Abstract:
The present invention relates to electroporation (EP) devices that are able to generate an electroporation causing electrical field at the mucosal layer, and preferably in a tolerable manner. Further, it includes the generation of a protective immune response, cellular and/or humoral, using the oral EP device along with a genetic construct that encodes an immunogenic sequence.

Description:
FIELD OF INVENTION 
       [0001]    The present invention relates to electroporation devices that enable the delivery of therapeutics to a subject. 
       BACKGROUND 
       [0002]    A vast majority of human pathogens are known to initiate infections at mucosal surfaces, thus, making the gastrointestinal, urogenital and respiratory tracts major routes of entry into the body. As a result, the other primary way to contract an infection is through blood-borne routes such injections, transfusions and bites. Examples of mucosally-infecting agents include cold viruses, influenza, food poisoning agents tuberculosis, sexually transmitted diseases, cholera, diphtheria and the plague. 
         [0003]    The mucous membranes are one of the largest organs of the body. Collectively, they cover a surface area of more than 400 m 2  (equivalent to one and half tennis courts) and comprise the linings of the gastrointestinal, urogenital and respiratory tracts. These mucosal surfaces, while located inside the body, are actually a physical barrier between the outside and the sterile interior cavity of the body known as the “systemic” environment. Critical nutrients, oxygen and other molecules are constantly taken up across these mucosal barriers; however, another important function of the mucous is to keep invading pathogens out. Daily these mucous membranes are bombarded by outside elements and it is up to the unique immune system of the mucous to determine what is potentially harmful and what is beneficial. 
         [0004]    The importance of mucosal immunology lies in the interplay between the mucosal response and the systemic immune response. Several studies have demonstrated that stimulating the immune system systemically (i.e. via injection or blood-borne routes) results in the production of protective antibody and T cells only within the sterile, internal environment of the body—no mucosal response is generated. On the other hand, stimulation of the mucosal immune response can result in production of protective B and T cells in both mucosal and systemic environments so that infections are stopped before they get into the body. 
         [0005]    The mucous membranes produce a special type of antibody called secretory IgA or sIgA. The mucous membranes are bathed in huge quantities of sIgA, which act as a first line of defense to neutralize invading pathogens. Experimental evidence shows that the presence of sIgA correlates with resistance to infection by various pathogens, including bacteria, viruses, parasites and fungi. It has also been shown to neutralize viruses and prevent their adherence to the epithelial cells lining the mucous (thereby preventing infection) as well as mediating excretion of pathogens and preventing the assembly of mature virus particles. 
         [0006]    Another important component of mucosal immunity is the T cell-mediated immune response. T-cells that specifically recognize pathogens can help antibodies to clear the infection or directly kill the invader themselves. T cells produced in the mucous are capable of traveling throughout the mucosal tissues through special “homing” receptors on their membranes. This means that if an immune response is generated in the gastrointestinal lining, T cells produced there can travel to other mucosal sites, for example, the lungs or nasal cavity, providing protection over a large area. 
         [0007]    Despite the important role of the mucosal surface, only a handful of vaccines specifically target this area of the immune system, thus there remains a need for vaccines that are directed toward the mucosal surface to provide protective immune responses at the mucosal tissue. 
       SUMMARY OF THE INVENTION 
       [0008]    There are provided electroporation devices capable of electroporating cells of a mucosal membrane of a mammal. Such devices include an electrode microneedle plate, a counter electrode plate, a main housing and an energy source. The main housing is in physical communication with said microneedle plate and counter electrode plate, wherein the main house is in fluid communication with a syringe capable of storing a pharmaceutical formulation for delivery. The energy source is in electrical communication with the microneedle plate and capable of generating an electric potential and delivering the electric potential to the cells through the microneedle plate. 
         [0009]    In another aspect, there are provided methods of administering a pharmaceutical formulation to cells of a mucosal membrane of a mammal with the provided devices. The methods comprise contacting said microneedle plate to said mucosal membrane, delivering said pharmaceutical formation to said mucosal membrane, and applying an electroporation causing electrical pulse to the mucosal membrane through the microneedle plate, which was generated by said energy source. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0010]      FIG. 1  shows an immunization (via standard injection) and challenge timeline to be performed in a mouse. 
           [0011]      FIG. 2  displays a graph that shows that chemokine adjuvants induce cellular immunity specific against influenza APR/8/34 in a mouse model of mucosal lung infection. 
           [0012]      FIG. 3   a  displays a graph that shows InfluenzaA/PR/8/34-specific serum long-lived IgA and IgG pre-challenge;  FIG. 3   b  displays a graph that shows InfluenzaA/PR/8/34 neutralizing antibody pre-challenge;  FIG. 3   c  displays a line graph that shows average weight loss over days; and  FIG. 3   d  displays a line graph that shows the various survival rates after challenge. 
           [0013]      FIG. 4  displays a timeline and additional information about Indian Rhesus Macaques Immunization Schedule. 
           [0014]      FIG. 5  displays a graph that shows ELISpot data from known IM (intramuscular)/EP (electroporation) delivery of DNA vaccine is superior to IM delivery alone. 
           [0015]      FIG. 6  displays graphs and images that show a strong immune response was generated:  FIG. 6   a  displays a graph that shows ELISpot data;  FIG. 6   b  displays a graph that shows levels of T cell proliferation;  FIG. 6   c  shows plates from R10 or SIV peptide cultures; and  FIG. 6   d  shows graphs that suggest CFSE Proliferation 
           [0016]      FIG. 7  displays graphs that show that CTACK Co-immunization Augments Cytokine Secretion by CD4+ T cells in the BAL:  FIG. 7   a  displays graphs that show the cellular response in the periphery;  FIG. 7   b  displays graphs that show the cellular response in BAL. 
           [0017]      FIG. 8  displays graph that show that CTACK Elicits High Levels of Cytokine Secreting CD8+ T cells in the Lung:  FIG. 8   a  displays a graph that shows the 107a+ CD8+ levels;  FIG. 8   b  displays a graph that shows the IFN-gamma CD+ levels;  FIG. 8   c  displays a graph that shows the TNF+CD8+ levels; and  FIG. 8   d  displays a graph that shows IL-2+ CD8+ levels. 
           [0018]      FIG. 9  displays a photo that shows positive GFP expression by way of fluorescence. 
           [0019]      FIG. 10  displays a 4×4 array (Inovio Pharmaceuticals, Inc., Blue Bell, Pa.) 
           [0020]      FIG. 11  displays graphs that show HAI titer levels in serum from macaques that were immunized with SynCon™ influenza vaccine. Results shown are two weeks post-second immunization:  FIG. 11   a  HAI titers with respect to A/H1N1/Mexico/2009 strain; and  FIG. 11   b  HAI titers with respect to A/H1N1/New Caledonia. 
           [0021]      FIG. 12   a  displays photos that show GFP expression in guinea pig oral mucosal tissue following shallow injection of GFP plasmid and electroporation Whole cheek mounts were harvested 3 days post-treatment and viewed under a fluorescent microscope to determine positive GFP expression. 
           [0022]      FIG. 12   b  displays a graph that shows H5-specific IgA titers following 3 immunizations in the guinea pig. 
           [0023]      FIG. 13  is a ¾ view of an oral electroporation/injection device comprising: a pulse generator, an injection and an electroporation device. 
           [0024]      FIG. 14  is a drawing showing a vertical cross-section A-A of the oral electroporation/injection device. 
           [0025]      FIG. 15  is an exploded assembly of the electroporation/injection device. 
           [0026]      FIG. 16  is the main electrode micro-needle plate of the electroporation/injection device. 
           [0027]      FIG. 17  shows the OM-I/EP device in relation to an open mouth. 
           [0028]      FIG. 18  displays graphs showing IgA titers in a) Saliva; b) Stool; c) Blood. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0029]    There are provided electroporation devices capable of electroporating cells of a mucosal membrane of a mammal. Such devices include an electrode microneedle plate, a counter electrode plate, a main housing and an energy source. The main housing is in physical communication with said microneedle plate and counter electrode plate, wherein the main house is in fluid communication with a syringe capable of storing a pharmaceutical formulation for delivery. The energy source is in electrical communication with the microneedle plate and capable of generating an electric potential and delivering the electric potential to the cells through the microneedle plate. In an embodiment, there is also a piston in physical communication between said main housing and said microneedle plate. The piston is actuatable and by actuating can cause even distribution of the pharmaceutical formulation through the microneedle plate. 
         [0030]    In one aspect of the invention, there are provided oral electroporation (EP) devices that are able to generate an electroporation causing electrical field at the mucosal layer, and preferably in a tolerable manner. In one embodiment of this aspect, there is an oral mucosal injection and electroporation device (OM-I/EP) that is adapted to perform delivery of therapeutic (or prophylactic) formulations, such as DNA vaccines, and the transfection into the mucosal tissue/cells on the inside of the mouth. During a DNA vaccination procedure the device would be affixed across the cheek area of the patient. The main body with the main electrode micro-needle plate feature on the inside of the mouth and the return electrode plate clamp feature adjacent, on the outside of the cheek. The DNA vaccine would be injected through the micro-needle plate; this would then be followed by low voltage EP pulses applied to that same electrode micro-needle plate, this design co-locates the DNA vaccine and the electroporation to the same area. Research has shown that the co-location of DNA vaccine and EP to be very important in the amount of DNA vaccine transfection into the surrounding cells. 
         [0031]    In some embodiments, the microneedles of the microneedle plate are made from electrically conductive materials comprising gold and silver plated brass, gold and silver plated copper, stainless steel, or titanium, or other commonly known conductive metal or metal-like material. In some embodiments, the energy source is capable of delivering through the microneedle plate to the cells of the mucosal membrane at least one pulse of electrical energy having characteristics of between 1V and 30V, 2 mA and 100 mA, or 1 mS and 250 mS. The mucosal membrane or mucosal tissue can be buccal, nasal, esophageal, rectal, vaginal, vulva, intestinal, bowel, stomach, bladder, urinary tract, or eye tissue, and preferably buccal tissue, e.g., the inner surface of the mouth. 
         [0032]    In another aspect, there are provided methods of administering a pharmaceutical formulation to cells of a mucosal membrane of a mammal with the provided devices. The methods comprise contacting said microneedle plate to said mucosal membrane, delivering said pharmaceutical formation to said mucosal membrane, and applying an electroporation causing electrical pulse to the mucosal membrane through the microneedle plate, which was generated by said energy source. 
         [0033]    During in vivo electroporation, electric pulses are applied directly to the tissue to enhance uptake of extracellular molecules. Present types of in vivo EP are done with very high volt/centimeter electrical field strengths, using such large electrical field strengths is would be painful to the patient in mucosal tissue due to the high density of nerves . With the current OM-I/EP devices , they can be equipped to deliver very low field strength EP, such as using the low energy electrical pulses that were applied at intradermal (ID) injection sites, which were described in an earlier filed, co-owned PCT application entitled, “CONTACTLESS ELECTROPERMEABILIZATION ELECTRODE AND METHOD” having application number PCT/US10/31431, filed Apr. 16, 2010, and incorporated by reference herein in its entirety. Such intradermal EP can be performed with very low voltages and with minimal to no pain to the patient. In early experiments on mucosal tissues these lower EP field strengths have shown transfection into mucosal tissue with similar results (data not shown). The EP parameters can include voltages ranging from 0.1 volts (V) to 30 V, 0.1 V to 20 V, 0.1 V to 15 V, 0.1 V to 10 V, 0.1 V to 9 V, 0.1 V to 8 V, 0.1 V to 7 V, 0.1 V to 6 V, 0.1 V to 5 V, 0.1 V to 4 V, 0.1 V to 3 V, 0.1 V to 2 V, 0.1 V to 1 V, 2 V to 30 V, 2 V to 20 V, 2 V to 15 V, 2 V to 10 V, 2 V to 9 V, 2 V to 8 V, 2 V to 7 V, 2 V to 6 V, 2 V to 5 V, 2 V to 4 V, 2 V to 3 V, 4 V to 30 V, 4 V to 20 V, 4 V to 15 V, 4 V to 10 V, 4 V to 9 V, 4 V to 8 V, 4 V to 7 V, 4 V to 6 V, 4 V to 5 V, 6 V to 30 V, 6 V to 20 V, 6 V to 15 V, 6 V to 10 V, 6 V to 9 V, 6 V to 8 V, 8 V to 30 V, 8 V to 20 V, 8 V to 15 V, 8 V to 10 V, 8 V to 9 V, 10 V to 30 V, 10 V to 20 V, or 10 V to 15 V; and currents ranging from 2 mA to 100 mA, 3 mA to 100 mA, 4 mA to 100 mA, 5 mA to 100 mA, 6 mA to 100 mA. 7 mA to 100 mA, 8 mA to 100 mA, 9 mA to 100 mA, 10 mA to 100 mA, 20 mA to 100 mA, 30 mA to 100 mA, 40 mA to 100 mA, 60 mA to 100 mA, 80 mA to 100 mA, 2 mA to 80 mA, 3 mA to 80 mA, 4 mA to 80 mA, 5 mA to 80 mA, 6 mA to 80 mA, 7 mA to 80 mA, 8 mA to 80 mA, 9 mA to 80 mA, 10 mA to 80 mA, 20 mA to 80 mA, 30 mA to 80 mA, 40 mA to 80 mA, 60 mA to 80 mA, 2 mA to 60 mA, 3 mA to 60 mA, 4 mA to 60 mA, 5 mA to 60 mA, 6 mA to 60 mA, 7 mA to 60 mA, 8 mA to 60 mA, 9 mA to 60 mA, 10 mA to 60 mA, 20 mA to 60 mA, 30 mA to 60 mA, 40 mA to 60 mA, 2 mA to 40 mA, 3 mA to 40 mA, 4 mA to 40 mA, 5 mA to 40 mA, 6 mA to 40 mA, 7 mA to 40 mA, 8 mA to 40 mA, 9 mA to 40 mA, 10 mA to 40 mA, 20 mA to 40 mA, 30 mA to 40 mA, 2 mA to 30 mA, 3 mA to 30 mA, 4 mA to 30 mA, 5 mA to 30 mA, 6 mA to 30 mA, 7 mA to 30 mA, 8 mA to 30 mA, 9 mA to 30 mA, 10 mA to 30 mA, 20 mA to 30 mA, 2 mA to 20 mA, 3 mA to 20 mA, 4 mA to 20 mA, 5 mA to 20 mA, 6 mA to 20 mA, 7 mA to 20 mA, 8 mA to 20 mA, 9 mA to 20 mA, 10 mA to 20 mA, 2 mA to 10 mA, 3 mA to 10 mA, 4 mA to 10 mA, 5 mA to 10 mA, 6 mA to 10 mA, 7 mA to 10 mA, 8 mA to 10 mA, 9 mA to 10 mA, 2 mA to 9 mA, 3 mA to 9 mA, 4 mA to 9 mA, 5 mA to 9 mA, 6 mA to 9 mA. 7 mA to 9 mA. 8 mA to 9 mA, 2 mA to 8 mA, 3 mA to 8 mA, 4 mA to 8 mA, 5 mA to 8 mA, 6 mA to 8 mA. 7 mA to 8 mA, 2 mA to 7 mA, 3 mA to 7 mA, 4 mA to 7 mA, 5 mA to 7 mA, 6 mA to 7 mA. 2 mA to 6 mA, 3 mA to 6 mA, 4 mA to 6 mA, 5 mA to 6 mA, 2 mA to 5 mA, 3 mA to 5 mA, 4 mA to 5 mA, 2 mA to 4 mA, or 3 mA to 4 mA. In some embodiments the EP parameters used range from 30 volts and 100 mA on the high end to 2 volts and 2 mA on the low end. For EP delivery, the desired tissue received two (2) pulses 100 ms each with a 100 ms delay between pulses. 
         [0034]    The OM-I/EP device has a main electrode micro needle plate (item # 3  &amp;  FIG. 15 ) fastened to the head of a main housing (item # 1 ). A voltage return electrode plate and arm (item # 5 and # 6 ) is placed adjacent and outside the mouth. The main housing (item # 1 ) can be mounted to a standard 1-ml lure-lock syringe (item # 8 ). In use, the micro needle plate array (item # 3  &amp;  FIG. 15 ) is placed on the inside of the mouth in intimate contact with the buccal mucosal lining (inner surface of cheek). The voltage return electrode (item # 5 ) would be in contact with the outside adjacent surface of the cheek. The micro needle plate array (item # 3  &amp;  FIG. 15 ), main housing (item # 1 ) and the attached syringe (item # 8 ) form the injection/electroporation device. The design of the large micro needle array allows for the injection of DNA vaccine over a large area. The small size and short length of the micro needles places the DNA vaccine to a controlled specific depth. A piston (item # 2 ) and its sealing o-ring (item # 9 ) form a common manifold area and driver that will insure even distribution of DNA vaccine through the micro-needle plate (item # 3  &amp;  FIG. 15 ). 
         [0035]    The requirement for the main electrode (item # 3  &amp;  FIG. 15 ) is that they have many micro-needles of a specific length and diameter. The electrode must also, be made from electrically conductive materials (such as gold/silver plated brass or copper, stainless steel and/or titanium). The DNA vaccine must be placed in the upper most layers of the mucosa membranes. The main electrode (item # 3  &amp;  FIG. 15 ) can be made by a few manufacturing techniques: such as Chemical etching, Electrical Discharge machining (EDM) and Electro-less nickel plating on a sacrificial pattern. The main housing and support parts could be made from injection molded materials (such as ABS, Polycarbonate and Polyolefin). 
         [0036]      FIGS. 4-8  show results that support the following: 
         [0037]    Optimized SIV DNA constructs+EP elicited IFN-g (˜12,000 SFC/106) and proliferative CD8+ T cell responses (˜20%) (no difference with CTACK). These responses were highest following the 4th immunization. The addition of optimized CTACK DNA did not further enhance the induced response in the periphery by:
       IFN-g ELISpot   CFSE Proliferation   PBMC cytokine secretion   IgA in the sera       
 
         [0042]    The addition of optimized CTACK DNA changes the phenotype of the response in the mucosa as measured by:
       BAL cytokine secretion   More Polyfunctional CD8+ T cells   Higher Frequencies of responding CD4+ and CD8+ T Cells   IgA in Fecal &amp; BAL samples       
 
       EXAMPLES 
       [0047]    The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. 
         [0048]    Experiments were performed to assess IgA titers in the blood, nasal secretions, saliva and stools of animals immunized via an EP enhanced mucosal (orally) route with Influenza HA antigens. Significant IgA titers observed in the saliva is indicative of a mucosal immune response being successfully raised in a local mucosal region. Detection of IgA responses in the stool samples indicates a mucosal response at a distant site was raised. Detection of IgA titers in the blood sera suggests a systemic response was also raised. 
       H5 IgA ELISA 
       [0049]    Following three mucosal EP-enhanced immunizations, positive H5 specific IgA titers were observed in the saliva of 3 out of 4 animal&#39;s electroporated with the 4×4 device (Inovio Pharmaceuticals, Inc., Blue Bell, Pa.) and 4 out of 4 animals electroporated with a caliper electroporation device. One animal was positive in the injection only group. See  FIGS. 18   a - 18   c.  
 
Two animals had target specific positive IgA titers in their blood samples following three immunizations with the 4×4 device.
 
One animal from both the 4×4 device and caliper groups had target specific IgA responses in their stools.
 
None of the negative controls or injection only group animals had positive IgA stool or blood samples.