MXPA99009026A - Method for introducing pharmaceutical drugs and nucleic acids into skeletal muscle - Google Patents

Abstract

A method of delivering a molecule to the skeletal muscle of a mammal in vivo comprising:injecting a molecule into a skeletal muscle of a mammal;positioning electrodes near the injection site such that current traveling through the electrodes passes through the injection site;and electrically stimulating the muscle with an electrical current having a field strength between 25 V/cm and 200 V/cm.

Description

METHOD FOR THE INTRODUCTION OF DRUGS AND NUCLEIC ACIDS WITHIN THE SKELETAL MUSCLE FIELD OF THE INVENTION The present invention relates to a method for making skeletal muscle semipermeable to drugs and nucleic acids. More specifically, skeletal muscle is made semipermeable by electrical stimulation of the muscle at low field strengths after injection of drugs and nucleic acids. 2. TECHNICAL FIELD Scientists are continually discovering genes that are responsible for many human diseases, such as the genes responsible for some forms of breast cancer, colon cancer, muscular dystrophy and cystic fibrosis. In addition, scientists are continually discovering genes that code for bacterial and viral antigens (eg, viral capsid proteins). Despite these new discoveries, a major obstacle facing the REF, 31291 medical profession is how to safely administer effective amounts of these agents to patients, to treat diseases or for genetic immunization. Currently, most pharmaceutical agents are taken orally or intravenously. However, methods of administering drugs and genes orally and intravenously have several drawbacks. First, a large percentage of the orally or intravenously administered drugs are degraded by the body before reaching the target organs or cells. Acids and enzymes in the stomach and intestine, for example, can break down many pharmaceutical products. Similarly, the genes could be destroyed quickly by the proteins found in the blood and liver, which break the DNA. In addition, drugs and genes administered intravenously are frequently sequestered by the liver or the immune system before reaching the diseased organ or cells. Second, the oral and intravenous administration of the drug and the gene is non-specific. That is, the drug or gene is administered to the target cells and non-target cells.

Skeletal muscle is a promising candidate for the administration of drugs, for gene therapy and for genetic immunization. Firstly, the skeletal muscle constitutes more than 50% of the body mass of a human, most of which is easily accessible in comparison to other tissues and organs of the body. Secondly, there are numerous inherited and acquired disorders such as Duchenne muscular dystrophy (DMD), diabetes mellitus, hyperlipidemia and cardiovascular diseases that are good candidate disorders for the administration of drugs and genes within the muscle. Third, the muscle is an ideal site for genetic immunization because it is easily accessible and the proteins made in the muscle are secreted, thereby causing an immune response. Finally, the skeletal muscle cells do not divide. Therefore, skeletal muscle cells are capable of expressing a protein encoded by a gene for a longer period of time than would be expected from other cell types that are continuously dividing. Because the protein is expressed for a longer time, smaller treatments would be necessary. Currently, however, there is no non-viral method to effectively administer drugs and DNA within skeletal muscle in vivo. There are several methods known in the art for the transfer of drugs and DNA within skeletal muscle, such as intramuscular injection of DNA. The ability of clinical application of direct injection into the muscle, however, is limited mainly due to the low transfection efficiency, typically a transfection efficiency of less than 1%. It has been shown that transfection efficiency can be improved if DNA injections are performed on the regenerating muscle. The injection is induced three days before the injection of the DNA with the drug Bivucine. While the injection in the muscles in regeneration induced by Bivucine shows higher efficiency, the method has limited application possibility in humans, due to the severe damage caused to the muscle. From the foregoing, it will be appreciated that it could be an advance in the art to provide a non-viral method of administering drugs and DNA only to diseased organs and cells. It could also be an advance in the art to provide an electroporation method of drug and DNA administration directly within the skeletal muscle. There could be another breakthrough in the art if the electroporation method could therapeutically administer effective amounts of drugs and DNA within the skeletal muscle at multiple sites, simultaneously. It could also be an advance if the method allowed management efficiencies to be regulated. Such method is described herein. 3. BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for administering or transfecting drugs and DNA within skeletal muscle. Without being compromised by any theory, it is thought that the method is similar to electroporation. Electroporation works on the principle that cells act as an electric capacitor in general unable to pass current. By attaching the cells to a high voltage electric field, therefore, transient permeable structures or micropores are created in the cell membrane. These pores are large enough to allow drugs, DNA and other polar compounds to gain access to the interior of the cell. Over time, the pores in the cell membrane close and the cell once again becomes impermeable. Conventional electroporation, however, employs high field strengths of 0.4 to several kV / cm. In contrast to conventional electroporation, the field strength used in the present invention is in the range of about 25 V / cm to 250 V / cm. It is thought that these lower field strengths cause less damage to the muscle without sacrificing, and of course increasing, the transfection efficiencies. In addition, by using the method of the present invention, the transfection efficiencies can be perfectly regulated by altering parameters such as frequency, pulse duration and number of pulses. The increase in DNA transfection efficiency is observed only if the muscle is electrically stimulated, or immediately, or shortly after the DNA injection. In this way, the semipermeable quality of the tissue induced by the stimulation is reversible. In addition, it is dependent on the current through the muscle; the activity induced through the nerve does not affect the efficiency of transfection. Once transfected, the muscle cells are able to express the proteins encoded by the nucleic acid. Therefore, the transfection method of the present invention can be used, for example, to transfect expression vectors for genetic immunization (e.g., DNA vaccines). In one embodiment, rabbits were transfected with a plasmid containing the cDNA for rat agrin. The transfected muscles produced and secreted agrin protein. Nineteen days after transfection, serum from the rabbits contained significant antibodies against rat agrin. In a second embodiment, rats and mice were transfected using the method of the present invention with one or more of the three different eukaryotic expression vectors containing the coding sequences for DH-CNTF, an agonistic variant of human ciliary neurotrophic factor, AADH -CNTF, an antagonistic variant of human ciliary neurotrophic factor and sec-DHCNTF, a secreted form of DH-CNTF. The muscles were either not electrically stimulated or stimulated immediately after the DNA injection. Blood was collected at various time points and antibody titers were determined. In rats and mice, electrical stimulation immediately after DNA injection leads to antibody titers approximately 5 to 10 times higher than the injection of single DNA. The transfection method of the present invention can also be used to systemically administer proteins to treat diseases. In a preferred embodiment, a DNA plasmid harboring the erythropoietin (EPO) gene was injected into the skeletal muscle and stimulated according to the method of the present invention. The controls were either unstimulated or transfected with a control vector that does not harbor the EPO gene. After 14 days, only the mice transfected with EPO according to the method of the present invention showed an increased hematocrit, indicating that the transfected muscles were capable of producing and secreting into the bloodstream substantial amounts of EPO.

The non-nucleic acids can also be transfected by the method of the present invention. In one embodiment, dextran conjugated to rhodamine was injected into the muscle, followed by electrical stimulation. Three to five days later the muscles were frozen in liquid nitrogen and sectioned in a cryostat. Fluorescence was observed in injected and stimulated cells, indicating that dextran conjugated to rhodamine was able to enter and remain in muscle cells. These and other objects and advantages of the present invention will become apparent after reference to the following drawings and graphs, and after reading the following detailed description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS A more particular description of the invention briefly described above will be made by reference to the accompanying drawings and graphs. These drawings and graphs only provide the information concerning the typical embodiments of the invention, and therefore do not have to be considered as limiting their scope.

Figure 1 graphically illustrates the method of administering drugs and DNA within the skeletal muscle of the present invention.

Figure 2 is a graphic illustration of an electrical stimulation administered according to the method of the present invention.

Figure 3 illustrates the total amounts of the muscles that have been injected with 50 μl of the RSV-Lac Z Plasmidic DNA solution at a concentration of 1 μg / μl. Muscles 3a and 3b were removed 15 days after the DNA injection. The muscles in 3c and 3d were removed 7 days after the injection of the DNA. All muscles are pairs of the same rat.

Figure 4 depicts a whole muscle and a 1 mm slice or slice of a transfected muscle. The dark stain indicates an o-nitrophenyl-b-D-galactopyranoside (ONPG) that has been catalyzed by the β-galactosidase in the muscle to produce a dark precipitate. The arrows illustrate the muscle fibers that were successfully transfected using the method of the present invention.

Figure 5 includes the average number of transfected fibers from each group of skeletal muscles shown in Figure 3.

Figure 6 is a bar diagram illustrating the average transfected fibers of muscles from several different experiments and different batches of DNA grouped together. In the columns marked SOL S and EDL S the muscles (16 in each group) have been stimulated directly after the DNA injection. In the columns marked SOL NS and EDL NS the muscles (10 in each group) have been stimulated by the nerve, not stimulated at all or stimulated directly 10 minutes before the DNA injection.

Figure 7 is a graph illustrating the number of transfected skeletal muscle fibers, versus the logarithm of the stimulation frequency. The duration of the stimulation train was kept constant to one second.

Figure 8 is a photograph of the transfected muscles from which the data was generated in Figure 7.

Figure 9 illustrates the results achieved when complete assemblies of muscles were transfected according to the method of the present invention using two different electrodes.

Figure 10 is a graph illustrating the number of skeletal muscle fibers transfected with increasing frequency compared to an increasing number of pulses.

Figure 11 is a graphic illustration of the number of skeletal muscle fibers transfected versus the number of pulses at constant frequency.

Figure 12 is a graph illustrating the mean luciferase activity versus the number of pulses.

Figure 13 is a graph illustrating the voltage dependence of the stimulation method of the present invention. Figure 13a illustrates muscle luciferase activity stimulated with varying volts. Figure 13b illustrates the average luciferase activity of muscles stimulated with an amplitude above 13 volts and below 5 volts.

Figure 14 is a graph illustrating the effect of pulse duration on transfection efficiency.

Figure 15 is a bar chart illustrating a comparison of transfection efficiencies for varying pulse durations and varying pulse numbers.

Figure 16 is a bar chart illustrating the effect of DNA concentration on transfection efficiency.

Figure 17 is a photograph of the transfected muscles illustrating the damage caused by the stimulation and regeneration of the muscle after a short period of time. Figure 17a illustrates an injected muscle that was not stimulated. Figure 17b illustrates muscle damage after muscle stimulation. Figure 17c illustrates the muscle stimulated under stronger stimulation conditions. Figure 17d illustrates that the muscles stimulated under the conditions of the muscles in 17c are completely regenerated and repaired after 14 days. Figure 17e illustrates the muscles transfected with the green fluorescent protein (GFP). Figure 17f illustrates that the transfected fibers can be observed in the vicinity of the damaged area.

Figure 18 is a photograph of cells stained with polyclonal anti-agrin antibodies derived from a rabbit genetically immunized with an expression vector encoding rat agrin using the stimulation technique of the present invention.

Figure 19 are graphs illustrating the improved genetic immunization of mice and rats using the stimulation technique of the present invention versus the injection of naked DNA.

Figure 20 is a photograph of muscles transfected with dextran conjugated to rhodamine and green fluorescent protein. Top: fluorescence of rhodamine from dextran conjugated with rhodamine. Intermediate: the same section as the previous one but with filters that reveal the fluorescence of GFP. Bottom: staining with hematoxylin and eosin from a neighboring section.

. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a novel method for increasing the permeability of skeletal muscle tissue, thereby allowing drugs and nucleic acids to enter or transfect the cells. The method of the present invention passes a predetermined amount of electrical current through the skeletal muscle tissue. Contrary to the electroporation methods previously described, however, the parameters of the method of the present invention are unique, particularly with respect to the low field strength used, and the amount of damage that occurs. Other parameters such as the number of trains, frequency, number of pulses and pulse duration can be varied in order to regulate the amount of drug or nucleic acid administered. As illustrated in Figure 1, in general, skeletal muscle is exposed and a predetermined amount of a molecule is injected into the muscle. In one embodiment, the DNA is dissolved in 0.9% sodium chloride (NaCl). The exact solvent, however, is not critical to the invention. For example, it is well known in the art that other solvents such as sucrose are capable of increasing the uptake of DNA in skeletal muscle. Other substances can also be cotransfected with the molecule of interest for a variety of beneficial reasons. For example, P188 (Lee, and collaborators PNAS., 4524-8, 10, 89 (1992)), which is known to seal the electropermeabilized membranes, can beneficially affect transfection efficiencies by increasing the survival rate of the transfected fibers. With continuous reference to Figure 1, the electrodes are placed on the muscle, approximately 1 to 4 mm apart, near the area where the molecule was injected. The exact position or design of the electrodes is not critical, as long as the current is allowed to pass through the muscle fibers perpendicular to its direction in the area of the injected molecule. Once the electrodes are in position, the muscle is subjected to electrophoresis or stimulated. As illustrated in Figure 2, the stimulation is administered as a square bipolar pulse having a predetermined amplitude and duration. In order to optimize transfection efficiencies, these parameters have been widely varied and transfection efficiencies have been compared. For example, voltages have been in the range of approximately 0 to 50 volts; pulse durations have been in the range of 5 μs to 5 ms; the number of pulses have been in the range from a single pulse to 30,000 pulses; and the frequency of pulses within the trains have been in the range of 0.5 Hz to 1000 Hz. The conclusion from these results is that as long as the field strength is above about 50 V / cm, the other parameters can be varied depending on the desired experimental conditions. While no upper limit was detected, effective transfection efficiencies were observed with much higher field strengths. The field strength of the stimulation can be calculated using the formula: E = V / (2r In (D / r)), which gives the electric field between the wires if D > > r. In the formula V = voltage 10 V, D = distance between the centers of the cables = 0.1-0.4 cm, r = diameter of electrode = 0.06 cm. See Hofmann, G.A. Cells in electric fields. In E. Neumann, A. E. So ers, & AC Jordan (Eds.), Electroporation and electrofusion in cell biology (pp. 389-407). Plenum Publishing Corporation (1989).

At 10 volts, the field strength is between 163 V / cm-43 V / cm (from 0.1 to 0.4 cm between the electrodes, respectively). Because D is not much greater than r, it may be more appropriate to use the formula for electric fields between large parallel plates: E = V / D this gives a similar field strength of between 100 V / cm - 25 V / cm (0.1-0.4 cm between electrodes, respectively). It will be appreciated that the field strength, as well as other parameters, are affected by the tissue that is transfected, and thus optimal conditions may vary. Using the parameters given in the present invention, however, the optimum parameters can easily be obtained by someone skilled in the art. As illustrated in Figures 3 and 5-8, the method of the present invention dramatically increases the efficiency of administration of the drug and DNA within the skeletal muscle. In one embodiment, the rat soleus or the EDL were injected with the plasmid DNA containing the β-galactosidase gene (lac Z). The ß-galactosidase gene produces a protein capable of converting a colorless substrate into a blue substrate that can be visually analyzed or measured spectrophotometrically. Figure 3 depicts the representative soleus and the representative EDL muscles that have been transfected with the β-galactosidase gene using various stimulation parameters.

Figure 3a illustrates the improved efficiency of DNA administration of soleus muscles and EDL that have been transfected according to the method of the present invention. The soleus muscles and EDL (n = 3) were first denervated by transfection of the sciatic nerve. This was done to eliminate any influence of nerve-induced activity, which could probably contribute to the increased transfection efficiency observed. Three days after denervation, the muscles were injected with the β-galactosidase gene as described above. After the DNA injection, the muscles were either untreated or, immediately after the DNA injection, the muscles were stimulated according to the method of the present invention. Fifteen days after the DNA injection, the soleus muscles and EDL were analyzed. As illustrated in Figure 3a, muscle cells that were stimulated immediately after DNA injection (lower panels) contain more blue product, indicating that more ß-galactosidase gene was introduced into the muscle cells. The transfection efficiency was quantified by counting the muscle fibers in a 1 mm cross section of the muscle containing the blue product as illustrated in Figure 4. As illustrated by the bar diagram in Figure 5a, the muscle Soleus transfected using the method of the present invention showed a 47-fold increase over the muscles that were not stimulated. Similarly, the EDL muscle transfected using the method of the present invention showed a 12-fold increase over muscles that were not stimulated. To determine whether nerve activity affected the transfection efficiency, the method of the present invention was performed on the innervated EDL and sole muscles (non-transfected sciatic nerve) and denervated (transfected sciatic nerve) as described above. As illustrated in Figure 3b, fifteen days after the DNA injection, the innervated and denervated muscles produced a generous amount of blue product, indicating the high efficiency transfer of the ß-galactosidase gene. As illustrated in Figure 5b, the quantification of the transfected muscle fibers confirms the high efficiency transfection of the innervated and denervated muscles. To verify the possibility that the increased transfection efficiency observed was due to muscle activity, direct stimulation of the sciatic nerve was compared to muscle stimulation (n = 5). If the increased transfection efficiency was due to muscle activity, the efficiency of transfection in muscles stimulated via the nerve should produce similar efficiencies such as direct muscle stimulation. As illustrated in Figure 3c, direct nerve stimulation did not significantly increase transfection efficiencies compared to direct muscle stimulation. As illustrated in Figure 5c, in the soleus muscles and EDL a 10-fold increase in transfection efficiency was observed with direct muscle stimulation. As illustrated in Figure 3d, the increased efficiency is transient, consistent with electroporation. The muscles stimulated directly after the DNA injection show significantly more blue dye than the muscles that were stimulated before the DNA injection. In fact, the muscles that were stimulated directly after the DNA injection showed transfection efficiencies between 10 and 25 times greater than the muscles that were stimulated 10 minutes before the DNA injection (Figure 5d). Figure 6 summarizes the results of the present invention. Muscles from several different experiments and several different batches of DNA are grouped together. In the columns marked SOL S and EDL S the muscles (16 in each group) have been stimulated directly after the DNA injection. In the columns marked SOL NS and EDL NS the muscles (10 in each group) have been stimulated by the nerve, not stimulated at all, or stimulated directly 10 minutes before the injection of the DNA. The electrical stimulator used for the experiments was manufactured by FHC (Brunswick, ME 04011). Pulsar 6bp and Pulsar 6bp-a / s stimulators were used. The Pulsar 6bp-a / s stimulator distributes a maximum voltage of 150 volts and a maximum current of 50 A. The maximum voltage that can be distributed requires a resistance between the electrodes greater than 3000 ohms. The stimulators have been operated as a constant voltage. Due to the low strength of the muscle, the voltages have been lower as discussed in the following Examples. In all the experiments the current has been maintained at 50 mA. It will be appreciated by those skilled in the art that numerous other electrode configurations may be employed. For example, Figure 9 illustrates the results obtained using two different electrode configurations. The electrode shown in (A) was placed perpendicular to the fibers of the muscle. This consisted of a silver wire with diameter (d) of 0.6 mm, (C) (this is the electrode that was used in all experiments except (B)). An electrode was placed on each side of the muscle. A short segment in the middle third of the muscle is positive for Lac Z staining (A), indicating localized expression. In (B) a 1.5 cm electrode made from an isolated silver wire was used (d = 0.3 mm). The insulation was removed from the short segments (0.5-1.0 mm) along the cable at 2 mm intervals (D). The electrode was penetrated into the muscle in parallel with the muscle fibers. One of the two wires of the electrode was penetrated into the muscle parallel with the muscle fibers. The second wire was placed on the muscular surface, also parallel with the fibers. Both types of electrodes (Figures 9c and 9d) gave a similar number of transfected fibers (approximately 250). Using the longer electrode in parallel with the muscle fibers, however, gave a more widely diffused staining indicating a transfection along a longer segment of the fibers and / or increased transfection. The muscles were stained for Lac Z in supports or complete assemblies by methods well known in the art. After staining, photographs were taken with the blue side of the muscle facing up. After this the muscle was cut into three pieces as seen in Figure 2. The number of blue fibers in a slice about 1 mm thick from the middle part of the muscle were counted (transfected fibers distally or proximally of the slice). they are not therefore counted). In order to count the transfected fibers, the slices were dissected in smaller clusters so that simple fibers could not be distinguished under a dissecting microscope. The pSV40-luc construct was used in four (4) muscles. This was injected into the soleus muscle, 3 days after the muscles were removed and the luciferase activity was measured using the Promega Luciferase Assay system (Daviset et al., 1993). The non-injected EDL of the same rats was used as control. It will be appreciated that any nucleic acid can be used with the method of the present invention, for example, plasmid DNA, linear DNA, antisense DNA and RNA. In a preferred embodiment, the nucleic acid is a DNA expression vector of the type well known in the art. In general, an expression vector contains a promoter operably linked to a DNA molecule encoding the protein of interest, followed by a termination signal such as a polyadenylation signal. Other elements required for bacterial development and appropriate processing by the mammal may be included, such as the ß-lactamase coding region, an origin fl and the origin of plasmid-derived replication of ColEl. Similar constructs containing a DNA coding region of interest to any person skilled in the art may be elaborated. As illustrated in the following examples, different nucleic acid molecules can be administered to the muscle using the technique of the present invention. In one embodiment, dextran conjugated to rhodamine injected into the muscles and stimulated according to the method of the present invention was able to enter the muscle cells. In addition, nucleic acid and proteins can be simultaneously introduced into a muscle undergoing electroporesis. In one embodiment, the nuclear localization signal of the large T antigen was mixed with a plasmid containing the DNA construction region for Lac Z. The nuclear localization signal of the large T antigen is a protein that binds to DNA and facilitates its transport within the nucleus of a cell. In other systems, the nuclear localization signal of the large T antigen has been shown to increase transfection efficiency. Using the method of the present invention, the nuclear localization signal of the large T antigen also increased the transfection efficiency of Lac Z, indicating that the protein was able to bind to DNA and enter the muscle cell. 6. EXAMPLES The following examples are given to illustrate the various embodiments that have been made of the present invention. It should be understood that the following examples are not limiting or exhaustive of the many types of embodiments that may be prepared in accordance with the present invention.

Example 1 - Stimulated Versus Unstimulated Muscles The transfection efficiencies were determined by injecting the skeletal muscles with the reporter construct pSV40-Luc into the soleus muscle. Three days after the injection, the muscles were removed and the luciferase activity was measured using the Luciferase Assay System Promega (Madison, Wl) according to the manufacturer's protocols. The unstimulated EDL muscles from the same rats were used as control. The data are shown in Table 1 below.

TABLE 1 STIMULATED MUSCLES VERSUS NON-STIMULATED Stimulated Muscle (Non-Stimulated Luciferase Activity (Relative Relative Luciferase Activity)) Percentage Soleo animal I 34.40 1.950 1664% Soleo animal II 21.50 0.250 8500% EDL animal I 0.045 EDL animal II 0.046 Example 2 Transfection Efficiency Versus Frequency: The rats were injected with 50 μl of 1 mg / μl of a plasmid possessing the Lac Z gene. Immediately after the injection, the electrodes were placed between 2-3 mm apart and the muscles were stimulated with the following stimulation parameters. : voltage = 30 volts; pulse duration = 0.2 ms (total 4.0 ms, bipolar); successions or trains = 30, 1 second off 1 second on for 1 minute. The transfected fibers were counted from a 1 mm slice from the middle part of the muscle. The number of transfected fibers is shown below in Table 2 and illustrated in Figure 7. These data also illustrate that the method of the present invention transforms more than just superficial muscle fibers; muscle fibers of several deep cell layers are also transfected.

TABLE 2 TRANSFER EFFICIENCY VERSUS FREQUENCY Frequency (Hz) Medium (Transfected Fibers) Percent Increase with Stimulation 0 22 1 83 277% 10 153 595% 100 215 877% 1000 315 1332% Example 3 Transfection Efficiency Versus Pulses: The vocal muscles of Wistar rats (200-270 grams) were injected with 50 μg of plasmid DNA of the RSV luciferase in 50 μl of 0.9% NaCl. Shortly after the injection, the muscles were electrically stimulated using the following parameters: 1000 Hz, between 0 - 1000 bipolar pulses of 200 μs duration in each train were applied to the muscle 30 times in a period of 1 minute. The muscles were removed three days after transfection and frozen in liquid nitrogen. Sections of cryostats were taken from the muscles and stained with Hematoxoline, Eosin and Safran (see Example 9). The remaining pieces were homogenized as described in Example 4 below. As illustrated in Figures 10-12, transfection efficiency increased with the number of pulses distributed to the muscle.

Example 4 - Determination of the Voltage Effect on Transference Efficiency EDL and Wistar rat sole muscles (245-263 grams) were injected with 25 μg of luciferase plasmid DNA promoted by RSV in 50 μl of 0.9% NaCl. Shortly after the injection, the injected muscles were electrically stimulated using the following parameters: 100 Hz, 100 bipolar pulses in each train of 200 μs duration, voltage varied between 0 to 47.5. The muscles were removed 4 days after injection and stimulation, homogenized in Promega luciferase assay buffer (Madison, Wl) and the luminescence was measured according to the manufacturer's protocols. A Macintosh computer and a LabWiev acquisition program were used to capture the first voltage pulses. Records were made in parallel with the stimulation electrodes. Voltage measurements were manually made on impressions as the average of the maximum voltage of 10 pulses approximately 100 ms after the start of the stimulation.

As illustrated in Figure 13a, there was a pronounced increase in transfection efficiency with the increased voltage. As illustrated in Figure 13b, under the conditions of this experiment, muscles stimulated with 13 volts or more showed luciferase activity 40 times higher compared to muscles stimulated with 5 volts or less.

Example 5 - Determination of Duration Optimum Pulse Wistar rat sole muscles (200-270 grams) were filled with 50 μg of plasmid DNA containing the β-galactosidase gene in 50 μl of 0.9% NaCl. Shortly after the injection, the muscles were electrically stimulated using the following parameters: 100 Hz, 25 volts, 100 bipolar pulses in each train having pulse durations in the range of 5-200 μs. The number of transfected fibers was counted in a 1 mm thick section from the middle part of the muscle under a dissecting microscope. A second group of rats was injected with 25 μg of the plasmidic DNA of the luciferase promoted by RSV in 50 μl of 0.9% NaCl and electrically stimulated with the same parameters as described above, except that the pulse durations were varied from 500 at 2000 μs. As illustrated in Table 3 below and in Figure 14, under these stimulation parameters, the optimal duration of the pulses was in the range of about 50 μs to about 200 μs. This method can be used to optimize the pulse duration of other stimulation parameters.

TABLE 3 TRANSFER EFFICIENCY VERSUS PULSE DURATION Pulse Duration Transfected Fibers Pulse Duration Activity (μs) (Medium) (μs) luciferase (average) 0 -. 0 - 0 52.7 5 51 50 631 20 107 200 536 50 228 500 348 200 272 2000 194 Example Current versus number of pulses Sole muscles of six Wistar rats (178-193 grams) were injected with 50 μg of plasmid DNA containing the β-galactosidase gene in 50 μl of 0.9% NaCl. Shortly after the injection, the muscles. they were electrically stimulated as described above, except that the duration of the pulses was varied. The following electroporation parameters were compared: (1) 100 pulses of duration of 50 μs versus 1 pulse of 5,000 μs; and (2) 10 trains of 100 pulses of 50 μs versus 10 pulses of 5,000 μs. The muscles were removed 14 days later and sectioned in a cryostat. The cross sections were stained as previously described. The number of transfected fibers was counted. As illustrated in Figure 15, longer pulse durations result in higher transfection efficiency.

Example 7 - DNA concentration The EDL muscles of six Wistar rats (178-193 grams) were injected with either 1 μg / μl or 5 μg / μl of plasmid DNA containing the β-galactosidase gene in 50 μl of 0.9% NaCl. Shortly after the injection, the muscles were electrically stimulated with 30 trains of 100 pulses of 200 μs in duration or not stimulated at all. The muscles were removed 14 days later and sectioned in a cryostat. The cross sections were stained as previously described and the transfected fibers were counted. As illustrated in Figure 16, higher transfection efficiencies were obtained with higher DNA concentrations.

Example 8 - Nuclear Localization Signal of T Antigen, Large: Wistar rat muscles were injected with plasmid DNA containing the β-galactosidase gene containing a molar excess of 100: 1 of the nuclear localization signal of the large T antigen. This has been shown in other transfection studies, which improves transfection (See, p.

Coll as and col aboradores. Transgenics Res. , 6: 451-8 (1996)). The muscles were stimulated with 10 trains or series of 100 pulses of 50 μs in duration. The muscles that contained the nuclear localization signal of the large T antigen had the highest number of transfected fibers. Specifically, the muscle contrasted with the nuclear localization signal of the large T antigen had 100 and 38 transfected fibers versus 7.3 and 4.7 for the muscles transfected only with DNA, respectively. These data illustrate that transfection efficiencies can be aided by mixing DNA with non-nucleic acid molecules. In addition, these data illustrate that the non-nucleic acid molecules can also be distributed to the muscle using the electroporation techniques of the present invention. No improvement was observed in the cells that were not stimulated after the injection.

Example 9 - Muscular Damage Resulting from Stimulation: The muscles of Example 3 that were sectioned and stained to evaluate muscle damage from electroporation. As illustrated in Figure 17a, some damage may occur with the injection alone, although most of the unstimulated muscles remained undamaged. In muscles stimulated with 300 pulses, more damage was observed (Figure 17b). As illustrated in Figure 17c, the muscle stimulated with 30 trains of 1000 pulses showed greater damage, indicating that the damage is proportional to the degree of stimulation. Figure 17d illustrates that the muscles stimulated under the conditions of the muscles in 17c are completely regenerated and repaired after 14 days. In another muscle that had the highest amount of stimulation (3 trains of 1000 pulses), the plasmid DNA encoding the green fluorescent protein (GFP) was also included. Figure 17e illustrates the muscles transfected with GFP. The transfected fibers can be observed in the vicinity of the damaged area (Figure 17f). The transfected regeneration fibers were never observed in cross sections three days after electroporation.

Example 10 Genetic Immunization of Rabbits: A female rabbit (4.5 kg) was injected into the right femuralis rectus with 2 milliliters of a 1 μg / μl solution of plasmid DNA containing the rat neural agrin cDNA promoted by the CMV promoter (Cohen et al., MCN, 9). , 237-53 1997)). The first milliliter was injected equally into ten superficial sites in the muscle, followed by ten sets of 1000 pulses distributed at a frequency of 1000 Hz. The second milliliter was placed lower in the muscle. To test the rabbit serum, the rat muscles and COS cells were transfected with the same product. The muscles were taken 5 days after transfection and the COS cells were stained 4 days after transfection. Bleeds were collected at days 0, 19, 50, 81 and 106 and diluted to 1: 100 and 1: 1000. After 19 days the blood contained sufficient antibodies in the serum to give a weak staining of the transfected fibers when diluted 1:10. As a positive control, the monoclonal antibody (mAb) AG-86 was used. See Hoch et al.

EMBO J, 12 (13): 2814-21 (1994). The preimmune serum did not show any staining of the transfected fibers. The subsequent bleeds had all antibodies against agrin in the serum. Blood collected on day 50 or later contained sufficient antibodies for sections stained at a dilution of 1: 1000. Figure 18a illustrates the COS cells transfected with agrin, stained with antiserum from immunized rabbit (diluted 1: 100) and the secondary antibody conjugated to fluorescein. The COS cells were first stained by fixing the cells in 1.5% paraformaldehyde for 10 minutes, followed by a 30 minute wash with phosphate buffered saline (PBS). The cells were then blocked with 0.2% bovine serum albumin, triton X-100, 0.1% in 0.1 M PBS, for 4 minutes. The serum diluted in the same solution was added to the cells and allowed to incubate for 20 minutes. The cells were washed for 4 minutes in PBS and incubated with the secondary antibody (Cappel, 55646) for 10 minutes followed by a wash with PBS. The primary mouse mAb, Agr-86, was included in the same antibody mixture and the rhodamine conjugated secondary antibody (Sigma T-5393, St. Louis MO) was used at a dilution of 1: 100. Figure 18b illustrates the same cells stained with the Ag-86 / rhodamine mAb conjugate. These data illustrate the potential of the technique of the present invention for genetic immunization or DNA vaccine technology.

Example 11 Genetic Immunization of Mice Groups of two-month-old male Sprague Dawley rats were inoculated bilaterally into the EDL and soleus muscles with a total of 200 micrograms (4 x 50 microliters of a 1 mg / ml solution of DNA in saline) of three expression vectors Different eukaryotes containing the cytomegalovirus immediate early promoter (CMV) and the coding sequences for the following proteins: DH-CNTF, an agonistic variant of human ciliary neurotrophic factor (Saggio et al., EMBO J. 14, 3045-3054, 1995 ); AADH-CNTF, an antagonistic variant of human ciliary neurotrophic factor (Di Marco et al Proc. Nati, Acad. Sci. USA 93, 9247-9252, 1996); sec-DHCNTF, a secreted form of DH-CNTF. The muscles were either not electrically stimulated or stimulated immediately after DNA injection using 30 series of 100 or 1000 square bipolar pulses (duration of 200 microseconds; amplitude adjustment to 150 Volts, effective voltage approximately 25 Volts) each, administered at a frequency of 1000 Hz with an interval of two seconds between series or successive trains. Groups of male CD1 mice, two months old, were inoculated bilaterally into the quadriceps muscles with 100 micrograms (2 x 50 microliters of a 1 mg / ml solution of DNA in saline) of the sec-DHCNTF plasmid, with or without Electrical stimulation of the muscle immediately after the DNA injection. The stimulation conditions were 10 series of 1000 square bipolar pulses (amplitude adjustment at 150 volts) administered at a frequency of 1000 Hz with an interval of two seconds between trains or successive series. Blood was collected from the retroorbital sinus at selected points of time and the serum was prepared and stored at -20 °. The presence of CNTF antibodies in rat and mouse sera was determined by ELISA. Microtitre plates coated with the human recombinant CNTF were incubated with serial dilutions of the sera, followed by the alkaline phosphatase conjugated antibody against rat or mouse IgG (Pierce). The plates were then incubated in the presence of p-nitrophenyl-phosphate and the absorbance at 405 nm was determined using a microplate reader. The antibody titers were defined as the dilution of the serum that produces an absorbance reading equal to 50% of that obtained with a saturation concentration of the anti-CNTF antiserum. The results are shown in Figure 19. Titers could not be averaged accurately, due to the fact that some animals did not develop detectable amounts of antibodies. The data are therefore presented for the individual animals, with a value of 1: 100 representing a low or non-detectable antibody titer (reciprocal titer 3/4 100). The results were similar for all plasmids used, as well as for rats and mice, as described in Figure 19. Similar results can be obtained in rats and mice with another plasmid encoding an unrelated viral protein (data not shown ). In rats and mice, electrical stimulation immediately after DNA injection led to antibody titers about 5 to 10 times higher than the injection of single DNA. This was true for stimulation with high and low numbers of pulses. These results demonstrate that the electroporation method increases the efficiency of DNA-mediated immunization.

Example 12 - Secreted Proteins with Systemic Biological Activity: Fifty micrograms (50 microliters of a 1 mg / ml solution in 0.9% NaCl) of a eukaryotic expression plasmid (CMV-EPO) containing the mouse erythropoietin cDNA under the control of the cytomegalovirus immediate early promoter was injected in the left quadriceps muscle of female mice 129xBalb / C three months old. In five mice (group I), the muscles were electrically stimulated immediately after the DNA injection using 10 trains or series of 1000 square bipolar pulses of 200 microseconds duration, with an interval of 2 seconds between series or successive trains. The frequency of the series was 1000 Hz and the amplitude was adjusted to 150 volts (effective voltage ~ 25 volts). In another group more than five mice (group 2) the muscles were not stimulated after the DNA injection. As a control, a group of 4 mice (group 3) was injected with a plasmid (CMV / GFP) containing the coding sequence for the green fluorescent protein under the control of the CMV promoter, followed by the electrical stimulation at the same conditions that group I. Group 4 consisted of 5 injected mice are only saline without electrical stimulation. Blood was collected from the retroorbital sinus at the selected time points and the hematocrit was measured by centrifugation in capillary tubes. Serum samples were analyzed for the presence of EPO using a commercial ELISA kit (R &D Systems). The results are shown in Table 4. In all groups of mice, except those that were injected with the EPO construct and electrically stimulated immediately thereafter, the circulating EPO levels were below the detection limit of the ELISA kit. (< 15 mU / ml). In contrast, mice injected with the EPO construct and electrically stimulated had serum EPO levels significantly elevated 5 days after injection (average of approximately 50 mU / ml). EPO serum concentration remained elevated for up to 28 days after DNA injection (last time point examined, data not shown). These EPO levels produced an increase in hematocrits, which increased 46.2% before injection to 70.0% and 76.7% at 14 and 28 days after DNA injection, respectively. These values were significantly different from those obtained with both control groups (groups 3 and 4) and those mice injected with the EPO expression vector without the muscle electrical stimulation (group 2). Of course, the latter had hematocrit levels not significantly different from those of the control groups (see Table 4). These results demonstrate that the electroporation method is superior to the injection of simple DNA in terms of the expression levels of a secreted protein and in the production of a biological effect mediated by the secreted protein.

TABLE 4 Concentrations and Serum EPO Activity Day 2 Day 5 Day 14 Mouse No. HCT% mEPO HCT 5% mEPO HCT% mEPO (mU / ml) (mU / ml) (mU / ml) 7 45 ND ND 55.7 71 72.4 8 48 ND ND 54.6 68 5.3 9 47 ND ND 59 75.5 48.7 Group 1 stimulated with 10 44 ND ND 62.2 69.5 62.9 CMV-EPO 11 47 ND ND 7.9 66 22.4 Desv. Est. 46.2 ND ND 47.9 70.0abc 48.3 Prom. 1.6 3.6 12 45 ND ND ND 50 < fifteen 13 45 ND ND ND 50 < fifteen 14 ND ND ND ND 48 < fifteen Group 2 without stimulation with 15 46 ND ND ND 49.5 < 15 CMV-EPO 16 44 ND ND Nd 52 < fifteen Desv. 45 ND ND ND 49.9 < fifteen Est. Prom. 0.8 2 ND TsTD ND < 15 43.5 < I5 3 ND ND ND < 15 48 < fifteen ND ND ND < 15 46 < fifteen Group 3 stimulated with 6 ND ND ND < 15 46 < 15 CMV-GFP Dev. Est. ND ND ND < 15 45.9 < 15 Prom. 1.8 17 45 ND ND < 15 45.5 ND 18 45 ND ND < 15 49 ND 19 43 ND ND < 15 48 ND 20 45 ND ND < 15 51.5 ND Group 4 with CMV-EPO 21 50 ND ND < 15 47 ND Dev. Est. 45.6 ND ND < 15 48.2 ND Prom. 2.6 2.3 ND = not determined. ap < 0.0001 vs. Group 2, bp < 0.0001 vs group 3, cp < 0.0001 vs. Group 4 (minor significant difference protected by Fisher).

Example 13 - Administration on Non-Nucleic Acid Molecules: The muscles were injected with 50 μl of a mixture of plasmid DNA or GPF at 1 μg / μl and 2 μg / μl of dextran conjugated to rhodamine (10 kD of Molecular Probes). Three to 5 days later the muscles (n = 6) were frozen in liquid nitrogen and sectioned on a cryostat. As illustrated in Figure 20, the stimulated (background) muscles were transfected with conjugated dextran to rhodamine (top) and GFP (middle part). As further illustrated, the same muscle fibers were transfected with GFP and dextran conjugated to rhodamine. These data indicate that non-nucleic acid molecules can be distributed to muscle cells using the technique of the present invention.

Figure 2 The entire muscle and a slice 1 mm thick were cut from the middle part of it. The numbers of transfected fibers were counted after they were divided into smaller clusters and the single fibers could be observed through a dissecting microscope. In some muscle areas, most of the fibers were transfected (black arrows). These areas were close to where the electrodes were located during the stimulation.

Figure 9 Two different electrodes have been used in order to improve transfection efficiency. The injection procedure and the stimulation pattern (100 Hz) were the same as previously described. The electrode shown in (A) was perpendicular to the muscle fibers. This consisted of a silver wire with diameter (d) of 0.6 mm, (C) (this is the electrode that was used in all the experiments except (B).) An electrode was placed on each side of the muscle. In the intermediate third of the muscle it is positive for (A) that LacZ stains, indicating localized expression In (B) a 1.5 cm electrode made of an isolated silver wire (d = 0.3 mm) was used. the short segments (0.5 - 1.0 mm) along the wire at intervals of 2 mm (D) The electrode was penetrated into the muscle parallel to the muscle fibers A second electrode was placed on the surface of the muscle. positive blue staining in approximately 250 fibers that were located towards the middle third of the muscle In (B) the fibers showed diffused staining, indicating transfection along a longer segment of the fiber and / or increased expression of the trans gen.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (48)

1. Having described the invention as above, the content of the following claims is claimed as property:. . , 1. A method for administering a molecule to the skeletal muscle of a mammal, in vivo, characterized in that it comprises: the injection of a molecule into a skeletal muscle of a mammal; the placement of electrodes near the injection site such that the current traveling through the electrodes passes through the site of the injection; and - electrical stimulation of the muscle with an electric current having a field strength between 25 V / cm and 200 V / cm.
2. 2. The method of administering a molecule according to claim 1, characterized in that the electrical stimulation is administered in the form of a simple square bipolar pulse.
3. 3. The method of administering a molecule according to claim 2, characterized in that the bipolar pulse has a duration between approximately 50 μs and 5000 μs.
4. 4. The method of administering a molecule according to claim 1, characterized in that the electrical stimulation is distributed in the form of between approximately 2 to 30,000 square bipolar pulses.
5. 5. The method of administering a molecule according to claim 4, characterized in that the bipolar pulses have a total duration of between about 10 ms to 12, 000 ms.
6. 6. The method of administering a molecule according to claim 5, characterized in that the bipolar pulses are distributed in the form of at least two trains or series.
7. 7. The method of administration of a molecule according to claim 6, characterized in that the frequency of the electrical stimulation is between approximately 0.5 Hz and 1,000 Hz.
8. 8. The method of administering a molecule according to claim 1, characterized in that the molecule is a nucleic acid, the nucleic acid is operably linked to a promoter that directs the expression in muscle cells of the protein encoded by the nucleic acid.
9. 9. A method for genetically immunizing a mammal by transfection of nucleic acid into the skeletal muscle of said mammal in vi, characterized in that it comprises: injection into a skeletal muscle of a mammal, with a nucleic acid operably linked to a promoter that directs the expression in said muscle of the protein encoded by the nucleic acid; the placement of electrodes near the nucleic acid injection site, such that the current traveling through the electrodes passes through the nucleic acid injection site; and - stimulating the muscle with an electric current having a field strength between about 25 V / cm and 200 V / cm.
10. 10. The method of administering a molecule according to claim 9, characterized in that the electrical stimulation is distributed in the form of a simple square bipolar pulse.
11. 11. The method of administering a molecule according to claim 10, characterized in that the bipolar pulse has a duration between approximately 50 μs and 5,000 μs.
12. 12. The method of administering a molecule according to claim 9, characterized in that the electrical stimulation is administered in the form of between approximately 2 to 30,000 square bipolar pulses.
13. 13. The method of administration of a molecule according to claim 12, characterized in that the sum of the durations of the bipolar pulses is between approximately 10 ms to 12, 000 ms.
14. 14. The method of administration of a molecule according to claim 13, characterized in that the bipolar pulses are distributed in the form of at least two trains or senes
15. 15. The method of administering a molecule according to claim 14, characterized in that the frequency of the electrical stimulation is between approximately 0.5 Hz and 1,000 Hz.
16. 16. A method for systemically administering a protein in a mammal, characterized in that it comprises: injection into a muscle of a mammal, with a nucleic acid operably linked to a promoter that directs the expression in said muscle of the protein encoded by the nucleic acid; - the placement of electrodes near the injection site of the nucleic acid, such that the current traveling through the electrodes passes through the injection site of the nucleic acid; and stimulation of the muscle with an electric current having a field strength of between 25 V / cm and 200 V / cm.
17. 17. The method of administering a molecule according to claim 16, characterized in that the electrical stimulation is distributed in the form of a simple square bipolar pulse.
18. 18. The method of administration of a molecule according to claim 17, characterized in that the bipolar pulse has a duration between approximately 50 μs and 5,000 μs.
19. 19. The method of administration of a molecule according to claim 18, characterized in that the electrical stimulation is distributed in the form of between approximately 2 to 30.00 square bipolar pulses.
20. 20. The method of administering a molecule according to claim 19, characterized in that the sum of the pulse durations of the bipolar pulses is between approximately 10 ms to 12,000 ms.
21. 21. The method of administering a molecule according to claim 20, characterized in that the bipolar pulses are distributed in the form of at least two trains or series.
22. 22. The method of administering a molecule according to claim 21, characterized in that the frequency of the electrical stimulation is between approximately 0.5 Hz and 1,000 Hz.
23. 23. The use of a drug for the manufacture of a medicament for use in a therapy comprising administration within the skeletal muscle in vi via: a) injection of the drug into a skeletal muscle of a mammal; b) the placement of electrodes near the injection site, such that the current traveling through the electrodes passes through the injection site; and c) electrical stimulation of the muscle with an electric current having a field strength between 25 V / cm and 200 V / cm.
24. 24. The use of a drug according to claim 23, wherein the electrical stimulation is distributed in the form of a simple square bipolar pulse.
25. 25. The use of a drug according to any of claims 23 to 24, characterized in that the bipolar pulse has a duration of between about 50 μs and 5,000 μs.
26. 26. The use of a drug according to any of claims 23 to 25, characterized in that the electrical stimulation is distributed in the form of between approximately 2 to 30,000 square bipolar pulses.
27. 27. The use of a drug according to any of claims 23 to 26, characterized in that the bipolar pulses have a total duration of between about 10 ms to 12, 000 ms.
28. 28. The use of a drug according to any of claims 23 to 27, characterized in that the bipolar pulses are distributed in the form of at least two trains or series.
29. 29. The use of a drug according to any of claims 23 to 28, characterized in that the frequency of the electrical stimulation is between approximately 0.5 Hz and 1,000 Hz.
30. 30. The use of a drug according to any of claims 23 to 29, characterized in that the drug comprises a nucleic acid, the nucleic acid being operably linked to a promoter that directs the expression in muscle cells of the protein encoded by said nucleic acid.
31. 31. The use of a drug according to claim 23 to 29, wherein the drug comprises a single-stranded nucleic acid.
32. 32. The use of a drug according to any of claims 23 to 31, wherein the drug comprises a detectable label.
33. 33. A device for making the cellular tissue susceptible to the administration of a pharmaceutical or therapeutic substance, the device is characterized in that it comprises: a) an apparatus that controls the voltage capable of distributing an electric field with a field strength of 25 V / cm at 200 V / cm. b) electrodes suitable for being placed around a part of the body of a mammal, and capable of providing the electric field to the relevant body part.
34. 34. The device according to claim 33, characterized in that the electric field is provided in a gradual or pulsed manner.
35. 35. The device according to claim 34, characterized in that the electric field is provided with a frequency within the range of 0.5 to 1,000 Hz.
36. 36. The device according to any of claims 33 to 35, characterized in that the electric field is distributed in the form of a simple square bipolar pulse.
37. 37. The device according to claim 36, characterized in that the electric field is distributed as a bipolar pulse, with a duration between 50 and 5,000 μs.
38. 38. The device according to any of claims 33 to 35, characterized in that the electric field is distributed in the form of a number of pulses, preferably between 2 and 30,000 square bipolar pulses.
39. 39. The device according to claim 38, characterized in that the duration of the square bipolar pulses falls between 10 ms and 12, 000 ms.
40. 40. The device according to claim 39, characterized in that the square bipolar pulses are distributed in the form of at least two trains or series.
41. 41. The use of a device according to any of claims 33 to 40, for the administration of a pharmaceutical or therapeutic substance to the cellular tissue.
42. 42. The use according to claim 41, wherein the cellular tissue is the skeletal muscle tissue.
43. 43. The use according to claim 41 or 42, wherein the cellular tissue is the tissue of mammalian skeletal muscle cells.
44. 44. The use according to any of claims 41 to 43, wherein the pharmaceutical or therapeutic substance is a nucleic acid molecule.
45. 45. The use according to claim 44, wherein the nucleic acid molecule is a nucleic acid molecule operably linked to a promoter that directs the expression of a protein encoded by the nucleic acid in a muscle cell.
46. 4. 6 The use according to claim 44, wherein the nucleic acid is a single-stranded nucleic acid.
47. 47. The use according to any of claims 41 to 46, wherein the electric field is placed with its field lines approximately through the muscle fibers.
48. 48. The use according to any of claims 41 to 47, wherein the stimulation of the cellular tissue is performed in association with the injection of the pharmaceutical or therapeutic substance into the cellular tissue.