CN1261812A - Device for the in vivo optimal electrotransfer of nucleic acid vectors into tissue - Google Patents

Abstract

The present invention relates to a system and device for significantly enhancing the in vivo transfer of nucleic acid vectors to cells, especially muscle cells and tumor cells, using a weak electric field to increase transfer efficiency. The device of the present invention is used to provide an optimal voltage gradient to enhance the migration of nucleic acid carriers into cells without damaging cells or tissues. These devices are characterized by a unique electrode arrangement, and a unique power limit defined by the maximum voltage setting.

Description

Device for optimal electrotransfer of nucleic acid vectors into tissues in vivo

Field of Invention

The present invention relates to the use of a weak electric field to increase the efficiency of transfer to significantly enhance the transfer of nucleic acid vectors into cells, especially in vivo in muscle cells. In particular, the invention relates to methods, devices and compositions for effecting such nucleic acid vector transfer for gene therapy. The device of the present invention is used to provide an optimal voltage gradient to enhance the migration of nucleic acid carriers into cells without damaging cells or tissues. These devices are characterized by a unique electrode arrangement, and a unique power limit defined by the maximum voltage setting.

Background of the Invention

Vectors and methods for gene delivery

The transfer of genes into specific cells is the basis of gene therapy. One problem, however, is the introduction of sufficient amounts of nucleic acid into the host cells to be treated. The gene of interest must be expressed in the transfected cells. One method employed in this regard is the integration of nucleic acids in viral vectors, especially in retroviruses, adenoviruses or adeno-associated viruses. These systems exploit the cellular invasion mechanisms that viruses develop, as well as the protection against degradation. However, this method also has problems. One is the risk of producing infectious virus particles that are prone to spread in the host organism and, in the case of retroviral vectors, of insertional mutagenesis. Furthermore, the ability to insert therapeutic or vaccine genes into viral genomes is still limited. Finally, an immune response is often generated against the viral vector, which renders re-administration of the virus ineffective due to immune clearance and can also cause inflammation.

Another method (Wolf et al., Science 247, 1465-68, 1990; Davis et al., Proc. Natl. Acad. Sci. USA 93, 7213-18, 1996; Felgner et al. U.S. Patent No. 5,580,859) involves administering a plasmid-type nucleic acid into muscle or bloodstream, whether or not it is linked to a compound that facilitates its transfection, such as a protein, liposome, charged lipid, or cationic polymer such as Polyethyleneimine, they are good in vitro transfection agents (Behr et al., Proc. Natl. et al., Proceedings of the National Academy of Sciences USA 92, 7297-301, 1995; US Patent 5,676,954 and European Patent 425475).

With regard to muscle, since the original publication by Wolff et al., supra, showing the ability of muscle tissue to take up DNA injected as episomal plasmids, many investigators have sought to refine this method (Manthorpe et al., 1993, Human Gene Therapy (Human Gene Ther.) 4, 419-431; Wolff et al., 1991, BioTechniques 11, 474-485). Certain trends have emerged from these trials, in particular:

· Application of mechanical means to get DNA into cells by attaching DNA to a ball and then propelling it into tissue ("gene gun") (Sanders Williams et al., 1991, Proc. Natl. Acad. Sci. USA 88, 2726-2730; Fynan et al., 1993, Biotechnology 11, 474-485). These methods have been shown to be effective in inoculation strategies, but only in contact with superficial tissue layers. For muscle, its application requires a surgical approach to provide access to the muscle, as the particles cannot penetrate the skin tissue;

• Injection of DNA, which is no longer in the form of an episomal plasmid, but linked to a molecule that can be used as a carrier to facilitate the entry of the complex into the cell. Cationic lipids used in many other transfection methods have so far been clearly disappointing in application to muscle, since those tested lipids proved to inhibit transfection (Schwartz et al., 1996, Gene Therapy (Gene Ther. )405-411). The same is true for cationic peptides and polymers (Manthorpe et al., 1993, Human Gene Therapy 4, 419-431). The only examples of favorable combinations appear to be mixtures of polyvinyl alcohol or polyvinylpyrrolidone with DNA. These combinations produced enhancements of only less than 10-fold relative to naked DNA injection (Mumper et al., 1996, Pharmaceutical Research 13, 701-709);

Pretreatment of the tissue into which the solution is injected in an attempt to enhance DNA diffusion and/or stability (Davis et al., 1993, Human Gene Therapy 4, 151-159) or to facilitate the entry of nucleic acids, for example, the induction of cellular proliferation or regenerative phenomena . The treatment involves inter alia the application of local anesthetics or cardiotoxins, vasoconstrictors, endotoxins or other molecules (Manthorpe et al., 1993, Human Gene Therapy 4, 419-431; Danko et al., 1994, Gene Therapy 1, 114-121 ; Vitadello et al., 1994, Human Gene Therapy 5, 11-18). These pretreatment regimens are difficult to control. Bupivacaine, in particular, must be used at concentrations very close to the lethal dose in order to be effective. Pre-injection of hypertonic sucrose in an attempt to enhance diffusion failed to increase transfection levels in muscle (Davis et al., 1993).

Other tissues have been transfected in vivo by using plasmid DNA alone or by ligation with synthetic vectors (review by Cotten and Wagner (1994), Current Opinion in Biotechnology 4, 705; Gao and Huang (1995), Gene Therapy, 2, 710; Ledley (1995), Human Gene Therapy 6, 1129). The primary tissues studied are the liver, respiratory epithelium, vessel walls, central nervous system, and tumors. In all these tissues, the level of transgene expression proved to be too low to foresee therapeutic applications (for example, for the liver, see Chao et al. (1996), Human Gene Therapy 7, 901), although recent studies of Metastasis has had some encouraging results (Iires et al. (1996) Human Gene Therapy 7, 959 and 989). Metastasis is very inefficient in the brain, as in tumors (Schwartz et al., 1996, Gene Therapy 3, 405; Lu et al., 1994, Cancer Gene Therapy 1, 245; Son et al., US National Proceedings of the Academy of Sciences 91, 12669).

        Electroporation and Iontophoresis for Gene Delivery

Electroporation, or the application of an electric field to permeabilize cells, is commonly used in vitro to facilitate DNA transfection in cultured cells. This phenomenon is dependent on reaching a threshold electric field strength. For animal cells, electropermeabilization is observed at relatively high electric fields of about 800-1200 volts/cm. It has also been proposed to perform this technique in vivo in order to enhance the efficacy of antineoplastic agents such as bleomycin in human solid tumors (US Patent No. 5,468,228, L.M. Mir). Applying pulses of very short duration (100 microseconds), these electrical conditions (800-1200 volts/cm) are well suited for intracellular transfer of small molecules. Application of these conditions (pulses of 100 microseconds) did not lead to enhanced in vivo transfer of nucleic acids into the liver, compared to DNA injection without electric pulses, demonstrating that electric fields below 1000 V/cm were completely ineffective or even inhibitory (patent WO 97/07826 and Heller et al., FEBS Letters, 389, 225-8, 1996).

Furthermore, for in vivo applications, this technique presents difficulties because the use of such strong electric fields can cause extensive tissue damage. Target tissue damage is not a problem in tumor treatment of cancer patients, but can be a major drawback when administering nucleic acids to non-tumor tissue, especially striated muscle.

There are three basic types of systems used to deliver electrical pulses in electroporation and iontophoresis: external electrodes, internal electrodes (including catheters), and a combination of external and internal electrodes.

External electrodes

In one type of device, electrodes are placed external to the patient. See, eg, U.S. 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; Using an external electrode arrangement, the electrodes are in contact with the patient's superficial tissue region. The use of electrodes in the device may be non-invasive on the patient's skin, or invasive through surgically exposed organ surfaces.

The Hofmann '514 patent discloses a device for implanting macromolecules such as genes, DNA or drugs into preselected areas of surface tissue in a patient. The device has a head assembly which, in a first embodiment, includes serpentine conductors on open cell elastomer, both supported on a generally planar support member. Adjacent parallel sections of the serpentine conductor serve as electrodes. To administer electrical pulses to a patient, the head assembly is placed in contact with a preselected area of surface tissue on the patient and the conductors are placed in contact with the skin. By delivering fluid to the elastomer that can be absorbed by the elastomer, the liquid medium carrying the macromolecules is transferred to the patient's skin. A switch is then pressed to deliver a high-voltage pulse from the signal generator to the electrodes, creating an electric field between them. The depth of the electric field into the skin is proportional to the gap between the electrodes. The electric field injects fluid into the tissue region.

In another embodiment, the head assembly includes a plurality of fine needles extending generally perpendicular to the planar support member. These pins are arranged in rows and staggered to connect to the output of the signal generator so that each pin is adjacent to another pin of opposite polarity. These needles penetrate the outermost layer of skin cells, allowing electrical pulses to be delivered to the targeted area.

The Hofmann '440 patent discloses a device comprising adjustable spacing electrodes for generating an electric field. The electrodes are mounted on a moveable linkage so that the user can manipulate the electrodes to move toward or away from each other like jaws. During operation, the electrode clips are opened and the selected tissue to be treated is clamped between the electrode clips. A suitable switching device is used to control a signal generator connected to the electrodes to generate an electric field in the tissue between the electrodes.

                               ,

A second type of electrotransfer system utilizes implantable or insertable electrodes that are placed within the patient's body to deliver an electric field to the area adjacent to the implanted/inserted electrode. See, eg, 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; the disclosures of which are incorporated herein by reference.

The Crandell '120 patent discloses a catheter that is inserted into a blood vessel selected by a patient. The catheter includes a plurality of axially extending, circumferentially spaced electrodes that contact the inner wall of the blood vessel. A liquid medium containing macromolecules is then injected into the blood vessel adjacent to the electrodes, and the electrodes are energized to apply predetermined electrical signals for electrotransfer. The spaced electrodes can be serpentine strips or parallel strips that generate the desired electric field when energized.

The Hofmann '724 patent is another example of a catheter-based electrotransfer device having spaced apart electrodes located on the outside of a catheter that is inserted into a blood vessel in contact with the vessel wall to be treated.

The Hofmann '662 patent discloses a pair of spaced apart electrodes mounted in a cylindrical dielectric carrier. The electrodes are positioned around the center of the blood vessel at a predetermined consistent distance from each other and close to the center of the blood vessel so that blood flow in the blood vessel passes between the electrodes. A cylindrical dielectric carrier is surgically implanted into the surrounding blood vessel. Applying a predetermined electrical signal to the electrodes creates an electric field in the blood flow between the electrodes.

The Hofmann '525 patent discloses a dual needle injector in which the needles serve as electrodes for electrotransfer. Once the needle is inserted into the target area, an electrical signal is applied to the electrodes to release an electric field to the target area. The Hofmann '359 patent also discloses needle-based electrodes for electrotransfer.

Combination of external and internal electrodes

A third class of electrotransfer devices combines features of the systems described above. These devices utilize at least one internal electrode and at least one external electrode to deliver an electric field to a desired tissue region. See, eg, US Patent Nos. 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 device with a drug delivery wall (e.g., a wall made of a permeable or semipermeable material capable of passing a drug or other macromolecule). ) with a drug lumen catheter and an electrode located inside the catheter opposite the drug delivery wall. A second electrode is placed on the distal end of the patient's skin. A liquid containing a macromolecule of interest is delivered into a drug chamber placed in an electric field generated between the two electrodes when an electric current is supplied. In this way, macromolecules are delivered to the target area.

Other features disclosed in the aforementioned patents include: reversing the polarity of the electrodes to expel excess macromolecules in the opposite direction from that in delivery (eg, the Shapland '238 and Shapland '785 patents); to avoid electrically induced arrhythmias or abnormal cardiac rhythm, a system that synchronizes the delivery of current to the electrodes with the depolarization of the heart's ventricles (eg, Feiring, U.S. Patent Nos. 5,236,413 and 5,425,703 and Shapland, U.S. Patent No. 5,634,899); and the use of an ultrasonic piezoelectric transducer instead of The electrodes generate sound waves as the driving force for macromolecular transport, known as acoustophoresis (eg, the Shapland '238 and Shapland '730 patents).

Invention Summary

Although all of the studies cited mention the need for high electric fields (approximately 1000 V/cm) to be effective in vivo, applicants have now demonstrated, most unexpectedly and strikingly, that by applying low intensities (below 600 V/cm cm) such as 100-200 volts/cm and relatively long duration electrical pulses substantially enhance the in vivo transfer of nucleic acids into tissues without undue effect. Furthermore, Applicants found that the method according to the invention significantly reduces the high variability in the expression of plasmid-borne transgenes observed in the prior art of DNA transfer into muscle.

Accordingly, the present invention relates to a method and device for in vivo nucleic acid transfer into a tissue, such as one or more striated muscles or a tumor, wherein tissue cells are contacted by direct administration into the tissue or by local or systemic administration. nucleic acid, and transfer is ensured by applying one or more electrical pulses to the tissue at an intensity of 1-400 volts/cm for muscle and 1-600 volts/cm for tissue such as a tumor.

Accordingly, the present invention provides a system, such as an improved device, for in vivo nucleic acid transfer into cells of a multicellular eukaryote, wherein the tissue cells are contacted by direct administration into the tissue or by local or systemic administration. The transferred nucleic acid is ensured by applying to the tissue one or more electrical pulses delivered by the device of the invention configured to provide a specific intensity. In particular, the electric field strength may be 1-600 volts/cm for delivery of nucleic acids to tumor cells and 1-400 volts/cm for delivery of nucleic acids to muscle cells. The system (or device) of the present invention comprises an electrical pulse generator (or means for generating electrical pulses), wherein the electrical pulse generator generates pulses with a duration of more than 1 millisecond and an intensity of 1-400 or 1-600 volts/cm, an electric pulse with a frequency of 0.1-1000 Hz; and an electrode connected to the electric pulse generator for generating an electric field in the tissue in contact with the electrode body. In a specific embodiment, the electrical pulse generator generates pulses at an intensity of 30-300 V/cm (for transfer into muscle) and 400-600 V/cm for transfer into tumor cells and other small cells. pulse. In another specific embodiment, the electrical pulse generator generates pulse times greater than 10 milliseconds. In yet another specific embodiment, the electrical pulse generator generates 2-1000 pulses.

According to the invention, the electrical pulse generators of the system or modified device are capable of pulsating irregularly with respect to each other, whereby the function describing the field strength as a function of the pulse duration is variable, with the proviso that the system or device Electric fields above (or below) the above parameters are never applied. For example, the integral of the function describing the change in electric field with time may be greater than 1 kV·msec/cm; in another embodiment greater than or equal to 5 kV·msec/cm.

The electrical pulse generator (pulse generator) is capable of generating pulses selected from square wave pulses, exponentially decaying waves, short-duration oscillating monopolar waves, and short-duration oscillating bipolar waves. Preferably, the electrical pulse generator generates square wave pulses.

The present invention contemplates a variety of electrode configurations. For example, the electrode may be an external electrode placed on the tissue to be treated, eg, for the transfer of nucleic acids into cells of the patient's surface tissue. Alternatively, the electrode may be an internal or tissue penetrating electrode that is implanted in the tissue to be treated. This internal electrode can be a needle and can be assembled as an injector system, enabling the simultaneous administration of nucleic acid and electric field. In another embodiment, the invention provides both external electrodes and internal electrodes.

An external electrode of the present invention may be sized to contact an external portion of the patient's body adjacent to a large muscle. In a particular embodiment, the electrode is a plate electrode; in another embodiment, it is a semi-cylindrical plate electrode.

In yet another embodiment, the electrode is an intra-arterial or intravenous electrode, eg, the improved flexible catheter device according to the invention.

A preferred material for the electrodes of the present invention is stainless steel.

The improved arrangement of the present invention can be produced by modifying prior art devices, particularly the means used to generate the electric field in these devices, to generate the electric field of the present invention. For example, the means for generating electrical pulses can be adapted to generate pulses of 1-400 or 1-600 volts/cm by modifying the voltage gate so that it does not exceed a voltage corresponding to 400 or 600 volts/cm. In a particular embodiment of this improved device, the voltage can be set at a constant voltage and the electrodes placed at a constant spacing. Furthermore, the electrical pulse generating means can be adapted to generate pulses of 1-400 or 1-600 volts/cm by labeling the device so that it does not exceed a voltage corresponding to 400 or 600 volts/cm.

It is therefore an object of the present invention to provide a system, or modification of existing devices, to provide an electric field with a voltage gradient, pulse width and pulse number that have been found to be optimal for nucleic acid transfer without damaging tissue.

A particular object of the present invention is the electrotransfer of nucleic acids under mild and low damaging conditions used for electroporation (voltage gradients exceeding 600 V/cm, usually exceeding 1000 V/cm).

Yet another specific object of the present invention is to provide much more efficient intracellular delivery of nucleic acids than can be achieved under the very low strength electric fields used in iontophoresis.

Yet another advantage of the present invention is to provide efficient, reproducible nucleic acid delivery to muscle cells.

These and other objects of the invention have been achieved as described above, and as described in more detail in the Detailed Description of the Invention and in the Examples with Drawings.

Brief description of the attached drawings

Figure 1: Effect of high-field-strength electrical pulses on transfection of plasmid DNA pXL2774 in mouse cranial tibialis muscle; mean ± SEM.

Figure 2: Effect of MSI pulses on transfection of plasmid DNA pXL2774 in mouse cranial tibialis muscle; mean ± SEM.

Figure 3: Effects of electric pulses of weak field strength and different durations on the transfection of plasmid DNA pXL2774 in the cranial tibialis muscle of mice; mean ± SEM.

Figure 4: Effects of electric pulses with weak field strength and different durations on the transfection of plasmid DNA pXL2774 in the cranial tibialis muscle of mice; mean ± SEM.

Figure 5: Efficiency of electrotransfer of plasmid DNA pXL2774 in mouse cranial tibialis muscle at low electric field strength; mean ± SEM.

Figure 6: Kinetics of luciferase expression in mouse cranial tibialis muscle. Administration of plasmid pXL2774 with (■) and without (x) electrotransfer; mean ± SEM.

Figure 7: Expression levels as a function of dose of DNA administered with (•) and without (□) electrotransfer.

Figure 8: Effect of different types of electrodes on electrotransfer efficiency.

Figure 9: Kinetics of serum concentrations of secreted alkaline phosphatase. Administration of plasmid pXL3010 with (■) and without (◆) electrotransfer; mean ± SEM.

Figure 10: Kinetics of aFGF expression in muscle with (open bars) or without (closed bars) electrotransfer.

Figure 11: Maps of plasmids pXL3179 and pXL3212.

Figure 12: Maps of plasmids pXL3388 and pXL3031.

Figure 13: Maps of plasmids pXL3004 and pXL3010.

Figure 14: Maps of plasmids pXL3149 and pXL3096.

Figure 15: Maps of plasmids pXL3353 and pXL3354.

Figure 16: Map of plasmid pXL3348.

                      Invention Details

As noted above, the present invention provides greatly enhanced in vivo nucleic acid transfer into tissue by applying low intensity electrical pulses to the tissue. For example, electric fields below 600 volts/cm have been found to enhance the transfer of nucleic acids into tumors, while in muscle it is below 400 volts/cm, preferably 100-200 volts/cm for electrodes placed about 0.5-1 cm apart. These electric fields are applied for relatively long durations. Furthermore, Applicants found that the method according to the invention significantly reduces the high variability of transgene expression observed in the prior art of DNA transfer into muscle. Finally, expression has been found to persist for extended periods of time, for example, more than 60 days. In a specific example, high levels of expression were detected within 63 days.

As readily ascertainable from the description provided herein, Applicants refer to the in vivo transfer of nucleic acid into cells under these conditions as "electrotransfer"; another suitable term used herein is "electroporation". These two terms distinguish the optimal conditions for nucleic acid transfer from "electroporation" (using electric fields above 800 V/cm) and iontophoresis (using electric fields of very low strength).

Accordingly, the present invention relates to methods, systems and devices (or devices) for in vivo nucleic acid transfer into tissues, especially striated muscle, and compositions wherein tissue cells are contacted by direct administration into the tissue or by local or systemic administration. Nucleic acid to be transferred, and transfer is ensured by applying one or more electrical pulses to the tissue at an intensity of 1-600 volts/cm (for example, for tumor cells) and 1-400 volts/cm for muscle cells. In other words, the present invention particularly relates to systems (ie, devices or devices) for electrotransfer.

According to a preferred embodiment, the method of the invention is applicable to tissues whose cells have a particular geometric shape, e.g. cells of large and/or elongated shape, and/or are naturally responsive to electrical potentials, and/ or have a special shape.

For muscle, the field strength is preferably 4-400 volts/cm, and for tumors up to 600 volts/cm, for a total duration of application in excess of 1 millisecond (msec), preferably 10 msec. In certain instances, the total duration is 8 msec or more. In many instances, the pulse duration is 20msec, and durations in excess of 40msec have been found to be effective. The number of pulses used is, for example, 1-1000 pulses, preferably 2-100, more preferably 4-20, and the pulse frequency is 0.1-1000 Hertz (Hz); more precisely 0.2-100 Hz. In certain embodiments, frequencies of 2 Hz, 3 Hz and 4 Hz were found to be effective. Pulses can also be released irregularly, and the function describing the time-dependent field strength is variable. The integral of the function describing the change of the electric field with time is greater than 1 kV·msec/cm. According to a preferred embodiment of the invention, this integral is higher than or equal to 5 kV·msec/cm. It should be noted, however, that those of ordinary skill in the art will readily understand that the integral function must be achieved at the sub-electrophoretic voltages described above.

In a particular example starting from the invention, the device of the invention can provide a combination comprising at least one high voltage pulse (higher than 400 V/cm, preferably 500-800 V/cm) of short duration (less than 1 msec) ), followed by one or more longer pulses (over 1 msec) of much lower electric field strength (below 200 V/cm).

According to a preferred embodiment of the invention, the field strength of the pulses is 30-300 V/cm.

The electrical pulses are selected from square wave pulses, generated exponentially decaying waves, short oscillating monopolar waves, short oscillating bipolar waves, or electric fields of other waveforms. According to a preferred embodiment of the invention, the electrical pulses are square wave pulses.

Administration of electrical pulses can be performed by any method known in the art, such as:

A system of external electrodes placed on both sides of the tissue to be treated, in particular, non-invasive electrodes in contact with the skin,

· Electrode systems implanted in tissue,

• Electrode/syringe system supporting simultaneous administration of nucleic acid and electric field.

For administration performed in vivo, it is sometimes necessary to resort to intermediate products to ensure electrical continuity of non-invasive external electrodes. For example, this includes an electrolyte in the form of a gel. Examples of suitable gels include the gels used in the Examples below, as well as gels commonly used in medicine to enhance electrical contact, such as for electrocardiograms or defibrillators.

The nucleic acid can be administered by any suitable method, but preferably is administered in vivo directly into the tissue, or by another local or systemic route which presents the nucleic acid at the site of application of the electric field. Nucleic acids may be administered with agents that permit or facilitate transfer, as previously described. In particular, these nucleic acids can be free in solution or linked to synthetic reagents or carried by viral vectors. Synthetic agents can be lipids or polymers known to the skilled person, or even targeting elements that enable immobilization on target tissue membranes. Among these elements, mention may be made of carriers carrying sugars, peptides, antibodies, receptors and ligands.

It is conceivable that, under the conditions of the present invention, the application of the nucleic acid may precede, be concurrent with or even follow the application of the electric field, of course continuing to apply the electric field after the application of the nucleic acid.

The invention also relates to a nucleic acid and an electric field with a strength of 1-600 V/cm (preferably 400 V/cm) as a combined product for simultaneous, separate or In vivo administration is time-staggered. For transfer into muscle, the field strength is preferably 4-400 Volts/cm, more preferably 30-300 Volts/cm. For transfer into tumors and cells with similar electrotransfer receptive properties, the preferred field strength is 400-600 V/cm; preferably about 500 (ie, 500 ± 10%, preferably 5%) V/cm. Those of ordinary skill in the art will readily appreciate that this combination defines a nucleic acid structure in which the nucleic acid adopts a specific orientation relative to an electric field in the presence of an electric field and possesses specific secondary and tertiary structures. In addition, the DNA is associated with extracellular components found in the target tissue, which can be distinguished from the DNA by low-field electrophoresis under agarose gel or other laboratory conditions.

                  Electrotransfer Systems and Devices

The essential elements of any electrotransfer system (ie, device or device; these terms are used interchangeably herein) consist of an electrical pulse generator and electrodes designed or modified to deliver pulses not exceeding 600 V/cm. In particular, systems or devices of the invention for delivering nucleic acids to muscle provide pulses of no more than 400 volts/cm. Naturally, the actual voltage depends on the distance between the electrodes. It is well known in the art that this distance affects the resistivity through the target tissue. Therefore, the actual voltage applied depends on the resistance and thus the current, keeping the total power within acceptable levels. The term "acceptable level" as used herein means that the total power does not cause irreversible tissue damage, especially tissue burns. Thus, in a preferred aspect, the device of the invention either controls acceptable current by setting the voltage and electrode distance, or includes a feedback device to prevent the application of a voltage that is too high for the distance between the electrodes, thereby applying too much large current. The system of the present invention may include an oscilloscope or other metering device to monitor voltage, current, or both.

In one embodiment, the system of the invention is prepared using commercially available equipment. Preferably, such devices are modified to provide the particular electrotransfer conditions determined herein to be optimal. In another embodiment, a new apparatus is designed and fabricated to accomplish the objects of the present invention. Design specifications for improved or manufactured pulse generators include, but are by no means limited to, the introduction of mechanical or electrical controls to maintain a desired voltage gradient, i.e., for application to muscle, below 600 or 400 V/cm, preferably low at 200V/cm. For example, a mechanical controller may include a stop on the voltage selection dial to prevent selection of a voltage that would generate an excessively high electric field. Furthermore, it is also possible to manufacture or modify the device so that this voltage cannot be selected. In yet another embodiment, the device may include a circuit breaker or fuse that opens when the voltage (and thus current) exceeds the parameters of the present invention. In yet another embodiment, the microprocessor controller can prevent or overcome the selection of excessive voltage. In yet another embodiment, the pulse generator is simply modified by using a label directing the use of a specific voltage range that provides the electric field strength of the present invention. All such modifications are routine in the art and utilize standard electrical and mechanical techniques.

As noted above, the actual voltage delivered by the system of the present invention to achieve the electric field strength determined herein to be optimal depends in part on the electrode spacing. If the electrodes are spaced apart in a fixed manner, the voltage (for a defined tissue, eg muscle, liver, heart or tumor) may be a predetermined constant value. However, if it is desired to vary the spacing of the electrodes, the voltage must be adjusted to maintain a constant voltage gradient. It can be determined by measuring the distance between the electrodes, introducing a counter-electrode measuring device which provides a (voltage) value for the adjusted spacing, or using an automated measuring device which feeds back to the pulse generator to automatically provide a corrected voltage (cf. Hofmann US Patent 5,439,440).

The following sections more fully describe prior art pulse generators, electrodes and devices that may be modified in accordance with the present invention.

                     

Pulse generators (also known as "voltage generators" and "pulse or voltage generating devices") are electrical devices that generate current at a specified voltage, duration, pulse width, duty cycle (pulse and total time of rest) and pulse frequency . Such devices are well known in the art and include commercially available pulse generators such as the ELECTRO CELL MANIPULATOR ECM600, 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 hereby incorporated by reference in its entirety. Additionally, the pulse generator may be an Electropulsator PS 15 available from Jouan, France, as disclosed in the Examples below. In yet another embodiment, a voltage generator capable of generating one or more waveforms as described in US Pat. No. 5,634,899, which is hereby incorporated by reference in its entirety, may be used. This voltage (generator) can be used to generate pulses of variable shape, intensity and duration. For example, a pulse of 200 V/cm or 400 V/cm, 5-20 msec, followed by a longer pulse of lower intensity. The device can further provide an iontophoresis electric field in combination with the electric field of the present invention.

The pulse generator of the present invention has the following specifications:

• Generating a voltage gradient of 1-600 or 1-400 V/cm, preferably 4-400 V/cm, more preferably 30-300 V/cm. For electrotransfer into muscle, the specific voltage gradient contemplated by the device of the present invention is approximately 100 V/cm and 200 V/cm; preferably below 200 V/cm. For electrotransfer into tumor cells or similar cells, it was surprisingly found that an electric field of 400-600 V/cm, optimally about 500 V/cm, is preferred.

• Pulse frequency from 0.1 to 1000 hertz (Hz); in a particular embodiment, about 2 Hz or higher, up to 10 Hz; preferably higher than 1 Hz. In a preferred embodiment, the frequency is 3 Hz or 4 Hz.

• Pulse time (duration) longer than 1 millisecond (msec), with variable on-time; preferably pulse time longer than 5 msec; more preferably longer than 10 msec; more preferably longer than 20 msec.

In a preferred aspect, the pulse generator of the present invention generates at least two, preferably 4, 6 or 8 pulses. For example, it can generate 8-1000 pulses. If the patient begins to feel unwell or if the field strength becomes uncontrollable, the pulse generator should be able to be stopped or turned off. This stopping can be manual or automatic, or both.

It will be readily apparent to those of ordinary skill in the art that an external signal, such as another device, computer, etc., can generate the pulse. For example, for the delivery of nucleic acids to cardiac tissue, the pulse generator is preferably linked to the patient's electrocardiogram to synchronize the pulses with the heartbeat. Such systems preferably include active pacing of the patient's heart rhythm, such as with a pacemaker (see US Patent No. 5,634,899 to Shapland).

Electrodes

The electrodes of the present invention provide an electric field in tissue. The cathode is negatively charged; the anode is positively charged. Generally, according to the present invention, there is a net flow or flow of ions from one electrode to the other (the flow will of course depend on the net charge of the ionic species and the polarization of the electrodes). In general, nucleic acids with a strong net negative charge will move towards the anode.

Electrodes used in accordance with the invention must conduct electricity efficiently and preferably be inert, non-reactive and non-toxic under the conditions used. In particular, electrodes used internally should not react to any appreciable extent with biological matter, for example, to avoid release of potentially harmful or toxic metal ions from the electrodes, or to avoid oxidation that reduces the efficiency of the electrodes. A preferred material for the electrodes of the present invention is stainless steel, which is relatively non-reactive, relatively efficient in conducting electricity and cheap enough to be manufactured at reasonable cost. More desirable electrodes, especially for internal use, are gold or platinum. However, such noble metal electrodes are very expensive. The cost can be reduced by plating these materials on 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 cannot be used internally. Electrodes can also be formed from zirconium, iridium, titanium and some forms of carbon.

Certain electrodes such as silver and copper have antimicrobial activity, which is desirable for internal application to inhibit infection.

Electrodes can be formed in any configuration suitable for the target tissue, including without limitation: straight wires, coiled wires (straight and coiled wire electrodes are ideal for catheter applications), conductive surfaces such as catheters or balloons Conductive surface of catheter; see US Patent No. 5,704,908, incorporated herein by reference in its entirety), metal strip, needle (or stylet), array 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. In a specific embodiment, a needle electrode can be used with a syringe to deliver DNA. Such a needle electrode may have a hole through its axial length, allowing nucleic acid solution to be delivered across its length. For delivery of nucleic acids to large organs, especially large muscles, two surface electrodes can be used. Surface electrodes are preferably used in combination with an electrolyte composition, as described above, to ensure good contact and conductivity, eg through the skin.

In a particular embodiment where there is an inner electrode and an outer electrode, the outer electrode may have "heads" disposed around the inner electrode. In fact, one electrode may have multiple "heads" in general for any of the configurations described above.

The present invention further contemplates an arrangement of electrodes; a needle with a hole along the axis; a needle with a defined and calibrated conductive length (for providing a constant and repeatable conductive zone into the tissue, regardless of the depth the needle penetrates into the tissue ) with its upper and lower halves electrically isolated; a needle with an isolated point to prevent point-to-point arcing into the tissue; and any kind of pouch/reservoir containing the product around one needle.

As described above for needle electrodes, plate electrodes may include insulated edges.

Typically, the electrodes are arranged such that the target tissue is directly between them. In this way, the nucleic acids are subjected to the highest field strength. However, since the electric field flows around the electrodes, it is possible to use electric fields generated at the periphery between the electrodes as well as electric fields generated directly between the electrodes.

Improved device of prior art

In a particular embodiment, a device in which electrodes are external to the patient is modified in accordance with the present invention (see, e.g., U.S. Pat. 5,688,233; and 5,019,034 by Weaver et al.), ie, providing an electric field under specified conditions to provide an improved device of the present invention. The improved device can be used non-invasively by applying electrodes to the patient's skin, or invasively by applying electrodes to the surface of an organ that has been surgically exposed.

Likewise, an electrotransfer system can be modified according to the present invention, resulting in an improved device of the present invention, which electrotransfer system utilizes implantable or insertable electrodes placed in the patient to implant/insert adjacent electrodes, especially The region of the catheter electrode releases an electric field (see, eg, Crandell et al., US Patent Nos. 5,304,120; Hofmann et al., 5,507,724; Hofmann et al., 5,501,662; Hofmann et al., 5,702,359; and Hofmann, et al., 5,273,525).

Naturally, an electrotransfer device can be modified according to the invention as follows, providing an improved device according to the invention, which combines the features of the system described above, for example, which uses at least one internal electrode and at least one external electrode to deliver an electric field to a desired tissue region (see, eg, U.S. Patent Nos. 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).

Gene therapy using electrotransfer

The method according to the invention can be used in gene therapy, ie where the expression of the gene is transferred and the regulation or blockade of the gene makes it possible to ensure the treatment of a particular disease.

Tissue cells are preferably treated for the purpose of gene therapy, which makes it possible to:

Correction of dysfunctional cells themselves (for example, for the treatment of diseases associated with genetic defects such as mucoid obstruction or muscular dystrophy);

Protection and/or regeneration of angiogenesis or innervation of tissues such as muscles, organs, or bones by transgenically produced trophic, neurotrophic, angiogenic, or anti-inflammatory factors;

Transformation of muscle into secretory organs that secrete products leading to a therapeutic effect, such as those of genes themselves (eg, regulators of thrombosis and hemostasis, trophic factors, growth factors, hormones like insulin, erythropoietin, and leptin ( leptin) etc.), or as active metabolites synthesized in muscle by adding therapeutic genes, for example, to correct genetic diseases by secretion of therapeutic products;

Delivery of antineoplastic genes such as tumor suppressor genes (retinoblastoma protein, p53, p71), suicide genes (eg, HSV-thymidine kinase), antiangiogenic genes (eg, angiostatin, endostatin, urinary N-terminal fragments of kinases), cell cycle blockers, apoptosis genes (such as BAX), intracellular single-chain antibodies, and immunostimulatory genes.

• Nucleic acid vaccine or immunostimulant genes.

The particular advantage of using electrotransfer in gene therapy for systemic problems expressed in muscle is due to a number of factors:

remarkable stability of transgene expression, over several months, thus enabling a stable and continuous production of intramuscular or secreted therapeutic proteins,

Easy access to muscle tissue, enabling direct, rapid and safe administration in non-critical organs,

• Large volume of muscle mass, enabling multiple application sites.

· Well-documented muscular secretory capacity.

An added advantage is the safety afforded by the local treatment associated with the use of localized and targeted electric fields.

Due to the safety associated with the use of weak electric fields, the present invention can be applied to the myocardium for heart disease treatment, for example, using artificial pacing to ensure safe electrical transfer (see US Patent No. 5,634,899). The present invention can also be used in the treatment of restenosis by inhibiting the expression of smooth muscle cell proliferation genes, such as GAX protein.

The combination of low-intensity, long-duration electric fields applied in vivo, in particular to tissue, enhances nucleic acid transfection without causing significant tissue deterioration. These results improve the efficiency of DNA transfer in gene therapy using nucleic acids.

Thus, an advantage associated with the present invention is the production of physiological and/or therapeutic doses of substances in or adjacent to tissues, or the systemic secretion in the bloodstream or lymphatic circulation. Furthermore, the present invention makes possible, for the first time, the fine regulation and control of the effective amount of the transgene expressed, in terms of the possibility to adjust the volume of the transfected tissue, for example, to apply multiple application sites, or even to adjust the number of electrodes, shape, Possibility of surfaces and arrangements. Another factor of control arises from the possibility to adjust the transfection rate by varying the field strength, the number, duration and frequency of pulses as well as the amount and volume of nucleic acid administered according to the prior art. A particular advantage of the present invention is the perfect dose-response curve obtained for DNA transfer, which has never been achieved by any of the prior art methods. Transfection levels comparable to desired tissue production or secretion can then be achieved. Finally, this method enables a particular safety compared to chemical or viral methods of gene transfer in vivo, where the complete elimination and control of reaching non-target organs cannot be achieved. In fact, the method according to the invention makes it possible to control the localization of the transfected tissue (closely related to the volume of the tissue subjected to local electric pulses) and thus creates the possibility of inhibiting the expression of the transgene by complete or partial resection of the tissue, This is possible because certain tissues are not essential and can regenerate or both, such as muscle. This greater flexibility in the method of use makes it possible to optimize the method according to the animal species (human and veterinary applications), the patient's age and his or her physiological and/or pathological condition.

The method according to the invention furthermore enables the transfection of large nucleic acids for the first time compared to viral methods, which are limited by the capsid-compatible viral genome size with regard to the size of the transgene. This possibility is necessary for the transfer of very large genes such as dystrophin or genes containing introns and/or large regulatory elements, for example, for the physiologically regulated production of hormones. This possibility is required for the transfer of artificial yeast episomes or chromosomes or minichromosomes.

Another object of the present invention is to combine electrical impulses of voltage fields with nucleic acid-containing compositions formulated for any mode of administration, making it possible to administer topical, dermal, oral, vaginal, parenteral, intranasal, intravenous, Enter the tissue through intramuscular, subcutaneous, intraocular, and percutaneous routes. The pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier for injectable formulations, in particular, for direct injection into a desired organ, or for any other mode of administration. It may include, inter alia, sterile isotonic solutions or dried, especially lyophilized compositions, to which, as the case may be, addition of sterile water or physiological saline results in injectable solutions. The nucleic acid dosage for injection as well as the number of administrations and the injection volume can be adapted to different parameters, in particular the method of administration, the pathology concerned, the gene to be expressed and even the duration of treatment sought.

target tissue

The present inventors found that the optimum conditions for gene transfer according to the present invention differ depending on the target tissue. For example, an electric field of 200 volts/cm was found to greatly enhance gene transfer into muscle cells. Efficient gene transfer into tumor cells also took place under these conditions (in a particular experiment a 3-fold enhancement of gene transfer was observed), but gene transfer into tumor cells was more efficient in an electric field of 400 volts/cm ( Gene transfer efficiency increased by 2 logs). In further experiments, an electric field strength of 500 volts/cm was optimal for gene transfer into tumor cells.

Thus, according to the present invention, a system or modified device can be produced for the delivery of nucleic acids into muscle cells (and other large cells), and a system or modified device can be developed with different electric field strength parameters for the delivery of nucleic acids into muscle cells (and other large cells). Gene delivery in tumor cells (and other small cells).

For the delivery of nucleic acids to muscle cells, the system or device of the invention will generate a voltage gradient of 1-400 volts/cm, preferably 4-400 volts/cm, more preferably 30-300 volts/cm. In a particular embodiment, the voltage gradient is 100-200 volts/cm. Particularly contemplated are systems or devices that provide a voltage gradient of no more than 200 volts/cm.

For delivery of nucleic acids to tumor cells, the system or device of the present invention will generate a voltage gradient of 1-600 Volts/cm, preferably 100-600 Volts/cm, more preferably 400-600 Volts/cm. In a particular embodiment, the voltage gradient is 400-500 Volts/cm, preferably about 500 V/cm. Particularly contemplated are systems or devices that provide a voltage gradient of no more than 600 volts/cm.

Nucleic acid

Nucleic acids may be of synthetic or biosynthetic origin, or extracted from viruses or prokaryotic or eukaryotic cells derived from unicellular organisms (eg, yeast) or multicellular organisms. They can be administered wholly or partly with components of biological and/or synthetic origin.

A nucleic acid may be deoxyribonucleic acid or ribonucleic acid. It may include sequences of natural or artificial origin, in particular genomic DNA, cDNA, mRNA, tRNA and rRNA, hybridization sequences or synthetic or semi-synthetic oligonucleotide sequences, whether modified or not. These nucleic acids can be obtained by any method known to the expert, in particular by cloning, by chemical synthesis or even by hybrid methods, including chemical or enzymatic modification of the sequence obtained by cloning. They can be chemically modified.

In particular, the nucleic acid may be sense or antisense or DNA or RNA having catalytic properties, such as ribozymes. "Antisense" refers to a nucleic acid having a sequence complementary to a target sequence (eg, mRNA) that blocks its expression by hybridizing to the target sequence. "Sense"refers to a nucleic acid that has a sequence that is homologous or identical to a target sequence, eg, a sequence that is linked to a protein transcription factor and is involved in the expression of a particular gene. According to a preferred embodiment, the nucleic acid contains a gene of interest and elements enabling the expression of the gene of interest. The nucleic acid fragments are advantageously in the form of plasmids.

Deoxyribonucleic acid can be single-stranded or double-stranded, such as short oligonucleotides or longer sequences. They may carry genes, sequences that regulate transcription or replication, or regions linked to other cellular components, among others. These genes may include marker genes, ie, genes that produce a detectable marker to study cell function, migration, or gene function; therapeutic genes; protective antigen or immunogenic genes; and the like. According to the present invention, "therapeutic gene" refers in particular to any gene that encodes an RNA or encodes a protein product that has a therapeutic effect. The encoded protein product can be a protein, peptide, and the like. The protein product may be homologous to the target cell (ie, a product that is normally expressed in the target cell when not exhibiting pathology). In this case, for example, transgenic expression makes it possible to overcome underexpression in cells or expression of an inactive or weakly active protein due to modification, or to overexpress the protein. Therapeutic genes can also encode mutants of cellular proteins with increased stability, improved stability, and the like. This protein product can also be heterologous to the target cell. In this case, for example, the expressed protein can perfect or introduce an activity that is defective in the cell (treatment of myopathy or enzyme deficiency), or it is possible to combat a pathology, or, for example, stimulate an immune response in tumor therapy . It may contain a suicide gene (herpes thymidine kinase) for the treatment of cancer or restenosis.

The nucleic acid preferably also includes sequences that support and/or facilitate expression of the therapeutic gene and/or the gene encoding the antigenic peptide in the tissue. It may contain sequences which are naturally responsible for the expression of the gene in question when they are functional in the transfected cell. Sequences of different origin (responsible for the expression of other proteins, or even synthetic sequences) may also be included. In particular, it may contain eukaryotic or viral gene promoter sequences. For example, it may contain a promoter sequence derived from the genome of the cell desired to be transfected. In eukaryotic promoters, an inducing or repressing sequence may be used to support specific expression of a gene. Strong or weak, constitutive or inducible promoters can be used. Ubiquitous (constitutive) promoters include HPRT, vimentin, alpha-actin, tubulin, etc. promoters. Tissue-specific promoters (including elongation factor-1-alpha, flt, flk) can be used. Inducible promoters include promoters that are responsive to hormones (eg, steroid receptors, retinoic acid receptors, etc.), or promoters regulated by antibiotics (tetracycline, rapamycin, etc.) or other natural or synthetic molecules. Promoter sequences derived from viral genomes may likewise be contained. In this regard, for example, promoters of EIA, MLP, CMV, RSV genes and the like can be mentioned. In addition, these expressed sequences can be modified by adding sequences that activate, regulate, allow conditional expression, transient or transient expression, tissue-specific expression or general expression, and the like.

Furthermore, especially upstream of the therapeutic gene, the nucleic acid may also contain a signal sequence that directs the synthesized therapeutic product into the secretory canal of the target cell. The signal sequence may be the natural signal sequence of the therapeutic product, but may also include any other functional or artificial signal sequence. Nucleic acids may also contain components that direct synthetic therapeutic products into specific cellular compartments, such as mitochondria for the treatment of mitochondrial genetic disorders.

Therapeutic genes and gene products

Among the therapeutic products according to the invention, mention may in particular be made of enzymes, blood proteins, hormones such as insulin or growth hormone, lymphokines: interleukins, interferons, tumor necrosis factor (TNF), etc. (French Patent No. 92 03120), growth Factors, such as angiogenic factors such as VEGF or FGF.

For the treatment of neuropathy, the system of the present invention can be used to deliver genes encoding neurotransmitters or their precursors or enzymes for synthesizing neurotransmitters, trophic factors, especially neurotrophic factors, for the treatment of neurodegenerative diseases, trauma or injury caused by nerve damage. Systemic damage, or retinal degeneration. For example, members of the neurotrophin family include, but are not limited to: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT4/5, NT6 (including allelic variants bodies and members of the same gene family). Other neurotrophins include members of the ciliary neurotrophic factor family, including ciliary neurotrophic factor (CNTF), axokine, leukemia inhibitory factor; other factors including IL-6 and related cytokines; cardiotrophins and their associated genes; 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 TGFβ; HARP/pleiotrophin, or bone growth factor; hematopoietic factors, etc.

Other genes of interest code for therapeutically helpful, secreted and non-secreted muscle proteins, such as dystrophin or mini-dystrophin (French Patent No. 91 11947) or alpha-1-antitrypsin.

Other target genes encode factors related to coagulation: factors VII, VIII, IX; suicide genes (thymidine kinase, cytosine deaminase); hemoglobin gene or other protein carriers.

In yet another embodiment, the corresponding genes of proteins involved in lipid metabolism, such as those selected from apolipoprotein A-I, A-II, A-IV, B, C-1, C-II, C-III, can be delivered , D, E, F, G, H, J, and a type of apo(a), and metabolic enzymes such as: lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase, 7-alpha - cholesterol hydroxylase, phosphatidic acid phosphatase, or even lipid transfer proteins, such as cholesteryl ester transfer protein and cholesteryl ester transfer protein and phospholipid transfer protein, HDL binding protein, or even selected from e.g. LDL receptor, chylomicron remnant receptor A receptor such as body and scavenger receptors. Leptin can be further added to treat obesity. The p53 anti-oncogene or other tumor suppressor genes such as HSV-tk or even GAX protein can also be added to limit cell proliferation in smooth muscle (treatment of restenosis).

Other genes of interest include angiogenic factors, including vascular endothelial growth factors (VEGF, VEGF-2, VEGF-3, platelet growth factor) and angiostatin. On the other hand, the delivery of genes encoding inhibitors of angiogenesis, especially tumor angiogenesis, such as soluble receptors of angiogenic factors, specific inhibitors of angiogenic factor receptors (Tie2, urokinase receptor Flt1, KDR), antibodies against angiogenic factors (including single-chain Fv antibodies) (eg, anti-VEGF or anti-FGF), anti-integrin antibodies, endothelial tumor-specific toxins, polypeptide inhibitors of angiogenesis (N-terminal fragment of urokinase-ATF, angiostatin, endostatin, interferon-α or β, interleukin-12, platelet factor 4, TNFα, thrombospondin, platelet-activating factor (PAI)-1, PAI2, TIMP1 , prolactin fragments, etc.

Among other proteins or peptides that may be secreted by a tissue or produced by that tissue, it is important to emphasize antibodies, variable fragments of single-chain antibodies (ScFv) or any other fragments of antibodies with the ability to recognize, for immunotherapy, e.g. , Treatment of infectious diseases, tumors and autoimmune diseases such as islet sclerosis (anti-idiotypic antibodies). Non-limiting other proteins of interest are: soluble receptors, for example, CD4 soluble receptors or TNF soluble receptors for anti-HIV therapy, soluble TNF receptors for rheumatoid arthritis (especially soluble TNFα receptors) body) and acetylcholine soluble receptors for the treatment of myasthenia; substrate peptides or enzyme inhibitors, or even receptor agonists or antagonists peptides or adhesive proteins, for example, for the treatment of asthma, thrombosis, restenosis, metastases or inflammation (such as IL-4, IL-10, and IL-13 to attenuate Th1 cell responses); and artificial, chimeric, or truncated proteins.

Among the important hormones of interest, mention may be made of insulin, growth hormone and calcitonin for diabetes.

To enhance anti-tumor or anti-infective immune responses, genes encoding immunostimulatory cytokines including IL-2, IL-12, colony-stimulating factors (GM-CSF, G-CSF, M-CSF), macrophages, etc. Phage inflammatory factors (MIP1, MIP2), dendritic cell activating factor (flt3 ligand), etc.

McKusick, V.A. (Mendelian Inheritance in man, catalogs of autosomal dominant, autosomal recessive, and X-linked phenotypes) 8th ed. , Johns Hopkins University Press (1988)) and Stanbury, J.B. et al. ("The metabolic basis of inherited disease" (The metabolic basis of inherited disease) 5th edition, McGraw-Hill (1983)) have described other genes of interest. Genes of interest include proteins involved in the metabolism of amino acids, lipids, and other cellular components.

Genes associated with disorders of carbohydrate metabolism can thus be mentioned, for example: fructose-1-phosphate aldolase, fructose-1,6-bisphosphatase, glucose-6-phosphatase, lysosomal alpha-1,4- Glucosidase, amyloid-1,6-glucosidase, amyloid-(1,4:1,6)-transglucosidase, myophosphorylase, myofructose phosphokinase, phosphorylase-b-kinase, half Lactose-1-phosphate uridine acyltransferase, all enzymes of the pyruvate dehydrogenase complex, pyruvate carboxylase, 2-oxoglutarate glyoxalase carboxylase, and D-glycerol dehydrogenase.

May also mention:

- Genes associated with disorders of amino acid metabolism, such as: phenylalanine hydroxylase, dihydrobiopterin synthase, tyrosine aminotransferase, tyrosinase, histidase, fumarylacetoacetase, Glutathione synthase, g-glutamylcysteine synthetase, ornithine-d-aminotransferase, carbamoyl phosphate synthase, ornithine carbamoyltransferase, argininosuccinate synthesis Enzymes, argininosuccinate lyase, arginase, L-lysine dehydrogenase, L-lysine ketoglutarate reductase, valine transaminase, leucine isoleucine transaminase, branch Chain 2-ketoacid decarboxylase, isovaleryl-CoA dehydrogenase, acyl-CoA dehydrogenase, 3-hydroxy-3-methylglutaryl-CoA lyase, acetoacetyl-CoA 3-ketothiolase , propionyl-CoA carboxylase, methylmalonyl-CoA mutase, ATP:cobalamin adenylyltransferase, dihydrofolate reductase, methylenetetrahydrofolate reductase, cystathionine β- Synthetase, sarcosine dehydrogenase complex, proteins belonging to the glycine breakdown system, beta-alanine aminotransferase, serum carnosinase and brain homocarnosinase.

- Genes associated with disorders of fat and fatty acid metabolism, such as: lipoprotein lipase, apolipoprotein C-II, apolipoprotein E, other apolipoproteins, lecithin cholesterol acyltransferase, LDL receptor, hepatosterol hydroxylation Enzyme and "phytanic acid" alpha-hydroxylase.

- Genes associated with lysosomal deficiencies such as: lysosomal α-L-iduronidase (iduronidase), lysosomal iduronate sulfatase, lysosomal heparin N-sulfate Enzymes, lysosomal N-acetyl-α-D-glucosaminidase, acetyl-CoA:lysosomal α-glucosamine N-acetyltransferase, lysosomal N-acetyl-α-D- Glucosamine 6-sulfatase, lysosomal galactosamine 6-sulfatase, lysosomal β-galactosidase, lysosomal arylsulfatase B, lysosomal β-glucuronidase Acidase, N-acetylglucosamine phosphotransferase, lysosomal α-D-mannosidase, lysosomal α-neuraminidase, lysosomal aspartyl glucosaminidase, lysosomal α -L-fucosidase, lysosomal acid lipase, lysosomal acid ceramidase, lysosomal sphingomyelinase, lysosomal glucocerebrosidase and lysosomal galactocerebrosidase, Alpha chains of lysosomal galactoside ceramidase, lysosomal arylsulfatase A, α-galactosidase A, lysosomal acid β-galactosidase, and lysosomal hexosaminidase A .

Mention may also be made, without limitation, of genes associated with diseases of steroid and lipid metabolism, genes associated with diseases of purine and pyrimidine metabolism, genes associated with diseases of porphyrin and heme metabolism, diseases of connective tissue, muscle and bone metabolism Genes involved, and genes involved in diseases of the blood and blood cell production, muscles (myopathy), nervous system (neurodegenerative diseases) or circulatory system (eg, treatment of ischemia and stenosis).

The numerous examples above and below illustrate the potential range of fields of application of the present invention. The examples below illustrate the expression of each of the following factors.

Electrotransfer of NT3. A highly promising gene for electrotransfer into muscle tissue is neurotrophin 3 (NT3). NT3 delivered intramuscularly with an adenoviral vector or a non-viral vector has been found to increase the survival of pmn mice (Haase et al., Nature 3:429-436, 1997). This is a useful animal model for amyotrophic lateral sclerosis (ALS), commonly known as "Lou Gehrig's disease." Treatment of this insidious disease with NT3 gene therapy is expected to be greatly facilitated by the delivery of NT3 by electrotransfer, which ensures adequate, reproducible expression of this trophic factor.

Electrotransfer of acidic FGF or VEGF. Another highly desirable gene for electrotransfer into muscle tissue is acidic FGF (aFGF; ECGF) or vascular endothelial growth factor (VEGF). Both have been found to be effective in the treatment of arterial occlusive disease. Treatment of this disease with aFGF or VEGF electrotransfer-enhanced gene therapy is expected to further improve the efficiency of vessel growth. Treatment of cardiac arterial occlusive diseases would also be possible by applying electric fields in a paced manner, in particular by active cardiac pacing.

A therapeutic nucleic acid can also be a gene or an antisense sequence whose expression in a target cell makes it possible to control the gene expression and mRNA transcription of the cell. For example, according to the method described in European Patent No. 140,308, these sequences can be transcribed into RNA in the target cell, which complements the cellular mRNA, thereby blocking its protein translation. Therapeutic genes also include sequences encoding ribozymes that selectively destroy target RNAs (European Patent No. 321,201).

Immunogenic Genes and Vaccines

As noted above, the nucleic acid may also contain one or more genes encoding immunogenic or antigenic peptides, which are capable of eliciting an immune response in a human or animal. In this particular embodiment, the invention thus makes possible vaccine or immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancer. In particular, specific antigenic peptides of Epstein-Barr virus, HIV virus, hepatitis B virus (European Patent No. 185,573), pseudorabies virus, "syncytia virus", other viruses, and even specific tumor antigens such as MAGE protein (European Patent No. 185,573) may be included. Patent No. 259,212) or antigens that stimulate anti-tumor responses, such as bacterial heat shock proteins.

Carrier

In the method according to the invention, the nucleic acid may be linked to any type of vector or any combination of these vectors, enabling enhanced gene transfer, for example, without limitation, to a vector such as a virus, a synthetic or biosynthetic substance ( For example, lipids, polypeptides, glycosides or polymers), or even on impelling or non-propelling spheres. Nucleic acids can also be injected into tissue that has been subjected to a treatment aimed at enhancing gene transfer, for example, pharmacological treatments in local or systemic applications, or enzymatic, permeabilization (use of surfactants), surgical , mechanical, thermal or physical treatment.

The following examples are intended to illustrate the invention in a non-limiting manner.

Example

Example 1: Standard Electroporation Conditions

Standard electroporation conditions were tested, such as those used in U.S. Pat. conditions, which were found to cause inefficiency or even inhibition of nucleic acid (plasmid DNA) transfer in striated muscle.

Materials and Methods

The following products were used in this example:

DNA pXL2774 (PCT/FR patent 96/01414) is a plasmid DNA containing a luciferase reporter gene. Other products are available from market suppliers: ketamine, xylazine, normal saline (NaCl 0.9%).

An oscilloscope and a commercial electrical pulse generator (rectangular or square) (Electropulsator PS 15, Jouan, France) were used. The electrodes used were stainless steel flat electrodes with a pitch of 5.3 mm.

This experiment was performed in C57B1/6 mice. Mice from different cages were assigned randomly ("randomization") before the experiment.

Anesthetize mice with a mixture of ketamine and xylazine. The plasmid solution (30 mg/ml solution containing 500 mg/ml NaCl 0.9%) was injected longitudinally through the skin into the cranial tibialis muscle of the left and right paws by means of a Hamilton syringe. Both electrodes were covered with conductive gel, and the injected paw was placed between the electrodes in contact with the latter.

Electrical pulses were applied perpendicular to the muscle axis by a square pulse generator one minute after the injection. The oscilloscope made it possible to check the intensity (volts) of the released pulses (the values shown in the examples represent the maximum value), the duration (milliseconds) and the frequency (hertz), which is 1 Hz. Releases 8 consecutive pulses.

To evaluate transfection of muscles, mice were euthanized 7 days after plasmid administration. The cranial tibial muscles of the left and right paws were then removed, weighed, placed in lysis buffer and ground. The suspension obtained was centrifuged to obtain a clear supernatant. 10 ml of the supernatant were assayed for luciferase activity by means of a commercial luminometer in which the substrate was automatically added to the solution. The intensity of the luminescence response is expressed in RLU (relative luminescence units) for muscles administered the total volume suspension. Ten points were tested per experimental condition: 5 animals were injected bilaterally. Statistical comparisons were performed with nonparametric tests.

Results and discussion

The results are illustrated with two graphs on a linear or log scale.

In the first experiment, the effect of an electric field of 800-1200 volts/cm was tested, which is the condition used for tumor electroporation (Mir et al., Eur. J. Cancer 27, 68, 1991; US Patent 5,468,223).

It was observed from Figure 1 that, compared to the control group injected with DNA when there was no electric pulse:

With 8 pulses of 1200 volts/cm and 0.1 msec duration, the mean value of luciferase activity was much lower;

With a pulse of 1200 volts/cm and 1 msec, three animals died and the mean value of luciferase activity was much lower;

• The mean value of luciferase activity was also significantly reduced with a pulse of 800 volts/cm and 1 msec.

Most muscles exposed to the electric field were visibly altered (brittle and pale in appearance).

Example 2: Electrotransfer of Nucleic Acids

C57B1/6 mice were used for this experiment. Except for the electric field intensity and duration of the pulse, the experimental conditions were the same as in Example 1.

The results are shown in Figure 2. The results of Example 1 were reproduced, ie the inhibition of luciferase activity by a series of 8 pulses at 800 volts/cm with a duration of 1 msec was detected in muscle. Applying an electric field of 600 volts/cm, the same inhibition and the same changes in muscle tissue were observed. However, shorter pulse widths at this voltage may enhance DNA transfer by avoiding tissue damage. On the other hand, quite clearly and surprisingly, it is possible that the reduction of the voltage no longer significantly changes the muscle, and, at 400 and 200 volts/cm, the transfection level of the muscle is on average higher than that obtained for the muscle not subjected to the electric field level. It should be noted that the dispersion of luciferase activity values at 200 volts/cm was significantly reduced (SEM = 20.59% of the mean value compared to 43.32% for no electric field) compared to the control group (no electric field). Figure 2A)).

Those skilled in the art can readily ascertain that these data demonstrate that prior art devices for electrotransfer can be modified in accordance with the findings of the present invention to yield systems or devices of the present invention. Despite the simplicity of this improvement, the results of this improvement were completely unexpected. The plasmid DNA transfer experiments reported in this example and in the following examples demonstrate that the systems or devices of the present invention lead to unexpected enhancements in both efficiency and reproducibility of nucleic acid transfer.

Example 3: Electrotransfer Enhances Transgene Expression

C57B1/6 mice were used for this experiment. Except for the electric field strength and duration of the pulses and the release of the pulses for 25 seconds after DNA injection, the experimental conditions were the same as in the previous examples.

The results are shown in FIG. 3 . Starting with a pulse duration of 5 msec at 200 volts/cm, with a pulse duration of 20 msec at 100 volts/cm there was a marked increase in the mean value of transgene expressed luciferase.

This experiment also clearly shows the mean value of luciferase activity obtained by DNA electrotransfection in muscle as a function of the duration of the electrical pulse when voltages of 200 and 100 V/cm were used. It was also found that the dispersion of values was significantly reduced for the electrotransfected muscle group (Fig. 3A). When there is no electric pulse (contrast), the SEM is 77.43% of the average value, and under the pulse time of 5msec and the electric field condition of 200 volts/cm, the relative SEM drops to 14%. The electric field condition drops to 41.27%, for 30%-48% electrotransfer at 100 volts/cm.

Under the optimal conditions for this experiment, transgene expression was enhanced 89.7-fold compared to controls injected without electrical pulses.

Example 4: 200-fold enhancement of expression

The experiments were performed in DBA2 mice using electrical pulses of 200 V/cm of variable duration. Other conditions of this experiment are the same as in Example 3.

This example demonstrates that at 200 volts/cm, the transfection of luciferase activity was enhanced when the pulse duration was extended from 5 msec to longer durations (Figure 4 and Figure 5). A reduction in inter-individual variability represented by SEM relative to non-electrotransfected controls was observed. The relative value of SEM was equal to 35% for the control, 25%, 22%, 16%, 18% and 26% for the pulse series of 1, 5, 10, 15, 20 and 24 msec, respectively. Under the optimal conditions used in this experiment, transgene expression was enhanced 205-fold compared to controls injected without electrical pulses. These results demonstrate that electrotransfer under the conditions described in these examples greatly improves efficiency and reproducibility.

Example 5: Quantification of Electrotransfer Efficiency

Figure 5 illustrates the importance of a parameter equivalent to the product of "number of pulses x field strength x duration of each pulse". In fact, this parameter corresponds to the time-dependent integral of the function describing the change of the electric field.

The data in Figure 5 are from the results obtained during experiments 2, 3 and 5. Field strengths of 200 V/cm and 100 V/cm or no field were evaluated. The data show that the transfection rate increases as the product of the total duration of exposure to the electric field and the field strength. An enhancement of nucleic acid transfer is obtained when the product of "electric field x total pulse duration" has a value exceeding 1 kV x msec/cm. According to a preferred embodiment, stimulation is obtained when the value of the product "electric field x total pulse duration" exceeds or is equal to 5 kV x msec/cm.

Example 6: Broad Applicability of Electrotransfer

Further experiments have been performed to confirm the results of Examples 2-5 above. Indeed, significant and reproducible enhancement of gene transfer was observed in mice, rats and rabbits using low voltage electrical pulses. The conditions used were previously thought to be ineffective in other cell types studied such as tumor, skin or liver cells.

The following parameters were varied and investigated in this example:

-Characteristics of the electric field: volts/cm, number of pulses, frequency, duration, it was determined that the optimal conditions for the mouse tibialis muscle were 8 pulses at 200 V/cm, 1-2 Hz, 20 ms.

- Electrode shape/type: Most experiments were performed with non-invasive flat plate electrodes; feasibility of needle electrodes was demonstrated in rabbit experiments.

- Amount of DNA (using optimal transfer conditions for mouse tibialis muscle).

-Different batches of DNA.

- Different reporter genes: luciferase, LacZ (mouse), FGF (rat).

- Different animal species: mice, rats, rabbits.

- Different injection sites: tibialis, gastrocnemius, quadriceps in mice; tibialis in rats; tibialis, quadriceps and triceps in rabbits.

- Distance from injection site to pulse site.

- Timing of injection and application of electrical pulses.

- Perform different experiments with injection and pulse application.

The observed results are as follows:

Significant enhancement of gene transfer compared to simple naked DNA injection: from 5-10-fold to 100-fold or even higher in mouse muscle, 100-250-fold enhancement in rats, 100-50,000-fold enhancement in rabbits, depending on at the control level.

• Significant reduction in inter-animal scatter/variability of results. The electrical pulse must be applied after the DNA injection and at the same site. The DNA vector can be administered up to 30 minutes before the pulse without significantly reducing the response. This allows multiple injections of DNA at adjacent sites followed by a single pulse.

• Experiments with LacZ in mice followed by histological analysis demonstrated that this approach resulted in approximately 10-fold more muscle fibers expressing the gene. Data using FGF in rats also showed a significant increase in the number of expressing cells.

• Experiments using secreted alkaline phosphatase, a secreted reporter gene, also showed a two-log increase for the secreted protein in serum.

• Inverse dependence of transfection efficiency on size. Smaller plasmids can be transferred more efficiently; however, the electrotransfer device dramatically increases DNA transfer efficiency regardless of DNA size, thereby overcoming an important obstacle to transfection with YACs, cosmids, or artificial chromosomes.

• Independence on promoter and protein processing. Electrotransfer efficiency is not dependent in any way on the transgene promoter, allowing another level of gene expression control. Furthermore, the presence of secretory or other regulatory/processing sequences in the gene product had no effect on electrotransfer efficiency.

These experiments showed that electrotransfer had some side effects, although these effects were minimal compared to electroporation conditions. A local inflammatory response and regenerative processes were demonstrated 7 days after the pulse. This inflammation is mild and reversible. Electrical pulses induce a general contraction in mice. In rats, this effect was more reduced and localized to the hind limbs. In rabbits, preliminary experiments showed that this contraction was restricted to the muscle group subjected to the pulse. Anesthetized rats or rabbits have no obvious pain response (no barking). During recovery from anesthesia, none of the animals exhibited any significant pain in the handled limb.

These experiments demonstrate the superiority of the electrotransfer device used under the conditions described in this application in terms of enhanced nucleic acid transfer, reduced intra-assay variability, and reduced or eliminated adverse side effects. These results are described in more detail in the Examples below.

Example 7: Transfection as a function of pulse duration

This example demonstrates the effect of prolonged pulse duration on transfection efficiency under electrotransfer conditions.

The experimental conditions were the same as in Example 1 using 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 is capable of applying square pulses with a duration of up to 100ms. Plasmid pXL2774 (WO 97/10343) (15 μg) was injected. Table 1 indicates that at a constant electric field strength of 200 V/cm, increasing the pulse duration (T) increased the transfection efficiency.

These data identify optimal parameters for the electrotransfer device used to deliver nucleic acids to muscle. Such means preferably provide pulses of 20 msec or longer, at least 4, more preferably 8 pulses.

Table 1: Dependence of electrotransfer efficiency on pulse duration Pulse duration (msec) 0 1 5 10 20 30 40 50 60 80 Test A8 pulses 11±5 39±6 211±26 288±46 1158±238 1487±421 2386±278 Test B4 pulses 11±5 26.8±6 123±17 246±32 575±88 704±130 3440±1077 Test C4 pulses 15±8 2885±644 2626±441 1258±309

Median +/- SEM of luciferase activity in million RLU per muscle, N = 10. Electrotransfer conditions: 200V/cm field strength, 1Hz frequency.

These data show that a device performing electrotransfer at the optimum field strength described in this application can further increase the efficiency of DNA transfer by extending the pulse duration. For example, extending the duration of a series of 8 pulses to at least 40 msec, or to 50 msec for a series of 4 pulses, significantly enhanced transfection efficiency at 200 V/cm. Similar optimizations of the device can also be achieved for other field strengths.

Example 8: Transfection as a function of pulse number

This example demonstrates the effect of increasing the number of pulses on transfection efficiency under electrotransfer conditions.

Experimental conditions were the same as described in Example 1 using C57B1/6 mice. Table 2 shows that at 200V/cm with a pulse duration of 20 ms, the transfection efficiency was significantly improved compared with the control group (no electric field applied), starting from a single pulse, when the number of pulses increased to 2, 4, 6, Continue to increase at 8, 12 and 16, and the best time is 8-16 pulses. It is also shown that the variance (S.E.M.) was reduced for all electrotransfected groups compared to the control (0 pulses).

Table 2: Transfection Efficiency vs. Pulse Number Pulse number 0 1 2 4 6 8 12 16 Average value (×10 ^-7 ) 69.57 147.91 281.02 438.62 678.44 818.73 929.20 889.75 SEM 80.09 17.76 16.38 11.44 19.05 8.87 18.20 15.44

Mean luciferase expression ± S.E.M. in millions RLU per muscle; N = 10 per group; 200 V/cm field strength; 1 Hz frequency.

These data show that the electrotransfer device improves DNA transfer efficiency with more pulses. At the field strength tested (200 V/cm), the device optimally delivers 4 or more, better 8 or more pulses. By changing the number of pulses, the electrotransfer device can adjust the efficiency of nucleic acid transfer and thus the expression level.

Example 9: Transfection as a function of frequency

This example shows that increasing the pulse frequency can enhance transfection efficiency. In clinical applications, electrotransfer devices that apply higher frequency pulses increase patient comfort by reducing the overall duration of the applied electric field. Thus, both efficiency and patient comfort are improved by increasing the frequency.

Experimental conditions were the same as described in Example 1 using C57B1/6 mice. Plasmid pXL2774 (15 μg) was injected. The frequency was varied from 0.1 Hz to 4 Hz under 8 or 4 pulses of 20 ms duration at a field strength of 200 V/cm. The results are shown in Table 3.

Table 3: Transfection Efficiency vs. Frequency (Hz) Frequency (Hz) 0 0.1 0.2 0.5 1 2 3 4 Test A8 pulses 5±2 54±13 95±16 405±60 996±156 1528±257 Test B4 pulses 114±14 163±24 175±26 337±53 587±90 Test C8 pulses 21±14 1294±189 2141±387 3634±868 2819±493 Test D4 pulses 1451±228 1572±320 1222±126 2474±646

The unit is the average luciferase activity±S.E.M. of each muscle in millions of RLU; N=10 for each group; the electric field intensity is 200V/cm; the pulse time is 20msec.

The results demonstrate that transfection efficiency increases at higher frequencies (greater than 1 Hz, preferably greater than 2 Hz) under the electrotransfer conditions tested.

Example 10: Electrotransfection using an exponentially decreasing electric field over time

This example shows the effect of applying an exponentially decreasing electric field on the efficiency of nucleic acid transfer in vivo.

C57B1/6 mice were used in this experiment. In this experiment, the plasmid pXL3031 (Figure 12) derived from plasmid pXL2774 was used, which was constructed by introducing a modified Photinus pyralis luciferase (pGL3; Genbank accession number CVU47295) in the cytomegalovirus immediate early stage (CMV-IE ) promoter (Genbank accession number HS5IEE) and polyadenylation signal derived from SV40 virus (Genbank accession number SV4CG). Inject 10 μg of DNA.

A commercial electric pulse generator used here (Equibio electric pulse generator, EasyjectT Plus type, Kent, UK) was equipped to deliver electric field pulses that decreased exponentially with time. Applied voltages were recorded as exponential peak voltages. The second adjustable parameter is capacitance (in μF), which controls the amount of energy released.

Table 4 shows that when an exponentially decreasing electric field pulse was applied, a very significant increase in transgene expression was obtained compared to the case where no electric field was applied. This result was obtained at different voltages corresponding to different time constants of the exponential and at different energies, the former being adjustable by an adjustable capacitance of the instrument. The parameters determined in this example can be applied to electrotransfer devices. Table 4: Transfection efficiency (relative to control luciferase activity) when a time-varying exponentially decreasing electric field is applied Capa μ F150 Capa μF300 Capa μ F450 Capa μF600 Capa μF1200 Capa μF2400 Capa μF3000 40V/cm 1,23 11 100V/cm 16,5 2,8 6,5 23,9 150V/cm 1,8 3,5 6,1 200V/cm 5,1 15,8 18,8 121,5 189,7 300V/cm 32,1 90,5 48,7 760,4 56,2 400V/cm 795 600V/cm 62 800V/cm 3,1 1,1

Increase in luciferase expression levels relative to control levels determined by injection of plasmid pXL3031 under electroless transfer conditions. Means for increased expression levels are given; N=4-6 mice per experiment.

By comparison, using a square wave pulse at 200 V/cm, 8 pulses of 20 msec each, at a frequency of 1 Hz, the increase in luciferase activity was 44.

These data show that electrotransfer devices applying an electric field that decreases exponentially over time can enhance expression at lower electric field strengths, higher capacitances (eg, 200 V/cm, 3000 μF capacitance).

Example 11: Combination of one short high-voltage pulse and several long low-voltage pulses

This example shows that the electric field released in this experiment can be a combination that includes at least one strong electric field of 500-800 V/cm for a short time, such as 50 or 100 μs, and at least one time for a longer time, such as longer than 1 ms. Weak electric field (<100V/cm) up to 90ms. The weak electric field value here is 80V/cm, applied with 4 pulses of 1 Hz, each with a duration of 90 ms. For this experiment, two commercial electrical pulse generators (Jouan and Gentronix) were used. The release of voltage to the pads by one instrument and then the other occurs in less than a second by manual modification of the mode of operation. The plasmid encoding luciferase used here was pXL3031, and the injection volume was 3 μg. The electric field strength was varied as reported in Table 5. In addition, the experimental conditions were the same as described in Example 1.

Table 5 summarizes the experiment. These data indicate that application of one short high voltage pulse or four long low voltage pulses, or a weak electric field pulse followed by a strong electric field pulse, did not increase transfection efficiency compared to the control group (no electric field applied). In contrast, in this experiment the combination of one short high voltage pulse followed by 4 pulses of 1 Hz, 90 ms duration, 80 V/cm enhanced the transfection very significantly compared to the control group. From these data, it appears that a preferred electrotransfer device will provide a series of shorter pulses of higher field strength.

Table 5: Combinations of pulses of different intensities and times Electric field conditions Experiment 1 (3 μg pXL3031) Experiment 2 (3 μg pXL3031) Control (no electric field applied) 320±126 75±27 A: 500V/cm, 1×0.1msec - 169±63 B: 800V/cm, 1×0.1msec 416±143 272±84 C: 80V/cm, 4×90msec, 1Hz 1282±203 362,21±85,17 Condition: A, followed by C - 1479±276 Condition: B, followed by C 3991±418 1426±209 Condition: C, followed by B - 347±66

As previously shown in Example 1, application of 8 pulses of 600, 800 or 1200 V/cm at a frequency of 1 Hz and a duration of 1 msec resulted in muscle damage and inhibition of transfection. The results obtained in this example show that, under certain conditions, it is possible to use high voltage electric fields without causing damage. Indeed, visual inspection of the muscles failed to demonstrate any visible changes. The use of a short duration of high voltage electric field followed by a longer duration of weak electric field provides another way to tune the efficiency of DNA transfer.

Example 12: Kinetics of Luciferase Expression in Muscle

The use of the electrotransfer device of the present application allows high level transfection and stable expression of nucleic acids for at least four months.

C67B1/6 mice were used in this experiment. Plasmid pXL2774 (15 μg) was injected intramuscularly into mice. DNA injection was followed by application of electric field under the following conditions: 200 V/cm; 8 pulses of 20 msec duration; frequency of 1 Hz. Other conditions are as described in Example 1. Groups of 10 mice sacrificed at different times after DNA injection were assayed for luciferase activity. Control mice were not exposed to the electric field.

The data in Figure 6 show that luciferase expression was detectable from the third hour after plasmid injection and increased until the third day (D3). Luciferase expression decreased from day 35 onwards. For the group treated with the electric field, it is noteworthy that the transfection was very significantly enhanced regardless of the time at which expression levels were measured. At day 121 (D121), the difference between control and treated groups was more pronounced, as the expression of the transfected DNA remained after electrotransfer, while expression decreased in control muscles.

More notably, the expression level of the transgene was stable up to D121. This result is particularly favorable from the point of view of applying therapeutic genes to long-term clinical treatment.

Example 13: Histology of Electrotransfected Muscle

Histological analysis during kinetic experiments confirmed the absence of a severe inflammatory response. Moderate inflammation indicated by the presence of macrophages and lymphocytes was observed. By D121, this inflammatory response was greatly reduced, while the expression level of the transgene remained stable and high, as shown in FIG. 6 .

Histological analysis was performed under these same conditions except that plasmid pXL3004 (Figure 13) was used. This plasmid is a pCOR plasmid (pXL2774; see WO97/10343) encoding β-galactosidase. The pXL2774 plasmid was modified by introducing the lacZ gene modified with a nuclear localization signal sequence (see Mouvel et al., 1994, Virology (Virology) 204:180-189), placed in the CMV obtained from the plasmid pCDNA3 (Invitrogen, the Netherlands). The promoter is under the control of the SV40 polyadenylation signal (Genbank accession number SV4CG). Animals were sacrificed 7 days after plasmid administration. Histological analysis allowed the detection of β-galactosidase-transfected cells (Xgal histochemistry) and inflammatory foci by aluminized carmine staining and the characterization of the muscle tissue condition by oxidized hematoxylin-eosin staining. Control mice were not exposed to the electric field.

Differences between electropermeabilized and non-electropermeabilized muscles are shown by:

9-fold the number of myofibrils expressing β-galactosidase in electrotransfected muscles (average 76, N=6 mice) relative to controls (average 8.5, N=8 mice) . Most of these myofibers are quiescent and contain peripherally located nuclei. Very few central nuclear myofibrils (regenerated) expressed β-galactosidase.

• The area of expression of β-galactosidase was up to 2 times (4 mm) larger in electropermeabilized muscles compared to controls, with a decreasing expression gradient from the injection site.

• In this study, it should be noted that electropermeabilized muscles showed a reversible number of infiltrates (macrophages and lymphocytes), a large number of regenerated myofibers with a nuclear concentration and a large number of necrotic fibers filled with phagocytic macrophages. The areas of inflammation, necrosis and regeneration correspond to the areas surrounding the transfected myofibrils. The reaction lasts for up to two weeks and reverses itself. The untransfected portion of the muscle is still in good condition.

• In non-electropermeabilized muscles, a few necrotic and otherwise regenerated myofibrils were located around the injection site, with few foci of inflammation.

In short, these data show that while electrotransfer conditions result in visible inflammation, inflammation is not evident, especially with regard to the surprising increase in nucleic acid transfer efficiency. Furthermore, data from kinetic studies demonstrate that inflammation reverses itself, even when transgene expression remains stable at high levels.

Example 14: Effect of plasmid injection time relative to electric field application time

This example demonstrates that nucleic acid can be injected into tissue (in this case, muscle) at least 30 minutes prior to application of the electric field, and even as long as one hour.

Plasmid pXL2774 (15 μg or 1.5 μg) was injected intramuscularly into C57B1/6 mice. DNA was injected up to 120 minutes before or 60 seconds after application of the electric field. Table 6 reports the time before or after injection. The electric field conditions used were: 200 V/cm; 8 pulses of 20 msec duration; 1 Hz frequency. Control mice received plasmid injections but were not exposed to electric fields. Other experimental conditions are the same as in Example 1.

Data are reported in Table 6. DNA injection up to one hour before field application resulted in the achievement of high transfection efficiencies, as detected by luciferase expression. The same trend observed for each intramuscular injection of 15 µg of plasmid was also observed with a 10-fold lower dose of 1.5 µg of DNA. No increase in DNA transfection efficiency was observed when the plasmid was injected after electric field application. Table 6: Electrotransfer efficiency of injected plasmids before and after electric field application 6A: DNA injected without electric field (control) Injection time relative to electric field application Experiment 1pXL2774 (15 μg) Experiment 2pXL2774 (15 μg) Experimental 3pXL2774 (1,5 μg) Experiment 4pXL2774 (15 μg) Experimental 5pXL2774 (1,5 μg) 0 7±4 8±6 0.4±0.2 22±15 1±1 6B: DNA Injection Before Electric Field Application time Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 -120 minutes 20±5 2±1 -60 minutes 106±22 10±3 -30 minutes 303±36 237±61 7±3 184±22 15±4 -5 minutes 410±7 -60 seconds 253±51 -20 seconds 492±122 201±43 9±3 123±23 12±2 6C: Injection of DNA after electric field application time Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 +10 seconds 7±7 +20 seconds 11±6 0.4±0.1 +60 seconds 8±7 17±15

In contrast to the results observed here, where injection of plasmid DNA up to one hour prior to field application provided high level expression of the plasmid, different authors observed in vitro that the presence of the plasmid at the time of field application was essential for electroporation.

Example 15: Dose-response of electrotransfer

The statistical analysis shown in this example allows for dose-response comparisons under electrotransfer conditions. This study demonstrates that the use of the electrotransfer device greatly reduces the variability in plasmid expression levels.

10 mice were used for each dose of DNA, and 5-week-old C57B1/6 mice were injected intramuscularly with a dose of 0.25-32 μg DNA (plasmid pCOR-pXL3031) on both sides of the cranial tibialis Transgenic luciferase for cytoplasmic expression under sub-CMVh. The DNA dose was 0.25-32 μg. Immediately after the injection, one of the two legs was exposed to an electric field of 250 V/cm with 4 pulses of 20 ms at a frequency of 1 Hz. Animals were sacrificed 5 days after treatment and the expression of the transgene was studied in tissue extracts of each muscle according to the protocol described in Example 1 . Under these electrotransfer conditions, macroscopic observation of the muscles revealed only two traces of minor thermal damage to the tissue among the 150 muscles subjected to the treatment.

For each series of mice (n=10), comparison of the change in variance as a function of the mean clearly shows that the distribution of transgene expression is lognormal. Graphical analysis of the computationally confirmed results (Figure 7) showed that the effect varied linearly with the log of the injected DNA dose.

Applying Kirkland's test, it was possible to demonstrate that for each regression (with and without electrotransfer) there was a homogeneity of variance, a fact that allowed all calculations to be performed with residual variance. Variations in linearity under electrotransfer conditions are not significant with a 5% confidence level. In contrast, under standard conditions (non-electrotransfer conditions), the variation in linearity was very significant (p<0.01), indicating significant heterogeneity in DNA transfer efficiency. The data showed a 5-fold higher residual variance without electrotransfer compared to the case with electrotransfer.

For the value of the estimated residual variance, it was necessary to use 5 times as many animals in the non-electrotransfer condition as in the electrotransfer condition in order to obtain the same power of the transfection efficiency comparison test. This analysis shows a clear advantage of using an electrotransfer device. To account for a 2-, 5- or 10-fold variation in transgene expression (95% confidence level), only approximately 33, 8 or 5 animals need be injected under electrotransfer conditions compared to 165, 40 or 25 animals under non-electrotransfer conditions. only animals. A table summarizing this calculation is shown in Table 7 below. Table 7: Calculation of the number of animals required for statistically significant plasmid expression using the electrotransfer device Efficiency ratio P=95% P=90% P = 85% P=75% 2510 3385 2875 2464 1964

These data show that the electrotransfer technique not only greatly increases the transfection rate, but also significantly reduces the variability of the reaction. The method and the devices used to practice it allow rigorous analytical studies of tissue transfection and reproducible delivery of therapeutic genes within the therapeutic window.

Comparison tests of the slopes obtained for each regression were not significant. There is thus a 5% risk of parallelism between the two regressions. Calculations of relative power showed that to obtain the same effect, approximately 250 times more DNA had to be injected per muscle under standard conditions (243 ± 85, 95% confidence interval) compared to using the electrotransfer device. This result can be interpreted as an approximately 500-fold enhancement of transgene expression compared to standard DNA injection for the same dose of DNA injected using electrotransfer.

This example shows that, using two luciferase-encoding plasmids, it is possible to establish a significant linear correlation between injected plasmid dose and electrotransfection. This correlation is very insignificant in the absence of electrotransfer. Statistical analysis also demonstrated a significant reduction in variance in the electrotransfected group. Therefore, it is possible to efficiently and predictably regulate the expression level of a transgene by varying the amount of injected plasmid using the electrotransfer device of the present invention.

Example 16: Electrotransfer Using Different Electrodes

This example compares the effect on nucleic acid transfer efficiency of an electrotransfer device equipped with one of two types of electrodes, plate electrodes and needle electrodes. Furthermore, needle electrodes were tested with different implant orientations.

Plasmid pXL2774 (150 μg) was injected into the triceps muscle of rats. Plate electrodes were placed as described in Example 1 with an electrode spacing of 1.2 cm. For needle electrodes, the electrode spacing was 0.9 cm. Insert the needle electrodes into the muscle tissue at equal lengths, either parallel or perpendicular to the axis of the muscle fibers surrounding the injection site. Regardless of the electrode type or its orientation, the electric field conditions were as follows: intensity of 200 V/cm; 8 pulses of 20 msec; frequency of 2 Hz.

The results of this experiment are shown in FIG. 8 . The data show that electrotransfection is comparable regardless of the way the electric field is applied. Similar levels of transfection were obtained with needle electrodes and plate electrodes. Furthermore, the efficiency of electrotransfer does not appear to depend on the orientation of the needle electrode relative to the direction of the muscle fiber.

These data show that an electrotransfer device can use plate or needle electrodes regardless of the orientation of the electrodes relative to the target tissue. For nucleic acid administration to large muscles, the application of needle electrodes may be preferred to ensure that the total voltage is moderate, eg 100 V for a field strength of 200 V/cm with a needle electrode placed at 0.5 cm. However, non-invasive plate electrodes may be preferable for small muscles such as the fingers, such as for the delivery of gene therapy for arthritis.

Example 17: Electrotransfer into different muscles and species

This example demonstrates that electrotransfer devices can be used to achieve the transfer of nucleic acids to a variety of different types of muscle in different animal species.

The electrotransfer apparatus was adjusted to provide the conditions described in Table 8 for each species. The results are also shown in Table 8. Table 8: Electrotransfer Enhancement of Nucleic Acid Transfection in Different Species and Muscles species plasmid Electric field conditions cranial tibialis muscle Gastrocnemius rectus femoris arm triceps quadriceps mouse 10 μg pXL3031 8×200V/cm20msec, 2Hz ×28 ×196 ×342 ×1121 the rat 150 μg pXL3031 8×200V/cm20msec, 2Hz ×31 ×160 ×13,2 rabbit 200 μg pXL2774 4×200V/cm20msec, 1Hz ×25417 ×724 ×3595

The relative increase in luciferase expression levels using the electrotransfer device relative to the control (no electrotransfer) is shown. Data are the average of 10 muscles per group. Luciferase activity was determined 7 days after plasmid administration.

Electrotransfer was also tested in monkeys (Macaca fascicularis). Plasmid pXL3179 (Fig. 11) containing the 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 the cDNA of aFGF ( sp-FGF1, Jouanneau et al., 1991, PNAS88: 2893-2897), its introduction was placed under the control of the human CMV-IE promoter and the SV40 polyadenylation signal. Expression of aFGF was determined by immunohistochemistry. The number of positive cells (cells expressing aFGF) was evaluated three days after intramuscular injection of 500 μg of plasmid pXL3179. The electric field conditions were 200 V/cm, 8 pulses of 20 msec each, and a frequency of 1 Hz. Controls were not treated with electric field (electrotransfer-).

The results of this experiment are shown in Table 9. The data clearly demonstrate that aFGF protein expression was detectable only after the efficiency of DNA transfer into muscle tissue was enhanced using the electrotransfer device. Interestingly, no expression was detected in the absence of electrotransfer under these conditions. Table 9: Immunohistochemical analysis of aFGF expression in monkey muscle electrotransfer- Electrotransfer+ triceps 0 0 cranial tibialis muscle 0 30 biceps 0 4 quadriceps 0 30

Immunohistochemical analysis of aFGF expression in different muscles of monkeys. Data represent the number of positives three days after intramuscular injection of 500 μg of plasmid pXL3179 encoding aFGF with (+) and without (-) application of an electric field.

Example 18: Electrotransfer in Rat Diaphragm

The ability to provide long-term, stable expression of transgenes using the electrotransfer device of the present invention has important implications in the treatment of degenerative diseases affecting diaphragm function, especially muscular dystrophy.

In these experiments, the diaphragm was exposed through an incision along the sternum following anesthesia (a mixture of 1 mg/kg largactyl and 150 mg/kg ketamine). Injections (50 μg plasmid pXL2774 in 50 μl 20 mM NaCl and 5% glucose) were performed in the hemidiaphragm. Then place the flat plate electrodes on either side of the diaphragm plane along the injection route, with an electrode spacing of 1 mm. The electric field conditions used were as follows: 160 V/cm or 300 V/cm; 8 pulses of 20 msec duration each; a frequency of 1 Hz. The electric field was applied to the muscle less than 1 min after the injection. The animal's incision is then closed.

The results are shown in Table 10. Table 10: Electrotransfer into rat diaphragm V/cm 0 160 300 Total number of RLUs 48±33 920±474 51±29

Values for luciferase expression are mean ± S.E.M. of luciferase activity in million RLU per muscle. N=12 per group. This example demonstrates a significant improvement in transgene expression in the diaphragm following application of eight 20 msec pulses at an electric field strength of 160 V/cm (p<0.003 using the Mann-Whitney non-parametric test).

Example 20: Electrotransfer of secreted alkaline phosphatase gene

This example demonstrates the ability to transfect and express genes encoding secreted proteins. Secreted proteins are used, for example, in systemic gene therapy and for generating an immune response (DNA vaccines). The secreted genes shown here were found in circulation at elevated concentrations and their presence was stable.

In this example, adult C57B1/6 mice were injected with plasmid pXL3010 encoding alkaline phosphatase ( FIG. 13 ) into one of the two cranial tibialis muscles. Plasmid pXL3010 was derived from ColE1, into which the gene encoding secreted alkaline phosphatase (SeAP) derived from pSEAP-basic (Clontech; Genbank accession number CVU09660) was introduced, placed in the CMV promoter (pCDNA3; Invitrogen, The Netherlands) and SV40 multi- under the control of adenylation signaling. The application of the electric field was carried out under standard conditions, ie, an 8th square wave pulse of 200 V/cm with a duration of 20 msec, a frequency of 1 Hz, and 20 seconds after plasmid injection. Serum alkaline phosphatase concentrations were measured 7 days later on blood samples taken from the eyes (puncture of the retro-orbital plexus) using a commercial chemiluminescent assay (Phosphalight, Tropix, Bedford, Massachusetts, US). Intramuscular injection of a non-coding plasmid (ballast DNA) into a small number of subjects exposed or not to the electric field confirmed that the lack of serum alkaline phosphatase was not due to transgene expression.

The effect of increasing transgene expression by applying an electric field under these conditions was clear for different amounts of injected plasmid (Table 11). It is possible to achieve high serum concentrations of phosphophosphatase by increasing the amount of plasmid injected. In this experiment, this result persisted for a long time after injection relative to conventional transfection. Table 11: Expression of SeAP from mouse muscle with and without electrotransfer Plasmid pXL30 10 μg Plasmid pUC19μg electrotransfer- Electrotransfer+ 0.1 0 0.03±0.01(n=5) 1.23±0.21(n=10) 0.3 0 0.05±0.02(n=5) 1.92±0,33(n=10) 1 0 0.16±0.04(n=5) 7.58±1.18(n=10) 10 0 1.52±0.59(n=10) 262.87±54.97 (n=10) 400 0 15.64±10.77(n=5) 2203.11±332.34 (n=5) 0.1 9.9 0.088±0.015(n=5) 21.39±3.54(n=10) 0.3 9.7 0.90±0.49(n=5) 95.67±16.15(n=10) 1 9 0.26±0.09(n=5) 201.68±32.38(n=10) 10 0 0.21±0.05(n=10) 357.84±77.02(n=10)

The mean ± S.E.M. of SeAP in serum is reported in this table in ng/ml.

The mean ± S.E.M. of SeAP in serum is reported in this table in ng/ml.

Injection of 400 μg of plasmid (two injections of 100 μg DNA in 54 μl flanked by 30 min intervals) resulted in a serum concentration of alkaline phosphatase of 2.2 μg/ml with electrotransfer compared to 0.016 μg for the control. Furthermore, the electrotransfection level of small amounts of plasmid pXL3010 (≤1 μg) was further enhanced by the use of ballasted DNA, which made it possible to act with a constant amount of DNA regardless of the amount of plasmid (total 10 μg of DNA per mouse) .

Kinetics of SeAP expression were also performed subsequently. In this case, the DNA dose was 15 μg, injected bilaterally into each muscle (30 μg per mouse). The results are shown in FIG. 9 . Seven days after transfection, significant and stable (at least two months) serum concentrations of SeAP due to pXL3010 electrotransfer were observed.

These results demonstrate that the transfer of nucleic acids in muscle using the device of the present invention enables high and stable levels of expression of secreted transgenes. Therefore, it is possible to use muscle as an organ for the production of secreted proteins of interest, as well as the direct expression of therapeutic genes (such as dystrophin genes or angiogenic factors) that act directly on the muscle itself.

Example 21: Electrotransfer of the Erythropoietin Gene

This example demonstrates that a therapeutic gene can be transferred to muscle using the device of the invention and that expression of the gene product elicits a detectable and meaningful physiological response. In this case, 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 unilaterally injected with the plasmid pXL3348 containing the gene encoding erythropoietin in the cranial tibialis muscle (Fig. 16). Plasmid pXL3348 was derived from plasmid pXL2774 in which the murine erythropoietin gene (NCBI: 193086) was introduced under the control of the human CMV-IE promoter and SV40 polyadenylation signal. An electric field (200 V/cm, 8 pulses of 20 msec, frequency of 1 Hz) was applied immediately after plasmid injection.

Table 12 reports the results of this experiment. Table 12: Expression and effect of erythropoietin Serum level of erythropoietin on D7 (mIU/ml) Serum level of erythropoietin on D24 (mIU/ml) plasmid electrotransfer electrotransfer electrotransfer- Electrotransfer+ pXL3348 (1 μg) 0 3.0±1.6 0 1.12±0.8 pXL3348 (10 μg) 0,9±0,9 61.8±15.8 0 74.1±28.9 pUC19 (1 μg) 0 0 % increase in hematocrit at D7 % increase in hematocrit at D24 plasmid electrotransfer- Electrotransfer+ electrotransfer- Electrotransfer+ pXL3348 (1 μg) 38.5±0.5 35.0±3.6 50.8±2.3 81±0.5 pXL3348 (10 μg) 32.0±3.2 26.0±4.1 69.0±5.1 83.0±1.0 pUC19 (1 μg) 30.8±2.3 43.2±0.9

Mean ± S.E.M.; N = 4-5 mice per group.

At D24, injection of 1 μg of plasmid was associated with a mild increase in hematocrit for conventionally transfected mice and very high for electrotransfected mice. Application of 10 μg of plasmid increased hematocrit in the control group. The hematocrit was significantly higher with lower variance for the electrotransfected group. Similar results were observed with a smaller amount of DNA (1 μg).

Example 22: Electrotransfer of VEGF (vascular endothelial growth factor) gene

C57B1/6 or SCID mice were injected bilaterally in the cranial tibialis muscle with 15 μg of plasmid pXL3212 ( FIG. 11 ), a plasmid pCOR hVEGF encoding VEGF. Plasmid pXL3212 was derived from plasmid pXL2774 into which cDNA encoding VEGF (Genbank accession number HUMEGFAA) was introduced under the control of the human CMV-IE promoter and SV40 polyadenylation signal. Electrotransfection was performed using a commercial electric pulse generator (Jouan) with 8 pulses of 20 msec duration, 200 V/cm, and 2 Hz frequency. Blood samples were collected from the retroorbital plexus in dry test tubes. Blood samples were collected one day before plasmid injection and 7 days after injection. Quantikine kit (R&D System) was used to quantify human VEGF by immunoenzyme. Supplemented human VEGF series in mouse serum. The results of these series are shown in Table 13. Table 13: Expression of human VEGF in mouse serum mouse strain sky Transfection method Human VEGF(ng/L) C57BL6 D-1 simple injection not detected C57BL6 D+7 electrotransfer 393±110 SCID D-1 simple injection not detected SCID D+7 electrotransfer 99±26

Serum concentrations of VEGF (ng/liter) in C57B1/6 or SCID mice. Control mouse serum was obtained from mice the day before plasmid injection. Example 23: Electrotransfection of coagulation factor IX gene Except that 15 μg of plasmid pCor hFIX (pXL3388; FIG. 12 ) encoding coagulation factor IX was injected per muscle in C57BL6 or SCID mice, the experimental conditions were the same as in Example 22. Plasmid pXL3388 was derived from plasmid pXL2774 into which cDNA encoding human factor IX (Chrismus factor; Genbank accession number HUMFIXA) was introduced under the control of the CMV-IE promoter and SV40 polyadenylation signal. Electrotransfer conditions were as follows: 8 pulses at 200 V/cm, frequency 2 Hz, duration 20 msec. Factor IX levels were determined 7 days after plasmid injection. Blood samples were collected from the retroorbital plexus in tubes containing trisodium citrate and the tubes were stored on ice.

The table below (Table 14) shows that human Factor IX was found only in the blood of C57BL6 or SCID mice that had been injected with pXL3388 plasmid in the cranial tibialis muscle and applied an electric field using the electrotransfer device of the present invention. Table 14: Expression of Human Factor IX mouse strain injection electrotransfer Human Factor IX(μg/L) C57BL/6 pXL3388 + 69±12 C57BL/6 pXL3388 - not detected C57BL/6 NaCl 0.9% + not detected SCID pXL3388 + 66±5 SCID pXL3388 - not detected

Factor IX concentrations in C57B1/6 or SCID mice.

Human Factor IX was undetectable in mouse blood without the use of the electrotransfer device of the present invention.

Example 24: Electrotransfer of the Acid Fibroblast Growth Factor (aFGF) Gene

The experimental conditions were the same as in Example 22, except that 15 μg of the plasmid pCorCMV aFGF (pXL3096; FIG. 14 ) encoding FGF was injected per muscle in C57BL6 or SCID mice. Plasmid pXL3096 was derived from plasmid pXL2774 (TH; Wils et al., 1997, Gene Therapy 4:323-330) in which the signal peptide encoding human fibroblast interferon was fused to the cDNA encoding FGF1 by introducing triple-helix forming sequences The gene for the FGF1 (sp-FGF1; Jouanneau et al., supra) is under the control of the CMV-IE promoter, followed by the transcribed, untranslated leader sequence of HSV thymidine kinase, and the SV40 polyadenylation signal. The following electrotransfer conditions were used: 8 pulses of 20 msec duration, 200 V/cm, 2 Hz frequency. The presence of FGF was shown by immunohistochemistry.

Figure 10 shows the results of transfection in C57B1/6 mice, and Table 15 shows the results in SCID mice. For electrotransfected muscles, the number of fibers expressing FGF in randomly selected sections was always significantly higher than that of control muscles that received simple injections of the pXL3096 plasmid alone. The expression of FGF reached the maximum at D8 after electrotransfection. At D21 and D35, the presence of FGF was virtually undetectable for the control group, whereas a large number of positive fibers were observed in the electrotransfected group. Table 15: Expression of aFGF in SCID mice electrotransfer cranial tibialis muscle (left) cranial tibialis muscle (right) pXL3096 (15 μg) ++ 600700 450300 pXL3096 (15 μg) --- 330 000 pXL3096 (1.5 μg) +++ 8020110 7035100 pXL3096 (1.5 μg) -- 00 01

The number of aFGF-positive fibers detected by immunohistochemistry in muscle sections was determined for each mouse. Muscle sections were obtained from the middle of the muscle.

Expression of aFGF was detected almost exclusively in mice treated with the electrotransfer device, as determined by the number of positive fibers shown by immunohistochemistry. Furthermore, aFGF expression was detectable at lower DNA doses as well as at higher doses.

Example 25: Electrotransfer of the Neurotrophic Factor-3 (NT3) Gene

5-week-old C57B1/6 and juvenile Xt/pmn mice were injected unilaterally in the cranial tibialis muscle with 12.5 μg of plasmid pXL3149 encoding neurotrophic factor NT3 ( FIG. 14 ). The pmn mouse is a model of amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), characterized by early and rapid motor neuron degeneration, and an average life expectancy of approximately 40 days. Plasmid pXL3149 was derived from plasmid pXL2774 into which the murine NT3 gene (Genbank accession number MMNT3) was introduced, placed under the control of the human CMV-IE promoter and SV40 polyadenylation signal. The expression of NT3 was studied in the supernatant prepared by grinding the muscle in PBS buffer and centrifuged (12000 xg) after 7 days of mouse treatment, and quantified by ELISA assay (Promega kit).

C57B1/6 mice received an injection of 12.5 μg of plasmid DNA. Half of the mice were subjected to an electric field (250 V/cm, 1 Hz frequency, 4 pulses of 20 ms) immediately after injection. The mean (calculated with 95% confidence interval) for 20 muscles was 77±11 pg/muscle without electrotransfer and 2.7±0.9 ng/muscle with electrotransfer. Endogenous NT3 levels were not determined.

Similar data were found for NT3 expression in 4-5 day old Xt/pmn heterozygous mice. These mice received 130 μg DNA per animal following multiple injections in different muscles (gastrocnemius, 25 μg; cranial tibialis, 12.5 μg). The following electrotransfer conditions were used: 4 pulses of 20 msec duration, 500 V/cm, 1 Hz. NT3 was detected in these mice 7 days after plasmid administration and electric field application. Table 16 reports the results of this experiment. Table 16: Expression of NT3 in Xt/pmn mice NaCl 0,9% (control) pXL3149 electrotransfer - + - + plasma 0(n=2) 0(n=2) 46±10(n=4) 1599±639 (n=4) Gastrocnemius 3619±102 (n=4) 1619±150(n=2) 3647±1078 (n=8) 19754±3818 (n=8) cranial tibialis muscle 1415±363(n=4) 1453±375(n=2) 1400±155(n=8) 16826±3135 (n=8)

Mean ± S.E.M. of NT3 (pg per muscle or pg/ml plasma).

Under the experimental conditions tested here, basal levels of NT3 were detected in the gastrocnemius and cranial tibialis muscles. Injection of plasmid pXL3149 increased NT3 expression levels under standard DNA transfer conditions. A tremendous increase in the amount of NT3 in tissue and plasma was observed when using the device of the invention. Thus, for any amount of DNA plasmid administered, application of the device of the present invention to increase transfection efficiency greatly increases the amount of transgene product expressed in muscle and plasma. This increase is particularly important for NT3 expression for enabling neurotrophic gene therapy.

Example 26: Electrotransfer of Human Growth Hormone Gene

C57B1/6 mice received unilateral injection (10 μg) of plasmid pXL3353 ( FIG. 15 ) or plasmid pXL3354 ( FIG. 15 ) in the cranial tibialis muscle. Plasmid pXL3353 was derived from plasmid pXL2274 into which the genomic human growth hormone gene (XbaI/Sph fragment of hGH extending from the transcription initiation signal to the BamHI site at 224 base pairs after the polyadenylation signal ), placed under the control of the human CMV-IE promoter and SV40 polyadenylation signal. hGH cDNA was obtained by reverse transcription from a human pituitary mRNA library after 30 cycles of amplification using the following primers:

5' complementary oligonucleotides:

5'-GGGTCTAGAGCCACCATGGCTACAGGCTCCCGGAC-3'

This oligonucleotide contains a Kozak XbaI sequence.

3' complementary oligonucleotides:

5'-GGGATGCATTTACTAGAAGCCACAGCTGCCTC-3'

The oligonucleotide contains an NsiI site and stop codon.

The amplified fragment was cloned into plasmid pCR2.1 (TA cloning kit, Invitrogen) and sequenced. The 681 base pair XbaI/NsiI fragment containing the hGH cDNA was ligated with the XbaI/NsiI 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 plasmid DNA injection. The presence of hGH in ground muscle supernatant buffered in PBS after centrifugation at 12000 xg was assayed 7 days after mouse treatment. The amount of hGH was determined by ELISA (Boehringer Manheim).

The results of this experiment are reported in Table 17. Table 17: Expression of Human Growth Hormone Genomic hGH injection (pXL3353) hGH cDNA injection (pXL3354) electrotransfer - + - + cranial tibialis muscle 87.1±9.3(n=10) 1477.6±67.6 (n=10) 2820.0±487.5(n=10) 15739.1±915.5 (n=10)

Mean (pg/muscle) ± S.E.M. of hGH expression.

These results show that the application of the electrotransfer device of the present invention enables higher expression of human growth hormone. This enhancement of expression was observed regardless of whether the genomic clone or the cDNA encoding hGH was administered.

Example 27: Electrotransfer of Vaccine Transgenes

This example reports that the electrotransfer device of the present invention enhances gene delivery for gene (or DNA) vaccination. In this example, the following products were used: VR-HA, a plasmid DNA containing the hemagglutinin gene of influenza virus (A/PR/8/34 strain). mVRgBDT is a plasmid DNA containing the glycoprotein B (gB) gene of human cytomegalovirus (Towne strain). Other products were obtained from market suppliers: ketamine, xylazine and physiological sodium chloride solution (NaCl 0.9%). Nine-week-old female Balb/c mice were used for this experiment. Mice from different cages were assigned randomly ("randomization") before the experiment.

An oscilloscope and a commercial electrical pulse generator (rectangular or square) (Electropulsator PS 15, Jouan, France) were used. The electrodes used were stainless steel plate electrodes with a pitch of 5 mm.

Anesthetize mice with a mixture of ketamine and xylazine. The plasmid solution (50 μl of 20 μg/ml or 200 μg/ml in NaCl 0.9%) was injected longitudinally through the skin with a Hamilton syringe into the cranial tibialis muscle of the left leg. Cover both electrodes with conductive gel, and place the injected leg between the electrodes in contact with it. Twenty seconds after the injection, an electrical pulse was applied perpendicular to the muscle axis with a square wave pulse generator. Monitor the electric field with an oscilloscope: 200V/cm, 20ms duration, 1Hz frequency, 8 consecutive pulses.

To assess the stimulation of the immune response, the following immunization protocol was followed:

D0→Collection of pre-immune serum samples

D1 → first injection, with or without electrotransfer

D21→Collection of immune serum samples

D22 → Intensive injection, with or without electrotransfer

D42→Collection of immune serum samples

D63→Collection of immune serum samples

Serum samples were collected from the retro-orbital sinus. The amount of specific antibody was determined by ELISA. Each experimental condition was tested at 10 points with 10 animals injected unilaterally.

The results of antibody titers against influenza hemagglutinin are shown in Table 18. Table 18: Anti-Influenza Hemagglutinin Antibody Responses electrotransfer D0 D21 D42 D63 VR-HA (1μg) - <50 132±739 1201±4380 1314±2481 VR-HA (1μg) + <50 1121±1237 10441±7819 8121±5619 (p) (0.0135) (0.0022) (0.0033) VR-HA (10μg) - <50 781±666 5113±16015 4673±8238 VR-HA (10μg) + <50 4153±2344 74761±89228 41765±52361 (p) (0.0002) (0.0005) (0.0007)

Antibody titers against influenza hemagglutinin after injection of 1 μg or 10 μg VR-HA DNA in the absence (-) or presence (+) of the electric field provided by the electrotransfer device. The results are the geometric mean of 10 animals per group (N=8 for the group injected with 1 μg DNA and exposed to the electric field, detected at D63) ± standard error. Values (p) were obtained by pairwise comparison of groups injected with DNA followed by electric field treatment or no treatment using the non-parametric Mann-Whitney test.

These results showed that antibody titers against influenza hemagglutinin were greatly increased, approximately 10-fold, in the group treated with the electrotransfer device. Indeed, mice that received only 1 μg of DNA and were treated with the electrotransfer device had higher antihemagglutinin titers than mice that received 10 μg of DNA but were not treated with an electric field.

Figure 19 shows the results of antibody responses against CMV glycoprotein B. Table 19: Anti-CMV Glycoprotein B Antibody Responses electrotransfer D0 D21 D42 D63 VR-gB(10μg)VR-gB(10μg)(p) -+ <50<50 73±138200±119(0.0558) 755±17663057±1747(0.0108) 809±13632112±1330(0.0479)

Antibody titers against CMV glycoprotein B obtained after injection of 10 μg VR-gB DNA in the absence (-) or presence (+) of the electric field provided by the electrotransfer device. The results are the geometric mean (N=9, exposed to electric field) ± standard error of 10 animals per group. Values (p) were obtained by pairwise comparison of groups injected with DNA followed by electric field treatment or no treatment using the non-parametric Mann-Whitney test.

These results showed a 4-fold increase in anti-gB titers by day 42 (D42) in the group treated with the electric field compared to the control group. Furthermore, as previously observed, the variability (standard error) was greatly reduced in mice treated with the electrotransfer device compared to untreated (control) mice.

Example 28: Electrotransfer device for tumor cell transfection

The following example illustrates one application of an electrotransfer device to enhance the delivery of nucleic acids into tumor tissue. In particular, efficient in vivo transfection of tumor cells (and most other cells) can be achieved by modifying the electrotransfer device of the present invention to provide higher voltages than are preferred for electrotransfer of nucleic acids into muscle. This example demonstrates the effect of electrotransfer on different tumors, either of human origin implanted in nude (immunodeficient) mice or of murine origin implanted in C57B1/6 (immune competent) mice. The effect of low intensity electric field pulses on both A) transfection of plasmid DNA by intratumoral injection and B) secretion of the protein encoded by the transgene into plasma after intratumoral injection has been demonstrated.

Materials and Methods

Tumor grafts were implanted into the side of female nude or C57B1/6 mice weighing 18-20 g. 20 mm ³ human lung cancer (H1299) or colon adenocarcinoma (HT29) tumors were implanted in nude mice. Murine fibrosarcoma (LBP) cells (10 ⁶ cells) or melanoma (B16) or lung cancer (3LL) tumors (20 mm ³ ) were implanted in C57B1/6 mice. Mice were sorted according to tumor size and divided into homogeneous groups.

Anesthetize mice with a mixture of ketamine and xylazine. Plasmid pXL3031 (cytosolic luciferase) or pXL3031 (secreted alkaline phosphatase) was injected intratumorally after tumors reached target volume. The plasmid solution (40 μl of 250 μg/ml DNA solution in 20 mM NaCl, 5% glucose) was injected longitudinally into the center of the tumor with a Hamilton syringe. Cover the lateral surface of the tumor with conductive gel, and place the tumor between two electrodes, and 20-30 seconds after injection, apply an electric pulse with a square pulse generator. Control the voltage intensity, duration in milliseconds and frequency in Hz with an oscilloscope at 200-800 V/cm, 8 pulses delivered at 20 msec and 1 Hz. An oscilloscope and a commercial electrical pulse (rectangular or square) generator (Electro-pulsateur PS15, Jouan, France) were used. The electrodes are stainless steel plate electrodes separated by 0.45-0.7cm.

To evaluate tumor transfection of luciferase, mice (typically 10 mice per experimental group, depending on conditions) were euthanized two days after plasmid injection. Tumors were removed, weighed, and crushed in lysis buffer. The obtained suspension was centrifuged to obtain a clear supernatant. The luciferase activity in 10 [mu]l of the supernatant was measured using a commercial luminometer to which the substrate was added automatically. Results are expressed as total RLU (relative light units) per tumor.

On days 1, 2 and 8 (D1, D2, D8) after DNA injection, plasma levels of secreted alkaline phosphatase (SeAp) were determined as described in Example 20 above.

Results and discussion

Electrotransfer into human lung cancer tumors. In the first experiment, use the conditions usually used for intramuscular gene electrotransfer: an electric field of 200 V/cm, 8 pulses at a frequency of 1 Hz, and compare the results obtained with higher voltages at 300-500 V/cm Compare the results obtained below. The purpose of the second experiment was to determine the optimal voltage conditions that had to be applied in order to obtain maximum transfection, or a voltage of 400-800 volts/cm. The results are shown in Table 20. Table 20: Electrotransfer into human lung cancer tumors RLU/Tumor test 1 test 2 Volt/cm Mean SEM Mean SEM 0200300400500600800 32 807 758 6 790 565129 744 454 39 119 425585 033 326 134 810 5345 266 632 345 1 473 785 460 44 723 317 10 163 3188 488 242 519 3 881 651 20114 201 644 540 6 162 551 2697 401 041 930 5 323 128 04711 884 115 963 40 48 4

Plasmid pXL3031 was injected into H1299 human lung cancer tumors reaching a ^target volume of 200-300 mm in female nude mice. Reported as mean and SEM of luciferase expression.

As can be seen from Table 20, with respect to the control group injected with DNA without subsequent application of an electric field:

Enhancing gene transfer in a manner dependent on the applied voltage of 200-400 volts/cm until reaching a plateau, which corresponds to the maximal transfection obtained, starting at 500 volts/cm;

• At higher voltages (600 and 800 Volts/cm), skin or deeper burns were caused, respectively; however, the expression of the transgene was not reduced.

• The enhancement of gene transfer by electrotransfer was approximately 240-320 fold.

Electrotransfer into human colon adenocarcinoma tumors. Table 21 illustrates the results of two experiments. Application of an electric field with an intensity of 600 V/cm resulted in an optimal transfection rate independent of the level of transfection in the absence of electrotransfer compared to the control without electrotransfer. Transfection was increased 6-23 fold, respectively, from 400 to 600 volts/cm. The results were quite similar. Table 21: Electrotransfer into human colon adenocarcinoma tumors RLU/Tumor test 1 test 2 Volt/cm Mean SEM Mean SEM 0400500600 4 043 062 1 827 23716 037 136 5 420 57214 096 640 7 629 21224 223 872 9 217 062 634 999 338 3115 537 359 3 571 43314 607 850 6 392 841

Plasmid pXL3031 was injected into HT29 human colon adenocarcinoma tumors reaching ^a target volume of 100-200 mm in female nude mice. Reported as mean and SEM of luciferase expression. The electrode spacing in this experiment was 0.45 cm.

Electrotransfer into murine fibrosarcoma tumors. Table 22 illustrates the results of two experiments. Application of an electric field with an intensity of 300-600 V/cm increased gene transfer by a factor of 30-70 compared to a control without electrotransfer, independent of the applied voltage. Table 22: Electrotransfer into murine fibrosarcoma tumors RLU/Tumor test 1 test 2 Volt/cm Mean SEM Mean SEM 0300400500600 581 270 348 64526 296 355 14 811 82642 498 832 31 152 74416 966 612 12 754 188 394 922 129 39511 579 942 4 615 33210 431 574 3 495 1186 034 954 1 818 46510 952 214 7 093 932

Plasmid pXL3031 was injected into murine LPB fibrosarcoma tumors reaching a target volume of 100-200 ^mm3 in female C57B1/6 mice. Reported as mean and SEM of luciferase expression.

Electrotransfer of murine melanoma tumors. The results are shown in Table 23. Application of an electric field with a strength of 500 volts/cm increased gene transfer 24-fold compared to a control without electrotransfer. Table 23: Electrotransfer of murine melanoma tumors RLU/Tumor Volt/cm Mean SEM 0300500600 1 318 740 667 58814 275 486 7 625 26232 249 218 12 605 04117 215 505 6 241 666

Plasmid pXL3031 was injected into murine B16 melanoma tumors reaching a target volume of 200-300 ^mm3 in female C57B1/6 mice. Reported as mean and SEM of luciferase expression.

Electrotransfer of murine lung cancer tumors. The results of this experiment are reported in Table 24. Table 24: Electrotransfer of Murine Lung Cancer Tumors RLU/Tumor Volt/cm Mean SEM 0300500600 121 080 37 3223 715 877 2 936 873470 499 612 237 588 44353 275 350 23 857 181

Plasmid pXL3031 was injected into murine 3LL lung cancer tumors that reached a target volume of 30 ^mm3 after 5 days of growth in female C57B1/6 mice. Reported as mean and SEM of luciferase expression.

Application of an electric field with a strength of 500 V/cm optimally increased gene transfer by a factor of 3885. These impressive results are related to the fact that these tumors were extremely difficult to be transfected with naked DNA under conditions without electrotransfer compared to other tumors previously tested.

Electrotransfer of secreted transgenes into human lung cancer tumors. The results of this experiment are shown in Table 25. Table 25: Electrotransfer of secreted transgenes into human lung cancer tumors Plasma alkaline phosphatase levels evaluation time 0 V/cm (mean ± SEM) 500 V/cm (mean ± SEM) D1D2D8 1.42±0.071.40±0.011.31±0.01 8.90±1.749.04±1.551.67±0.12

Plasmid pXL3031 (expressing SeAP) was injected into human H1299 lung cancer tumors reaching a target volume of 200-300 ^mm3 in female nude mice. Mean and SEM of luciferase expression are reported. A single electric field of 500 V/cm was applied, and SeAP levels in plasma were detected 1 day, 2 days and 8 days after plasmid injection.

The results of this experiment demonstrate a striking, transient increase in SeAp levels in plasma following tumor cell transfection under electrotransfer conditions. Administration of immunostimulatory genes such as GM-CSF or IL-2 to tumors may result in the production of effective amounts of cytokines. Furthermore, these data, combined with the luciferase expression data reported above, suggest that administration of secreted cytokines containing suicide genes such as HSV-thymidine kinase will elicit a strong antitumor response. Furthermore, data from SeAP suggest that electrotransfer-mediated transfection of anti-angiogenic genes such as the amino-terminal fragment of urokinase (ATF) or angiostatin (or endostatin) is also an effective tumor gene therapy.

This data further demonstrates that the device for electrotransfer of therapeutic genes into tumor cells provides an optimum electric field strength of 400-600 volts/cm, possibly optimally 500 V/cm ± 10% (i.e., 450-550 V/cm) .

The scope of the invention is not limited to the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are within the scope of the appended claims.

It is further understood that all base sizes or amino acid sizes and all molecular weights or molecular weight values specified for nucleic acids or polypeptides are approximate and are provided for description.

A number of publications are cited herein, the disclosures of which are incorporated by reference in their entirety.

Claims (37)

1. one kind is used for carrying out the system that nucleic acid in vivo shifts to the many cells eukaryotic cells, wherein by directly using or histiocyte contacted with nucleic acid to be transferred by part or systemic administration to tissue, and by tissue being applied one or many intensity is that the electric pulse of 1-600 volt/cm is guaranteed to shift, and this device comprises:

A) a kind of electric pulse generator, wherein this electric pulse generator produces burst length to be longer than 1 millisecond and intensity is that 1-600 volt/cm, frequency are the electric pulse of 0.1-1000Hz; With

B) electrode that is connected with electric pulse generator, be used for tissue that electrode contacts in produce electric field.

2. according to the system of claim 1, wherein this electric pulse generator generation intensity is the pulse of 1-400 volt/cm.

3. according to the system of claim 1, wherein this electric pulse generator generation intensity is the pulse of 30-300 volt/cm.

4. according to the system of claim 1, wherein this electric pulse generator produces the burst length of being longer than 10 milliseconds.

5. according to the system of claim 1, wherein this electric pulse generator produces 2-1000 time pulse.

6. according to the system of claim 1, wherein this electric pulse generator produces pulse each other erratically, and the function of describing the field intensity that depends on the burst length thus is variable.

7. according to the system of claim 6, the integration of wherein describing the time dependent function of electric field surpasses 1kVmsec/cm.

8. according to the system of claim 7, wherein said integration surpasses or equals 5kVmsec/cm.

9. according to the system of claim 1, wherein electric pulse generator produces and is selected from square-wave pulse, Exponential Decay Wave, the vibrate pulse of the one pole ripple and the bipolar ripple that vibrates in short-term in short-term.

10. according to the system of claim 1, wherein this electric pulse generator produces square-wave pulse.

11. according to the system of claim 1, one of them electrode is to be used for placing pending structural outer electrode.

12. according to the system of claim 1, one of them electrode is the internal electrode in the implantable pending tissue.

13. according to the system of claim 1, one of them electrode is to be used for placing pending structural outer electrode, electrode is the internal electrode in the implantable pending tissue.

14., determine that wherein the size of this outer electrode makes it to contact the exterior section of the contiguous big muscle of patient body according to the system of claim 13.

15. according to the system of claim 14, wherein this electrode is a kind of flush end plate electrode.

16. according to the system of claim 14, wherein this electrode is a kind of half-cylindrical plate electrode.

17. according to the system of claim 1, one of them electrode is intra-arterial or intravenous electrode.

18. according to the system of claim 12, wherein said internal electrode is a kind of injector system, it makes can while administration of nucleic acid and electric field.

19. according to the system of claim 18, one of them electrode is to be used for placing pending structural outer electrode.

20. according to the system of claim 1, wherein said electrode is a kind of stainless steel electrode.

21. one kind is used for carrying out the improved device that nucleic acid in vivo shifts to the many cells eukaryotic cells, wherein this device comprises the device that is used to produce electric pulse, it is connected with the electrode of generation electric field in the body in tissue, wherein this improvement comprises and improves the device be used to produce electric pulse, and surpassing 1 millisecond, intensity with generation time is that 1-600 volt/cm, frequency are the pulse of 0.1-1000Hz.

22. the device of claim 21, it is the pulse of 1-400 volt/cm that the device that wherein is used to produce electric pulse produces intensity.

23. the device of claim 21, it is a kind of pipe guide of flexibility.

24. the device of claim 21, it is a kind of by organizing the through type electrode to implant the device of nucleic acid in tissue.

25. the device of claim 24, wherein this tissue through type electrode is a kind of pin.

26. the device of claim 21, it is a kind of device that moves nucleic acid to patient's surface texture transit cell.

27. the device of claim 21 wherein makes it to be no more than the voltage that is equivalent to 600 volts/cm by adjusting the voltage door, makes the pulse that the device that is used to produce electric pulse is suitable for producing 1-600 volt/cm.

28. the device of claim 27, wherein voltage is set to constant voltage, and electrode is settled with constant space.

29. the device of claim 21 wherein by labelling and make it to be no more than the voltage that is equivalent to 600 volts/cm, and makes the pulse that the device that is used to produce electric pulse is suitable for producing 1-600 volt/cm on device.

30. according to the device of claim 21, it is the pulse of 30-300 volt/cm that the device that wherein is used to produce electric pulse produces intensity.

31. according to the device of claim 21, it is the pulse of 400-600 volt/cm that the device that wherein is used to produce electric pulse produces intensity.

32. according to the device of claim 21, the device that wherein is used to produce electric pulse produces the burst length of being longer than 10 milliseconds.

33. according to the device of claim 21, the device that wherein is used to produce electric pulse produces 2-1000 time pulse.

34. according to the device of claim 21, the device that wherein is used to produce electric pulse produces pulse each other erratically, thereby the function of describing the field intensity that depends on the burst length is variable.

35. according to the device of claim 21, the device that wherein is used to produce electric pulse produces and is selected from square-wave pulse, Exponential Decay Wave, the vibrate pulse of the one pole ripple and the bipolar ripple that vibrates in short-term in short-term.

36., wherein be used to produce the device generation square-wave pulse of electric pulse according to the device of claim 21.

37. according to the device of claim 21, wherein electrode is a kind of stainless steel electrode.

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