Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
  • A61N1/325—Applying 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

Definitions

• This invention is directed to remarkable enhancement of in vivo transfer into cells, particularly muscle cells, of nucleic acid vectors using weak electric fields, to increase the efficiency of such transfers.
• the invention specifically relates to methods, devices, and compositions that effect such nucleic acid vector transfer for gene therapy.
• the devices of the invention are designed to provide an optimum voltage gradient to enhance migration of nucleic acid vectors into cells, without damaging the cells or tissue.
• Such devices are characterized by unique arrangements of electrodes, and by unique power limits defined by maximum voltage settings.
• pretreatment of the tissue to be injected with solutions intended to enhance DNA diffusion and/or stability (Davis et al., 1993, Hum. Gene Ther. 4, 151-159) or to favor the entry of nucleic acids, e.g., the induction of cell multiplication or regeneration phenomena.
• the treatments have concerned, in particular, the use of local anesthetics or cardiotoxin, vasoconstrictors, endotoxin or other molecules (Manthorpe et al., 1993, Human Gene Ther. 4, 419-431; Danko et al., 1994, Gene Ther. 1, 114-121; Vitadello et al., 1994, Hum. Gene Ther. 5, 11-18).
• These pretreatment protocols are difficult to manage.
• Bupivacaine in particular, must be used at very close to lethal doses in order to be effective.
• the pre- injection of hyperosmotic sucrose, intended to enhance diffusion, does not increase the level of transfection in the muscle (Davis et al., 1993).
• Electroporation and Iontophoresis for Gene Delivery Electroporation is commonly used in vitro to promote DNA transfection in culture cells. This phenomenon depends on achieving a threshold electric field strength. Electropermeabilization was observed at electric fields of relative high intensity, in the order of 800 to 1,200 volts/cm for animal cells. This technique was also proposed in vivo in order to enhance the efficacy of antitumoral agents, like bleomycin, in solid tumors in man (US patent No. 5,468,228, L.M. Mir). With pulses of very short duration (100 microseconds), these electric conditions (800 to 1,200 volts/cm) are very well adapted to the intracellular transfer of small molecules.
• Electrodes are positioned externally with respect to the patient. See, for example, US Patent Nos. 5,318,514 to Hofmann; 5,439,440 to Hofmann; 5,462,520 to Hofmann; 5,464,386 to Hofmann; 5,688,233 to Hofmann et al.; and 5,019,034 to Weaver et al.; the disclosures of which are inco ⁇ orated herein by reference.
• an external electrode device the electrodes are in contact with a surface tissue region of a patient. The device can be used non- invasively by applying the electrodes to the skin of the patient or invasively by applying the electrodes to the surface of an organ that has been exposed surgically.
• the Hofmann '514 patent discloses a device that is used to implant macromolecules such as genes, DNA or pharmaceuticals into a preselected surface tissue region of a patient.
• the device has a head assembly which includes, in a first embodiment, a se ⁇ entine conductor positioned on an open-pore elastomer, both of which are carried on a generally planar support member. Adjacent parallel segments of the serpentine conductor serve as electrodes.
• the head assembly is placed in contact with the preselected surface tissue region of the patient, placing the conductor in contact with the skin.
• a liquid medium carrying the macromolecules is transferred to the skin of the patient by delivery to the elastomer of the liquid which is absorbed or soaked up by the elastomer.
• a switch is then engaged to deliver a high voltage pulse from a signal generator to the electrodes, causing an electric field to be generated across the electrodes.
• the depth of the electric field into the skin is proportional to the gap between the electrodes. The electric field injects the liquid into the tissue region.
• the head assembly includes a plurality of fine needles extending generally perpendicular to the planar support member.
• the needles are arranged in rows and are connected alternatively to the output of the signal generator so that each needle is adjacent another needle of opposite polarity.
• the needles penetrate the outer most layers of skin cells and facilitate the administration of the electric pulses into the target area.
• the Hofmann '440 patent discloses an apparatus which includes adjustably spaced electrodes for generating an electric field.
• the electrodes are mounted on a moveable linkage so that the electrodes can be manipulated by the user to move toward and away from one another like the jaws of a clamp.
• the electrode jaws are opened and a selected tissue to be treated is gripped between the electrode jaws.
• a signal generator connected to the electrodes is operated by a suitable switching device to generate the electric fields in the tissue between the electrodes.
• Internal Electrodes A second type of electrotransfer system utilizes implantable or insertable electrodes which are placed inside the patient to deliver an electric field to the area adjacent to the implanted/inserted electrode. See, for example, US Patent Nos.
• the Crandell '120 patent discloses a catheter that is inserted into a selected blood vessel of a patient.
• the catheter includes a plurality of axially extending, circumferentially spaced electrodes that are placed in contact with the inner wall of the blood vessel.
• a liquid medium containing the macromolecules is then infused into the blood vessel adjacent the electrodes and the electrodes are energized to apply the predetermined electrical signal for electrotransfer.
• the spaced electrodes can be se ⁇ entine or parallel strips which are energized to create the desired electric field.
• the Hofmann '724 patent is another example of a catheter-based electrotransfer device having spaced apart electrodes which are positioned on the outside of a catheter that is inserted into a blood vessel to contact the wall of the vessel to be treated.
• the Hofmann '662 patent discloses a pair of spaced apart electrodes mounted within a cylindrical dielectric carrier.
• the electrodes are positioned around the center of the blood vessel a predetermined uniform distance apart from each other and near the center of the vessel so that blood flowing in the vessel passes between the electrodes.
• the cylindrical dielectric carrier is implanted surgically within a surrounding blood vessel.
• a predetermined electrical signal is applied to the electrodes to create electric fields in the blood flowing between the electrodes.
• the Hofmann '525 patent discloses a dual-needle syringe in which the needles serve as the electrodes for carrying out the electrotransfer. Once the needles have been inserted into the target area, an electrical signal is applied to the electrodes to direct the electric field to the target area.
• the Hofmann '359 patent discloses also needle-based electrodes used for electrotransfer.
• a third type of electrotransfer device combines features of the above-mentioned systems. These devices utilize at least one internally placed electrode and at least one externally placed electrode to deliver the electric field to the desired tissue area. See, for example, US Patent No. 5,286,254 to Shapland et al.; 5,499,971 to Shapland et al.; 5,498,238 to Shapland et al.; 5,282,785 to Shapland et al.; and 5,628,730 to Shapland et al.; the disclosures of which are incorporated herein by reference.
• Typical of these devices is the one described in the Shapland '785 patent which discloses a catheter having a drug chamber with a drug delivery wall (for example, a wall made of permeable or semipermeable material which can pass drugs or other macromolecules therethrough) and an electrode located inside of the catheter in an opposed relation to the drug delivery wall.
• a second electrode is located at a remote site on a patient's skin.
• a liquid containing desired macromolecules is delivered to the drug chamber to be placed in the electric field generated between the two electrodes when they are provided with current. In this manner, the macromolecules are delivered to the target area.
• this invention concerns a process and device for nucleic acid transfer into tissues in vivo, e.g. , one or more striated muscles or tumors, in which the tissue cells are brought in contact with the nucleic acid to be transferred by direct administration into the tissue or by topical or systemic administration, and in which transfer is ensured by application to the tissues of one or more electric pulses of intensity ranging between 1 and 400 volts/cm for muscle, and 1 and 600 volts/cm for tissues such as tumors.
• the present invention provides a system, such as an improved apparatus, for in vivo nucleic acid transfer into cells of multicellular eukaryotic organisms, in which the tissue cells are brought in contact with the nucleic acid to be transferred by direct administration into the tissue or by topical or systemic administration, and in which transfer is ensured by application to the tissue of one or more electric pulses delivered by an apparatus of the invention set to provide the specified intensity.
• the electric field strength can range between 1 and 600 volts/cm for delivery of a nucleic acid to tumor cells, and between 1 and 400 volts/cm for delivery of a nucleic acid to muscle cells.
• the system (or apparatus) of the invention comprises an electrical pulse generator (or means for generating an electrical pulse), wherein the electrical pulse generator produces electric pulses with pulse times of greater than 1 millisecond and of intensity ranging between 1 and 400 or 600 volts/cm at a frequency of between 0.1 and 1000 Hz; and electrodes connected to the electrical pulse generator for generating an electric field in a tissue in vivo in contact with the electrodes.
• the electrical pulse generator produces pulse of intensity ranging between 30 and 300 volts/cm (for transfer into muscle) and between 400 and 600 V/cm for transfer into tumor cells and other small cells.
• the electrical pulse generator produces pulse times of greater than 10 milliseconds.
• the electrical pulse generator produces between 2 and 1000 pulses.
• the system or improved apparatus electrical pulse generator can produce pulses irregularly in relation to one another, whereby a function describing the intensity of the field dependent on time of an pulse is variable, with the proviso that at no time does the system or apparatus supply an electric field greater (or less) than the parameters set forth above.
• the integral of the function describing variation of the electric field with time can exceed 1 kV » msec/cm; and in a further embodiment exceeds or is equal to 5 kV* msec/cm.
• the electrical pulse generator (pulse generating means) can produce pulses selected from the group consisting of square-wave pulses, exponential decay waves, oscillating unipolar waves of limited duration, and oscillating bipolar waves of limited duration.
• the electrical pulse generator produces square- wave pulses.
• the electrode can be an external electrode for placement on a tissue to be treated, e.g. , for transferring nucleic acids into cells of a surface tissue of a subject.
• the electrode can be an internal electrode or tissue penetrating electrode, which is implantable in a tissue to be treated.
• Such an internal electrode can be a needle, and may be configured as an injector system making possible the simultaneous administration of nucleic acids and of the electric field.
• the invention provides both an external electrode and an internal electrode.
• An external electrode of the invention can be dimensionsed to contact an external portion of a subjects body in close proximity to a large muscle.
• such an electrode is a flat plate electrode; in another embodiment, it is a semi-cylindrical plate electrode.
• the an electrode is an intra-arterial or intravenous electrode, for example a flexible catheter apparatus modified according to the invention.
• the preferred material for an electrode of the invention is stainless steel.
• the improved apparatus of the invention can be produced by modifying prior art devices, and particularly the means for generating an electric field of such devices, to generate an electric field of the invention.
• the means for generating an electric pulse can be adapted to produce pulses ranging between 1 and 400 or 600 volts/cm by modifying the voltage gate not to exceed a voltage corresponding to 400 or 600 volts/cm.
• the voltage can be set at a constant voltage and the electrodes can be set at a constant spacing distance.
• the means for generating an electric pulse can be adapted to produce pulses ranging between 1 and 400 or 600 volts/cm by labeling the device not to exceed a voltage corresponding to 400 or 600 volts/cm.
• an object of the invention is to provide a system, or improve existing devices, to supply an electric field having a voltage gradient, pulse width, and number of pulses that have been found to be optimum for transfer of nucleic acids without damaging tissue.
• Yet another advantage of the invention is to provide for efficient, reproducible delivery of nucleic acids to muscle cells.
• FIGURE 1 Effects of electric pulses of high field intensity on the transfection of plasmid DNA pXL2774 in the cranial tibial muscle in the mouse; mean values ⁇ SEM.
• FIGURE 2 Effects of electric pulses of intermediate field intensity on the transfection of plasmid DNA pXL2774 in the cranial tibial muscle in the mouse; mean values ⁇ SEM.
• FIGURE 3 Effects of electric pulses of weak field intensity and of different duration on the transfection of plasmid DNA pXL2774 in the cranial tibial muscle in the mouse; mean values ⁇ SEM.
• FIGURE 4 Effects of electric pulses of weak field intensity and of different duration on the transfection of plasmid DNA pXL2774 in the cranial tibial muscle in the mouse; mean values ⁇ SEM.
• FIGURE 5 Effectiveness of electrotransfer of plasmid DNA pXL2774 in the cranial tibial muscle of the mouse at low electric field intensities; mean values ⁇ SEM.
• FIGURE 6 Kinetics of expression of luciferase in mouse tibial cranial muscle. Administration of plasmid pXL2774 with (B)and without (X) electrotransfer; average values + SEM.
• FIGURE 7 Level of expression as a function of the dose of DNA administered with (•) and without (D)electrotransfer.
• FIGURE 8 Effect of different types of electrodes on electrotransfer efficiency.
• FIGURE 9 Kinetics of serum concentration of secreted alkaline phosphate. Administration of plasmid ⁇ XL3010 with ( ⁇ ) et without ( ⁇ ) electrotransfer; average values + SEM.
• FIGURE 10 Kinetics of expression of aFGF in muscle with (open histogram bars) or without (solid histogram bars) electrotransfer.
• FIGURE 11 Map of plas ids pXL3179 and pXL3212.
• FIGURE 12 Map of plasmids pXL3388 and pXL3031.
• FIGURE 13 Map of plasmids pXL3004 and pXL3010.
• FIGURE 14 Map of plasmids pXL3149 and pXL3096.
• FIGURE 15 Map of plasmids pXL3353 and pXL3354.
• FIGURE 16 Map of plasmid pXL3348.
• the present invention provides greatly enhanced in vivo nucleic acid transfer into tissues by subjecting the tissue to electric pulses of low intensity.
• electric fields of less than 600 volts/cm have been found to enhance nucleic acid transfer into tumors, and less than 400 volts/cm, and preferably, 100 to 200 volts/cm for electrodes placed about 0.5 to 1 cm apart in muscle. These fields are applied for relatively long duration.
• the applicants discovered that the wide variability of transgene expression observed in the prior art of DNA transfer into the muscle was notably reduced by the process according to the invention.
• expression persists for a long period of time, e.g., greater than 60 days. In a specific example, high level expression was detected 63 days.
• electrotransfer transfer of nucleic acids into cells in vivo under these conditions
• electrotransfection an appropriate alternative term used herein is “electrotransfection”. Both of these terms distinguish the optimized conditions for nucleic acid transfer from “electroporation” (using electric fields greater than 800 V/cm) and iontophoresis (using very low strength electric fields).
• this invention concerns processes, systems and devices (or apparatus), and compositions for in vivo nucleic acid transfer into tissues, particularly striated muscle, in which the tissue cells are brought in contact with the nucleic acid to be transferred by direct administration into the tissue or by topical or systemic administration, and in which transfer is ensured by application to said tissues of one or more electric pulses of intensity ranging between 1 and 600 volts/cm (e.g. for tumor cells) or between 1 and 400 volts/cm for muscle cells.
• the present invention particularly concerns systems ⁇ i.e., devices or apparatus) for electrotransfer.
• the process of the invention is applied to tissues whose cells have particular geometries, like, for example, cells of large size and/or of elongated shape and/or naturally responding to electric potentials and/or having a specific mo ⁇ hology.
• the intensity of the field preferably ranges between 4 and 400 volts/cm for muscle, and up to 600 volts/cm for tumors, and the total duration of application exceeds 1 millisecond
• the total duration is 8 msec or longer.
• the pulse duration is 20 msec, and durations greater than 40 msec were found to be effective.
• the number of pulses used is, for example, from 1 to 1,000 pulses, preferably 2 to 100 and more preferably 4 to 20, and the pulse frequency ranges between 0.1 and 1,000 hertz (Hz); more precisely between 0.2 and 100 Hz. In specific embodiments, frequencies of 2, 3, and 4 Hz were found to be effective.
• the pulses can also be delivered irregularly and the function describing the intensity of the field dependent on time can be variable. The integral of the function describing the variation of the electric field with time is greater than 1 kV»msec/cm.
• that integral is higher than or equal to 5 kV » msec/cm.
• this integrated function must be achieved at the sub-electrophoretic voltages described supra.
• the device of the invention can supply a combination of at least one higher voltage pulse (greater than 400 V/cm, and preferably between 500 and 800 V/cm) for short duration (less than 1 msec), followed by one or more longer pulses (greater than 1 msec) at much lower electric field strength (less than 200 V/cm).
• the field intensity of the pulses ranges between 30 and 300 volts/cm.
• the electric pulses are chosen among square-wave pulses, the electric fields generating exponential decay waves, oscillating unipolar waves of limited duration, oscillating bipolar waves of limited duration or other wave-forms.
• the electric pulses are square wave pulses.
• the administration of electric pulses can be carried out by any method known in the art, e.g.:
• Nucleic acids can be administered by any appropriate means, but are preferably injected in vivo directly into the tissues or administered by another route, local or systemic, which makes them available on the site of application of the electric field.
• the nucleic acids can be administered with agents permitting or facilitating transfer, as previously mentioned.
• Those nucleic acids can, notably, be free in solution or linked to synthetic agents or carried by viral vectors.
• the synthetic agents can be lipids or polymers known to the expert, or even targeting elements making possible fixation on the membrane of the target tissues. Among those elements, vectors carrying sugars, peptides, antibodies, receptors, and ligands, can be mentioned.
• nucleic acids can precede, be simultaneous or even follow the application of electric fields, provided of course that the electric field continues to be applied after the nucleic acid is administered.
• This invention also concerns a nucleic acid and an electric field of intensity ranging between 1 and 600 volts/cm (preferably 400 volts/cm), as a combination product for their simultaneous, separate or time-staggered in vivo administration into mammal cells and, in particular, into human cells.
• the intensity of the field preferably ranges between 4 and 400 volts/cm and, even more preferably, the field intensity ranges between 30 and 300 volts/cm for transfer into muscle.
• a preferred electric field intensity is 400-600 V/cm; preferably about 500 (i.e., 500 ⁇ 10%, preferably 5%) V/cm .
• such a combination defines a nucleic acid structure, in which the nucleic acid adopts a specific orientation relative to the electric field, as well as specific secondary and tertiary structure in the presence of the electric field.
• the DNA will be associated with the extracellular components found in the target tissue, which is in distinction to DNA undergoing low field electrophoresis in an agarose gel or other laboratory conditions.
• any electrotransfer system consist of an electric pulse generator that is designed or modified to provide pulses of no more than 600 V/cm, and electrodes.
• a system or apparatus of the invention for delivery of a nucleic acid specifically to muscle provides pulses of no more than 400 volts/cm.
• the actual voltage will depend on the distance between the electrodes. As is well known in the art, this distance affects the specific resistance (resistivity) through the target tissue. Accordingly, the actual voltage applied will depend on the resistance so that current, and thus the total power, is kept within acceptable levels.
• accepted levels as used herein means that the total power does not result in irreversible tissue damage, particularly tissue burning.
• the apparatus of the invention either controls for acceptable current by fixing voltage and electrode distance, or includes a feedback means to prevent applying too high a voltage for the distance between the electrodes, and thus too much current.
• a system of the invention may include an oscilloscope or other metering device to monitor voltage, current, or both.
• the system of the invention is prepared using commercially available equipment. Preferably, such equipment is modified to provide the specific electrotransfer conditions defined herein as optimal. In another embodiment, a new apparatus is designed and built to achieve the objectives of the invention.
• Design specifications of the modified or built pulse generator include, but are by no means limited to, incorporation of a mechanical or electrical controller to maintain the desired voltage gradient, i.e., less than 600 or 400 V/cm, and preferably less than 200 V/cm for administration to muscle.
• a mechanical control could include, for example, a stop on the voltage selection dial that prevents selection of a voltage that would yield too high an electric field.
• the device can be built or modified to that such voltages cannot be selected.
• the device can include a breaker or fuse that will trip when the voltage (and thus current) exceeds the parameters of the invention.
• microprocessor controls can prevent or override selection of too great a voltage.
• a pulse generator is modified simply by applying a label directing use of a particular voltage range that provides the electric field strength of the invention. All of these modifications are routine in the art, and employ standard electrical and mechanical technologies.
• the actual voltage delivered by a system of the invention to achieve the electric field strength defined herein as optimal will depend, in part, on the electrode spacing. If the electrodes are spaced apart in a fixed manner, then the voltage (for a defined tissue, e.g., muscle, liver, heart, or a tumor) may be a pre-defined constant. However, if it is desirable to provided for varying the spacing of the electrodes, then the voltage may have to be adjusted to maintain a constant voltage gradient. This can be determined by measuring the distance between the electrodes, by including measuring means on the electrodes that provides a value for their spacing after adjustment, or by automated measuring means that feedback to the pulse generator to automatically provide the correct voltage (see US Patent 5,439,440 to Hofmann).
• Pulse generators are electrical devices that produce a current of defined voltage, duration, pulse width, duty cycle (the total time of the pulsing and resting), and pulse frequency. Such devices are well known in the art, and include commercially available pulse generators such as the ELECTRO CELL MANIPULATOR Model ECM 600, T800L and T820 voltage generators available from the BTX Instruments Division of Genetronics, Inc. of San Diego, California, e.g. , as described in US Patent No. 5,704,908, which is inco ⁇ orated herein by reference in its entirety.
• the pulse generator can be an Electropulsator PS 15, available from Jouan, France, as disclosed in the Examples, infra.
• a voltage generator that can produce one or more of the wave forms described in US Patent No. 5,634,899, which is incorporated herein by reference in its entirety, can be used.
• the voltage can be designed to generate pulses of variable shape, intensity, and duration. For example, a pulse of 200 V/cm or 400 V/cm, 5-20 msec, could be followed by a lower intensity, longer pulse.
• the device could further supply iontophoretic electric fields in combination with electric fields of the invention.
• a pulse generator of the invention will have the following specifications:
• pulse time greater than 1 millisecond (msec), with variable duty times; preferably the pulse time is greater than 5 msec; more preferably greater than 10 msec; and more preferably still greater than 20 msec.
• the pulse generator of the invention produces at least two, and preferably four, six or eight pulses. It can produce, for example, between 8 and 1,000 pulses.
• the pulse generator should permit an override or cut-off if the patient begins to experience an adverse event or the electric field strength is out of control. Such an override could be manual or automatic, or both.
• pulses can be generated by an external signal, such as another device, a computer, etc.
• the pulse generator will optimally interface with the subject's electrocardiogram so that pulses are synchronized with the heart beat.
• Such a system preferably includes active pacing of the subject's cardiac rhythm, e.g. with a pacemaker (see US Patent No. 5,634,899 to Shapland).
• Electrodes The electrodes of the invention provide the electric field in the tissue. One electrode, the cathode, is negatively charged; the anode is positively charged.
• an electrode for use according to the invention must conduct electricity efficiently, and preferably is inert, non-reactive and non-toxic under the conditions used.
• an electrode for internal use should not react with biological materials to any appreciable degree, e.g., to avoid releasing metal ions from the electrode that could be harmful or poisonous, or to avoid oxidation that reduces electrode efficiency.
• a preferred material for electrodes of the invention is stainless steel, which is fairly non-reactive, reasonably efficient for conducting electricity, and inexpensive enough to be manufactured at a reasonable cost. More ideal electrodes, particularly for internal use, are gold or platinum. However, such noble metal electrodes are very expensive. The cost of these materials can be reduced by plating them over other conductors. Other conductive metals include copper, silver or silver chloride, tin, nickel, lithium, aluminum, and iron, and amalgams thereof. However, certain materials, such as aluminum, should not be used internally. Electrodes can also be formed from zirconium, iridium, titanium, and certain forms of carbon.
• Some electrodes such as silver and copper, have antibacterial activity, which could be desirable for internal administration to suppress infection.
• the electrodes can be formed in any configuration appropriate for the target tissue, including, without limitation, straight wires, coiled wires (straight and coiled wire electrodes are ideal for catheter applications) , conductive surfaces ⁇ e.g., of catheters or balloon catheters; see US Patent No. 5,704,908, inco ⁇ orated herein by reference in its entirety), metal strips, needles (or probes), arrays of needles, surface electrodes, or combinations thereof.
• Contemplated electrode combinations include (1) a catheter electrode and a needle electrode; (2) a catheter electrode and a surface electrode; and (3) a needle electrode and a surface electrode.
• a needle electrode can be used with a syringe to deliver DNA.
• Such a needle electrode can have holes through the length of its shaft to permit delivery of the nucleic acid solution throughout its length.
• two surface electrodes can be used.
• Surface electrodes are preferably used in combination with an electrolytic composition to ensure good contact and conductance, e.g. , through the skin, as described above.
• the external electrode can have multiple "heads" placed around the internal one. Indeed, in general for any of the configurations set forth above, one of the electrodes can have multiple "heads.”
• the invention further contemplates arrays of electrodes; needles with holes along the shaft; needles with defined and calibrated conductive length (to provide constant and reproducible conductive area into a tissue, whatever the depth of the penetration of the needle into a tissue), with the upper and lower parts being electrically isolated; needles with isolated points, to prevent point to point electrical arcs into a tissue; and any kind of pouch / reservoir containing the product around one needle.
• needle electrodes can comprise isolated margins.
• the electrodes are arranged so that the target tissue is directly between them. That way, the nucleic acids are subject to the maximum field strength. However, since the electric field flux is all around the electrode, it is possible to use the field generated peripherally between the electrodes as well as the field generated directly between the electrodes.
• an apparatus in which electrodes are positioned externally with respect to the patient is modified in accordance with the present invention, i.e., to supply an electric field under the defined conditions, to provide an improved apparatus of the invention.
• the improved apparatus can be used non-invasively by applying the electrodes to the skin of the patient or invasively by applying the electrodes to the surface of an organ that has been exposed surgically.
• an electrotransfer system that utilizes implantable or insertable electrodes placed inside the patient to deliver an electric field to the area adjacent to the implanted/inserted electrode, particularly a catheter electrode (see, for example, US Patent Nos. 5,304,120 to Crandell et al.; 5,507,724 to Hofmann et al.; 5,501,662 to Hofmann; 5,702,359 to Hofmann et al.; and 5,273,525 to Hofmann) can be modified in accordance with the present invention to yield an improved apparatus of the invention.
• an electrotransfer device that combines features of the above-mentioned systems, e.g., that utilize at least one internally placed electrode and at least one externally placed electrode to deliver the electric field to the desired tissue area (see, for example, US Patent No. 5,286,254 to Shapland et al.; 5,499,971 to Shapland et al.; 5,498,238 to Shapland et al.; 5,282,785 to Shapland et al.; and 5,628,730 to Shapland et al)can be modified in accordance with the present invention to provide an improved apparatus of the invention.
• the process according to this invention is useful for gene therapy, that is, therapy in which the expression of a transferred gene, but also the modulation or blocking of a gene, makes it possible to ensure treatment of a particular pathology.
• tissue cells are preferably treated with a view to gene therapy making possible:
• the protection and/or regeneration of vascularization or innervation of the tissue such as muscles, organs or bone, by trophic, neurotrophic, angiogenic factors, or by anti- inflammatory factors produced by the transgene; the transformation of muscle into an organ secreting products leading to a therapeutic effect, such as the product of the gene itself (for example, thrombosis and hemostasis regulation factors, trophic factors, growth factors, hormones like insulin, erythropoietin, and leptin, etc.) or such as an active metabolite synthesized in the muscle by addition of the therapeutic gene e.g., to correct a genetic disease by secretion of a therapeutic product;
• a therapeutic effect such as the product of the gene itself (for example, thrombosis and hemostasis regulation factors, trophic factors, growth factors, hormones like insulin, erythropoietin, and leptin, etc.) or such as an active metabolite synthesized in the muscle by addition of the
• anti-tumor genes such as tumor suppressors (retinoblastoma protein, p53, p71), suicide genes ⁇ e.g., HSV-thymidine kinase), anti-angiogenesis genes ⁇ e.g., angiostatin, endostatin, amino terminal fragement of urokinase), cell cycle blockers, apoptosis genes (such as BAX) intracellular single chain antibodies, and immunostimulatory genes.
• tumor suppressors retinoblastoma protein, p53, p71
• suicide genes ⁇ e.g., HSV-thymidine kinase
• anti-angiogenesis genes e.g., angiostatin, endostatin, amino terminal fragement of urokinase
• cell cycle blockers e.g., apoptosis genes (such as BAX) intracellular single chain antibodies, and immunostimulatory genes.
• apoptosis genes such as BAX
• this invention could be applied to the cardiac muscle for the treatment of heart diseases, e.g., using cardiac pacing to ensure safe electrotransfer (see US Patent No. 5,634,899). It could also be applied to the treatment of restenosis by the expression of genes inhibiting smooth muscle cell proliferation like the GAX protein.
• the combination of fields of low intensity and long duration of administration enhances the transfection of nucleic acids, without causing notable tissue deterioration. These results enhance the efficiency of DNA transfers in gene therapy employing nucleic acids. Consequently, the advantages associated with the invention are the production of an agent at physiologic and/or therapeutic doses either in the tissues or in proximity thereto, or secreted systemically in the blood stream or lymph circulation. Furthermore, the invention makes possible, for the first time, fine modulation and control of the effective quantity of transgene expressed by the possibility of modulating the volume of the tissue to be transfected, for example, with multiple administration sites, or even the possibility of modulating the number, shape, surface and arrangement of the electrodes.
• a particular advantage of the present invention is the excellent dose-response curve achieved for DNA transfer, which none of the prior art methods have achieved. One can thus obtain a level of transfection appropriate to the level of intra-tissue production or secretion desired. The process makes possible, finally, extra safety in relation to the chemical or viral methods of in vivo gene transfer, in which reaching organs other than the target organ cannot be totally ruled out and controlled.
• the process according to the invention makes possible the control of localization of the tissues transfected (strictly linked to the volume of tissue subjected to local electric pulses) and therefore introduces the possibility of suppressing transgene expression by the total or partial ablation of the tissue, which is possible because certain tissues are not critical or can regenerate, or both, as in the case of muscle. That great flexibility of use makes it possible to optimize the process according to the animal species (human and veterinary applications), the subject's age and his or her physiological and/or pathological condition.
• the process according to the invention further makes it possible, for the first time, to transfect large-sized nucleic acids, in contrast to viral methods, which are limited with respect to the size of a transgene by the size of the viral genome that can fit within the capsid.
• This possibility is essential for the transfer of very large-sized genes, like that of dystrophin or genes with introns and/or large-sized regulator elements, which is necessary, for example, for a physiologically regulated production of hormones. That possibility is essential for the transfer of artificial yeast episomes or chromosomes or minichrornosomes.
• Another object of the invention is linking of the electric pulses of a voltage field to compositions containing nucleic acids formulated with a view to any administration, making it possible to access the tissue by topical, cutaneous, oral, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal route, etc.
• the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, notably, for a direct injection into the desired organ, or for any other administration. It can involve, in particular, sterile isotonic solutions or dry compositions, notably lyophilized, which, with addition, as the case may be, of sterilized water of physiological saline, make possible the composition of injectable solutions.
• the nucleic acid doses used for injection as well as the number of administrations and volume of injections can be adapted to different parameters and, notably, to the method of administration, the pathology involved, the gene to be expressed or even the duration of treatment sought.
• Target Tissues The present inventors have discovered that optimum conditions for gene transfer according to the invention differ depending on the target tissue. For example, an electric field of 200 volts/cm has been found to greatly enhance gene transfer into muscle cells. Under these conditions, significant gene transfer proceeds into tumor cells as well (in specific experiments, a 3-fold enhancement of gene transfer was observed), but gene transfer into tumor cells is much more efficient in an electric field of 400 volts/cm (2 log increase in gene transfer efficiency). In further experiments, an electric field strength of 500 volts/cm was optimum for gene transfer into tumor cells.
• a system or improved apparatus can be made for delivery of nucleic acids to muscle cells (and other large cells), and a system or improved apparatus having different electric field strength parameters can be developed for delivery of genes to tumor cells (and other small cells).
• a system or apparatus of the invention will generate a voltage gradient of between 1 and 400 volts/cm, preferably 4 to 400 volts/cm, more preferably 30 to 300 volts/cm. In specific embodiments, the voltage gradient is between 100 and 200 volts/cm. Particularly contemplated are systems or apparatus that provide a voltage gradient that does not exceed 200 volts/cm.
• a system or apparatus of the invention will generate a voltage gradient of between 1 and 600 volts/cm, preferably 100 to 600 volts/cm, more preferably 400 to 600 volts/cm.
• the voltage gradient is between 400 and 500 volts/cm, and preferably about 500 V/cm.
• Particularly contemplated are systems or apparatus that provide a voltage gradient that does not exceed 600 volts/cm.
• nucleic acids can be of synthetic or biosynthetic origin, or extracted from viruses or prokaryotic cells or eukaryotic cells originating from unicellular organisms ⁇ e.g., yeasts) or multicellular organisms. They can be administered linked in whole or in part to components of the original organisms and/or system of synthesis.
• the nucleic acid can be a deoxyribonucleic acid or a ribonucleic acid. It can involve sequences of natural or artificial origin and, notably, genomic DNA, cDNA, mRNA, tRNA and rRNA, hybrid sequences or synthetic or semisynthetic sequences of oligonucleotides, whether modified or not. Those nucleic acids can by obtained by any method known to the expert and, notably, by cloning, by chemical synthesis or even by mixed methods, including chemical or enzymatic modification of sequences obtained by cloning. They can be modified chemically.
• the nucleic acid can be a DNA or an RNA with sense or antisense or catalytic property like a ribozyme.
• Antisense refers to a nucleic acid having a sequence complementary to a target sequence, e.g. , an mRNA sequence whose expression it is sought to block by hybridization on the target sequence.
• Sense refers to a nucleic acid having a sequence homologous or identical to a target sequence, e.g. , a sequence linked to a protein transcription factor and involved in the expression of a given gene.
• the nucleic acid contains a gene of interest and elements making possible the expression of said gene of interest.
• the nucleic acid fragment is advantageously in the form of a plasmid.
• Deoxyribonucleic acids can be single or double-strand, like short oligonucleotides or longer sequences. They can carry genes, sequences regulating transcription or replication or regions of linkage to other cell components, etc. Such genes can include marker genes, i.e. , genes that produce a detectable marker to study cell function, migration, or gene function; a therapeutic gene; a protective antigen or immunogen gene; and the like. According to the invention, "therapeutic gene” refers, notably, to any gene coding for an RNA or for a protein product having a therapeutic effect.
• the coded protein product can be a protein, a peptide, etc.
• This protein product can be homologous with the target cell (that is, a product which is normally expressed in the target cell when it presents no pathology).
• the transgene expression makes it possible, for example, to overcome an inadequate expression in the cell or the expression of a protein inactive or weakly active by reason of a modification, or also makes it possible to overexpress said protein.
• the therapeutic gene can also code for a mutant of a cellular protein, having an increased stability, a modified stability, etc.
• the protein product can likewise be heterologous to the target cell.
• an expressed protein can, for example, complete or introduce a deficient activity in the cell (treatment of myopathies or enzyme deficiencies), or make it possible to fight against a pathology, or stimulate an immune response, for example, in the treatment of tumors. It can involve a suicide gene (thymidine kinase of he ⁇ es) for the treatment of cancers or restenosis.
• a suicide gene thymidine kinase of he ⁇ es
• the nucleic acid preferably includes also sequences making possible and/or favoring expression in the tissue of the therapeutic gene and/or gene coding for the antigenic peptide. It can involve sequences which are naturally responsible for the expression of the gene considered when those sequences are capable of functioning in the transfected cell. It can also involve sequences of different origin (responsible for the expression of other proteins, or even synthetic ones). Notably, it can involve eukaryotic or viral gene promoter sequences. For example, it can involve promoter sequences originating from the genome of the cell it is desired to transfect. Among the eukaryotic promoters, one can use an inducer or represser sequence to provide for specific expression of the gene.
• Ubiquitous (constitutive) promoters include HPRT, vimentin, ⁇ -actin, tubulin, etcl. promoters.
• Tissue-specific promoters include (elongation factor-1- ⁇ , fit, flk) may be used.
• Inducible promoters include promoters responsive to hormones (such as steroid receptors, retinoic acid receptors, etc.), or promoters regulated by antibiotics (tetracycline, rapamycine, etc.) or other natural or synthetic molecules. It can likewise involve promoter sequences originating from the genome of a virus.
• the nucleic acid can also contain, particularly above the therapeutic gene, a signal sequence directing the therapeutic product synthesized into the secretory ducts of the target cell.
• This signal sequence can be the natural signal sequence of the therapeutic product, but it can also involve any other functional signal, or an artificial signal sequence.
• the nucleic acid can also contain a signal sequence directing the synthesized therapeutic product to a particular cellular compartment, such as, for example, mitochondria for treatment of a mitochondrial genetic disease.
• Therapeutic Genes and Gene Products Among the therapeutic products according to the invention, one can particularly mention enzymes, blood proteins, hormones such as insulin or growth hormone, lymphokines: interleukins, interferons, tumor necrosis factors (TNF), etc. (French patent No. 92 03120), growth factors, e.g. , angiogenic factors such as VEGF or FGF.
• trophic factors For treatment of neuropathies, genes encoding neurotransmitters or their precursors or enzymes that synthesize neurotransmitters, trophic factors, particularly neurotrophic factors for the treatment of neurodegenerative diseases, damage to the nervous system caused by trauma or injury, or retinal degeneration, can be delivered with a system of the invention.
• members of the family of neurotrophic factors include, but are not limited to, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), NT4/5, NT6 (including allelic variants, and members of the same gene family).
• neurotrophins include members of the ciliary neurotrophic factor family, including ciliary neurotrophic factor (CNTF), axokine, leukemia inhibitory factor; other factors include IL-6 and related cytokines; cardiotrophin and its related genes; glial-derivcd neurotrophic factor (GDNF) and related genes; and members of the insulin-like growth factor (IGF) family, such as IGF-1, IFGF-2; members of the fibroblast growth factor family, such as FGF1 (acidic FGF), FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, etc.; members of the tumor growth factor family, such as TGF ⁇ ; HARP/pleiotrophin, or bone growth factors; hematopoietic factors, etc.
• CNTF ciliary neurotrophic factor
• axokine leukemia inhibitory factor
• other factors include IL-6 and related cytokines; cardiotrophin and its related genes; glial-derivc
• genes of interest code for muscle proteins of therapeutic benefit, both secreted and non-secreted, such as dystrophin or a minidystrophin (French patent No. 91 11947), or ⁇ -1- antitrypsin.
• genes of interest code for factors involved in coagulation: factors VII, VTH, IX; suicide genes (thymidine kinase, cytosine deaminase) ; hemoglobin genes or other protein carriers.
• genes corresponding to the proteins involved in lipid metabolism can be delivered, such as an apolipoprotein type chosen among the apolipoproteins A-I, A-II, A-IV, B, C-l, C-II, C-III, D, E, F, G, H, J and apo(a), and metabolic enzymes such as, for example, lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase, 7-alpha- cholesterol hydroxylase, phosphatidyl acid phosphatase, or even lipid transfer proteins like the transfer protein of cholesterol esters and the transfer protein of cholesterol esters and the transfer protein of phospholipids, an HDL-binding protein or even a receptor chosen, for example, among the LDL receptors, chylomicron remnant receptors and scavenger receptors, etc.
• leptin for the treatment of obesity.
• One can also add the p53 anti-oncogene or other tumor suppress
• angiogenic factors include vascular endothelial growth factors (VEGF, VEGF-2, VEGF-3, platelet growth factors), and angiostatin.
• delivery can be effected of genes encoding inhibitors of angiogenesis, particularly tumor angiogenesis, such as soluble receptors of angiogenic factors, specific inhibitors of the angiogenic factor receptors (Tie2, urokinase receptor, fltl, KDR), antibodies (including single chain Fv antibodies) against angiogenic factors ⁇ e.g., anti-VEGF or anti-FGF), anti-integrin antibodies, endotheliul tumor-specific toxins, polypeptide inhibitors of angiogenesis (amino terminal fragment of urokinase - ATF, angiostatin, endostatin, interferon- ⁇ or ⁇ , interleukin-12, platelet factor 4, TNF , thrombospondin, platelet activating factor (PAI)-l, PAI2,
• variable fragments of single-chain antibodies ScFv
• any other antibody fragment possessing recognition capacities for use in immunotherapy e.g., for the treatment of infectious diseases, tumors, and autoimmune diseases such as insular sclerosis (anti-idiotype antibodies).
• proteins of interest are, without limiting them, soluble receptors like, for example, the CD4 soluble receptor or the TNF soluble receptor for anti-HIV therapy, soluble TNF receptor (particularly soluble TNF ⁇ receptor) for treatment of rheumatoid arthritis, and the acetylcholine soluble receptor for the treatment of myasthenia; substrate peptides or enzyme inhibitors, or even receptor agonist or antagonist peptides or adhesion proteins like, for example, for the treatment of asthma, thrombosis, restenosis, metastases, or inflammation (for example LL-4 to diminish Thl cells responses, IL-10 and IL-13); and artificial, chimerical or truncated proteins.
• soluble receptors like, for example, the CD4 soluble receptor or the TNF soluble receptor for anti-HIV therapy, soluble TNF receptor (particularly soluble TNF ⁇ receptor) for treatment of rheumatoid arthritis, and the acetylcholine soluble receptor for the treatment of myas
• insulin in the case of diabetes, growth hormone and calcitonin.
• immunostimulatory cytokines including IL-2, IL-12, colony stimulating factors (GM- CSF, G-CSF, M-CSF), macrophage inflammatory factors (MIP1, MIP2), dendritic cell activating factors (flt3 ligand), etc.
• genes of interest have been described by McKusick, V.A. Mendelian (Inheritance in man, catalogs of autosomal dominant, autosomal recessive, and X-linked phenotypes. Eighth edition. Johns Hopkins University Press (1988)), and in Stanbury, J.B. et al. (The metabolic basis of inherited disease. Fifth edition, McGraw-Hill (1983)).
• the genes of interest cover the proteins involved in the metabolism of amino acids, lipids and other cell constituents.
• genes related to diseases of carbohydrate metabolism like, for example, fructose- 1 -phosphate aldolase, fructose- 1 ,6-diphosphatase, glucose-6-phosphatase, lysosomal a-l,4-glucosidase, amylo-l,6-glucosidase, amylo-(l,4:l,6)-transglucosidase, muscular phosphorylase, muscular phosphofructokinase, phosphorylase-b-kinase, galactose-1-phosphate uridyl transferase, all of the enzymes of the pyruvic dehydrogenase complex, pyruvic carboxylase, 2-oxoglutarate glyoxylase carboxylase, and D-glyceric dehydrogenase.
• fructose- 1 -phosphate aldolase fructose- 1 ,6-diphosphatase
• the genes related to diseases of amino acid metabolism like, for example, phenylalanine hydroxylase, dihydrobiopterine synthetase, tyrosine aminotransferase, tyrosinase, histidinase, fumarylaceto-acetase, glutathion synthetase, g-glutamylcysteine synthetase, ornithine-d-aminotransferase, carbamoylphosphate synthetase, omithine carbamoyltransf erase, argininosuccinate synthetase, arginosuccinate lyase, arginase, L-lysine dehydrogenase, L-lysine ketoglutarate reductase, valine transaminase, leucine isoleucine transaminase, branched-chain 2- keto-acid decarboxylase, isovaleryl-
• the genes related to diseases of fat and fatty acid metabolism like, for example, lipoprotein lipase, apolipoprotein C-II, apolipoprotein E, other apolipoproteins, lecithin cholesterolacetyltransferase, LDL receptor, liver sterol hydroxylase, and "phytanic acid" a- hydroxylase.
• lysosomal deficiencies like, for example, lysosomal a-L- iduronidase, lysosomal iduronate sulfatase, lysosomal heparan N-sulfatase, lysosomal N-acetyl- a-D-glucosaminidase, acetyl-CoA : lysosomal a-glucosamine N-acetyltransf erase, lysosomal N- acetyl-a-D glucosa ine 6-sulfatase, lysosomal galactosa ine 6-sulfate sulfatase, lysosomal ⁇ - galactosidase, lysosomal arylsulfatase B, lysosomal ⁇ -glucuronidase, N-acetylglucosaminyl-
• lysosomal aspartylglucosaminidase lysosomal a-L-fucosidase, lysosomal acid lipase, lysosomal acid ceramidase, lysosomal sphingomyelinase, lysosomal glucocerebrosidase and lysosomal galactocerebrosidase, lysosomal galactosylceramidase, lysosomal arylsulfatase A, a- galactosidase A, lysosomal acid ⁇ -galactosidase, and a chain of lysosomal hexosaminidase A.
• genes related to diseases of steroid and lipid metabolism the genes related to diseases of purine and pyrimidine metabolism, the genes related to diseases of po ⁇ hyrine and heme metabolism, and the genes related to diseases of connective tissue, muscle and bone metabolism as well as the genes related to diseases of the blood and hematopoiesis, muscles (myopathy), nervous system (neurodegenerative diseases) or circulatory system (treatment of ischemia and stenosis, for example).
• myopathy myopathy
• nervous system neurodegenerative diseases
• circulatory system treatment of ischemia and stenosis, for example.
• NT3 neurotrophin 3
• ALS amyotrophic lateral sclerosis
• Treatment of this insidious disease with NT3 gene therapy is expected to be greatly facilitated by delivering the NT3 by electrotransfer, which ensures adequate, reproducible expression of this trophic factor.
• aFGF acidic FGF
• VEGF vascular endothelial growth factor
• a therapeutic nucleic acid can also be a gene or an antisense sequence, whose expression in the target cell makes it possible to control the gene expression of cell mRNA transcription.
• Such sequences can, for example, be transcribed in the RNA target cell complementing cell mRNAs and thus block their protein translation, according to the method described in European patent No. 140,308.
• the therapeutic genes also include sequences coding for ribozymes, which are capable of selectively destroying target RNAs (European patent No. 321,201).
• Immunogenic Genes and Vaccines can also contain one or more genes coding for an immunogenic or an antigenic peptide, capable of generating an immune response in man or in the animal.
• the invention therefore makes possible vaccines or immunotherapeutic treatments applied to man or to the animal, notably, against microorganisms, viruses or cancers. It can involve, notably, specific antigenic peptides of the Epstein-Barr virus, HIV virus, hepatitis B virus (European patent No. 185,573), pseudo-rage virus, "syncitia forming virus,” other viruses or even specific antigens of tumors like MAGE proteins (European patent No. 259,212), or antigens capable of stimulating an antitumoral response, such as bacterial heat shock proteins.
• the nucleic acid can be linked to any type of vectors or any combination of those vectors making it possible to enhance gene transfer, e.g. , without limitation, to vectors such as viruses, synthetic or biosynthetic agents ⁇ e.g., lipid, polypeptide, glycoside or polymer), or even balls, propelled or not.
• the nucleic acids can also be injected into a tissue which has undergone a treatment aimed at enhancing gene transfer, e.g. , a treatment of a pharmacological nature in local or systemic application, or an enzyme, permeabilizing (use of surfactants), surgical, mechanical, thermal or physical treatment.
• a treatment aimed at enhancing gene transfer e.g. , a treatment of a pharmacological nature in local or systemic application, or an enzyme, permeabilizing (use of surfactants), surgical, mechanical, thermal or physical treatment.
• EXAMPLE 1 STANDARD ELECTROPORATION CONDITIONS Standard electroporation condition, e.g., as employed in the US Patent Nos. 5,468,223,
• DNA pXL2774 (PCT/FR patent 96/01414) is a plasmid DNA containing the reporter gene of luciferase.
• the other products are available at suppliers' on the market: ketamine, xylazine, physiological saline (NaCl 0.9%).
• mice were anesthetized with a ketamine and xylazine mixture.
• the plasmid solution (30 mg/ml of a solution with 500 mg/ml of NaCl 0.9%) was injected longitudinally through the skin into the cranial tibial muscle of the left and right paws by means of a Hamilton's syringe.
• the two electrodes were coated with a conductive gel and the paw injected was placed between the electrodes in contact with the latter.
• the electric pulses were applied pe ⁇ endicular to the axis of the muscle by means of a square pulse generator one minute after the injection.
• An oscilloscope made it possible to check the intensity in volts (the values indicated in the examples represent maximum values), the duration in milliseconds and the frequency in hertz of the pulses delivered, which was 1 Hz. Eight consecutive pulses were delivered.
• mice were euthanized seven days after administration of the plasmid.
• the cranial tibial muscles of the left and right paws were then removed, weighed, placed in the lysis buffer and ground.
• the suspension obtained was centrifuged in order to obtain a clear supernatant.
• Measurement of luciferase activity was carried out on 10 ml of supernatant by means of a commercial luminometer, in which the substrate was added automatically to the solution.
• the intensity of the luminous reaction is given in RLU (Relative Luminescence Units) for a muscle experiencing the total volume of suspension.
• RLU Relative Luminescence Units
• EXAMPLE 2 ELECTROTRANSFER OF NUCLEIC ACIDS This experiment was carried out with C57 Bl/6 mice. Apart from the electric field intensity of the pulses and their duration, the performance conditions were those of Example 1. The results are shown on Figure 2. The result of Example 1 was reproduced, that is, the inhibiting effect of a series of 8 pulses at 800 volts/cm with duration of 1 msec on the luciferase activity detected in the muscle. With a field of 600 volts/cm, the same inhibition and the same alteration of the muscular tissue was observed. However, shorter pulse widths at this voltage are likely to avoid tissue damage while enhancing DNA transfer.
• the transgene expression was enhanced by a factor of 89.7 in relation to the control injected in the absence of electric pulses.
• EXAMPLE 4 200-FOLD INCREASE IN EXPRESSION This experiment was carried out in DBA 2 mice with electric pulses of 200 volts/cm of variable duration. The other conditions of this experiment are the same as in Example 3. This example confirms that at 200 volts/cm the transfection of luciferase activity increased when the pulse duration was increased from 5 msec to longer duration ( Figures 4 and 5) . A reduction of the interindividual variability indicated by the SEM in relation to the control not electrotransfected was observed. The relative value of the SEM is equal to 35% for the control and 25, 22, 16, 18, and 26% for series of pulses of 1, 5, 10, 15, 20 and 24 msec respectively. Under the optimal conditions used in this experiment, the transgene expression was enhanced by a factor of 205 in relation to the control injected in the absence of electric pulses. These results confirm that electrotransfer under the conditions described in these examples greatly improves both efficacy and reproducibility.
• Figure 5 exemplifies the importance of the parameter corresponding to the product "number of pulses x field intensity x duration of each pulse.” That parameter corresponds, in fact, to the integral dependent on time of the function which describes the variation of the electric field.
• mice mice, rats, rabbits.
• Electric pulses have to be administered after DNA injection and at the same site.
• the DNA vector can be administered up to 30 min before the pulse without noticeably decreasing the response. This allows multiple DNA injections at nearby sites followed by one single pulse.
• Electrotransfer efficiency does not depend in any way on the transgene promoter, which permits another level of control of gene expression.
• the presence of secretory or other regulatory/processing sequences in the gene product has no effect on electrotransfer efficiency.
• EXAMPLE 7 TRANSFECTION AS A FUNCTION OF PULSE DURATION This example demonstrates the effect of increasing the pulse duration on transfection efficiency under electrotransfer conditions.
• the experimental conditions were the same as those of Example 1 with C57B1/6 mice, except that a Gentronics/BTX T820 pulse generator (BTX, a division of Genetronics, San Diego, California) was used.
• the BTX pulse generator enabled application of square pulses of durations up to 100 ms.
• Plasmid pXL2774 (WO 97/10343) was injected (15 ⁇ g). It is noted in Table 1 that at a constant electric field strength of 200 V/cm, increasing the duration (T) of the pulses improves the efficiency of the transfection.
• Such a device preferably provides a pulse of 20 msec or greater, with at least 4, and more preferably 8, pulses.
• Electrotransfer conditions 200 V/cm field strength, 1 Hz frequency.
• a device for electrotransfer under the optimized field strength described in this application can further enhance the efficiency of DNA transfer by increasing the duration of the pulse. For example, increasing the duration to at least 40 msec with a series of 8 pulses, or 50 msec for a series of 4 pulses, significantly enhanced transfection efficiency at 200 V/cm. Similar optimizations of the device can be effected for other field strength.
• EXAMPLE 8 TRANSFECTION AS A FUNCTION OF THE NUMBER OF PULSES
• Example 2 The experimental conditions were the same as described in Example 1, using C57B1/6 mice. Table 2 shows that at 200 V/cm with a pulse duration of 20 ms, the efficiency of the transfection was clearly improved compared to the control group (no electric field applied), starting from a single pulse, then continues to increase when the number of pulses is increased to 2, 4, 6, 8, 12, and 16, with the optimum between 8 and 16 pulses. Also to be noted is a reduction in the variance (S.E.M.) for all the electrotransfected groups compared to the control (0 pulses).
• S.E.M. variance
• an electrotransfer device enhances the efficiency of DNA transfer with more pulses.
• a device optimally provides 4 or more, and better yet, 8 or more, pulses.
• an electrotransfer device can modulate the efficiency of nucleic acid transfer, and thus adjust the level of expression.
• EXAMPLE 10 ELECTROTRANSFECTION WITH AN EXPONENTIALLY DECREASING TIME- VARYING ELECTRIC FIELD. This example shows the effect of application of an electric field that decreases exponentially on the efficiency of nucleic acid transfer in vivo.
• Plasmid pXL3031 ( Figure 12), derived from plasmid pXL2774 by introducing modified Photinus pyralis luciferase (pGL3; Genbank accession no. CVU47295) under control of the cytomegalovirus immediate early (CMV-EE) promoter (Genbank accession no. HS5LEE) and a polyadenylation signal from SV40 virus (Genbank accession no. SV4CG), was used in this experiment. Ten ⁇ g of DNA were injected.
• CMV-EE cytomegalovirus immediate early
• the commercial electropulser (Equibio electropulsater, model EasyjectT “ Plus, Kent, UK) used here was configured to deliver exponentially decreasing time-varying electric field pulses.
• the recorded voltage applied is the voltage at the peak of the exponential.
• the second adjustable parameter is the capacitance (in ⁇ F), which controls the quantity of energy delivered.
• Table 4 shows that, when an exponentially decreasing field pulse is applied, it is possible to obtain a very clear increase in the expression of the transgene compared to the case when no field is applied. This result is obtained at different voltages and for different energies corresponding to different time constants of the exponential, which may be modulated by the adjustable capacitance of the instrument.
• the parameters established in this example can be applied to an electrotransfer device.
• EXAMPLE 11 COMBINATION OF A SHORT HIGH- VOLTAGE PULSE AND SEVERAL
• the electric field delivered may be a combination of at least one strong field between 500 and 800 V/cm for a short period, for example 50 or 100 us, and at least one weak field ( ⁇ 100 V/cm) for a longer time, for example, more than 1 ms up to 90 ms in this experiment.
• the weak electric field values here are 80 V/cm applied in 4 pulses at 1 Hz with a duration of 90 ms each.
• two commercial electropulsaters were used (Jouan and Gentronix). The delivery of the electrical voltage by one and then the other instrument to the electrode plates occurs in less than one second by modifying the operation con iguration manually.
• the luciferase-encoding plasmid used here was pXL3031 and the quantity injected was 3 ⁇ g.
• the values for the electric field strength were varied, as reported in Table 5. Otherwise, the experimental conditions were the same as described in Example 1.
• Table 5 summarizes the experiments. These data indicate that, compared to the control group (no electric field applied), one short high-voltage pulse or 4 long low- voltage pulses, or the application of weak electric field pulses before the high field pulse, did little to improve transfection efficiency. In contrast, in the experiments, the combination of a short high voltage pulse followed by 4 pulses of 80 V/cm of 90 ms duration at 1 Hz very clearly increased transfection compared to the control group. From these data, it appears that a preferred electrotransfer device would supply a series of shorter pulses at higher electric field strength
• Example 1 As shown in Example 1, supra, application of 8 pulses of 600, 800, or 1200 V/cm for 1 msec at a frequency of 1 Hz caused muscle lesions and inhibited transfection.
• the results obtained in this Example show that, under the specified conditions, it is possible to use an high voltage electric field without causing lesions. Indeed, macrospopic examination of the muscle did not evidence any visible atleration.
• Use of a high voltage field for a short time, followed by weak fields for longer time periods, provides an alternative means for modulating the efficiency of DNA transfer.
• EXAMPLE 12 KINETICS OF LUCIFERASE EXPRESSION IN MUSCLE Use of an electrotransfer device of the application permits transfection and stable expression of a nucleic acid at a high level for at least four months.
• mice C67B1/6 mice were used in this experiment.
• the mice were injected intramuscularly with plasmid pXL2774 (15 ⁇ g). Injection of the DNA was followed by application of an electric field under the following conditions: 200 V/cm; 8 pulses of 20 msec duration; 1 Hz frequency. Other conditions were as described in Example 1. Luciferase activity was determined for groups of 10 mice sacrificed at different times after injection of the DNA. Control mice were not exposed to the electric field.
• the level of expression of the transgene was stable to D121. This result is especially advantageous from the perspective of long term clinical treatment with therapeutic genes.
• the pXL2774 plasmid was modified by introduction of the lacZ gene modified with a nuclear localization signal sequence (see Mouvel et al, 1994, Virology 204:180- 189) under control of the CMV promoter obtained from plasmid pCDNA3 (Invitrogen, Netherlands), with the SV40 polyadenylation signal (Genbank accession no. SV4CG).
• the animals were sacrificed seven days after administration of the plasmid. Histologic analysis allowed detection of ⁇ -galactosidase transfected cells (Xgal histochemistry) and the inflammatory foci by alumized carmine staining and the characterization of the muscle tissue condition by hematein-eosin staining. Control mice were not exposed to the electric field. The differences between the electropermeabilized and non-electropermeabilized muscles were shown by:
• the expression area of ⁇ -galactosidase was 2 times larger in the electropermeabilized muscles (4 mm) compared to controls, with an expression gradient which decreases from the injection site. •
• the electropermeabilized muscles present a reversible number of infiltrates (macrophages and lymphocytes), numerous muscular fibers in regeneration with a nuclear centralization, and numerous necrotic fibers filled with phagocytic macrophages.
• the inflammation, necrosis, and regeneration zone corresponds to the zone around the transfected myofibrils. This response lasted up to two weeks, and reversed itself. The non-transfected part of the muscle remains in good condition.
• EXAMPLE 14 ROLE OF TIME OF INJECTION OF THE PLASMID RELATIVE TO THE TIME OF APPLICATION OF THE ELECTRICAL FIELD
• nucleic acid can be injected into tissue (in this case, muscle) at least 30 minutes, and even as long as one hour, prior to application of the electric field.
• C57B1/6 mice were injected intramuscularly with plasmid pXL2774 (15 or 1.5 ⁇ g).
• the DNA was injected up to 120 minutes before or 60 seconds after the electric field was applied.
• the time before or after injection is reported in Table 6.
• the electric field conditions used were: 200 V/cm; 8 pulses of 20 msec duration; 1 Hz frequency.
• Control mice received an injection of the plasmid but were not exposed to the electric field.
• Other experimental conditions were the same as those of Example 1.
• the data are reported in Table 6. Injection of the DNA up to one hour prior to application of the electric field resulted in achievement of increased transfection efficiency, as detected by luciferase expression.
• mice Five-week-old C57BI16 mice were injected intramuscularly bilaterally in the tibial cranial muscles with doses varying from 0.25 to 32 ⁇ g of DNA (plasmid pCOR-pXL3031) bearing the transgene luciferase for cytoplasmic expression, under the promoter CMVh at a rate of 10 mice per dose of DNA.
• the dose of DNA varied from 0.25 to 32 ⁇ g.
• one of the two legs was exposed to a field of 250 V/cm, with 4 pulses of 20 ms at a frequency of 1 Hz.
• the animals were sacrificed 5 days after the treatment and the expression of the transgene was studied in the tissue extract of each muscle according to the protocol described in Example 1.
• EXAMPLE 16 ELECTROTRANSFER WITH DIFFERENT ELECTRODES
• This Example compares the effect of electrotransfer devices equipped with one of two types of electrodes, flat plate electrodes and needle electrodes, on the efficiency of nucleic acid transfer.
• needle electrodes were tested in different orientations of implantation. Plasmid pXL2774 (150 ⁇ g) was injected in the tricep muscle of the rat. The plate electrodes were placed as described in Example 1 at an inter-electrode distance of 1.2 cm. For the needle electrodes, the inter-electrode distance was 0.9 cm. The needle electrodes were inserted for an equal length in the muscle tissue, either parallel to or perpendicular to the axis of the muscle fibers around the injection site. Regardless of the type of electrodes or their orientation, the electric field conditions were as follows: intensity of 200 V/cm; 8 pulses of 20 msec; frequency of 2 Hz.
• an electrotransfer device can employ either plate or needle electrodes, regardless of the electrode orientation relative to the target tissue.
• Use of needle electrodes may be preferred to administer nucleic acids to muscles of large size to ensure that the total voltage is moderate, for example 100 V with placement of the needle electrodes within 0.5 cm for an electric field strength of 200 V/cm.
• the plate electrodes which are noninvasive, may be preferred with small muscles, e.g., the fingers, such as for delivery of a gene therapy for arthritis.
• EXAMPLE 17 ELECTROTRANSFER INTO DIFFERENT MUSCLES AND SPECIES
• This Example illustrates that the electrotransfer device can be used to effect nucleic acid transfer into many different types of muscles in different species of animals.
• the electrotransfer device was adjusted to provide the conditions for each species as defined in Table 8. The results are shown in Table 8 as well.
• the relative increase in the level of luciferase expression using an electrotransfer device relative to control (no electrotransfer) is indicated.
• the data are the average of 10 muscles per group. Luciferase activity was determined seven days after administration of the plasmid.
• Figure 11 comprising a gene encoding fibroblast growth factor 1 (acidic fibroblast growth factor) (FGF1 or aFGF) was derived from plasmid pXL2774 in which the human fibroblast interferon signal peptide was fused to cDNA for aFGF (sp-FGFl, Jouanneau et al. , 1991, PNAS 88:2893-2897) was introduced under control of the human CMV-IE promoter and the SV40 polyadenylation signal.
• aFGF expression was determined by immuno-histochemistry. The number of positive cells (cells expressing aFGF) were evaluated three days after intramuscular injection with 500 ⁇ g of plasmid pXL3179.
• an electrotransfer device of the invention to provide for long term, stable expression of a transgene has important implications in the treatment of degenerative diseases that affect function of the diaphram, notably muscular dystrophy.
• the diaphragm was rendered accessible by an incision along the sternum after anesthesia (mixture of 1 mg/kg largactyl and 150 mg/kg ketamine).
• the injection was made in the hemidiaphragm (50 ⁇ g of plasmid pXL2774 in 50 ⁇ l of NaCl 20 mM and glucose 5%).
• the plate electrodes were then placed one on either side of the plane of the diaphragm along the injection path at an inter-electrode distance of 1 mm.
• the electric field conditions used were as follows: 160 V/cm or 300 V/cm; 8 pulses of 20 msed duration each; 1 Hz frequency.
• the electric field was applied to the muscle less than one minute after the injection.
• the incision in the animal was then closed.
• Table 10 The results are shown in Table 10.
• Table 10 Electrotransfer into rat diaphragm muscle.
• This example demonstrates a significant amelioration of expression of the transgene in the diaphram after application 820 msec pulses at a field strength of 160 V/cm (p ⁇ 0.003 using the Mann- Whitney non parametric test).
• EXAMPLE 20 ELECTROTRANSFER OF A SECRETED ALKALINE PHOSPHATASE GENE This example demonstrates the ability to transfect and express a gene encoding a secreted protein.
• Secreted proteins are used, for example, in a systemic gene therapy approach, and for generating an immune response (DNA vaccine).
• the secreted gene presented here was found in the circulation at an elevated concentration, and its presence was stable.
• the alkaline phosphatase-encoding plasmid pXL3010 ( Figure 13) was injected into one of the two tibial cranial muscles of an adult C57B1 6 mouse.
• the plasmid pXL3010 was derived from ColEl in which the gene coding for secreted alkaline phosphatase (SeAP) from pSEAP-basic (Clontech; Genbank accession no. CVU09660) was introduced under control of the CMV promoter (pCDNA3; Invitrogen, Netherlands) and the SV40 polyadenylation signal.
• the application of the electric field was performed under standard conditions, i.e., 8 square pulses of 20 msec duration, 1 Hz frequency, and 200 V/cm applied 20 seconds after injection of the plasmid.
• Measurement of the concentration of the blood serum serum alkaline phosphatase was carried out on a blood sample from the eye (retro-orbital plexus puncture) 7 days later using a commercial chemiluminescence assay (Phosphalight, Tropix, Bedford, Massachusetts, US).
• the injection of a few muscles subjected or not to the electric field with a non-encoding plasmid (ballast DNA) allowed verification of the absence of serum alkaline phosphatase that does not come from the expression of the transgene.
• This example demonstrates that a therapeutic gene can be transferred to muscle using an apparatus of the invention, and that expression of the gene product effects a detectable and meaningful physiologica response.
• the expression of erythropoietin can be detected, and the expressed protein induces an increase in the hematocrit of the recipient animal.
• C57B1/6 mice were injected in the tibial cranial muscle unilaterally with plasmid pXL3348 (Figure 16), comprising the gene coding for erythropoietin.
• the plasmid pXL3348 was derived from plasmid pXL2774 by introducing the murine erythropoietin gene (NCBI: 193086) under control of the human CMV-EE promoter and SV40 polyadenylation signal.
• the electric field 200 V/cm, 8 pulses of 20 msec duration, 1 Hz frequency was applied immediately after injection of the plasmid.
• the injection of 1 ⁇ g of plasmid was associated with a moderate increase of the hematocrit for mice conventionally transfected, and was very high for the electrotransfected mice.
• the hematocrit increased for the control group.
• the electrotransfected group the hematocrit was clearly greater, with less variance. Similar results were observed with a lower amount of DNA (1 ⁇ g).
• EXAMPLE 22 ELECTROTRANSFER OF VEGF (VASCULAR ENDOTHELIAL GROWTH FACTOR) GENE
• Plasmid pXL3212 was derived from plasmid pXL2774 by introducing the cDNA coding for VEGF (Genbank accession no. HUMEGFAA) under control of the human CMV-ES promoter and the SV40 polyadenylation signal. The electrotransfection was carried out using a commercial electropulser (Jouan) at a rate of 8 pulses of 20 msec duration, 200 V/cm, at a frequency of 2 Hz.
• Serum concentration (ng/liter) of VEGF in C57B1/6 and SCID mice was obtained from mice one day prior to injection of the plasmid.
• the experimental conditions were the same as in Example 22, except that 15 ⁇ g of the clotting factor IX-encoding plasmid pCor hFIX (pXL3388; Figure 12) was injected per muscle in C57BL6 or SCID mice.
• the pXL3388 plasmid was derived from plasmid pXL2774 by introducing the cDNA encoding human factor IX (Christmas factor; Genbank accession no. HUMFLXA) under control of the CMV-LE promoter and SV40 polyadenylation signal.
• the electrotransfer conditions were as follows: 8 pulses of 20 msec duration at 200 V/cm, 2 Hz frequency. Factor IX levels were measured seven days after injection of the plasmid. Blood samples were taken from the retro-orbital plexus in tubes containing trisodium citrate, and the tubes stored in ice.
• Table 14 shows that the human factor IX was found only in the blood of mice C57BL6 and SCID whose tibial cranial muscles were injected with pXL3388 plasmid and subjected to the application of an electric field using an electrotransfer apparatus of the invention.
• Human factor IX is not detectable in mouse blood in the absence of use of an electrotransfer apparatus of the invention.
• EXAMPLE 24 ELECTROTRANSFER OF AN ACIDIC FIBROBLAST GROWTH FACTOR (aFGF) GENE
• the experimental conditions are like those in Example 22, except for the fact that 15 ⁇ g of the FGF-encoding plasmid pCor CMV a FGF (pXL3096; Figure 14) was injected per muscle in C57BL6 or SCID mice.
• the pXL3096 plasmid was derived from plasmid pXL2774 by introduction of a triple helix forming sequence (TH; Wils et al, 1997, Gene Ther.
• the number of fibers expressing FGF in randomly-selected sections was always clearly superior for the electrotransfected muscles than for the control muscles, which received only an injection of the ⁇ XL3096 plasmid alone.
• the expression of FGF after electrotransfection reaches a maximum at D8.
• D21 and D35 the presence of FGF for the control groups is virtually undetectable whereas a large number of positive fibers were observed in the electrotransfected groups.
• the number of aFGF positive fibers, detected by immunohistochemistry, in a muscle section were determined for individual mice.
• the muscle sections were obtained from the middle of the muscle.
• aFGF as determined by the number of positive fibers revealed by immunohistochemistry, was detected almost exclusively in mice that had received treatment with an electrotransfer apparatus. Moreover, aFGF expression was detectable at the lower DNA dose as well as the higher dose.
• EXAMPLE 25 ELECTROTRANSFER OF A NEUROTROPHIC FACTOR-3 (NT3) GENE
• NT3 NEUROTROPHIC FACTOR-3
• Thepmn mice are a model for amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease) characterized by an early and rapid degeneration of motoneurons and by an average life expectancy of about 40 days.
• ALS amyotrophic lateral sclerosis
• Plasmid pXL3149 was derived from plasmid pXL2774 by introduction of the murine NT3 gene (Genbank accession no. MMNT3) under control of the human CMV-IE promoter and the SV40 polyadenylation signal. The expression -of the NT3 was studied in the supernatant prepared by centrifugation (12,000 x g) of the ground muscle in PBS buffer 7 days after the treatment of the mice, and quantified by an ELISA assay (Promega Kit). With the C57B1/6 mice received injections of 12.5 ⁇ g of plasmid DNA.
• mice Half of the mice were subjected to an electric field (250 V/cm with 4 pulses of 20 ms at a frequency of 1 Hz) immediately after injection.
• the respective 95% confidence intervals calculated for an average of 20 muscles are 77 ⁇ 11 pg/muscle when there was no electrotransfer and 2.7 ⁇ 0.9 ng/muscle with electrotransfer.
• the endogenous NT3 level was not determined.
• Similar data were found for expression of NT3 in 4 to 5 day old Xtlpmn heterozygous mice. These mice received injections of 130 ⁇ g of DNA per animal after multisite injection in different muscles (gastrocnemien, 25 ⁇ g; tibial cranial muscle, 12.5 ⁇ g).
• NT3 a basal level of NT3 was detected in the gastrocnemien and tibial cranial muscles.
• injection of plasmid pXL3149 increases the level of NT3 expression.
• utilization of an apparatus of the invention to increase transfection efficiency greatly increases the amount of transgene product expressed, both in the muscle and in plasma. This increase is especially important for NT3 expression, to achieve a neurotrophic gene therapy.
• EXAMPLE 26 ELECTROTRANSFER OF A FOR HUMAN GROWTH HORMONE GENE
• Plasmid pX13353 is derived from plasmid pXL2774 by introduction of a genomic human growth hormone gene (fragment Xbal Sph of hGH that extends from the transcription initiation signal to a BamHI site, which is 224 basepairs after the polyadenylation signal) under control of the human CMV-IE promoter and the SV40 polyadenylation signal.
• the hGH cDNA was obtaned by reverse transcription from a human pituitary mRNA library after 30 cycles of amplification using the following primers: 5' complementary oligo:
• This oligonucleotide contains a kozak Xbal sequence.
• This oligonucleotide contains an Nsil site and the stop codon.
• the amplified fragment was cloned into plasmid pCR2.1 (TA Cloning Kit, Invitrogen) and sequenced.
• An Xbal/Nsil fragment of 681 basepairs containing the hGH cDNA was ligated with the Xbal Nsil fragment of pSL3353 to generate plasmid pXL3354.
• Electrotransfer conditions were as follows: 200 V/cm; 8 pulses of 20 msec duration; 1 Hz frequency. The electric field was applied immediately after injection of the plasmid DNA. The presence of hGH was detected seven days after treatment of the mice in ground muscle supernatant in buffered PBS after centrifugation at 12,000 x g. The quantity of hGH was measured by ELISA (Boehringer Manheim).
• This Example reports that an apparatus for electrotransfer of the invention enhances delivery of genes for gene (or DNA) vaccination.
• the following products were used: VR-HA, a plasmid DNA including the gene of the hemagglutinin of the flu virus (strain A/PR/8/34).
• mVRgBDT is a plasmid DNA including the gene of the glycoprotein B (gB) of the human cytomegalovirus (Towne strain).
• the other products are available from commercial suppliers: Ketamine, Xylazine and physiological sodium chloride solution (NaCl 0.9%).
• the experiment was performed in 9- week-old female mice Balb/c. Mice originating in different cages were distributed randomly before the experiment (randomization).
• An oscilloscope and a commercial generator of electrical pulses (rectangular or square) (Electropulser PS 15, Jouan, France) were used.
• the electrodes used were stainless steel flat electrodes spaced 5 mm apart.
• the mice were anesthetized using a mixture of ketamine and xylazine.
• the plasmid solution (50 ⁇ l of a solution at 20 ⁇ g/ml or 200 ⁇ g/ml in NaCl 0.9%) was injected longitudinally through the skin into the tibial cranial muscle of the left leg using a Hamilton syringe.
• the two electrodes are coated with a conductive gel and the injected leg was placed between the electrodes in contact with them.
• the electrical pulses were applied perpendicularly to the axis of the muscle using a square pulse generator, 20 seconds after the injection.
• Antibody titers directed against flu hemagglutinin obtained after injection of 1 or 10 ⁇ g of VR-HA DNA in the absence (-) or presence (+) of an electric field provided by an electrotransfer apparatus.
• the value (p) was obtained by comparison two by two of the groups injected with DNA, then treated or not with the electric field using the non parametric Mann- Whitney test.
• Antibody titers directed against CMV glycoprotein B obtained after injection of 10 ⁇ g of VR-gB DNA in the absence (-) or presence (+) of an electric field provided by an electrotransfer apparatus.
• the value (p) was obtained by comparison two by two of the groups injected with DNA, then treated or not with the electric field using the non parametric Mann-Whitney test.
• EXAMPLE 28 ELECTROTRANSFER APPARATUS FOR TRANSFECTION OF TUMOR CELLS
• Example illustrates use of an electrotransfer apparatus to enhance delivery of nucleic acids into tumor tissue.
• an electrotransfer apparatus of the invention to provide higher voltages than are preferred for electrotransfer of nucleic acids into muscle, efficient transfection of tumor cells (and most other cells) in vivo can be effected.
• This example demonstrates effects of electrotransfer on different tumors of either human origin implanted on nude (immunodeficient) mice, or of murine origin implanted on C57B1/6 (immunocompetent) mice.
• the effects of low-intensity electric-field pulses have been demonstrated: A) on plasmid DNA transfection by intratumoral injection, and B) on secretion of a protein encoded by a transgene into plasma following intratumoral injection.
• Tumor grafts were implanted on one side of either female nude or C57B3/6 mice weighing 18-20 g.
• Human lung carcinoma (HI 299) or colon adenocarcinoma (HT29) tumors of 20 mm 3 were implanted in nude mice.
• Murine fibrosarcoma (LBP) cells (10 6 cells), or melanoma (B16) or lung carcinoma (3LL) tumors (20 mm 3 ) were implanted in C57B1/6 mice. The mice were classified according to the size of their tumors and divided into homogeneous lots.
• mice were anesthetized with a mixture of ketamine and xylazine.
• Either plasmid pXL3031 (cytoplasmic luciferase) or pXL3010 (secreted alkaline phosphatase) were injected intratumorally after the tumors reached the target volume.
• the plasmid solution (40 ⁇ l of a 250 ⁇ g ml solution of DNA in 20 mM NaCl, 5% glucose) was injected lengthwise into the center of the tumor with a Hamilton syringe.
• the lateral surfaces of the tumor were coated with a conductive gel and the tumor was placed between the two electrodes. Electrical pulses were applied using a square pulse generator, 20 to 30 seconds after the injection.
• An oscilloscope controlled the voltage intensity, the duration in milliseconds and the frequency in hertz of the 8 pulses delivered at 200 to 800 volts/cm, 20 msec and 1 hertz.
• An oscilloscope and a commercial electric-pulse (rectangular or square) generator (Electro-pulsateur PS 15, Jouan, France) were used.
• the electrodes were stainless steel plate electrodes separated by 0.45 to 0.7 cm.
• mice (generally 10 mice per experimental group, depending on conditions) were euthanized 2 days after the injection of the plasmid.
• the tumors were removed, weighed, and crushed in a lysis buffer.
• the suspension obtained was centrifuged to obtain a clear supernatant.
• Luciferase activity was measured in 10 ⁇ l of supernatant using a commercial luminometer in which the substrate was added automatically. The results were expressed in total RLUs (Relative Light Units) per tumor.
• Plasma levels of secreted alkaline phosphatase (SeAP) were measured as described in Example 20, supra, at days 1, 2, and 8 (Dl, D2, D8) after injection of the DNA.
• Results and Discussion Electrotransfer into a human lung carcinoma tumor.
• the conditions generally used for intramuscular gene electrotransfer were used: an electric field of 200 v/cm, 8 pulses at a frequency of 1 hertz, and the results obtained were compared with those obtained at higher voltages ranging from 300 to 500 volts/cm.
• the purpose of a second experiment was to determine the optimal voltage conditions that must be applied to obtain maximum transfection, or voltages ranging from 400 to 800 volts/cm. The results are shown in Table 20.
• Plasmid pXL3031 was injected into H1299 human lung carcinoma tumors that had reached the target volume of 200-300 mm 3 in female nude mice. Average values of luciferase expression with the SEM are reported.
• Electrotransfer into a human colon adenocarcinoma tumor The results of the two experiments are illustrated in Table 2.
• Table 2 the application of an electrical field with an intensity of 600 volts/cm made it possible to reach an optimal rate of transfection regardless of the transfection level without electrotransfer.
• Transfection was improved by a factor of 6 to 23-fold, respectively, and it was relatively similar from 400 to 600 volts/cm.
• Table 21 Electrotransfer into a human colon adenocarcinoma tumor
• Plasmid pXL3031 was injected into HT29 human colon adenocarcinoma tumors that had reached the target volume of 100-200 mm 3 in female nude mice. Average values of luciferase expression with the SEM are reported. The inter-electrode distance in this experiment was 0.45 cm.
• Electrotransfer into a murine fibrosarcoma tumor The results of two experiments are illustrated in Table 22. Compared to the control groups without electrotransfer, the application of an electrical field with an intensity of 300 to 600 volts/cm improved gene transfer by a factor of 30 to 70-fold, regardless of the voltage applied.
• Table 22 Electrotransfer into a murine fibrosarcoma tumor.
• Plasmid pXL3031 was injected into murine LPB fibrosarcoma tumors that had reached the target volume of 100-200 mm 3 in female C57B]/6 mice. Average values of luciferase expression with the SEM are reported.
• Electrotransfer of murine melanoma tumors The results are illustrated in Table 23. Compared to the control group without electrotransfer, the application of an electrical field with an intensity of 500 volts/cm improves gene transfer by a factor of 24-fold.
• Table 23 Electrotransfer of murine melanoma tumors.
• Plasmid pXL3031 was injected into murine B16 melanoma tumors that had reached the ttaarrggeett vvoolluummee ooff 220000--330000 r mr m 3 in female C57B1 6 mice. Average values of luciferase expression with the SEM are reported.
• Plasmid pXL3031 was injected into murine 3LL lung carcinoma tumors that had reached the target volume of 30 mm 3 after 5 days of growth in female C57B1/6 mice. Average values of luciferase expression with the SEM are reported.
• Table 25 Electrotransfer of a secreted transgene into a human lung carcinoma tumor
• Plasmid pXL3010 (expressing SeAP) was injected into human H1299 lung carcinoma tumors that had reached the target volume of 200-300 mm 3 in female nude mice. Average values of luciferase expression with the SEM are reported. A single electric field of 500 V/cm was applied, and the level of SeAP in plasma detected 1, 2, and 8 days after injection of the plasmid.
• an anti-angiogenesis gene such as the amino terminal fragment of urokinase (ATF) or angiostatin (or endostatin) would also be an effective tumor gene therapy.
• ATF amino terminal fragment of urokinase
• angiostatin or endostatin

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