSm pcr~ls~ns~~

Table 3 cage primary secondary cage animal ~g DNA p~~ 2 week 4 week 9 week #

6 1 50 - 300 t 115 8 5 50 + 150 200 1680 6 50 + 40 0 0 7 50 t 140 120 581 8 50 + 280 560 1442 7 9 50 ++ 0 0 0 10 50 ++ 0 0 27 II 50 ++ 0 0 13 12 50 ++ 0 0 0 I3 1 50 + 120 200 3868 2 50 + 310 1000 57000 3 50 + 40 240 17087 4 50 + 50 1000 8941 Table 4 cage primary secondary cage animal ~g pNp pulse 2 week 4 week 6-8 # week 3 50 - 0 t 0 15 5 50 + 200 > >300 16000 6 20 + 105 80 2000 7 20 + 160 >300 7800 8 20 + 160 320 2700 16 9 20 + 180 160 1800 10 20 + >300 300 23800 11 5 + 80 200 1500 12 5 + >300 140 1450 17 13 5 + 60 60 1100 14 5 + >300 120 900 WO OO/OZ62t PC17US99/15755 Table 5 cage primary secondary cage animal ~8 DNA pulse 2 week 7 week #

2 20 - t 0 3 20 - t 55 11 5 + 160 315 12 5 + 170 154 wo ooras~mcrnts~ns~ss Table 6 DNA primary secondary titration Cage animal Etg P~ 2 weck 4 weck 5 woek # DNA

27 1 3 + 300 120 336 2 3 + 0 70 13.2 3 3 + 0 0 51 4 3 + 200 0 81_5 28 5 3 + 30 0 771 6 1 + 0 0 0 7 1 + 0 0 0 8 1 + 0 0 0 29 9 1 + 0 0 0 10 1 + 0 0 0 11 0.5 + 0 0 < 10 12 0.5 + 0 0 <10 30 13 0.5 + 0 0 0 14 0.5 + 0 0 0 15 0.5 + 0 0 <10 16 0.1 + 0 0 0 31 17 0.1 + 0 0 0 18 0.1 + 0 0 0 we t PcrNS99nsrss 19 ~ 0.1 + 0 0 0 20 0.1 + 0 0 0 Liample 8: Systemic delivery of SEAP Plasmid DNA via electroporatioa to ale moose stun.
In order to demonsmte that naked DNA can be transfected into skin cells with resulting expression and systemic delivery of grate product, fifty micrograms of SEAP DNA
(plasmid DNA wherein SEAP is driven by CMV promoter) in 1X PBS was injected i.d. into ~~ ~ followed by either ela~roporstion or no elxtropmation. A square wave pulse at settings of 100V, 20 ms was applied using a meander electrode. Six pulses were applied with the polarity reww~ after the first throe pulses, and an interval of 100 ms bawxn pulses.
Figure 18 shows that there is a draa>atic increase in SEAP expression in blood that is greater than two logs. The length of expression is still high after 21 days in the nude mouse that is immune compromised.
lEiample 9: Shallmv ssxdle army and micropatch deliver S1LAP plasma DNA
to nude mouse skin In order to test the effecdveaess of diet eloctro~ typos, efectroporation experimatts were conducted using the shallow needle array depicted in Fig. 20 and the micropatch array described in US Patent Application No. 081905,240.
F.xporinnental ~cdnditibns were as described in F,xample 8. Fig. 19 shown the dramatic increases in SEAP
expression in blood by using different configurations of shallow needle arrays as well as the ZO micropatch surface-type electrode. Square wave purses were used with micropatch el~trodes and 2-needle array (the higher bar in Fig. 19). The exponential pulses wero used for 2- or 3- needle arrays (100 V, 20 ms, with polarity reversed after first throe pulses). N=3 for micropatch and i.d. alone; n=2 for needle arrays; n~l for control.
Overall, electcoporation again gave a two order of magnihde increase for SEAP acpression. In the case of needle arrays (3 bars), only one needle iqjected DNA. However, DNA may be injecxed by more than one needle from the array.

1~~~~~;~~

Erample 10: Use of caliper electrodes to diver CMV-lucifee~aae plasmid DNA
to hnman akin ienogranod nude mice In order to demonstrate the capability if caliper alectrodo-basod EPT to augment transfection of naked DNA into human skin (much thicker than routine skin), fifty micrograms of naked plasmid DNA eoataining the luciferasa gene driven by the CMV
promoter was i.d, inja~ed iMo human skin xaiograt~ed unto nude mice.
Luciferase expression was assayed 24 hours post DNA injection, ph~s and minus electroporation.
Electroporation was as described in Example 8, employing a caliper electrode.
The human skin grafts were excised from nude mice. The enzyme activ'tty was measurod by the spectrophotometer after homogenization and extraction. Fig. 21 shows the relative expression of luciferase (ItLU) 24 hours after gone delivery. There was 7-fold increase for RhU in the pulsed group compared to i.d. injoction alone.
Ezamplo 11: Comparison of caliper vs. meander electrodes to deliver CMV
Incfferase plasmid DNA to hairless mouse skin In order to ovaluate the relative effoetiveneaa of caliper v. meander type electrodes, each was tested as follows.
Mice were anesthetmed by isoflurane inhalriion, weighed, numbered with permanent ink, and swabbed with alcohol at the site of eleeh~opaation. The skin area treated by caliper electrodes was about 2 cm'. 50 pg of naked plaunid DNA containing the reporter gene (luciferase dcivct~ by CMV promote), at a conca~cation of 3.5 mg/ml in water, was admnaistaed onto the mouse skin. The DNA in solution, being slightly viscous, was moderately adsorbed by the skin and stayod in place. The electrode was then positioned at the site of DNA application, followed by eloctroporati~, ua~dOr the same ekarporation conditions as described for Example 8. Lucifa~ase ~ expression was assayed at day 1 (24 hrs) a8er gene delivery.
In order to investigate the locati~ of gene expremion in the skin layers, frozen sections of excised mouse tin were processed ao that the full thickness skin (0.5 mm) was divided into two parts. The top part that was taken from outer most 150 um regions of the skin, the bottom part was the remainder of the :>ci:t. The enzyme activity was measurod by the we ooros6z~ r~crrit,rs~ns~ss spectrophotometer after homogenixstion and extraction. The results wee corrected for skin sampk weight and the results are indi~ed gr~bi~lly in Fig. 2Z. Bars AI and A2 show meander electrode results, while bars BI and B2 show caliper electrode results. A1 and B1 results were obtained from the outer 150 microns of skin, while A2 and B2 results were 5 obtained from the rcmainda of the akin. Use ~' meander electrodes for electroporation resulted in much greater incorpoca~i~lexpression of ltmiferase (i.e., two orders of magnitude). Fig. 22 further indicates that (1) a aignifieant amount of luciferase DNA are delivered and expressed in the upper layers of the skin.
Emmple 12: Meander elechndea delfver C'MV beta-gal and iuvoiucrlu beta-gal 10 DNA in arde mice and tairkss ~
In order to test the relative exprossion achieved in mouse skin using genes driven by a CMV promoter as compared to the human involucrin promoter, the following experiment was conducted.
Fifty micrograms of plasmid DNA was administered to the skin via i.d.
injection.
I S 1'wo sets of plasmids were used, containing the beta-gala~ctosidase gene either driven by the CMV promoter or the human involuerin promoter. Pulsed (DNA (+) EP (+) and DNA
(-) EP
(+)) skin, and non-pulsed skin (DNA (+) EP (-)) was excised 24 hours after gene delivery.
Ele~oration was conducted as described in Bxampk 8. Untreated skin (DNA (-) EP
(-)) was also excised as a control. X-gal histachemical staining was used to illustrate the beta-20 galactosidase gene product.
Photomicrographs of skin dross sections wars taken under the light microscope and intensive beta-galactosidase staining was obsacvod with CMV beta-gal DNA
delivery (i.d.
iujectbn + pulsing). It was distributed iiom epidermis to dermis. The expression of beta-gal DNA driven by the imrolucrin promote was much weaker than with CMV promote, and it 25 was mostly located in the epidermal sod upper dermal regions. No gene Vision was obsenrod with untreated skin, i.d. DNA injection alone, and pulsing without DNA.
These results demonstrate that it is feasible to target skin with a skin-specific promoter (i.e., involucrin). However, the involucrin promoter is much weaker than the CMV
promoter.

wo ooro~sm pcrNS99nsnss Although the invention hss been described with rofetence to the presently preferred embodiment, it should be understood that various modifications can be made without 8 from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims (3)

THE EMBODIMENTS OF THE INVENTION FOR WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A micropatch electrode for use with an electroporation apparatus, said micropatch electrode comprising a substantially planar array of patch elements, each patch element comprising two sets of electrodes, wherein each set of electrodes comprises a first electrode and a second electrode electrically insulated from one another, such that when different electric potentials are applied to said first and second electrodes; a voltage is produced therebetween.
2. A micropatch electrode according to claim 1, wherein said patch elements are separated by electrically insulating material.
3. A micropatch electrode according to claim 2, wherein said electrically insulating material can be pierced by an injection needle.
CA2337652A 1998-07-13 1999-07-13 Skin and muscle-targeted gene therapy by pulsed electrical field Expired - Lifetime CA2337652C (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US9254498P 1998-07-13 1998-07-13
US60/092,544 1998-07-13
US10932498P 1998-11-20 1998-11-20
US60/109,324 1998-11-20
US12605899P 1999-03-25 1999-03-25
US60/126,058 1999-03-25
PCT/US1999/015755 WO2000002621A1 (en) 1998-07-13 1999-07-13 Skin and muscle-targeted gene therapy by pulsed electrical field

Publications (2)

Publication Number Publication Date
CA2337652A1 CA2337652A1 (en) 2000-01-20
CA2337652C true CA2337652C (en) 2013-03-26

Family

ID=27377224

Family Applications (1)

Application Number Title Priority Date Filing Date
CA2337652A Expired - Lifetime CA2337652C (en) 1998-07-13 1999-07-13 Skin and muscle-targeted gene therapy by pulsed electrical field

Country Status (6)

Country Link
US (2) US6697669B2 (en)
EP (3) EP2428249B1 (en)
JP (1) JP2003505114A (en)
AU (1) AU770092B2 (en)
CA (1) CA2337652C (en)
WO (1) WO2000002621A1 (en)

Families Citing this family (266)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1261808A (en) 1997-06-30 2000-08-02 罗纳-布朗克罗莱尔股份有限公司 Improved method for transferring nucleic acid into multicelled eukaryotic organism cells and combination therefor
JP2002507985A (en) * 1997-06-30 2002-03-12 ローヌ−プーラン・ロレ・エス・アー Improved method for introducing nucleic acid into striated muscle and its combination
AU733053C (en) 1998-02-24 2001-11-29 Boston Scientific Limited High flow rate dialysis catheters and related methods
US6972013B1 (en) * 1998-07-13 2005-12-06 Genetronics, Inc. Enhanced delivery of naked DNA to skin by non-invasive in vivo electroporation
US6678556B1 (en) * 1998-07-13 2004-01-13 Genetronics, Inc. Electrical field therapy with reduced histopathological change in muscle
US7922709B2 (en) 1998-07-13 2011-04-12 Genetronics, Inc. Enhanced delivery of naked DNA to skin by non-invasive in vivo electroporation
US6300108B1 (en) * 1999-07-21 2001-10-09 The Regents Of The University Of California Controlled electroporation and mass transfer across cell membranes
AU3672000A (en) 1999-10-14 2001-04-23 Pola Chemical Industries Inc. Compositions for electroporation
US20020095134A1 (en) * 1999-10-14 2002-07-18 Pettis Ronald J. Method for altering drug pharmacokinetics based on medical delivery platform
US7565205B2 (en) * 2000-02-17 2009-07-21 Standen Ltd. Treating a tumor or the like with electric fields at different orientations
DK1259265T3 (en) * 2000-03-03 2011-07-11 Genetronics Inc Nucleic acid formulations for delivery of genes
AU2001264759B2 (en) * 2000-05-22 2006-06-01 Merck & Co., Inc. System and method for assessing the performance of a pharmaceutical agent delivery system
US20040175360A1 (en) * 2000-06-29 2004-09-09 Pettis Ronald J. Method for altering drug pharmacokinetics based on medical delivery platform
US6892099B2 (en) 2001-02-08 2005-05-10 Minnesota Medical Physics, Llc Apparatus and method for reducing subcutaneous fat deposits, virtual face lift and body sculpturing by electroporation
US6795728B2 (en) 2001-08-17 2004-09-21 Minnesota Medical Physics, Llc Apparatus and method for reducing subcutaneous fat deposits by electroporation
US6697670B2 (en) 2001-08-17 2004-02-24 Minnesota Medical Physics, Llc Apparatus and method for reducing subcutaneous fat deposits by electroporation with improved comfort of patients
US8251986B2 (en) 2000-08-17 2012-08-28 Angiodynamics, Inc. Method of destroying tissue cells by eletroporation
BR0116972A (en) * 2001-04-13 2004-12-21 Becton Dickinson Co Methods and devices for administering substances to the intradermal skin layer for systemic absorption
US7130697B2 (en) * 2002-08-13 2006-10-31 Minnesota Medical Physics Llc Apparatus and method for the treatment of benign prostatic hyperplasia
US6994706B2 (en) * 2001-08-13 2006-02-07 Minnesota Medical Physics, Llc Apparatus and method for treatment of benign prostatic hyperplasia
USRE42016E1 (en) 2001-08-13 2010-12-28 Angiodynamics, Inc. Apparatus and method for the treatment of benign prostatic hyperplasia
AU2002360540A1 (en) * 2001-12-04 2003-06-17 University Of Southern California Method for intracellular modifications within living cells using pulsed electric fields
EP1474153A4 (en) * 2001-12-14 2005-12-14 Genetronics Inc Methods for particle-assisted polynucleotide immunization using a pulsed electric field
US6952604B2 (en) * 2001-12-21 2005-10-04 Becton, Dickinson And Company Minimally-invasive system and method for monitoring analyte levels
GB0201736D0 (en) * 2002-01-25 2002-03-13 Glaxo Group Ltd DNA dosage forms
US7697981B2 (en) * 2002-03-22 2010-04-13 The Regents Of The University Of California Method and apparatus for using an electromagnetic field in cellular transplantation
US6912417B1 (en) 2002-04-05 2005-06-28 Ichor Medical Systmes, Inc. Method and apparatus for delivery of therapeutic agents
US7162303B2 (en) 2002-04-08 2007-01-09 Ardian, Inc. Renal nerve stimulation method and apparatus for treatment of patients
US7756583B2 (en) 2002-04-08 2010-07-13 Ardian, Inc. Methods and apparatus for intravascularly-induced neuromodulation
US7853333B2 (en) 2002-04-08 2010-12-14 Ardian, Inc. Methods and apparatus for multi-vessel renal neuromodulation
US7653438B2 (en) 2002-04-08 2010-01-26 Ardian, Inc. Methods and apparatus for renal neuromodulation
US20080213331A1 (en) 2002-04-08 2008-09-04 Ardian, Inc. Methods and devices for renal nerve blocking
US8774913B2 (en) 2002-04-08 2014-07-08 Medtronic Ardian Luxembourg S.A.R.L. Methods and apparatus for intravasculary-induced neuromodulation
US9636174B2 (en) 2002-04-08 2017-05-02 Medtronic Ardian Luxembourg S.A.R.L. Methods for therapeutic renal neuromodulation
US8347891B2 (en) 2002-04-08 2013-01-08 Medtronic Ardian Luxembourg S.A.R.L. Methods and apparatus for performing a non-continuous circumferential treatment of a body lumen
US8150519B2 (en) 2002-04-08 2012-04-03 Ardian, Inc. Methods and apparatus for bilateral renal neuromodulation
US20070129761A1 (en) 2002-04-08 2007-06-07 Ardian, Inc. Methods for treating heart arrhythmia
US20070135875A1 (en) 2002-04-08 2007-06-14 Ardian, Inc. Methods and apparatus for thermally-induced renal neuromodulation
US20140018880A1 (en) 2002-04-08 2014-01-16 Medtronic Ardian Luxembourg S.A.R.L. Methods for monopolar renal neuromodulation
US9308043B2 (en) 2002-04-08 2016-04-12 Medtronic Ardian Luxembourg S.A.R.L. Methods for monopolar renal neuromodulation
US7617005B2 (en) 2002-04-08 2009-11-10 Ardian, Inc. Methods and apparatus for thermally-induced renal neuromodulation
US8145316B2 (en) 2002-04-08 2012-03-27 Ardian, Inc. Methods and apparatus for renal neuromodulation
US6978174B2 (en) 2002-04-08 2005-12-20 Ardian, Inc. Methods and devices for renal nerve blocking
US8145317B2 (en) 2002-04-08 2012-03-27 Ardian, Inc. Methods for renal neuromodulation
US7620451B2 (en) 2005-12-29 2009-11-17 Ardian, Inc. Methods and apparatus for pulsed electric field neuromodulation via an intra-to-extravascular approach
US9308044B2 (en) 2002-04-08 2016-04-12 Medtronic Ardian Luxembourg S.A.R.L. Methods for therapeutic renal neuromodulation
US8131371B2 (en) 2002-04-08 2012-03-06 Ardian, Inc. Methods and apparatus for monopolar renal neuromodulation
US8774922B2 (en) 2002-04-08 2014-07-08 Medtronic Ardian Luxembourg S.A.R.L. Catheter apparatuses having expandable balloons for renal neuromodulation and associated systems and methods
US8150520B2 (en) 2002-04-08 2012-04-03 Ardian, Inc. Methods for catheter-based renal denervation
US20040082859A1 (en) 2002-07-01 2004-04-29 Alan Schaer Method and apparatus employing ultrasound energy to treat body sphincters
US7328064B2 (en) 2002-07-04 2008-02-05 Inovio As Electroporation device and injection apparatus
US20060148737A1 (en) * 2002-12-31 2006-07-06 The John Hopkins University Wound healing method and kits
US7211083B2 (en) * 2003-03-17 2007-05-01 Minnesota Medical Physics, Llc Apparatus and method for hair removal by electroporation
TW200424214A (en) * 2003-04-21 2004-11-16 Advisys Inc Plasmid mediated GHRH supplementation for renal failures
US8067371B2 (en) * 2003-05-09 2011-11-29 The Trustees Of Columbia University In The City Of New York RAGE G82S-related methods and compositions for treating inflammatory disorders
CN1798502A (en) * 2003-05-30 2006-07-05 南佛罗里达大学 Method for the treatment of malignancies
US8802643B1 (en) 2003-05-30 2014-08-12 University Of South Florida Method for the treatment of malignancies
WO2005016111A2 (en) 2003-08-08 2005-02-24 Abgenix, Inc. Antibodies directed to parathyroid hormone (pth) and uses thereof
US7318925B2 (en) * 2003-08-08 2008-01-15 Amgen Fremont, Inc. Methods of use for antibodies against parathyroid hormone
US20090076391A1 (en) * 2003-10-15 2009-03-19 Der-Yang Tien Method and system for leading macromolecule substances into living target cells
TWI243696B (en) * 2003-10-15 2005-11-21 Der-Yang Tien System for introducing macromolecule substance into living target cell
US20110071381A1 (en) * 2003-10-15 2011-03-24 Robert Tien Method and system for leading macromolecule substances into living target cells
ES2729378T3 (en) 2003-12-24 2019-11-04 Univ California Tissue ablation with irreversible electroporation
US8298222B2 (en) 2003-12-24 2012-10-30 The Regents Of The University Of California Electroporation to deliver chemotherapeutics and enhance tumor regression
US20050261210A1 (en) * 2004-02-19 2005-11-24 Bhatnagar Rajiv S Nonsense suppressor agents in treatment of cutaneous and gastrointestinal disorders
US7840263B2 (en) * 2004-02-27 2010-11-23 Cardiac Pacemakers, Inc. Method and apparatus for device controlled gene expression
WO2005089810A1 (en) * 2004-03-22 2005-09-29 Kansai Technology Licensing Organization Co., Ltd. Method of inducing bone by transferring human osteogenetic factor gene with the use of electroporation method
JP4443278B2 (en) * 2004-03-26 2010-03-31 テルモ株式会社 Catheter with expansion body
JP5065005B2 (en) 2004-04-01 2012-10-31 ザ ジェネラル ホスピタル コーポレイション Method and apparatus for dermatological treatment and tissue remodeling
US7764995B2 (en) 2004-06-07 2010-07-27 Cardiac Pacemakers, Inc. Method and apparatus to modulate cellular regeneration post myocardial infarct
FR2880808A1 (en) * 2005-01-20 2006-07-21 Yves Scherman Active material e.g. nucleotide, delivering device for providing e.g. chemotherapy, has electric current generator, electrodes connected to respective terminals of current generator and injection needle centered between electrodes
JP5197006B2 (en) * 2004-06-24 2013-05-15 スフェルゲン Devices that move molecules to cells using electrical force
US7729761B2 (en) * 2004-07-14 2010-06-01 Cardiac Pacemakers, Inc. Method and apparatus for controlled gene or protein delivery
US20060165668A1 (en) * 2004-12-10 2006-07-27 Liu Linda N Genetically modified tumor cells as cancer vaccines
WO2007012907A2 (en) * 2005-02-08 2007-02-01 Allen, John Electric therapy machine
EP1865969A4 (en) * 2005-03-19 2009-07-15 Univ California Ultra low strength electric field network-mediated ex vivo gene, protein and drug delivery in cells
US20060264752A1 (en) * 2005-04-27 2006-11-23 The Regents Of The University Of California Electroporation controlled with real time imaging
US20060293731A1 (en) * 2005-06-24 2006-12-28 Boris Rubinsky Methods and systems for treating tumors using electroporation
US20060293730A1 (en) 2005-06-24 2006-12-28 Boris Rubinsky Methods and systems for treating restenosis sites using electroporation
US20060293725A1 (en) * 2005-06-24 2006-12-28 Boris Rubinsky Methods and systems for treating fatty tissue sites using electroporation
US8114070B2 (en) * 2005-06-24 2012-02-14 Angiodynamics, Inc. Methods and systems for treating BPH using electroporation
EP1913578B1 (en) * 2005-06-30 2012-08-01 LG Electronics Inc. Method and apparatus for decoding an audio signal
US20070021803A1 (en) 2005-07-22 2007-01-25 The Foundry Inc. Systems and methods for neuromodulation for treatment of pain and other disorders associated with nerve conduction
US20070156135A1 (en) * 2006-01-03 2007-07-05 Boris Rubinsky System and methods for treating atrial fibrillation using electroporation
US20100168501A1 (en) * 2006-10-02 2010-07-01 Daniel Rogers Burnett Method and apparatus for magnetic induction therapy
US9339641B2 (en) 2006-01-17 2016-05-17 Emkinetics, Inc. Method and apparatus for transdermal stimulation over the palmar and plantar surfaces
US9610459B2 (en) 2009-07-24 2017-04-04 Emkinetics, Inc. Cooling systems and methods for conductive coils
US20070232984A1 (en) * 2006-03-30 2007-10-04 Michael Lovell Hand-held electrical stimulation device
KR101445401B1 (en) * 2006-06-16 2014-09-26 디에스엠 아이피 어셋츠 비.브이. Compositions based on hyperbranched condensation polymers and novel hyperbranched condensation polymers
US9005102B2 (en) 2006-10-02 2015-04-14 Emkinetics, Inc. Method and apparatus for electrical stimulation therapy
US11224742B2 (en) 2006-10-02 2022-01-18 Emkinetics, Inc. Methods and devices for performing electrical stimulation to treat various conditions
US10786669B2 (en) 2006-10-02 2020-09-29 Emkinetics, Inc. Method and apparatus for transdermal stimulation over the palmar and plantar surfaces
JP2010505471A (en) * 2006-10-02 2010-02-25 エムキネティクス, インコーポレイテッド Method and apparatus for magnetic induction therapy
US8273080B2 (en) 2006-10-16 2012-09-25 Syneron Medical Ltd. Methods and devices for treating tissue
US8007493B2 (en) 2006-10-16 2011-08-30 Syneron Medical Ltd. Methods and devices for treating tissue
US8133216B2 (en) 2006-10-16 2012-03-13 Syneron Medical Ltd. Methods and devices for treating tissue
US8142426B2 (en) 2006-10-16 2012-03-27 Syneron Medical Ltd. Methods and devices for treating tissue
WO2008048620A2 (en) * 2006-10-16 2008-04-24 The Regents Of The University Of California Gels with predetermined conductivity used in irreversible electroporation of tissue
WO2008048632A1 (en) * 2006-10-17 2008-04-24 Vgx Pharmaceuticals, Inc. Electroporation devices and methods of using same for electroporation of cells in mammals
US20080132884A1 (en) * 2006-12-01 2008-06-05 Boris Rubinsky Systems for treating tissue sites using electroporation
US8845630B2 (en) 2007-06-15 2014-09-30 Syneron Medical Ltd Devices and methods for percutaneous energy delivery
TW200903724A (en) * 2007-07-09 2009-01-16 Ind Tech Res Inst Phase change memory device and method of fabricating the same
JP5461427B2 (en) * 2007-12-17 2014-04-02 ニューワールド ファーマシューティカルズ,エルエルシー Integrated intradermal delivery, diagnosis and communication system
EP4328297A2 (en) * 2008-01-17 2024-02-28 Inovio Pharmaceuticals, Inc. Variable current density single needle electroporation system and method
US20100004623A1 (en) * 2008-03-27 2010-01-07 Angiodynamics, Inc. Method for Treatment of Complications Associated with Arteriovenous Grafts and Fistulas Using Electroporation
US20090248012A1 (en) * 2008-03-27 2009-10-01 The Regents Of The University Of California Irreversible electroporation device and method for attenuating neointimal
JP5873717B2 (en) 2008-04-04 2016-03-01 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア Vaccine and immunotherapeutic agent using IL-28 and composition and method of use thereof
CN107083391A (en) * 2008-04-04 2017-08-22 宾夕法尼亚大学托管会 Chikungunya virus albumen consensus sequence, the nucleic acid molecules and composition and its application method for encoding the chikungunya virus albumen consensus sequence
US9283051B2 (en) 2008-04-29 2016-03-15 Virginia Tech Intellectual Properties, Inc. System and method for estimating a treatment volume for administering electrical-energy based therapies
US10448989B2 (en) 2009-04-09 2019-10-22 Virginia Tech Intellectual Properties, Inc. High-frequency electroporation for cancer therapy
US10238447B2 (en) 2008-04-29 2019-03-26 Virginia Tech Intellectual Properties, Inc. System and method for ablating a tissue site by electroporation with real-time monitoring of treatment progress
US10245098B2 (en) 2008-04-29 2019-04-02 Virginia Tech Intellectual Properties, Inc. Acute blood-brain barrier disruption using electrical energy based therapy
US9198733B2 (en) * 2008-04-29 2015-12-01 Virginia Tech Intellectual Properties, Inc. Treatment planning for electroporation-based therapies
US11272979B2 (en) 2008-04-29 2022-03-15 Virginia Tech Intellectual Properties, Inc. System and method for estimating tissue heating of a target ablation zone for electrical-energy based therapies
US8992517B2 (en) 2008-04-29 2015-03-31 Virginia Tech Intellectual Properties Inc. Irreversible electroporation to treat aberrant cell masses
US9867652B2 (en) 2008-04-29 2018-01-16 Virginia Tech Intellectual Properties, Inc. Irreversible electroporation using tissue vasculature to treat aberrant cell masses or create tissue scaffolds
US9598691B2 (en) 2008-04-29 2017-03-21 Virginia Tech Intellectual Properties, Inc. Irreversible electroporation to create tissue scaffolds
US10117707B2 (en) 2008-04-29 2018-11-06 Virginia Tech Intellectual Properties, Inc. System and method for estimating tissue heating of a target ablation zone for electrical-energy based therapies
US10272178B2 (en) 2008-04-29 2019-04-30 Virginia Tech Intellectual Properties Inc. Methods for blood-brain barrier disruption using electrical energy
US10702326B2 (en) 2011-07-15 2020-07-07 Virginia Tech Intellectual Properties, Inc. Device and method for electroporation based treatment of stenosis of a tubular body part
US11254926B2 (en) 2008-04-29 2022-02-22 Virginia Tech Intellectual Properties, Inc. Devices and methods for high frequency electroporation
US20090281477A1 (en) * 2008-05-09 2009-11-12 Angiodynamics, Inc. Electroporation device and method
WO2009155526A2 (en) * 2008-06-20 2009-12-23 Angiodynamics, Inc. Device and method for the ablation of fibrin sheath formation on a venous catheter
WO2010008834A2 (en) * 2008-06-23 2010-01-21 Angiodynamics, Inc. Treatment devices and methods
CN101318054B (en) * 2008-07-18 2012-01-25 圣太科医疗科技(上海)有限公司 Microelectro field net guided transfer apparatus for medicament of liver target cell
KR101013581B1 (en) * 2008-08-06 2011-02-14 라종주 Electric Skin Treatment Device
WO2010064991A1 (en) * 2008-12-01 2010-06-10 Nanyang Polytechnic Electroporation apparatus and method
US20100152725A1 (en) * 2008-12-12 2010-06-17 Angiodynamics, Inc. Method and system for tissue treatment utilizing irreversible electroporation and thermal track coagulation
US8652129B2 (en) 2008-12-31 2014-02-18 Medtronic Ardian Luxembourg S.A.R.L. Apparatus, systems, and methods for achieving intravascular, thermally-induced renal neuromodulation
EP2376011B1 (en) 2009-01-09 2019-07-03 ReCor Medical, Inc. Apparatus for treatment of mitral valve insufficiency
WO2010085765A2 (en) * 2009-01-23 2010-07-29 Moshe Meir H Therapeutic energy delivery device with rotational mechanism
US8231603B2 (en) * 2009-02-10 2012-07-31 Angiodynamics, Inc. Irreversible electroporation and tissue regeneration
CN101829396B (en) * 2009-03-27 2013-01-30 清华大学 Micro-needle array chip and percutaneous administration patch using same and preparation method thereof
WO2010118387A1 (en) * 2009-04-09 2010-10-14 Virginia Tech Intellectual Properties, Inc. Integration of very short electric pulses for minimally to noninvasive electroporation
US11382681B2 (en) 2009-04-09 2022-07-12 Virginia Tech Intellectual Properties, Inc. Device and methods for delivery of high frequency electrical pulses for non-thermal ablation
US11638603B2 (en) 2009-04-09 2023-05-02 Virginia Tech Intellectual Properties, Inc. Selective modulation of intracellular effects of cells using pulsed electric fields
USD630321S1 (en) 2009-05-08 2011-01-04 Angio Dynamics, Inc. Probe handle
US8903488B2 (en) 2009-05-28 2014-12-02 Angiodynamics, Inc. System and method for synchronizing energy delivery to the cardiac rhythm
US9895189B2 (en) 2009-06-19 2018-02-20 Angiodynamics, Inc. Methods of sterilization and treating infection using irreversible electroporation
US8624011B2 (en) 2009-09-14 2014-01-07 The Trustees Of The University Of Pennsylvania Vaccines and immunotherapeutics comprising IL-15 receptor alpha and/or nucleic acid molecules encoding the same, and methods for using the same
JP2013508119A (en) 2009-10-26 2013-03-07 エムキネティクス, インコーポレイテッド Method and apparatus for electromagnetic stimulation of nerves, muscles and body tissues
CA3177356A1 (en) 2009-11-02 2011-05-05 The Trustees Of The University Of Pennsylvania Foot and mouth disease virus (fmdv) consensus proteins, coding sequences therefor and vaccines made therefrom
US20110118732A1 (en) 2009-11-19 2011-05-19 The Regents Of The University Of California Controlled irreversible electroporation
US8298820B2 (en) 2010-01-26 2012-10-30 The Trustees Of The University Of Pennsylvania Influenza nucleic acid molecules and vaccines made therefrom
EP2539354B1 (en) 2010-02-08 2017-04-05 The Trustees Of The University Of Pennsylvania Nucleic acid molecules encoding rantes, and compositions comprising and methods of using the same
KR101914152B1 (en) * 2010-03-01 2018-12-28 이노비오 파마수티컬즈, 인크. Multiple tissue layer electroporation applicator and device
US8588884B2 (en) 2010-05-28 2013-11-19 Emkinetics, Inc. Microneedle electrode
EP2600901B1 (en) 2010-08-06 2019-03-27 ModernaTX, Inc. A pharmaceutical formulation comprising engineered nucleic acids and medical use thereof
CA2812789A1 (en) 2010-09-27 2012-04-12 The Trustees Of The University Of Pennsylvania Consensus antigen constructs and vaccines made therefrom, and methods of using same to treat malaria
US20120089009A1 (en) * 2010-10-11 2012-04-12 Omary Reed A Methods and apparatus to deliver nanoparticles to tissue using electronanotherapy
US9700368B2 (en) 2010-10-13 2017-07-11 Angiodynamics, Inc. System and method for electrically ablating tissue of a patient
US8409807B2 (en) 2010-10-22 2013-04-02 T2 Biosystems, Inc. NMR systems and methods for the rapid detection of analytes
EP2630517B1 (en) 2010-10-22 2016-03-16 T2 Biosystems, Inc. Nmr systems and methods for the rapid detection of analytes
US8563298B2 (en) 2010-10-22 2013-10-22 T2 Biosystems, Inc. NMR systems and methods for the rapid detection of analytes
EP2632373B1 (en) 2010-10-25 2018-07-18 Medtronic Ardian Luxembourg S.à.r.l. System for evaluation and feedback of neuromodulation treatment
BR112013011705B1 (en) 2010-11-12 2022-04-05 The Trustees Of The University Of Pennsylvania Consensus prostate antigens, nucleic acid molecule encoding the same, and vaccine and uses comprising the same
EA033467B1 (en) 2011-01-31 2019-10-31 Univ Pennsylvania Nucleic acid molecule for inducing an immune response against herpes virus and use thereof
US9238679B2 (en) 2011-02-11 2016-01-19 The Trustees Of The University Of Pennslyvania Nucleic acid molecule encoding hepatitis B virus core protein and surface antigen protein and vaccine comprising the same
HUE049669T2 (en) 2011-02-11 2020-10-28 Univ Pennsylvania Nucleic acid molecule encoding hepatitis b virus core protein and vaccine comprising the same
CA2831613A1 (en) 2011-03-31 2012-10-04 Moderna Therapeutics, Inc. Delivery and formulation of engineered nucleic acids
CA2839196A1 (en) 2011-06-15 2012-12-20 Chrontech Pharma Ab Injection needle and device
KR102171142B1 (en) * 2011-06-28 2020-10-28 이노비오 파마수티컬즈, 인크. A miniminally invasive dermal electroporation device
ES2758713T3 (en) 2011-07-11 2020-05-06 Inovio Pharmaceuticals Inc Lassa virus DNA vaccine
KR101314091B1 (en) 2011-07-26 2013-10-04 연세대학교 산학협력단 Electro-microneedle assembly for cutaneous gene transfer in-situ and process for preparing the same
US9078665B2 (en) 2011-09-28 2015-07-14 Angiodynamics, Inc. Multiple treatment zone ablation probe
EP2763701B1 (en) 2011-10-03 2018-12-19 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
KR20220132652A (en) 2011-12-12 2022-09-30 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 Compositions, comprising improved il-12 genetic constructs and vaccines, immunotherapeutics and methods of using the same
EP2791362B1 (en) 2011-12-12 2019-07-03 The Trustees Of The University Of Pennsylvania Proteins comprising mrsa pbp2a and fragments thereof, nucleic acids encoding the same, and compositions and their use to prevent and treat mrsa infections
JP2015501844A (en) 2011-12-16 2015-01-19 モデルナ セラピューティクス インコーポレイテッドModerna Therapeutics,Inc. Modified nucleosides, nucleotides and nucleic acid compositions
US9414881B2 (en) 2012-02-08 2016-08-16 Angiodynamics, Inc. System and method for increasing a target zone for electrical ablation
EP3348220A1 (en) 2012-03-08 2018-07-18 Medtronic Ardian Luxembourg S.à.r.l. Biomarker sampling in the context of neuromodulation devices and associated systems
US9750568B2 (en) 2012-03-08 2017-09-05 Medtronic Ardian Luxembourg S.A.R.L. Ovarian neuromodulation and associated systems and methods
WO2013155205A1 (en) 2012-04-10 2013-10-17 The Trustees Of The University Of Pennsylvania Human respiratory syncytial virus concensus antigens, nucleic acid constructs and vaccines made thereform, and methods of using same
EP4190350A1 (en) 2012-04-12 2023-06-07 The Trustees Of The University Of Pennsylvania Filovirus consensus antigens, nucleic acid constructs and vaccines made therefrom, and methods of using same
EP2839038B1 (en) 2012-04-20 2019-01-16 T2 Biosystems, Inc. Compositions and methods for detection of candida species
US20140110296A1 (en) 2012-10-19 2014-04-24 Medtronic Ardian Luxembourg S.A.R.L. Packaging for Catheter Treatment Devices and Associated Devices, Systems, and Methods
KR102258021B1 (en) 2012-12-13 2021-06-01 더 트러스티스 오브 더 유니버시티 오브 펜실베니아 Wt1 vaccine
US11419925B2 (en) 2013-03-15 2022-08-23 The Trustees Of The University Of Pennsylvania Cancer vaccines and methods of treatment using the same
CA2895806C (en) 2013-03-15 2023-02-21 The Trustees Of The University Of Pennsylvania Foot and mouth disease virus (fmdv) consensus proteins, coding sequences therefor and vaccines made therefrom
US10272144B2 (en) 2013-07-31 2019-04-30 Bioventures, Llc Compositions for and methods of treating and preventing targeting tumor associated carbohydrate antigens
CN105829341A (en) 2013-12-13 2016-08-03 宾夕法尼亚大学理事会 DNA antibody constructs and method of using same
US10166321B2 (en) 2014-01-09 2019-01-01 Angiodynamics, Inc. High-flow port and infusion needle systems
US9980766B1 (en) 2014-03-28 2018-05-29 Medtronic Ardian Luxembourg S.A.R.L. Methods and systems for renal neuromodulation
US10194980B1 (en) 2014-03-28 2019-02-05 Medtronic Ardian Luxembourg S.A.R.L. Methods for catheter-based renal neuromodulation
US10194979B1 (en) 2014-03-28 2019-02-05 Medtronic Ardian Luxembourg S.A.R.L. Methods for catheter-based renal neuromodulation
JP6594901B2 (en) 2014-05-12 2019-10-23 バージニア テック インテレクチュアル プロパティース インコーポレイテッド Selective modulation of intracellular effects of cells using pulsed electric fields
CN114010802A (en) 2014-12-01 2022-02-08 宾夕法尼亚大学理事会 DNA antibody constructs and methods of use thereof
WO2016100325A1 (en) 2014-12-15 2016-06-23 Virginia Tech Intellectual Properties, Inc. Devices, systems, and methods for real-time monitoring of electrophysical effects during tissue treatment
US11519016B2 (en) 2016-01-21 2022-12-06 T2 Biosystems, Inc. NMR methods and systems for the rapid detection of bacteria
JP7123800B2 (en) 2016-02-05 2022-08-23 イノビオ ファーマシューティカルズ,インコーポレイティド Cancer vaccine and method of treatment therewith
EA038691B1 (en) 2016-03-28 2021-10-05 Айкор Медикэл Системс, Инк. Apparatus for delivery of therapeutic agents
WO2017180770A1 (en) 2016-04-13 2017-10-19 Synthetic Genomics, Inc. Recombinant arterivirus replicon systems and uses thereof
CN105943043A (en) * 2016-05-11 2016-09-21 苏州海神联合医疗器械有限公司 Surface-modified needle-type electromyographic electrode and manufacturing method thereof
CA3040264A1 (en) 2016-10-17 2018-04-26 Synthetic Genomics, Inc. Recombinant virus replicon systems and uses thereof
JP7158820B2 (en) 2016-10-26 2022-10-24 ロレアル Electroporation device and method for controlling electroporation device
US10905492B2 (en) 2016-11-17 2021-02-02 Angiodynamics, Inc. Techniques for irreversible electroporation using a single-pole tine-style internal device communicating with an external surface electrode
CN110352247A (en) 2016-12-05 2019-10-18 杨森制药公司 For enhancing the composition and method of gene expression
JP6884037B2 (en) * 2017-05-29 2021-06-09 株式会社ジェイメック Pulse voltage therapy device
RS63202B1 (en) 2017-06-30 2022-06-30 Inscripta Inc Automated cell processing methods, modules, instruments, and systems
US11590345B2 (en) 2017-08-08 2023-02-28 Pulse Biosciences, Inc. Treatment of tissue by the application of energy
US10850095B2 (en) 2017-08-08 2020-12-01 Pulse Biosciences, Inc. Treatment of tissue by the application of energy
US10738327B2 (en) 2017-08-28 2020-08-11 Inscripta, Inc. Electroporation cuvettes for automation
CN107440906A (en) * 2017-08-31 2017-12-08 中国农业科学院兰州畜牧与兽药研究所 A kind of animal Houhai acupoint single cave position annular electrode and single point electrode
US10857347B2 (en) 2017-09-19 2020-12-08 Pulse Biosciences, Inc. Treatment instrument and high-voltage connectors for robotic surgical system
US10443074B2 (en) 2017-09-30 2019-10-15 Inscripta, Inc. Modification of cells by introduction of exogenous material
US11607537B2 (en) 2017-12-05 2023-03-21 Virginia Tech Intellectual Properties, Inc. Method for treating neurological disorders, including tumors, with electroporation
BR112020011443A2 (en) 2017-12-13 2020-12-01 Inovio Pharmaceuticals, Inc. nucleic acid molecule and its use, vector and its use, composition, protein and vaccine
US11235044B2 (en) 2017-12-13 2022-02-01 Inovio Pharmaceuticals, Inc. Cancer vaccines targeting MUC16 and uses thereof
CA3083528C (en) 2017-12-13 2023-09-12 Inovio Pharmaceuticals, Inc. Cancer vaccines targeting mesothelin and uses thereof
US11021692B2 (en) 2017-12-19 2021-06-01 Janssen Sciences Ireland Unlimited Company Hepatitis B virus (HBV) vaccines and uses thereof
US11389531B2 (en) 2017-12-19 2022-07-19 Janssen Sciences Ireland Unlimited Company Methods and apparatus for the delivery of hepatitis B virus (HBV) vaccines
EP4272833A3 (en) 2017-12-19 2024-01-10 Janssen Sciences Ireland Unlimited Company Hepatitis b virus (hbv) vaccines and uses thereof
EA202091516A1 (en) 2017-12-19 2020-11-03 Янссен Сайенсиз Айрлэнд Анлимитед Компани METHODS AND COMPOSITIONS FOR INDUCING IMMUNE RESPONSE AGAINST HEPATITIS B VIRUS (HBV)
WO2019123250A1 (en) 2017-12-19 2019-06-27 Janssen Sciences Ireland Unlimited Company Methods and compositions for inducing an immune response against hepatitis b virus (hbv)
CN111902163B (en) 2018-01-19 2024-02-13 杨森制药公司 Induction and enhancement of immune responses using recombinant replicon systems
US11925405B2 (en) 2018-03-13 2024-03-12 Virginia Tech Intellectual Properties, Inc. Treatment planning system for immunotherapy enhancement via non-thermal ablation
US11311329B2 (en) 2018-03-13 2022-04-26 Virginia Tech Intellectual Properties, Inc. Treatment planning for immunotherapy based treatments using non-thermal ablation techniques
US10435662B1 (en) 2018-03-29 2019-10-08 Inscripta, Inc. Automated control of cell growth rates for induction and transformation
US10376889B1 (en) 2018-04-13 2019-08-13 Inscripta, Inc. Automated cell processing instruments comprising reagent cartridges
US10557216B2 (en) 2018-04-24 2020-02-11 Inscripta, Inc. Automated instrumentation for production of T-cell receptor peptide libraries
WO2019209926A1 (en) 2018-04-24 2019-10-31 Inscripta, Inc. Automated instrumentation for production of peptide libraries
US10858761B2 (en) 2018-04-24 2020-12-08 Inscripta, Inc. Nucleic acid-guided editing of exogenous polynucleotides in heterologous cells
EP4070802A1 (en) 2018-06-30 2022-10-12 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US11142740B2 (en) 2018-08-14 2021-10-12 Inscripta, Inc. Detection of nuclease edited sequences in automated modules and instruments
US10532324B1 (en) 2018-08-14 2020-01-14 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US10752874B2 (en) 2018-08-14 2020-08-25 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US20200188610A1 (en) * 2018-12-18 2020-06-18 Joseph Fretta Medical tool having needle hub
US11571569B2 (en) 2019-02-15 2023-02-07 Pulse Biosciences, Inc. High-voltage catheters for sub-microsecond pulsing
WO2020255017A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and dihydropyrimidine derivatives as capsid assembly modulators
US20220249647A1 (en) 2019-06-18 2022-08-11 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and dihydropyrimidine derivatives as capsid assembly modulators
WO2020255013A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and capsid assembly modulators being amide derivatives
CA3140588A1 (en) 2019-06-18 2020-12-24 Helen Horton Combination of hepatitis b virus (hbv) vaccines and small molecule pdl1 or pd1 inhibitor
TW202114731A (en) 2019-06-18 2021-04-16 愛爾蘭商健生科學愛爾蘭無限公司 COMBINATION OF HEPATITIS B VIRUS (HBV) VACCINES AND HBV-TARGETING RNAi
WO2020255022A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and aminopyridine derivatives as hpk1 inhibitors
WO2020255042A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and a pyrimidine derivative
US20220305116A1 (en) 2019-06-18 2022-09-29 Janssen Sciences lreland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and capsid assembly modulators being sulfonamide derivatives
EP3986915A1 (en) 2019-06-18 2022-04-27 Janssen Sciences Ireland Unlimited Company Recombinant interleukin 12 construct and uses thereof
WO2020255035A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and pyrimidine derivatives
US20220296703A1 (en) 2019-06-18 2022-09-22 Janssen Sciences Ireland Unlimited Company Cobmination of hepatitis b virus (hbv) vaccines and anti-pd-1 or anti-pd-l1 antibody
WO2020255016A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and dihydropyrimidine derivatives as capsid assembly modulators
WO2020254876A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Virus-like particle delivery of hepatitis b virus (hbv) vaccines
WO2020255010A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of recombinant interleukin 12 construct and hepatitis b virus (hbv) vaccines
WO2020255018A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Hepatitis b virus (hbv) vaccines and uses thereof
WO2020255038A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and pyridopyrimidine derivatives
CA3143634A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and anti-pd-1 antibody
US20220233684A1 (en) 2019-06-18 2022-07-28 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and pd-l1 inhibitors
US20220241402A1 (en) 2019-06-18 2022-08-04 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and quinazoline derivatives
WO2020255019A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Combination of hepatitis b virus (hbv) vaccines and a quinazoline derivative
CN114340663A (en) 2019-06-18 2022-04-12 爱尔兰詹森科学公司 Combination of Hepatitis B Virus (HBV) vaccine and HBV-targeted RNAi
EP3986563A1 (en) 2019-06-20 2022-04-27 Janssen Sciences Ireland Unlimited Company Lipid nanoparticle or liposome delivery of hepatitis b virus (hbv) vaccines
AU2020297019A1 (en) 2019-06-20 2022-02-17 Janssen Sciences Ireland Unlimited Company Carbohydrate nanocarrier delivery of hepatitis B virus (HBV) vaccines
US10907125B2 (en) 2019-06-20 2021-02-02 Inscripta, Inc. Flow through electroporation modules and instrumentation
CN114008070A (en) 2019-06-21 2022-02-01 因思科瑞普特公司 Whole genome rationally designed mutations leading to increased lysine production in E.coli
US10927385B2 (en) 2019-06-25 2021-02-23 Inscripta, Inc. Increased nucleic-acid guided cell editing in yeast
US10689669B1 (en) 2020-01-11 2020-06-23 Inscripta, Inc. Automated multi-module cell processing methods, instruments, and systems
UY39020A (en) * 2020-01-23 2021-08-31 Yae Llc MINIMALLY INVASIVE DEVICE AND A METHOD FOR TENSIONING FAMILY SKIN THROUGH LINEAR TENSIONING AND THE STIMULATION OF COLLAGEN PRODUCTION, WHERE ANESTHESIA, HEAT AND THE INDUCTION OF ADDITIONAL COLLAGEN OR MISCELLANEOUS ANTI-INFLAMMATORY MISCELLANEOUS MISCELLANEOUS FLUIDS IN THE MIXED-APPLICATED MISCELLANEOUS MISCELLANEOUS FLUIDS.
CN115135370A (en) 2020-01-27 2022-09-30 因思科瑞普特公司 Electroporation module and instrument
US20210332388A1 (en) 2020-04-24 2021-10-28 Inscripta, Inc. Compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells
US11787841B2 (en) 2020-05-19 2023-10-17 Inscripta, Inc. Rationally-designed mutations to the thrA gene for enhanced lysine production in E. coli
CN116133668A (en) 2020-06-12 2023-05-16 罗切斯特大学 Coding and expression of ACE-tRNA
KR20230050328A (en) 2020-07-08 2023-04-14 얀센 사이언시즈 아일랜드 언리미티드 컴퍼니 RNA replicon vaccine against HBV
WO2022099067A1 (en) 2020-11-05 2022-05-12 Rynerson James M Methods and devices for treating skin conditions
WO2022133230A1 (en) 2020-12-18 2022-06-23 Janssen Pharmaceuticals, Inc. Combination therapy for treating hepatitis b virus infection
WO2022146654A1 (en) 2020-12-28 2022-07-07 Janssen Pharmaceuticals, Inc. Transcription activator-like effector nucleases (talens) targeting hbv
WO2023150753A1 (en) 2022-02-07 2023-08-10 University Of Rochester Optimized sequences for enhanced trna expression or/and nonsense mutation suppression
WO2023233290A1 (en) 2022-05-31 2023-12-07 Janssen Sciences Ireland Unlimited Company Rnai agents targeting pd-l1

Family Cites Families (82)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1653819A (en) 1926-08-07 1927-12-27 Northcott Ephraim Electrotherapeutical apparatus
DE863111C (en) 1951-07-03 1953-01-15 Walter Hallegger Instrument for transcutaneous and subcutaneous heating and iontophoresis and method of its use
DE2800039C2 (en) 1978-01-02 1984-06-20 Horst Dr.Med. 6700 Ludwigshafen Kief Acupuncture device
US5421819A (en) 1992-08-12 1995-06-06 Vidamed, Inc. Medical probe device
US4699880A (en) 1984-09-25 1987-10-13 Immunomedics, Inc. Method of producing monoclonal anti-idiotype antibody
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US5749847A (en) 1988-01-21 1998-05-12 Massachusetts Institute Of Technology Delivery of nucleotides into organisms by electroporation
US5547467A (en) 1988-01-21 1996-08-20 Massachusettes Institute Of Technology Method for rapid temporal control of molecular transport across tissue
US5389069A (en) 1988-01-21 1995-02-14 Massachusetts Institute Of Technology Method and apparatus for in vivo electroporation of remote cells and tissue
DE68925030T2 (en) 1988-01-21 1996-07-25 Massachusetts Inst Technology MOLECULE TRANSPORT THROUGH FABRICS WITH THE USE OF ELECTROPORATION.
IL86076A (en) * 1988-04-14 1992-12-01 Inventor S Funding Corp Ltd Transdermal drug delivery device
WO1989010690A1 (en) * 1988-05-02 1989-11-16 University Of South Florida Method for electrofusing biological particles to tissues
EP0346513A1 (en) 1988-06-15 1989-12-20 Etama Ag Assembly for electrotherapy
ES2012944A6 (en) 1989-01-09 1990-04-16 Tomas Justribo Jose Ramon A device for the administration of medication by iontopheresis for local - regional treatment.
CS275292B2 (en) 1989-02-22 1992-02-19 Cvut Fakulta Elektrotechnick Private rehabilitation apparatus with ion transcutaneous acceleration
US5703055A (en) 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
US4991578A (en) * 1989-04-04 1991-02-12 Siemens-Pacesetter, Inc. Method and system for implanting self-anchoring epicardial defibrillation electrodes
US6001617A (en) 1989-06-07 1999-12-14 Queen's University At Kingston Electroporation device and method of use
US4950229A (en) * 1989-09-25 1990-08-21 Becton, Dickinson And Company Apparatus for an electrode used for iontophoresis
US5215088A (en) 1989-11-07 1993-06-01 The University Of Utah Three-dimensional electrode device
DE4000893A1 (en) 1990-01-15 1991-07-18 Bosch Gmbh Robert Multichannel appts. for electro-simulation - provides several current circuits for patient with electrodes applying pulse signals
US5279544A (en) * 1990-12-13 1994-01-18 Sil Medics Ltd. Transdermal or interdermal drug delivery devices
US5328451A (en) 1991-08-15 1994-07-12 Board Of Regents, The University Of Texas System Iontophoretic device and method for killing bacteria and other microbes
PT101031B (en) 1991-11-05 2002-07-31 Transkaryotic Therapies Inc PROCESS FOR THE SUPPLY OF PROTEINS BY GENETIC THERAPY
US5425752A (en) 1991-11-25 1995-06-20 Vu'nguyen; Dung D. Method of direct electrical myostimulation using acupuncture needles
US5309909A (en) * 1992-05-22 1994-05-10 Physio-Control Corporation Combined skin preparation and monitoring electrode
US5304120A (en) 1992-07-01 1994-04-19 Btx Inc. Electroporation method and apparatus for insertion of drugs and genes into endothelial cells
US5507724A (en) 1992-07-01 1996-04-16 Genetronics, Inc. Electroporation and iontophoresis apparatus and method for insertion of drugs and genes into cells
US5514131A (en) 1992-08-12 1996-05-07 Stuart D. Edwards Method for the ablation treatment of the uvula
US5273525A (en) 1992-08-13 1993-12-28 Btx Inc. Injection and electroporation apparatus for drug and gene delivery
US6009345A (en) 1992-08-17 1999-12-28 Genetronics, Inc. Method and apparatus for a combination of electroporation and iontophoresis for the delivery of drugs and genes
US5688233A (en) * 1992-08-17 1997-11-18 Genetronics, Inc. Electronincorporation enhanced transdermal delivery of molecules
US5318514A (en) 1992-08-17 1994-06-07 Btx, Inc. Applicator for the electroporation of drugs and genes into surface cells
US5462520A (en) 1992-08-17 1995-10-31 Genetronics, Inc. Transsurface drug delivery by electrofusion of microbubbles to the tissue surface
US5464386A (en) 1992-08-17 1995-11-07 Genetronics, Inc. Transdermal drug delivery by electroincorporation of vesicles
US5322520A (en) * 1992-11-12 1994-06-21 Implemed, Inc. Iontophoretic structure for medical devices
US5468223A (en) 1992-11-30 1995-11-21 C.N.R.S. Paris Electrochemotherapy
JPH08506259A (en) 1993-02-02 1996-07-09 ヴィーダメッド インコーポレイテッド Transurethral needle excision device and method
US5306236A (en) * 1993-02-18 1994-04-26 Vickers Plc Needle electrode for use with hypodermic syringe attachment
FR2703253B1 (en) * 1993-03-30 1995-06-23 Centre Nat Rech Scient APPLICATOR OF ELECTRIC PULSES FOR TREATING BIOLOGICAL TISSUES.
US5702359A (en) * 1995-06-06 1997-12-30 Genetronics, Inc. Needle electrodes for mediated delivery of drugs and genes
US5439440A (en) 1993-04-01 1995-08-08 Genetronics, Inc. Electroporation system with voltage control feedback for clinical applications
US5993434A (en) 1993-04-01 1999-11-30 Genetronics, Inc. Method of treatment using electroporation mediated delivery of drugs and genes
WO1994027670A1 (en) 1993-06-02 1994-12-08 Cardiac Pathways Corporation Catheter having tip with fixation means
US5536267A (en) 1993-11-08 1996-07-16 Zomed International Multiple electrode ablation apparatus
US5814599A (en) 1995-08-04 1998-09-29 Massachusetts Insitiute Of Technology Transdermal delivery of encapsulated drugs
IL108775A (en) 1994-02-25 2003-09-17 Univ Ramot Method for efficient incorporation of molecules into cells
US6277116B1 (en) * 1994-05-06 2001-08-21 Vidaderm Systems and methods for shrinking collagen in the dermis
US5728094A (en) 1996-02-23 1998-03-17 Somnus Medical Technologies, Inc. Method and apparatus for treatment of air way obstructions
US5786454A (en) 1994-09-16 1998-07-28 Washington University School Of Medicine Modified SH2 domains
US5720921A (en) 1995-03-10 1998-02-24 Entremed, Inc. Flow electroporation chamber and method
US5868740A (en) 1995-03-24 1999-02-09 Board Of Regents-Univ Of Nebraska Method for volumetric tissue ablation
US5810762A (en) 1995-04-10 1998-09-22 Genetronics, Inc. Electroporation system with voltage control feedback for clinical applications
US5780052A (en) 1995-04-24 1998-07-14 Northeastern University Compositions and methods useful for inhibiting cell death and for delivering an agent into a cell
US5789213A (en) 1995-05-11 1998-08-04 Health Research Inc. Method and compositions for high efficiency loading, transfection and fusion of cells by electric pulses
US6041253A (en) * 1995-12-18 2000-03-21 Massachusetts Institute Of Technology Effect of electric field and ultrasound for transdermal drug delivery
US6002961A (en) 1995-07-25 1999-12-14 Massachusetts Institute Of Technology Transdermal protein delivery using low-frequency sonophoresis
US5983131A (en) 1995-08-11 1999-11-09 Massachusetts Institute Of Technology Apparatus and method for electroporation of tissue
US6330478B1 (en) * 1995-08-15 2001-12-11 Rita Medical Systems, Inc. Cell necrosis apparatus
US5980517A (en) 1995-08-15 1999-11-09 Rita Medical Systems, Inc. Cell necrosis apparatus
WO1997007826A1 (en) 1995-08-29 1997-03-06 Cbr Laboratories, Inc. In vivo electroporation of cells
AU3309097A (en) 1996-06-12 1998-01-07 Alza Corporation Reduction of skin sensitization in electrotransport drug delivery
GB2314274A (en) 1996-06-20 1997-12-24 Gyrus Medical Ltd Electrode construction for an electrosurgical instrument
US5911223A (en) 1996-08-09 1999-06-15 Massachusetts Institute Of Technology Introduction of modifying agents into skin by electroporation
US5869326A (en) 1996-09-09 1999-02-09 Genetronics, Inc. Electroporation employing user-configured pulsing scheme
DE69718495T2 (en) * 1996-09-17 2003-11-20 Deka Products Lp SYSTEM FOR DISPOSAL OF MEDICINE BY TRANSPORT
US5874268A (en) 1996-09-23 1999-02-23 Duke University Method of introducing exogenous compounds into cells by electroporation and apparatus for same
US5908753A (en) 1996-10-04 1999-06-01 Merck & Co., Inc. Transformation of Candida albicans by electroporation
US5845646A (en) 1996-11-05 1998-12-08 Lemelson; Jerome System and method for treating select tissue in a living being
US6206004B1 (en) * 1996-12-06 2001-03-27 Comedicus Incorporated Treatment method via the pericardial space
DE69835761T2 (en) 1997-04-03 2007-09-13 Electrofect As. PROCESS FOR THE ADMINISTRATION OF PHARMACEUTICAL PREPARATIONS AND NUCLEIC ACIDS ON THE SKELETON MUSCLE
US5873849A (en) 1997-04-24 1999-02-23 Ichor Medical Systems, Inc. Electrodes and electrode arrays for generating electroporation inducing electrical fields
US6055453A (en) 1997-08-01 2000-04-25 Genetronics, Inc. Apparatus for addressing needle array electrodes for electroporation therapy
US5879891A (en) 1997-09-17 1999-03-09 Merck & Co., Inc. Transformation of saccharomyces cerevisiae by electroporation
US5972013A (en) * 1997-09-19 1999-10-26 Comedicus Incorporated Direct pericardial access device with deflecting mechanism and method
TW368420B (en) 1997-11-04 1999-09-01 Genetronics Inc Apparatus and method for transdermal molecular delivery by applying sufficient amplitude of electric field to induce migration of molecules through pores in the stratum corneum
US6120493A (en) 1998-01-27 2000-09-19 Genetronics, Inc. Method for the introduction of therapeutic agents utilizing an electroporation apparatus
US6022316A (en) 1998-03-06 2000-02-08 Spectrx, Inc. Apparatus and method for electroporation of microporated tissue for enhancing flux rates for monitoring and delivery applications
NZ507185A (en) 1998-04-14 2002-09-27 Gmp Drug Delivery Inc Iontophoresis, electroporation and combination catheters for local drug delivery to arteries and other body tissues
US20020102729A1 (en) 1998-06-08 2002-08-01 Maclaughlin Fiona Formulations for electroporation
EP1102608A4 (en) * 1998-07-13 2008-09-17 Genetronics Inc Method and apparatus for electrically assisted topical delivery of agents for cosmetic applications
US6040184A (en) * 1998-10-09 2000-03-21 Stratagene Method for more efficient electroporation

Also Published As

Publication number Publication date
EP1100579A1 (en) 2001-05-23
EP2428249A1 (en) 2012-03-14
EP2428249B1 (en) 2015-10-07
US6697669B2 (en) 2004-02-24
AU4988399A (en) 2000-02-01
EP1100579A4 (en) 2008-12-10
JP2003505114A (en) 2003-02-12
EP2428250A1 (en) 2012-03-14
US6654636B1 (en) 2003-11-25
US20020099323A1 (en) 2002-07-25
AU770092B2 (en) 2004-02-12
WO2000002621A9 (en) 2000-03-23
CA2337652A1 (en) 2000-01-20
EP1100579B1 (en) 2015-09-02
WO2000002621A1 (en) 2000-01-20

Similar Documents

Publication Publication Date Title
CA2337652C (en) Skin and muscle-targeted gene therapy by pulsed electrical field
US7570992B2 (en) Electrical field therapy with reduced histopathological change in muscle
US6972013B1 (en) Enhanced delivery of naked DNA to skin by non-invasive in vivo electroporation
US7922709B2 (en) Enhanced delivery of naked DNA to skin by non-invasive in vivo electroporation
US7171264B1 (en) Intradermal delivery of active agents by needle-free injection and electroporation
US20200001076A1 (en) Device and method for single-needle in vivo electroporation
US8455228B2 (en) Method to facilitate directed delivery and electroporation using a charged stream
AU2004277376B2 (en) Apparatus for generating electrical pulses and methods of using the same
US20050277868A1 (en) Electroporation Device and Method for Delivery to Ocular Tissue
EP1183068A1 (en) Method of electroporation-enhanced delivery of active agents
US20040092860A1 (en) Skin and muscle-targeted gene therapy by pulsed electrical field
CA2686855C (en) Device and method for single-needle in vivo electroporation

Legal Events

Date Code Title Description
EEER Examination request
MKEX Expiry

Effective date: 20190715