Dynamic vacuum treatment devices, systems, and methods

Abstract

A method for enhancing drug delivery into tissue includes positioning a housing that defines a chamber adjacent to the surface of the tissue, thereby positioning the chamber adjacent to an injection site where the drug is injected into the tissue; applying vacuum pressure to the chamber, thereby drawing a portion of the tissue through the opening of the chamber and into the chamber; and moving the housing relative to the tissue while vacuum pressure is applied to enhance drug delivery within the tissue.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 498,690, filed Apr. 27, 2023, the entire content of which is incorporated herein by reference.

[0002] The present invention relates to devices, assemblies, and systems for gripping and / or reshaping tissue using vacuum pressure and delivering fluid into the tissue.

Background Art

[0003] A number of different techniques have been developed to enhance drug delivery to tissue in vivo. Some of these techniques involve modes of injecting drugs into the tissue. Such modes include certain types of needle injection (e.g., Mantoux injection, side - port injection), and other injection types such as jet injection. Other techniques for enhancing drug delivery in vivo involve modes of diffusing or dispersing the injected drug within the tissue and / or across various tissue layers or within the same tissue layer. Iontophoresis, micro - needle arrays, suction cups, or vacuum cup treatments are examples of such modes. Still other techniques for enhancing drug delivery in vivo involve modes of directly increasing the uptake of drugs by target cells within the tissue. Electroporation and sonoporation are examples of such modes. Additional techniques for enhancing drug delivery in vivo involve modes that include pairing the drug with an adjuvant known to enhance the immune response(s) induced by the drug. Some techniques achieve multiple of the aforementioned modes for enhancing drug delivery in vivo. For example, vacuum - assisted electroporation (VEP) treatments have been shown to enhance drug delivery in vivo by modes that increase the dispersion of injectates within the tissue and modes that increase the direct uptake of drugs by target cells within the tissue.

Summary of the Invention

[0004] According to one embodiment of the present disclosure, a method for enhancing drug delivery into tissue includes arranging a housing that defines a chamber adjacent to the surface of the tissue, thereby positioning the chamber adjacent to an injection site where the drug is injected into the tissue; applying vacuum pressure to the chamber, thereby drawing a portion of the tissue through and into the opening of the chamber; and moving the housing relative to the tissue while vacuum pressure is applied to enhance drug delivery within the tissue.

[0005] According to another embodiment of the present disclosure, a method for enhancing drug delivery into tissue includes injecting the drug into the target tissue, thereby defining an injection site on the surface of the tissue; positioning a housing that defines a chamber at or adjacent to the injection site; and applying vacuum pressure to the chamber, thereby drawing a portion of the tissue through and into the opening of the chamber. While the vacuum pressure is applied, the housing is moved relative to the tissue to enhance drug delivery within the tissue.

[0006] According to additional embodiments of the present disclosure, a system for vacuum-enhanced drug delivery into tissue in vivo includes a housing defining a chamber and an opening into the chamber, and at least one port extending through the housing. The at least one port is separated from the at least one opening and is connectable to a vacuum source, thereby configuring the at least one port to communicate vacuum pressure from the vacuum source to the chamber. The housing is configured to communicate a vacuum field to a portion of tissue, thereby drawing the portion of tissue through the opening and holding the portion of tissue in the chamber at least momentarily. The housing is further configured to move relative to the tissue while the vacuum field is communicating with the tissue, and the relative motion deforms at least a portion of the tissue. The system includes one or more features that can be disposed between the distal end of the housing and the surface of the tissue to reduce sliding friction between the housing and the tissue.

[0007] This patent or application document includes at least one drawing made in color. A copy of this patent or patent application publication containing the color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fees.

[0008] The above summary and the following detailed description of exemplary embodiments of this application will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the features of this application, the drawings show exemplary embodiments. However, it should be understood that this application is not limited to the exact arrangements and fixtures shown. The drawings are as follows: [Brief explanation of the drawing]

[0009] [Figure 1A] This is a schematic diagram of a dynamic vacuum treatment system using a vacuum cup according to one embodiment of the present disclosure. [Figure 1B] Figure 1A is a perspective view of a vacuum cup, showing the vacuum chamber of a cup according to one embodiment of the present disclosure. [Figure 1C] Figure 1B is a cross-sectional side view of the vacuum cup shown, cut along a cross-section extending along the central axis of the cup. [Figure 1D] Figure 1B is another cross-sectional side view of the vacuum cup shown, illustrating the tissue bulge drawn into the vacuum chamber in response to the application of vacuum pressure, and also showing the fluid injection agent dispersed within the skin layer of the tissue. [Figure 2A] This is a perspective view of a vacuum cup having a central support located within a vacuum chamber, according to another embodiment of the present disclosure. [Figure 2B] Figure 2A is a cross-sectional side view of the vacuum cup shown, cut along a section extending along the central axis of the cup. [Figure 2C]This is another cross-sectional side view of a vacuum cup similar to the one shown in Figure 2A, showing a tissue protrusion that is drawn into the vacuum chamber and in contact with the central support in response to the application of vacuum pressure, and also showing the fluid injection material dispersed within the skin layer of the tissue. [Figure 3A] This is a cross-sectional side view of a vacuum cup similar to the cups shown in Figures 1A-1D, having an injection channel for an injection needle, according to another embodiment of the present disclosure. [Figure 3B] This is a cross-sectional side view of a vacuum cup similar to the cup shown in Figures 2A-2C, having a jet syringe extending into a vacuum chamber, according to another embodiment of the present disclosure. [Figure 4A] This is a perspective view of a vacuum cup having electrodes for electroperforating tissue drawn into a vacuum chamber, according to another embodiment of the present disclosure. [Figure 4B] Figure 4A is a cross-sectional side view of the vacuum cup shown. [Figure 5A] This is a perspective view of a handle assembly including a vacuum cup according to another embodiment of the present disclosure. [Figure 5B] Figure 5A is a partially exploded view of the handle assembly shown in the diagram, according to one embodiment of the present disclosure, showing the vacuum cup removed from the mounting configuration of the handle assembly, with the vacuum cup rotated to a bottom view for illustrative purposes. [Figure 6A] These are cross-sectional side views of tissue, showing typical stages of an exemplary dynamic vacuum treatment using the vacuum cups illustrated in Figures 1A to 1D, according to one embodiment of the present disclosure. [Figure 6B] These are cross-sectional side views of tissue, showing typical stages of an exemplary dynamic vacuum treatment using the vacuum cups illustrated in Figures 1A to 1D, according to one embodiment of the present disclosure. [Figure 6C] These are cross-sectional side views of tissue, showing typical stages of an exemplary dynamic vacuum treatment using the vacuum cups illustrated in Figures 1A to 1D, according to one embodiment of the present disclosure. [Figure 6D] These are cross-sectional side views of tissue, showing typical stages of an exemplary dynamic vacuum treatment using the vacuum cups illustrated in Figures 1A to 1D, according to one embodiment of the present disclosure. [Figure 6E] A cross-sectional side view of tissue showing representative stages of an exemplary dynamic vacuum treatment using the vacuum cup illustrated in FIGS. 1A-1D, according to one embodiment of the present disclosure. [Figure 6F] A cross-sectional side view of tissue showing representative stages of an exemplary dynamic vacuum treatment using the vacuum cup illustrated in FIGS. 1A-1D, according to one embodiment of the present disclosure. [Figure 6G] A cross-sectional side view of tissue showing representative stages of an exemplary dynamic vacuum treatment using the vacuum cup illustrated in FIGS. 1A-1D, according to one embodiment of the present disclosure. [Figure 6H] A cross-sectional side view of tissue showing representative stages of another exemplary dynamic vacuum treatment. [Figure 7A] A plan view showing an exemplary dynamic movement of a vacuum cup for providing a dynamic vacuum treatment, according to an embodiment of the present disclosure, showing the movement of left and right translational cups. [Figure 7B] A plan view showing an exemplary dynamic movement of a vacuum cup for providing a dynamic vacuum treatment, according to an embodiment of the present disclosure, showing the movement of translational cups in the up-down or front-back direction. [Figure 7C] A plan view showing an exemplary dynamic movement of a vacuum cup for providing a dynamic vacuum treatment, according to an embodiment of the present disclosure, showing the movement of a meandering translational cup. [Figure 7D] A plan view showing an exemplary dynamic movement of a vacuum cup for providing a dynamic vacuum treatment, according to an embodiment of the present disclosure, showing the movement of a rotating (e.g., twisted) cup. [Figure 7E] A plan view showing an exemplary dynamic movement of a vacuum cup for providing a dynamic vacuum treatment, according to an embodiment of the present disclosure, showing a specific exemplary order of movement of left and right translational cups. [Figure 7F] A plan view showing an exemplary dynamic movement of a vacuum cup for providing a dynamic vacuum treatment, according to an embodiment of the present disclosure, showing the movement of an exemplary unidirectional translational cup. [Figure 8]An image diagram showing gene expression in the skin of guinea pigs after intradermal injection of a plasmid encoding the gene for green fluorescent protein (GFP), followed by treatment with various techniques and devices using vacuum treatment respectively. [Figure 9] An image diagram showing gene expression in the skin of guinea pigs after intradermal injection of a plasmid encoding the gene for green fluorescent protein (GFP), followed by various dynamic vacuum (KV) treatments (and one static vacuum (SV) treatment) using a vacuum cup design. [Figure 10A] A chart showing binding ELISA immunogenicity data in guinea pigs at 2 weeks (Figure 10A) and 4 weeks (Figure 10B) after intradermal injection of plasmid, after various treatments including dynamic vacuum (KV) treatment, static vacuum (SV) treatment, vacuum electropermeation (VEP) treatment, and injection-only (INJ) treatment. [Figure 10B] A chart showing binding ELISA immunogenicity data in guinea pigs at 2 weeks (Figure 10A) and 4 weeks (Figure 10B) after intradermal injection of plasmid, after various treatments including dynamic vacuum (KV) treatment, static vacuum (SV) treatment, vacuum electropermeation (VEP) treatment, and injection-only (INJ) treatment. [Figure 10C] Including photographs showing the treatment effect on skin tissue at the treatment site for the study shown in Figures 10A - 10B at immediately after treatment (Figure 10C) and 7 days after treatment (Figure 10D). [Figure 10D] Including photographs showing the treatment effect on skin tissue at the treatment site for the study shown in Figures 10A - 10B at immediately after treatment (Figure 10C) and 7 days after treatment (Figure 10D). [Figure 11A] A chart showing binding ELISA immunogenicity data in guinea pigs at 2 weeks (Figure 11A) and 4 weeks (Figure 11B) after intradermal injection of plasmid, after various treatments including dynamic vacuum (KV) treatment, static vacuum (SV) treatment, vacuum electropermeation (VEP) treatment, and injection-only (INJ) treatment, shown as a follow-up investigation of the study shown in Figures 10A - 10B to confirm the ELISA results. [Figure 11B] This chart shows binding ELISA immunogenicity data in guinea pigs at 2 weeks (Figure 11A) and 4 weeks (Figure 11B) after intradermal plasmid injection, following various treatments including dynamic vacuum (KV) treatment, static vacuum (SV) treatment, vacuum electroporation (VEP) treatment, and injection-only (INJ) treatment, as a follow-up of the studies shown in Figures 10A-10B to confirm the ELISA results. [Figure 12A] This chart shows binding ELISA immunogenicity data in guinea pigs at 2 weeks (Figure 12A) and 4 weeks (Figure 12B) after intradermal injection of plasmids at different volumes following dynamic vacuum (KV) treatment. [Figure 12B] This chart shows binding ELISA immunogenicity data in guinea pigs at 2 weeks (Figure 12A) and 4 weeks (Figure 12B) after intradermal injection of plasmids at different volumes following dynamic vacuum (KV) treatment. [Figure 13] This chart shows binding ELISA immunogenicity data in untreated guinea pigs at two weeks after intradermal plasmid injection, following various treatments including vacuum electroporation (VEP), dynamic vacuum (KV), static vacuum (SV), and injection-only (INJ) treatment. [Figure 14] This chart compares binding ELISA immunogenicity data in guinea pigs at 2 weeks post-intradermal plasmid injection with various dynamic vacuum treatments using different amounts of cup translation across the injection site, compared to one such treatment including hyaluronidase. [Figure 15] This chart compares ELISA immunogenicity data in guinea pigs after intradermal plasmid injection following various dynamic vacuum (KV) treatments using different vacuum pressures. [Figure 16A] This chart compares ELISA immunogenicity data in rabbits at day 0 (Figure 16A) and week 2 (Figure 16B) after intradermal plasmid injection, following various procedures including dynamic vacuum (KV), static vacuum (SV), vacuum electroporation (VEP), needle electroporation (NEP), and injection-only (INJ) procedures. [Figure 16B] This chart compares ELISA immunogenicity data in rabbits at day 0 (Figure 16A) and week 2 (Figure 16B) after intradermal plasmid injection, following various procedures including dynamic vacuum (KV), static vacuum (SV), vacuum electroporation (VEP), needle electroporation (NEP), and injection-only (INJ) procedures. [Figure 17A] The charts in the follow-up study shown in Figures 16A-16B again compare ELISA immunogenicity data in rabbits at day 0 (Figure 17A) and week 2 (Figure 17B) after intradermal plasmid injection, after various treatments including dynamic vacuum (KV) treatment. [Figure 17B] Figures 16A and 16B show charts of follow-up studies, again comparing ELISA immunogenicity data in rabbits at day 0 (Figure 17A) and week 2 (Figure 17B) after intradermal plasmid injection, following various treatments including dynamic vacuum (KV) treatment. [Figure 18] This chart compares ELISA immunogenicity data in guinea pigs two weeks after intradermal plasmid injection, following various vacuum procedures, including dynamic vacuum (KV) and static vacuum (SV), with and without hyaluronidase. [Figure 19] This chart compares ELISA immunogenicity data in guinea pigs two weeks after treatment to evaluate the effects of the number of dynamic vacuum (KV) cup movements and skin thickness at the treatment site on the immune response. [Figure 20A] This table shows gene expression in guinea pig skin after intradermal injection of plasmids encoding the gene for green fluorescent protein (GFP), according to different injection volumes following dynamic vacuum (KV) or static vacuum (SV) treatment, with or without hyaluronidase, and displays images showing visible GFP expression. [Figure 20B]This chart shows gene expression in guinea pig skin after intradermal injection of plasmids encoding the gene for green fluorescent protein (GFP), according to different injection volumes following dynamic vacuum (KV) or static vacuum (SV) treatment, with or without hyaluronidase, and displays the quantified values ​​of the measured fluorescence. [Figure 21] This chart compares ELISA immunogenicity data in guinea pigs two weeks after treatment to evaluate the effect of high infusion volume on the immune response (ELISA expression) generated by dynamic vacuum (KV) and static vacuum (SV) treatments, regardless of the presence or absence of hyaluronidase. [Figure 22] This chart compares ELISA immunogenicity data in guinea pigs two weeks after treatment to evaluate the effect of combining dynamic vacuum (KV) treatment with vacuum electroporation (VEP) on the immune response (ELISA expression). [Figure 23A] This chart compares bound ELISA data (Figures 23A and 23B), ELISpot data (Figure 23C), and SARS-CoV-2 pseudovirus neutralization data (Figure 23D) generated by dynamic vacuum (KV) treatment with mRNA treatment and needle electroporation (NEP) treatment. [Figure 23B] This chart compares bound ELISA data (Figures 23A and 23B), ELISpot data (Figure 23C), and SARS-CoV-2 pseudovirus neutralization data (Figure 23D) generated by dynamic vacuum (KV) treatment with mRNA treatment and needle electroporation (NEP) treatment. [Figure 23C] This chart compares bound ELISA data (Figures 23A and 23B), ELISpot data (Figure 23C), and SARS-CoV-2 pseudovirus neutralization data (Figure 23D) generated by dynamic vacuum (KV) treatment with mRNA treatment and needle electroporation (NEP) treatment. [Figure 23D]This chart compares bound ELISA data (Figures 23A and 23B), ELISpot data (Figure 23C), and SARS-CoV-2 pseudovirus neutralization data (Figure 23D) generated by dynamic vacuum (KV) treatment with mRNA treatment and needle electroporation (NEP) treatment. [Figure 24] This is a table of images showing gene expression in guinea pig skin after various treatments, including injection fragmentation using multiple injection (multiple blister) sites enhanced by a single dynamic vacuum (KV) treatment. [Figure 25] This is a chart of operator studies showing binding ELISA immunogenicity data in guinea pigs two weeks after intradermal plasmid injection, following the same dynamic vacuum (KV) treatment performed by four different individuals (operators). [Figure 26A] This chart shows a nearly one-year study comparing binding ELISA data (Figure 26A) and SARS-CoV-2 pseudovirus neutralization data (Figures 26B-26B) generated by dynamic vacuum (KV) treatment using DNA launching nanoparticles (DNLP) with mRNA treatment. [Figure 26B] This chart shows a nearly one-year study comparing binding ELISA data (Figure 26A) and SARS-CoV-2 pseudovirus neutralization data (Figures 26B-26B) generated by dynamic vacuum (KV) treatment using DNA launching nanoparticles (DNLP) with mRNA treatment. [Figure 26C] This chart shows a nearly one-year study comparing binding ELISA data (Figure 26A) and SARS-CoV-2 pseudovirus neutralization data (Figures 26B-26B) generated by dynamic vacuum (KV) treatment using DNA launching nanoparticles (DNLP) with mRNA treatment. [Figure 27] This chart shows comparable spreading effects (measured as the ratio of blister diameters before and after treatment) for dynamic vacuum (KV) treatment (with and without hyaluronidase injection) and static vacuum (SV) treatment. [Figure 28A]This chart shows a vacuum cup device study comparing bound ELISA data in guinea pigs over two weeks receiving the same dynamic vacuum (KV) treatment (e.g., migration pattern) administered by different devices, including vacuum cups with a central support and four ready-made (OTS) vacuum cup devices. [Figure 28B] This chart compares bound ELISA data in guinea pigs at week 2, generated by performing the same dynamic vacuum (KV) procedure using different vacuum cup devices with different cup sizes and geometric shapes. [Figure 29] This chart compares binding ELISA data in guinea pigs at week 2, generated by dividing the injection volume into multiple blisters, after performing the same dynamic vacuum (KV) treatment on the divided blisters within each group. [Figure 30] This chart compares 2-week binding ELISA data in guinea pigs generated by injection with reduced DNA plasmid doses after dynamic vacuum (KV) treatment versus needle electroporation (NEP) treatment. [Figure 31] This chart compares binding ELISA data in guinea pigs at week 2, generated by reducing the injection volume and DNA dose across the test groups after performing the same dynamic vacuum (KV) treatment using the same vacuum cup device for each group. [Figure 32A] This chart compares the immune response, particularly the ELISA response (Figures 32A-32C) and T-cell response (Figures 32D and 32E), in rabbits after procedures involving dynamic vacuum (KV) versus needle electroporation (NEP) and intramuscular electroporation (IM-EP). [Figure 32B] This chart compares the immune response, particularly the ELISA response (Figures 32A-32C) and T-cell response (Figures 32D and 32E), in rabbits after procedures involving dynamic vacuum (KV) versus needle electroporation (NEP) and intramuscular electroporation (IM-EP). [Figure 32C]This chart compares the immune response, particularly the ELISA response (Figures 32A-32C) and T-cell response (Figures 32D and 32E), in rabbits after procedures involving dynamic vacuum (KV) versus needle electroporation (NEP) and intramuscular electroporation (IM-EP). [Figure 32D] This chart compares the immune response, particularly the ELISA response (Figures 32A-32C) and T-cell response (Figures 32D and 32E), in rabbits after procedures involving dynamic vacuum (KV) versus needle electroporation (NEP) and intramuscular electroporation (IM-EP). [Figure 32E] This chart compares the immune response, particularly the ELISA response (Figures 32A-32C) and T-cell response (Figures 32D and 32E), in rabbits after procedures involving dynamic vacuum (KV) versus needle electroporation (NEP) and intramuscular electroporation (IM-EP). [Figure 33] This chart shows a dynamic vacuum (KV) transfer pattern study comparing the effects of simplified KV transfer patterns and repetitions (cycles) on coupled ELISA data in guinea pigs during the second week. [Figure 34A] This chart compares bound ELISA data (Figure 34A) and SARS-CoV-2 pseudovirus neutralization data (Figure 34B) generated in guinea pigs by dynamic vacuum (KV) treatment versus needle electroporation (NEP) treatment. [Figure 34B] This chart compares bound ELISA data (Figure 34A) and SARS-CoV-2 pseudovirus neutralization data (Figure 34B) generated in guinea pigs by dynamic vacuum (KV) treatment versus needle electroporation (NEP) treatment. [Figure 35A] This paper presents a study exploring the effect of extravesical dynamic vacuum (KV) transfer on gene expression. The plan view shows the dynamic movement of the vacuum cup used in this study. [Figure 35B]This study explores the effect of extravesical dynamic vacuum (KV) transfer on gene expression. The chart shows quantified values ​​of visible gene expression in guinea pig skin after intradermal injection of a plasmid encoding the gene for green fluorescent protein (GFP), followed by similar KV transfer further progressively separated from the injected blister. [Figure 36A] This chart compares the binding ELISA data at week 3 (Figure 36A) and week 4 (Figure 36B) in mice, as well as the T cell response at week 4 (Figure 36V), after dynamic vacuum (KV) treatment with hyaluronidase, resulting in uniform plasmid injection volume (and DNA dose), with injection-only (INJ), static vacuum (SV), and needle electroporation (NEP) treatments. [Figure 36B] This chart compares the binding ELISA data at week 3 (Figure 36A) and week 4 (Figure 36B) in mice, as well as the T cell response at week 4 (Figure 36V), after dynamic vacuum (KV) treatment with hyaluronidase, resulting in uniform plasmid injection volume (and DNA dose), with injection-only (INJ), static vacuum (SV), and needle electroporation (NEP) treatments. [Figure 36C] This chart compares the binding ELISA data at week 3 (Figure 36A) and week 4 (Figure 36B) in mice, as well as the T cell response at week 4 (Figure 36V), after dynamic vacuum (KV) treatment with hyaluronidase, resulting in uniform plasmid injection volume (and DNA dose), with injection-only (INJ), static vacuum (SV), and needle electroporation (NEP) treatments. [Figure 37] This chart compares binding ELISA data in guinea pigs at 2 weeks, following uniform dynamic vacuum (KV) treatment and subsequent addition of different doses of hyaluronidase to plasmids with uniform DNA dose injections by other methods. [Figure 38]This chart shows the quantified immune cell migration in guinea pigs after dynamic vacuum (KV) treatment versus needle electroporation (NEP) treatment, and specifically the number of GFP-positive cells in the target lymph nodes 3 days post-treatment for the KV-treated group versus the NEP-treated group. [Figure 39] This chart shows binding ELISA data in rabbits at 2 weeks after low-dose plasmid DNA injection following dynamic vacuum (KV) treatment, and specifically compares immunogenicity outcomes resulting from the addition of hyaluronidase to low-dose injections. [Figure 40] This chart shows binding ELISA data in rabbits at 2 weeks after medium-dose plasmid DNA injection following dynamic vacuum (KV) treatment, and specifically compares the immunogenicity outcomes resulting from the addition of hyaluronidase to the medium-dose injection. [Figure 41] This chart shows the quantified immune cell migration in rabbits after dynamic vacuum (KV) treatment, specifically the number of GFP-positive cells in the target lymph nodes at days 1, 2, 3, and 7 post-treatment, thereby indicating the time period over which GFP-positive cell migration occurs. [Figure 42] This chart shows binding ELISA data in guinea pigs at 2 weeks after low-dose DNA infusion and subsequent dynamic vacuum (KV) treatment, and specifically compares the "trade-offs" arising from diluting low-dose DNA infusions with various hyaluronidase dosages. [Figure 43] This chart compares the effect on guinea pig binding ELISA data at week 2, generated by adjusting the number of repetitions (cycles) of the dynamic vacuum (KV) transfer pattern. [Figure 44]This chart compares the effect on binding ELISA data in guinea pigs at week 2, provided by combining intradermal (ID) injection of mRNA-1273 (an mRNA vaccine formulated using lipid nanoparticles) at normal, low doses with dynamic vacuum (KV) treatment with intramuscular (IM) injection of mRNA-1273 at normal, low doses without KV treatment. [Figure 45] This chart compares the effects on binding ELISA data in guinea pigs at week 2, provided by combining intradermal (ID) injection of mRNA-1273 (an mRNA vaccine formulated using lipid nanoparticles) at normal, moderate, and low doses with dynamic vacuum (KV) treatment with ID injection of mRNA-1273 at normal and low doses without KV treatment. [Figure 46] This chart shows binding ELISA data in guinea pigs at 2 weeks after high-dose DNA injection and subsequent dynamic vacuum (KV) treatment, and particularly compares the "trade-offs" arising from diluting high-dose DNA injections with various hyaluronidase dosages. [Modes for carrying out the invention]

[0010] This disclosure may be more readily understood by referring to the following detailed description provided in connection with the accompanying figures and examples that form part of this disclosure. It should be understood that this disclosure is not limited to any specific devices, methods, uses, conditions, or parameters described and / or shown herein, and that the terminology used herein is for illustrative purposes only to illustrate specific embodiments and is not intended to limit the scope of this disclosure.

[0011] Furthermore, as used herein, including in the attached claims, the singular forms "a," "an," and "the" include the plural forms, and references to specific numbers include at least that specific value unless the context clearly indicates otherwise.

[0012] As used herein, the term “multiple” means more than one. Where a range of values ​​is expressed, an alternative embodiment includes one value and / or a specific value of the other. Similarly, where values ​​are expressed as approximations, by using the antecedent “about,” it will be understood that a particular value forms an alternative embodiment. All ranges are inclusive and combinable.

[0013] The terms “approximately,” “about,” and “substantially” as used herein with respect to dimensions, angles, ratios, and other geometric shapes take into account manufacturing tolerances. Furthermore, the terms “approximately,” “about,” and “substantially” may include values ​​that are 10% greater or less than the stated dimensions, ratios, or angles. Additionally, the terms “approximately,” “about,” and “substantially” may be applied equally to specific values ​​stated.

[0014] As used herein, the term “dynamic vacuum” (KV) means a region of vacuum pressure movement caused by moving a vacuum applicator (e.g., a vacuum cup) against the surface of a target tissue.

[0015] As used herein, the term “static vacuum” (SV) means a vacuum pressure field that does not move relative to the surface of a target tissue. An example of a static vacuum may include a vacuum cup that applies vacuum pressure to tissue while remaining stationary relative to the tissue.

[0016] As used herein, the term “electropermeation” (EP) means the use of an electric field within a tissue to temporarily and reversibly increase the permeability and / or porosity of the cell membrane of cells within the tissue, thereby enabling the introduction of a drug into the cell.

[0017] When used herein as a postposition to a primary reference character, the character "n" (e.g., Pn) indicates that the primary reference character ("P") may have an unlimited number of copies. For example, when referring to various spatial locations P1, P2, P3, etc., shown in a figure, the combined reference character "Pn" as used below indicates that there may be additional locations not shown in each figure.

[0018] Terms containing numerical prepositions (e.g., "first," "second") may be used herein to describe various features, but it should be understood that such features should not be limited by these terms. These terms are instead used to distinguish one feature from another. For example, a first element may be referred to as a second element in a different context, and similarly, a second element may be referred to as a first element in a different context without departing from the scope of the embodiments described herein.

[0019] The embodiments described herein relate to systems and devices for performing dynamic vacuum treatment on target tissues, particularly skin tissue, but the treatments herein can be adapted to adipose tissue and / or muscle tissue. These embodiments subject a target region of tissue to a moving region of vacuum pressure (i.e., dynamic vacuum) and impose mechanical stress and strain on the target tissue in a manner suitable for enhancing drug delivery in vivo (e.g., by deforming the tissue), particularly in modes that increase the fluid dispersion (diffusion) of the injector within the tissue, in modes that increase the direct cellular uptake of the drug into target cells of the tissue (e.g., nucleic acid transfection), and in modes that increase the immune response to the injector (i.e., the injected drug). Thus, it can be said that the embodiments described herein provide a number of modes for enhancing drug delivery. Examples of agents deliverable by dynamic vacuum treatment as described herein include, but are not limited to, plasmids (e.g., DNA vaccine plasmids), peptides, small molecules, nucleic acids, synthetic DNA-encoding monoclonal antibodies (DMAbs), synthetic DNA-encoding proteins, cancer antigens, viral antigens associated with chronic infections, bacterial or other microorganism antigens or proteins, and combinations thereof.

[0020] In embodiments of this specification, the open end of a vacuum device, such as a vacuum cup, is positioned in contact with the outer surface of tissue (e.g., "skin") covering a tissue volume, and vacuum pressure is applied inside the cup, thereby drawing a portion of the tissue in a target area into the vacuum cup and instantaneously holding that portion of the tissue inside the cup. While the vacuum field is applied to the tissue, the cup is moved relative to the tissue surface, which is subjected to mechanical stress and strain (i.e., deformation) on at least the portion of the tissue being instantaneously and / or mutually drawn into the vacuum cup.

[0021] The inventors have observed that such dynamic vacuum treatment generates a predictable and substantially uniform zone of cell uptake (e.g., a transfection zone) within the target tissue region. Dynamic vacuum treatment provided by the embodiments described below has also been observed to result in favorable redistribution of fluids within the target tissue region, including favorable in vivo dispersion of the injector within the tissue volume, as well as favorable in vivo inflow and outflow of fluids into and from the target zone. For example, dynamic vacuum treatment has been observed to enhance the dispersion of the injector throughout the tissue, expand the transfection zone, and draw more in vivo fluids into the target tissue region, thereby increasing the amount of cells exposed to transfected cells.

[0022] The inventors have observed that the dynamic vacuum procedure described throughout this disclosure elicits an increased response of the target to the injector. Compared to a “static vacuum” procedure in which vacuum pressure is applied and then removed without translating or rotating the vacuum applicator, or otherwise moving the vacuum applicator, the dynamic vacuum procedure produces stronger and more widespread transfection of nucleic acids, more widespread dispersion of the injector throughout the target tissue, and a stronger resulting immune response when the injector is a vaccine, particularly a nucleic acid vaccine.

[0023] The inventors have also observed, surprisingly and unexpectedly, that the dynamic vacuum treatment described herein can induce cell uptake and immune responses comparable to, and even exceeding, those resulting from treatment using electroporation, without the use of electroporation. While not wishing to be constrained by any particular theory, the inventors believe that the gradient fluctuations and movement of vacuum pressure imparted by the dynamic use of the vacuum cup (i.e., moving the vacuum pressure field) impart mechanical stress and strain to the cell membrane within the tissue volume, which increases cell membrane permeability and thus increases cell uptake observed within the tissue volume. The inventors further believe that the aforementioned fluid redistribution and mechanical stress likely interact with each other to create a favorable environment within the tissue volume for the uptake of external drugs into cells (e.g., transfection). The use of dynamic vacuum also appears to independently benefit from the addition of “diffusing agents” (e.g., hyaluronidase) that allow the injectable to flow more freely throughout the tissue, which the inventors believe is due to the aforementioned fluid redistribution capability of dynamic vacuum. For example, the inventors observed that adding hyaluronidase to an injectable dramatically enhances the effect of dynamic vacuum on increasing the dispersion of the injectable throughout the skin tissue.

[0024] Referring to Figures 1A and 1B, the vacuum treatment system 100 for treating a patient according to this disclosure includes a vacuum applicator 2, which includes a housing body 4 defining an internal vacuum chamber 6. The vacuum applicator 2 may also be referred to as the “vacuum cup” or simply the “cup.” The housing body 4 may also be referred to as the “cup housing.” The cup housing 4 extends from a proximal end 8 to a distal end 9 along the transverse direction Z. The distal end 9 is spaced apart from the proximal end 8 in the distal direction D along the transverse direction Z, while the proximal end 8 also extends along the transverse direction Z and is spaced apart from the distal end 9 in the proximal direction P, opposite to the distal direction D. It should be noted that the proximal direction P and the distal direction D are bidirectional, unidirectional components of the transverse direction Z. The cup housing 4 also defines a central axis Z1 oriented along the transverse direction Z. The central axis Z1 can also be characterized as the central axis of the vacuum chamber 6 and / or the central axis of the vacuum cup 2.

[0025] The vacuum cup 2 is configured so that a user (such as a physician) can position the distal end face 10 of the vacuum cup 2 at its distal end 9 on the outer surface of the tissue to be targeted for treatment (e.g., skin), for example, on a portion of the skin surface that overlaps with a fluid injection (previously injected into the tissue). At such a position of the vacuum cup 2 on the skin surface, the user can apply vacuum pressure to the vacuum chamber 6 to draw, pull, or otherwise guide the tissue (e.g., skin tissue) into the vacuum chamber 6. Using the tissue drawn into the vacuum chamber 6, the user can move the vacuum cup 2 along the tissue surface, thereby manipulating the tissue along the path of the cup's movement, which is referred to herein as “dynamic vacuum treatment”. Target tissues that can be manipulated according to the dynamic vacuum treatment described below include the dermis (epidermis and dermis) and may include additional layers such as subcutaneous fat (i.e., the adipose layer), as will be described in more detail below.

[0026] The cup housing 4 may include a proximal surface 15 at or adjacent to the proximal end 8. In the shown embodiment, the cup housing 4 includes a peripheral annular lip 17 that extends proximal from the proximal surface 15 to the proximal end 8. In other embodiments, the cup housing 4 does not need to include a peripheral annular lip 17 so that the proximal surface 15 also defines the proximal end 8 of the cup housing 4. Additionally, the proximal surface 15 may be substantially planar as shown, but the geometric shapes of other surfaces are also within the scope of this disclosure.

[0027] The vacuum cup 2 includes one or more couplings, such as ports, for connection to one or more external components. For example, the vacuum cup 2 has a port 12 for providing fluid communication between the vacuum chamber 6 and a vacuum source 106, such as a vacuum pump. This port 12 can be defined along a port coupling 14, such as a stem 14, for connection to a tube 16 that provides fluid communication between the vacuum chamber 6 and the vacuum source 106. The stem 14 may extend proximal from the proximal surface 15 of the housing body 4. The vacuum source 106 can communicate electrically with a control unit 114 (also referred to herein as the “controller”), which may include a processor 116 configured to control the operation of the electroporation system 100, including the operation of the vacuum source 106. The processor 116 can communicate electronically with computer memory 118 and can be configured to run software and / or firmware containing one or more algorithms for controlling the operation of the system 100. The processor 116 can also electrically communicate with a user interface 120, which may include a display 122 for presenting information related to the operation of the system 100, and a keypad 124 that allows an operator, such as a user, to input information such as commands related to the operation of the system 100. It should be understood that the display 122 may be a touchscreen display that allows an operator to directly input information on the display 122. It should also be understood that the interface 120 may be a computer interface such as a tabletop computer or laptop computer, or a handheld electronic device such as a smartphone.

[0028] Referring here to Figures 1B and 1C, the distal end 9 of the vacuum cup 2 defines at least one opening 20 leading to the vacuum chamber 6. The opening 20 may be circular as shown, but other opening shapes are also within the scope of this disclosure. The distal end 9 of the vacuum cup 2 (and therefore the opening 20 as well) may be defined by the housing 4. In the illustrated embodiment, the distal end 9 of the vacuum cup 2 also defines the distal end of the vacuum chamber 6. Within the vacuum chamber 6, the housing 4 defines an inner surface 22 extending from the distal end surface 10 of the housing 4 to the proximal end surface 24 within the chamber 6. The proximal end surface 24 may define the proximal end 26 of the chamber 6. In the illustrated embodiment, the chamber 6 is defined by the inner surface 22 and the proximal end surface 24. Additionally, the vacuum cup 2 of this embodiment may be referred to as a “dome-shaped vacuum cup” 2 due to the generally dome-shaped geometry of the vacuum chamber 6. Chamber 6 has a chamber diameter D1 measured between opposing portions of its inner surface 22 along the radial direction R, perpendicular to and intersecting the central axis Z1. Chamber 6 also defines a chamber depth L1 measured along the transverse direction Z, from the distal end 9 to the proximal end 26 of chamber 6. The chamber diameter D1 may be in the range of approximately 1.0 mm to approximately 50.0 mm, more specifically in the range of approximately 3.0 mm to approximately 20.0 mm, and more specifically in the range of approximately 5.0 mm to approximately 15.0 mm. The chamber depth L1 may be in the range of approximately 1.0 mm to approximately 50.0 mm, more specifically in the range of approximately 3 mm to approximately 20 mm, and more specifically in the range of approximately 5 mm to approximately 17 mm.

[0029] The housing 4 also defines an outer surface 28 spaced apart from the inner surface 22 along the radial direction R. The housing 4 also defines a wall 30 extending from the inner surface 22 to the outer surface 28 along the radial direction R. In the illustrated embodiment, the housing 4 has a substantially circular geometric shape within a reference plane extending along a first direction X and a second direction Y, which are perpendicular to each other and also perpendicular to the transverse direction Z. Thus, as shown, the inner surface 22 and the outer surface 28, as well as the wall 30, can rotate uniformly around a central axis Z1. However, it should be understood that other housing geometric shapes, including the housing (and chamber) geometric shapes described in U.S. Patent Publication No. 2021 / 0290941A1 (hereinafter, "Reference '941"), published on September 23, 2021, entitled "VACUUM-ASSISTED ELECTROPORATION DEVICES, AND RELATED SYSTEMS AND METHODS," are also within the scope of this disclosure, and the entire disclosure is incorporated herein by reference as if it were described herein as a whole.

[0030] The housing 4 may be formed of a material that is preferably transparent or translucent, thereby allowing visualization of the tissue drawn into the vacuum chamber 6 during use. As shown, the housing 4 may include a scale 32 along the outer surface 28 and may be configured to provide a visual indication of the depth to which the tissue is drawn into the chamber 6. The material of the housing 4 may also have a certain degree of flexibility, particularly through elastic deformation, which can reduce patient discomfort during use. The material of the housing 4 may be a polymer material, including, as non-limiting examples, polyether ether ketone (PEEK), polyphthalamide (PPA), polyethylene, polycarbonate, polyetherimide (PEI), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyamide, polyimide, polysiloxane (silicone), polyethylene terephthalate, polyurethane, crosslinked or non-crosslinked rubber (elastomer), and polyester. It should be understood that other biocompatible materials and / or medical-grade materials may be used for the housing 4. The housing 4 may be a monolithic structure defining the vacuum cup 2, as in the embodiments shown herein. However, in other embodiments, the housing 4 does not have to be a monolithic structure and instead may include two or more body components joined together to define the housing 4. Additionally, the distal end face 10 of the vacuum cup 2 may be subjected to one or more finishing processes to reduce surface finish roughness, or otherwise to smooth and / or polish the distal end face 10, thereby reducing friction with the skin surface during dynamic vacuum treatment, and thereby improving patient comfort during and after treatment.

[0031] Referring here to Figure 1C, the inner surface 22 preferably has a generally bell-shaped geometric form. The inner surface 22 has a primary surface portion 22a that extends midway between the distal end 10 and the proximal end 26 of the chamber 6. This primary surface portion 22a may have a linear profile in an axial reference plane, but other profile geometric forms are also within the scope of this disclosure. As shown in Figure 1C, the primary surface portion 22a may have a linear profile oriented at an acute taper angle A0 with respect to a linear axis oriented along the transverse Z in an axial reference plane. In this way, the inner diameter of the chamber 6 decreases toward the proximal end 26. The taper angle A0 can be in the range of about 0 to about 60 degrees, more specifically in the range of about 0.5 to about 15 degrees, and more specifically in the range of about 5 to about 10 degrees.

[0032] The inner surface 22 also preferably includes a distal retraction portion 22b extending from the distal end face 10 to the primary surface portion 22a. The distal retraction portion 22b preferably has a tapered curved contour to reduce or otherwise mitigate tissue damage such as bruising around the tissue being retracted into the chamber 6 during use of the vacuum cup 2. In the embodiments described herein, the chamber diameter D1 is measured at the interface between the distal retraction portion 22b and the primary surface portion 22a. The chamber diameter D1 may optionally be measured at other locations along the inner surface 22. The inner surface 22 may include a proximal relaxation portion 22c extending from the primary surface portion 22a to the proximal end face 24. The proximal relaxation surface 22c is preferably curved or otherwise molded to reduce stress concentration within the housing 4 during use. The interface between the primary surface portion 22a and the distal retraction portion 22b and the proximal relaxation surface 22c is shown in Figure 1C by reference mark 23.

[0033] The distal end 9 of the vacuum cup 2 also preferably has a rounded, or otherwise chamfered, outer relieving surface 29 extending between the distal end face 10 and the outer surface 28. In this way, the outer relieving surface 29 is configured to reduce friction and / or abrasion (e.g., abrasions) with the skin at the outer edge of the cup 2 during the dynamic movement of the vacuum cup, thereby enhancing patient comfort. The interface between the outer relieving surface 29 and the distal end face 10 and outer surface 28 of the cup 2 is indicated in Figure 1C by a reference mark 25. The cup wall 30 defines the wall thickness W1 between the inner surface 22 and the outer surface 28. In the embodiments described herein, the wall thickness W1 is measured along the radial direction R between: (1) the interface 23 between the distal retraction portion 22b and the primary surface portion 22a, and (2) the interface 25 between the outer relieving surface 29 and the outer surface 28. The wall thickness W1 can optionally be measured at other locations along the wall 30. It should be understood that the outer surface 28 of the vacuum cup 2 can be used as a visual reference when performing dynamic vacuum cup movement, particularly at the interface 25 with the outer relaxation surface 29. For example, this interface 25 can be used to visually determine when the vacuum cup 2 has translated across and / or away from the injection site, as will be described in more detail below.

[0034] Referring here to Figure 1D, the vacuum cup 2 is positioned on a tissue surface, such as the surface 3 of the skin tissue 1, and rests on the fluid injector 7 within the tissue. The distal end surface 10 of the cup 2 is configured to contact the tissue surface 3, or an intermediate substance such as a gel, oil, lotion, or lubricant to facilitate the smooth movement of the cup along the tissue surface 3. Using the vacuum cup 2 positioned in this manner, a vacuum pressure sufficient to draw the tissue 1 into the chamber 6 can be supplied to the chamber 6 via the port 12. As shown, the tissue 1 can take on a mountain-like or dome-like shape within the chamber 6 in response to the vacuum pressure. This tissue can be drawn into the chamber 6 to various depths L2, measured from the apex of the tissue to the distal end 9 of the chamber 6, in response to the magnitude of the vacuum pressure communicating with the chamber 6. The vacuum pressure connected to chamber 6 may be in the range of approximately -0.1 psi to approximately -14.7 psi (approximately -0.70 kPA to approximately -100 kPA) (approximately -5 mmHg to approximately -760 mmHg), more specifically in the range of approximately -3 psi to approximately -14.7 psi (approximately -20 kPA to approximately -100 kPA) (approximately -155 mmHg to approximately -760 mmHg), more specifically in the range of -7.7 psi to approximately -14.7 psi (approximately -53 kPa to approximately -100 kPa) (approximately -400 mmHg to approximately -760 mmHg), and even more specifically in the range of approximately -7.7 psi to approximately -11.6 psi (approximately -53 kPa to approximately -80 kPA) (approximately -400 mmHg to approximately -600 mmHg).

[0035] Referring here to Figures 2A to 2C, it is shown that another embodiment of the vacuum cup 102 has a different internal vacuum chamber geometric shape than that of the vacuum cup 2 described above. In particular, the vacuum cup 102 of this embodiment includes a projection 34 that extends into the chamber 6 from the proximal end face 24 toward the opening 20. The vacuum cup 102 of this embodiment can otherwise be the same as that of the vacuum cup 2 described above. Therefore, the same reference letters as above can be used for features of the vacuum cup 102 that are the same as those of the vacuum cup 2 described above. The projection 34 preferably extends centrally along the central axis Z1. Therefore, the projection 34 may be referred to herein as the “central support” 34. Additionally, the vacuum cup 102 of this embodiment may be referred to as the “central support vacuum cup” 102.

[0036] As shown in Figure 2B, the central strut 34 has a length L3 measured from the proximal end face 24 to the distal end 36 of the strut 34. The vacuum cup 102 also defines a strut depth L4 measured from the distal end 9 of the chamber 6 to the distal end 36 of the strut 34. The central strut 34 in the illustrated embodiment has a rounded distal end geometric shape. For example, as shown, the central strut 34 has a rounded distal end face with a radius R1. As shown in Figure 2C, the central strut 34 may be configured to contact the surface 3 of the tissue drawn into the chamber 6 and impart additional mechanical stress and strain to the injected tissue, such as by deforming the tissue that contacts the central strut 34, as will be described in more detail below. It should be understood that various other geometric shapes may be used within the chamber 6 to increase the mechanical stress and strain on the tissue drawn into the chamber 6. For example, the central support 34 may have a textured outer surface (e.g., raised and / or recessed). Additionally or alternatively, the vacuum cup may have multiple supports extending distally within the chamber 6, these supports may have varying sizes and geometric shapes and may be arranged in various patterns. Additionally or alternatively, the vacuum cup may have one or more supports that can move relative to the cup housing 4, such as by being compressible or extendable along the lateral Z direction.

[0037] Referring here to Figure 3A, an additional embodiment of the vacuum cup 202 has an injection channel 38 that extends through the cup housing 4 into the vacuum chamber 6. Such a vacuum cup 202 may be referred to as an "injection channel vacuum cup" 202. The injection channel 38 is configured for the passage of an injection material from the proximal side of the vacuum cup 202 through the cup housing into the vacuum chamber 6. The injection channel 38 can be centrally located along the central axis Z1 of each vacuum cup 202, as shown, or it can be offset from the central axis.

[0038] The vacuum cup 202 can be similar to the vacuum cup 2 described above with reference to Figures 1A to 1D, and the injection channel 38 is configured to allow the injection needle 40 to pass into the vacuum chamber 6. In this embodiment, the vacuum cup 202 preferably includes a partition 42 or other device for forming a seal with the injection needle 40, while the needle 40 extends into the vacuum chamber 6. In this way, the partition 42 or other device can maintain a desired vacuum pressure in the chamber 6 while the injection needle 40 extends through the injection channel 38.

[0039] Referring here to Figure 3B, in a further embodiment, the vacuum cup 302 can be configured for use with a jet syringe 44 and otherwise be the same as the vacuum cups 2 and 102 described above. In this embodiment, the injection channel 38 is wide enough to receive the nozzle 48 of the jet syringe 44. As shown, the nozzle 48 may extend into the vacuum chamber 6 and may optionally be positioned to contact tissue drawn into the vacuum chamber 6. In this way, the nozzle 48 can deform the tissue drawn into the chamber 6 in a manner similar to that of the central support 34 described above. In this embodiment, the vacuum cup 302 may include a mount or other features for guide coupling with the jet syringe 44.

[0040] It should be noted that the vacuum cups 202 and 302 shown in Figures 3A and 3B can be adapted so that their injection channels 38 are configured for use with other types of injection devices. For example, the vacuum cup 202 shown in Figure 3A can be adapted so that its injection channel 38 is configured for use with a jet syringe or other types of injection devices. Similarly, the vacuum cup 302 shown in Figure 3B can be adapted so that its injection channel 38 is configured for use with an injection needle or other types of injection devices.

[0041] Referring here to Figures 4A and 4B, another embodiment of the vacuum cup 402 having one or more electrodes for electroperforating tissue during vacuum treatment, which may be referred to herein as “vacuum electroperforation” (VEP) treatment. Thus, the vacuum cup 402 of this embodiment may be referred herein as the VEP cup 402. The VEP cup 402 of the illustrated embodiment was primarily used for testing to compare treatment outcomes resulting from KV treatment and SV treatment with VEP treatment. However, it should be understood that the VEP cup 402 may be used to provide dynamic and / or static VEP treatment within the scope of this disclosure.

[0042] The VEP cup 402 of this embodiment is similar to the vacuum cup 102 disclosed above with reference to Figures 2A-2C, except that the VEP cup 402 has a central electrode 50 at the central support 34 and a concentric ring electrode 52 at the distal end 9 of the cup 402. Therefore, the VEP cup 402 of this embodiment may also be referred to as the “central support electrode VEP cup” 402. The central electrode 50 and the ring electrode 52 are configured to deliver one or more electroporation pulses to the tissue drawn into the vacuum chamber 6 during vacuum treatment. The electrical parameters of these one or more electroporation pulses (e.g., potential (voltage), current magnitude (amperes), pulse duration, inter-pulse delay, and pulse amount) may include those more fully described in Reference 941.

[0043] In the illustrated example, the ring electrode 52 extends inward from a first outer electrode end 54 located near the interface with the outer surface 28 of the cup housing 4 to a second inner electrode end 56 located on the primary surface portion 22a of the inner surface 22. Thus, the ring electrode 52 can extend along the distal end 9 and inner surface 22 of the VEP cup 402 and can also define the distal end face 10 of the vacuum cup 402. A transmission member, such as a wire or conductive strut 55, extends from the ring electrode 52 to the outside of the cup housing 4 and is configured to transmit one or more electroporation pulses to the ring electrode 52. As shown, the conductive strut 55 can extend proximal through the cup housing 4 to a contact 57 on the proximal surface 15. In the illustrated embodiment, the central electrode 38 defines the central strut 34, but in other embodiments, the central electrode 38 can extend along the outer surface of the central strut 34. The proximal end of the central electrode 38 defines a contact 59 that can be located on the proximal surface 15 of the cup housing 4. In other embodiments, it should be noted that the VEP cup can be used with a variety of other electrode configurations, including any of those described in '941 References. It should also be noted that the VEP cup 402 can be configured to have an injection channel 38 for an injection needle 40 and / or a jet injector 44, similar to the vacuum cups 202 and 302 described above with reference to Figures 3A-3B.

[0044] Referring here to Figures 5A and 5B, any of the vacuum cups 2, 102, 202, 302, and 402 described above can be adapted to be mounted on the handle assembly 58 of the vacuum treatment system 100. For illustrative purposes, the handle assembly 58 is shown coupled to the vacuum cup 402 disclosed above with reference to Figures 4A and 4B. The handle assembly 58 includes a handle member 60 having a first distal end 62 and a second proximal end 64 opposite the distal end 62. At the distal end 62, the handle member 60 has a mounting configuration 66 configured to releasably couple to the proximal end 8 of the vacuum cup. For example, one or more of the vacuum cups 2, 102, 202, 302, and 402 described herein, each at most, can be configured to have a complementary mounting structure 68 configured to releasably couple (i.e., repeatedly and nondestructively couple and uncouple) to the mounting configuration 66 of the handle assembly 58. In this way, the handle assembly 58 can be configured to be interchangeably coupled to one or more, up to all, of the vacuum cups 2, 102, 202, 302, and 402 described herein. The handle assembly 58 may also include, as a non-limiting example, at least one button or trigger 67 that can be configured to control the operation of the vacuum cup 402, such as for controlling the start and end of the application of vacuum pressure in the vacuum chamber 6.

[0045] Here, with reference to Figure 5B, an exemplary handle mounting configuration 66 and a complementary cup mounting structure 68 are described. For illustrative purposes, the vacuum cup 402 is rotated to a bottom view, while the portion of the handle member 60 being depicted remains in a side view. The handle mounting configuration 66 may include a base surface 70 and a peripheral landing surface 72 recessed proximal to the base surface 70. The peripheral landing surface 72 extends annularly around the periphery of the handle mounting configuration 66. The base surface 70 of the handle mounting configuration 66 is configured to align with the proximal surface 15 of the vacuum cup 402 when the cup 402 is attached to the handle mounting configuration 66. Additionally, when the cup 402 is attached to the handle mounting configuration 66, the peripheral landing surface 72 of the handle mounting configuration 66 is configured to align with the proximal end 8 of the cup 402, and the cup peripheral annular lip 17 extends proximal from the base surface 70 to the peripheral landing surface 72. In this way, engaging the cup periphery, the annular lip 17, and the peripheral landing surface 72 provides a centering mechanism for the vacuum cup 402 in the handle mounting configuration 66.

[0046] The handle mounting configuration 66 includes one or more mounting members 74, such as mounting prongs 74, which extend from the base surface 70 and are configured to mount to one or more complementary mounting configurations 76, such as keyed slots 76, of the cup mounting structure 68. The keyed slots 76 are defined within the cup housing 4 along the proximal surface 15, and each has a first wide slot portion 78 and a second narrow slot portion 80 extending circumferentially from each wide slot portion 78. The mounting prongs 74 have an extension portion 82 and a latch portion 84 projecting laterally from the extension portion 82. While the cup 402 is coupled to the handle assembly 58, the latch portion 84 of the mounting prongs 74 is configured to advance distally through the wide slot portion 78 of the keyed slot 76 (while the cup periphery, the annular lip 17 advances to the peripheral landing surface 72 of the handle mounting configuration 66). Subsequently, the vacuum cup 402 is rotated around the central axis Z1 such that the latch portion 84 protrudes into the housing body 4 alongside the narrow slot portion 80, thereby providing mechanical interference in the proximal direction P between the latch portion 84 and the housing body 4 in a manner that maintains the attachment of the cup 402 to the handle mounting structure 66. To disengage the vacuum cup 402 from the handle mounting structure 66, the vacuum cup 402 can be rotated in the opposite direction around the central axis Z1 until the latch portion 84 aligns with the wide slot portion 78, and then the cup 402 can be moved distally D away from the handle mounting structure 66.

[0047] Continuing to refer to Figure 5B, the handle member 58 includes a vacuum channel 86 configured to connect to the port 12 of the vacuum cup 402 and to provide fluid communication with the vacuum cup 402 in order to communicate vacuum pressure to the vacuum chamber 6 when the vacuum cup 402 is coupled to the handle assembly 58. The distal end of the vacuum channel 86 and the proximal end of the port 12 may have one or more complementary port coupling members to provide a sealed port connection between them when the vacuum cup 402 is coupled to the handle assembly 58.

[0048] Additionally, as shown, the handle assembly 58 can be adapted for use with the VEP cup 402. Thus, the handle mounting structure 66 may include one or more electrical contacts 88, 90 configured to provide electrical communication with the contacts 57, 59 of the vacuum cup 402 when the vacuum cup 402 is coupled with the handle assembly 58. As shown, the handle mounting structure 66 may include a first contact 88 for contacting the contact 59 of the center electrode 50 and a second contact 90 for contacting the contact 57 of the ring electrode 52. The contacts 88, 90 of the handle assembly 58 can electrically communicate with an electronic circuit, which may include a printed circuit board (PCB) 92, and the PCB 92 can electronically communicate with one or more electronically controlled devices for controlling electroporation pulses, such as a controller 114, and the controller 114 may be located on-board or off-board of the handle assembly 58.

[0049] Referring again to Figure 5A, the handle assembly 58 may include one or more conduits 94 extending away from the handle member 60. One or more conduits 94 may include a tube 16 that provides fluid communication between the vacuum channel 86 (and therefore the vacuum chamber 6 as well) and the vacuum source 106. One or more conduits 94 may also include electrical cables(s) for providing electrical communication between the handle assembly 58 and off-board electrical devices.

[0050] The vacuum cup handle assembly 58 and its handle mounting configuration 66 described above, as well as the complementary mounting structure 68, are provided as non-limiting examples of such components and features, and it should be understood that various other such designs and configurations are also within the scope of this disclosure.

[0051] Herein, with reference to Figures 6A–6G, a non-limiting example of dynamic vacuum treatment is described. In this example, the dynamic vacuum treatment is shown to be performed by vacuum cup 2 described above with reference to Figures 1A–1D, but it should be understood that similar vacuum treatment can be performed using any of the other vacuum cups 102, 202, 302, 402 described above. For illustrative purposes, the structures in Figures 6A–6G have superimposed graduated markings (-4 to +4) to provide a visual reference for the movement and distance of the cup shown in this non-limiting example of dynamic vacuum treatment. Each interval of these graduated markings is equivalent to the radius R2 of the vacuum chamber 6 (R2 = 1 / 2D1).

[0052] As shown in Figure 6A, the drug-containing fluid injector is delivered to the intradermal tissue 1 (i.e., skin tissue) by an injection needle 40 via a Manto injection or the like, which generates a bolus of the injected fluid 7 in the intradermal tissue beneath the skin surface 3. The bolus of the injected fluid 7 can form a mass or "blister" 11 on the skin surface 3. As shown in Figure 6B, after injection, the needle 40 is removed, and optionally, a substance 13 is applied to the skin surface 3 along the target treatment site around the injected fluid 7 to facilitate smooth cup movement along the skin surface 3 during dynamic vacuum treatment. The substance 13 can be a gel (e.g., ultrasonic gel), oil, lotion, or other lubricant.

[0053] As shown in Figure 6C, the vacuum cup 2 can be positioned on the substance 13 on the skin surface 3 at a first position P1 adjacent to the injected fluid 7. In this example, the first position P1 is centered on the injected fluid 7 (i.e., the blister 11). Alternatively, at the first position P1, the cup 2 can be positioned at least partially on the blister 11, but not centered on it, or completely offset from the blister 11.

[0054] As shown in Figure 6D, vacuum pressure can be supplied to the vacuum chamber 6, thereby applying a vacuum field sufficient to draw tissue into the chamber 6 to the tissue below the vacuum chamber 6 (in particular, the skin surface 3). In this non-limiting example, the vacuum pressure can be supplied at approximately -500 mmHG (approximately -66.7 kPa). In the illustrated example, the tissue drawn into the chamber 6 includes intradermal tissue 1 (e.g., epidermis and dermis) and some subcutaneous tissue 5. It should be understood that the amount and type of tissue drawn into the chamber 6 can be determined by various factors, including the supplied vacuum pressure.

[0055] As shown in Figure 6E, the vacuum cup 2 is translated along the skin surface 3 from a first position P1 to a second position P1 by a first distance in a first direction, thereby also effectively pulling the underlying tissue in and out of the chamber 6 as the chamber 6 passes overhead. As shown in Figure 6F, the vacuum cup 2 is translated along the skin surface 3 from a second position P2 to a third position P3 by a second distance in a second direction opposite to the first direction, again effectively pulling the underlying tissue in and out of the chamber 6 as the chamber 6 passes overhead. It should be understood that, as a non-limiting example, the vacuum cup 2 can be translated back and forth across the skin surface 3, including three or more times between positions P1-P3 shown in Figures 6E and 6F. As shown in Figure 6G, the vacuum cup 2 can be removed from the skin surface 3, and the procedure can be completed.

[0056] As shown in the example above, the translational distance from the first position P1 to the second position P2 is substantially equal to the chamber diameter D1 of cup 2, thereby causing the rear side of the inner surface 18 at P2 to be in the position occupied by the leading side of the inner surface 18 at P1. Thus, if the width of the blister 11 is substantially equal to the chamber diameter D1 and the first position P1 is centered on the blister 11, such movement from P1 to P2 moves the rear side of the inner surface 18 from one side edge of the blister 11 to the opposite side edge of the blister 11. In these states, such movement also shifts the chamber 6 from substantially completely encompassing the blister 11 at P1 to substantially completely offset from the blister 11 at P2 and P3. Thus, such movement at these distances can effectively shift the edge of the chamber 6 between the opposite edges of the blister 11. In this way, the user can refer to or "index" the movement of the dynamic vacuum cup by the spatial relationship between the inner surface 18 and the edge of the blister 11. Therefore, during each translation in this example, the user can use the trailing edge of the inner surface 18 as a reference for when to stop the translation. In embodiments where the housing body 4 is transparent or translucent, the inner surface 18 can be used as a visual reference governing the translation.

[0057] Referring here to Figure 6H, in another example of dynamic vacuum treatment, the outer surface 28 of the vacuum cup 2 can be used as a visual reference governing the cup translation. For illustrative purposes, the tissue in Figure 6H has superimposed graduated markings (-6 to +6) to provide a visual reference for the movement and distance of the exemplary cup. In this example, the cup 2 is translated from a first position P1 at the center of the blister 11 to a second position P2 offset from the blister 11, then to a third position P3 offset on the opposite side of the blister 11, and then back to a fourth position P4 coinciding with the first position P1. At the offset positions P2, P3 in this example, the outer surface 28 of the vacuum cup 28 is separated from the proximal edge of the blister 11 by a distance substantially equivalent to one of a stepped interval (i.e., 1 / 2D1). In other examples, the offset positions P2, P3 can be such that, at these positions, the proximal side of the outer surface 28 of the cup 2 is substantially aligned with the proximal edge of the blister 11. In other such examples, the user can translate the cup 2 from P1 to P2 and from P2 to P3 until the rear side of the outer surface 28 reaches the opposite edge of the blister 11, as guided by visual observation. In these examples discussed with reference to Figure 6H, the translational distance between positions P1 to P4 is determined by the width of the blister 11 and the wall thickness W1 of the vacuum cup 2.

[0058] Referring to Figures 7A–7D, exemplary cup movements are described according to various techniques for providing dynamic vacuum treatment. It should be understood that the exemplary examples shown in these figures are provided for illustrative purposes and for consideration of exemplary types of dynamic vacuum cup movements within the scope of this disclosure. It should also be recognized that these dynamic vacuum treatments are indicated in Figures 7A–7D by circles labeled with reference numeral 2z, which generally represent the vacuum cup 2z at the various positions described. In these illustrated examples, the reference circle 2z more accurately represents the position of the vacuum chamber 6, and therefore these exemplary illustrated movements are indexed to the inner surface 18 of the cup that defines the lateral boundary of the chamber 6. However, as mentioned above, the cup movements can alternatively be indexed to the outer surface 28 of the cup 2, in which case the reference circle 2z shown in Figures 7A–7D can similarly represent the outer surface 28 of the cup 2z, depending on how the user decides to index the position of the cup. It should also be recognized that the vacuum cup 2z shown in these examples may represent any of the vacuum cups 2, 102, 202, 302, and 402 described herein.

[0059] Referring here to Figures 7A and 7B, the exemplary movement of the dynamic vacuum cup involves translating the cup 2z linearly back and forth along a first direction X, for example, laterally left and right, as shown in Figure 7A, or vertically up and down or horizontally back and forth along a second direction Y, as shown in Figure 7B. The translation can occur between various positions P1 to Pn along the translation axis, such as a linear axis X3 oriented along the first direction X (Figure 7A), or a linear axis Y3 oriented along the second direction Y (Figure 7B). In these illustrated examples, the cup 2z starts at a first position P1, which can be substantially centered in the injection site or blister 11. From a first position P1, cup 2z can be translated in a first direction (X1 in Figure 7A, Y1 in Figure 7B) to a second position P2 on one side of blister 11, and then translated in a second opposite direction (X2 in Figure 7A, Y2 in Figure 7B) to a third position P3 on the opposite side of blister 11. In the illustrated example, the second and third positions P2 and P3 represent the lateral boundaries or ends of the translational movement and are spaced apart from the first position P1 such that the chamber 6 is completely offset from blister 11 at the second and third positions P2 and P3. Alternatively, cup 2z may remain partially on top of blister 11 at the second and / or third positions P3, as described below. As a non-limiting example, it should be recognized that the user may select the first translational direction based on various factors, e.g., moving with or against hair particles on the skin surface.

[0060] As shown, the translational distance T1 between the first position P1 and the second and third positions P2 and P3 may be substantially equal to the chamber diameter D1, and the translational distance T2 between the second and third positions P2 and P3 may be twice the translational distance T1 (x2). Thus, in the illustrated example, cup 2z translates starting around blister 11 and moving to lateral positions P2 and P3 away from blister 11 (these positions may be referred to as “off-blister”). For consistency in this specification, each translational movement in a consistent direction (e.g., X1, X2, Y1, or Y2) may be referred to as a “path” or “swipe,” while each such movement between terminal boundaries P2 and P3 may be referred to as a “full path” or “full swipe,” and each such movement from an intermediate position (e.g., P1) to an end position (e.g., P2, P3) may be referred to as a “partial” or “half” path or swipe.

[0061] It should be recognized that the aforementioned translational parameters can be adjusted as needed. For example, the translational distances T1 and T2 can be changed as needed. Thus, the translational distance T1 does not need to be equal to the chamber diameter D1; for example, the translational distance T1 may be greater or less than the chamber diameter D1. Additionally or alternatively, the translational distances T1 and T2 can be such that the cup 2 remains at least partially over the blister 11 during translation. Additionally or alternatively, the first position P1 does not need to be equally spaced from the second position P2 and the third position P3. Alternatively, the translational movement can be restricted to only the first position P1 located at the end of the translation and only the second position P2 (i.e., the first position P1 does not need to be midway between the ends). It should also be recognized that the user can perform various numbers of swipes and / or partial swipes during dynamic vacuum treatment and can pause the movement of the cup for various durations between swipes.

[0062] Referring here to Figure 7C, additional exemplary cup movement (dynamics) may include translating cup 2 along a detour path C1 to various positions P1-Pn. The detour path C1 may be repeated or partially repeated. Translating cup 2 along the entire detour path C1 may be referred to as a “circuit,” and translating cup 2 along a portion thereof may be referred to as a “partial circuit.” In the illustrated example, the detour path C1 is circular and rotates around an axis Z1 that intersects the skin surface in a direction perpendicular thereto (i.e., a “trajectory”). Axis Z1 may intersect the injection site or blister 11, or it may be offset from the injection site, as shown. However, it should be recognized that a variety of other translational paths are within the scope of this disclosure, including eccentric (not centered on axis Z1), elliptical, helical, triangular, or other polygonal, zigzag, and substantially countless others. Additionally, the detour translation path C1 may include a reversal of direction; for example, referring to the illustrated example, the detour path C1 may include translating cup 2 along a circular trajectory path in a first rotational direction R1 (e.g., counterclockwise) around axis Z1, and reversing the translation in a second opposite rotational direction R2 (e.g., clockwise) around axis Z1. It should be understood that numerous variations of the rotational translation path C1 may be used as needed during dynamic vacuum treatment. Additionally or alternatively, during any or part of any of the aforementioned trajectory movements, cup 2z may also be tilted with respect to an axis oriented perpendicular to the underlying tissue so that the cup chamber 6 remains substantially facing the injection site during the trajectory movement. For example, during the trajectory, cup 2z may optionally be manipulated to undergo a rotational tilt such that the cup central axis Z1 remains substantially intersecting the geometric center of the injected bolus 7.

[0063] Referring here to Figure 7D, additional exemplary cup movement (dynamics) may include rotating, swirling, or "twisting" cup 2 back and forth between various angular positions P1–Pn about an axis Z1 that intersects the skin surface in a direction perpendicular to the skin surface. As shown, axis Z1 may be located at the center of the injection site or blister 11, but alternatively, it may be offset from the center. Each rotational movement between positions may be referred to as a "pivot". Cup 2 may pivot back and forth between angular positions at various pivot angles A1, A2, A3, An as needed. In the illustrated example, cup 2 is shown pivoting from a first position P1 to a second position P2 at a pivot angle A1 of approximately 90 degrees, from a second position P2 to a third position P3 at a pivot angle A2 of approximately 180 degrees, and from a third position P3 to a first position P1 at a pivot angle A3 of approximately 90 degrees. However, it should be understood that the above are provided as non-limiting examples, and a number of deformations for torsional motion may be used as needed during dynamic vacuum treatment.

[0064] Furthermore, users should understand that various combinations of the aforementioned translational (forward / backward, circumvention) and rotational (twisting or pivoting) movements can be used during dynamic vacuum procedures.

[0065] Referring again to Figures 7A to 7D, the various positions of cup 2z can also be defined by referring to a coordinate system. For example, the linear anterior-posterior translation shown in Figures 7A and 7B can be defined by referring to a coordinate system such as a two-dimensional Cartesian coordinate system (x,y) having x and y axes extending along the X and Y directions, respectively. In the examples illustrated herein, the x and y axes intersect each other at the injection site, and therefore the coordinate system in these examples is located at the center of the injection site. Thus, in the examples illustrated in Figures 7A and 7B, when cup 2z is located at the center of the blister 11, such as at a first position P1, this cup position can also be represented as the (0,0) position.

[0066] Referring again to Figure 7A, an exemplary sequence of forward and backward dynamic movement along the x-axis is further shown in Figure 7E and can be characterized as follows (a pattern also referred to herein as “Pattern A”): Starting from the (0,0) position centered on the blister 11, cup 2z can undergo a first translation to a second position P2, which can also be represented as the (t,0) position, at a distance “t” in a first direction X1 along the x-axis. From the (t,0) position, cup 2z can undergo a second translation to return to the (0,0) position at a distance t in a second direction X2, and then cup 2z can undergo a third translation to a third position P3, which can also be represented as the (-t,0) position, at a distance t in the second direction X2. From the (-t,0) position, cup 2z can undergo a fourth translation to return to the (0,0) position at a distance t along the first direction X1, which can be the final position in the translation sequence. Therefore, the aforementioned translational order can be characterized as (0,0)→(t,0)→(0,0)→(-t,0)→(0,0). It should be recognized that the third translation (0,0)→(-t,0) can occur after the second translation (t,0)→(0,0) without discontinuity or delay. In such cases, the second and third translations can be characterized as two parts of a single translational movement from (t,0)→(-t,0) (i.e., from P2 to P3). It should also be recognized that cup 2z can remain at any of the positions (0,0), (t,0), (0,0), (-t,0), (0,0) in the order, and / or any intermediate position between them.

[0067] Referring again to Figure 7B, the exemplary sequence of forward and backward dynamic movement along the y-axis can be characterized as follows: From the (0,0) position, cup 2z can undergo a first translation to a second position P2, which can also be represented as the (0,t) position, at a distance t in the first direction Y1 along the y-axis. From the (0,t) position, cup 2z can undergo a second translation, returning to the (0,0) position at a distance t in the second direction Y2, and then cup 2z can undergo a third translation to a third position P3, which can also be represented as the (0,-t) position, at a distance t in the second direction Y2. From the (0,-t) position, cup 2z can undergo a fourth translation, returning to the (0,0) position at a distance t along the first direction Y1, which can be the final position in the translation sequence. Therefore, the aforementioned translational order can be characterized as (0,0)→(0,t)→(0,0)→(0,-t)→(0,0). As described above, the third translation (0,0)→(0,-t) can occur after the second translation (0,t)→(0,0) without discontinuity or delay. In such cases, the second and third translations can be characterized as two parts of a single translational movement from (0,t)→(0,-t) (i.e., from P2 to P3). It should also be recognized that cup 2z can remain at any of the positions (0,0), (0,t), (0,0), (0,-t), (0,0) in the order, and / or at any intermediate position between them.

[0068] The linear forward-to-back translational order described above can be repeated any number of times, each of which may be referred to as a “cycle.” It should be understood that the order of positions can be maintained or reversed from one cycle to the next. Additionally, cup 2z may be allowed to remain between cycles, or alternatively, the subsequent cycle may begin immediately after the completion of the previous cycle. It should also be recognized that the linear forward-to-back translational order can be adapted in many ways without departing from the scope of the disclosed embodiments.

[0069] Referring again to Figure 7C, the rotation (orbit) detour path C1 around axis Z1 can also be defined by referring to a coordinate system such as a two-dimensional polar coordinate system (r,θ) centered on axis Z1, where "r" is the radial distance from axis Z1 and "θ" is the polar coordinate (angle from the zero-angle position). In the illustrated example, the (0,0) position coincides with axis Z1. Therefore, in the illustrated example, cup 2z would be at position (0,0) if it is placed at the center on the blister 11. Using these polar coordinates, an exemplary sequence of circular rotation (orbit) translation is described here. Cup 2z can be placed at a first position P1, which is offset by a distance t from axis Z1 and is located at the zero-angle polar position, which can be indicated by position (t,0). From this position, the exemplary sequence consists of translating cup 2z along the circular path C1, which results in a complete rotation (orbit) around axis Z1. Therefore, the exemplary rotation involves translating cup 2z along a circular path C1 in a first rotational direction R1 from a first position P1(t,0) to a second position P2(t,π / 2), then to a third position P3(t,π), then to a fourth position P4(t,3π / 2), and back to the first position P1(t,0), which can also be represented as (t,2π). Thus, the aforementioned rotational translation sequence can be characterized as (t,0)→(t,π / 2)→(t,π)→(t,3π / 2)→(t,0). In this example, each of the positions (t,0), (t,π / 2), (t,π), and (t,3π / 2) are uniformly spaced apart from each other, but in other embodiments, the rotational translation sequence can use non-uniformly spaced angular positions.

[0070] Additionally, the rotational translation of this sequence can be made to occur at a substantially constant velocity (i.e., without discontinuities or deceleration between positions), except perhaps some acceleration (from the initial (t,0)) and deceleration (to the final (t,0)) of the sequence. It should also be recognized that cup 2z may be able to remain at any of the positions (t,0), (t,π / 2), (t,π), (t,3π / 2), (t,0) within the sequence, and / or at any intermediate position between them. The sequence can be repeated any number of times, each of which may be referred to as a “cycle”. As considered above, the direction of rotation (and thus the order of positions) may be maintained or reversed from one cycle to the next. Additionally, cup 2z may be able to remain between cycles, or alternatively, a subsequent cycle may begin immediately after the completion of the previous cycle. It should also be recognized that the rotational (orbital) translation can be adapted in many ways without departing from the scope of the disclosed embodiments.

[0071] Referring again to Figure 7D, the torsional motion of cup 2z can also be characterized by reference to a polar coordinate system. Thus, the first, second, and third positions P1, P2, and P3 in the illustrated example can also be represented as (0,π / 2), (0,0), and (0,π), respectively. Similarly, as stated above, a series of torsional motions can be repeated any number of times, each of which may be referred to as a “cycle”. Additionally, the direction of rotation (and therefore the order of positions) can be maintained or reversed from one torsional cycle to the next. Furthermore, cup 2z may remain at any position along a cycle and / or at any position between cycles, or alternatively, a subsequent cycle can begin immediately after the completion of the previous cycle. It should be recognized that the torsional motion can be adapted in many ways without departing from the scope of the disclosed embodiments.

[0072] Referring here to Figure 7F, an exemplary unidirectional cup translation movement is shown. In this exemplary movement, cup 2 can be positioned at a first position P1, offset from the center Z2 of blister 11 by a first offset distance along a certain direction. From the first position P1, the cup can be translated across the blister center Z2 along that direction to a second position P2, which can be separated from the blister center Z2 by a second offset distance measured along that direction. Preferably, both the first and second offset distances are greater than (i.e., "not less than") the maximum internal dimension of the chamber (e.g., the chamber diameter D1 for a circular cup shape). This offset spacing ensures that the entire vacuum chamber passes over the blister center Z2 during the unidirectional swipe. However, other offset distances may also be used. When the first and second offset distances are substantially equivalent and both are greater than the maximum internal dimensions of the chamber, the unidirectional KV movement across the blister center Z2 may be referred to herein as “Pattern C,” which can also be expressed as (-t,0)→(t,0).

[0073] Unidirectional dynamic vacuum (KV) movement patterns, such as pattern C, can be repeated for various numbers of cycles. During such repetitions, the vacuum field does not need to be maintained over the tissue. For example, after performing a unidirectional swipe across the blister, cup 2 can be removed from the tissue (or the vacuum pressure can be stopped), and cup 2 can be repositioned to a first position P1 (or a different positional offset from the blister center Z2), from which another unidirectional swipe can be performed. It should be recognized that the above example represents a non-limiting example of unidirectional translational dynamic vacuum (KV) movement.

[0074] In any of the aforementioned examples shown in Figures 6A to 7D, it should be noted that the cup can optionally be made to lose suction during movement (e.g., by removing the cup from the tissue). In other words, dynamic vacuum (KV) procedures do not require the application of active vacuum pressure throughout the entire duration of the procedure. This can enhance ease of operation for the user, who can choose to remove the cup from the tissue or "pop" it to replace the cup on top of it (including in a different position) as needed during a dynamic vacuum (KV) procedure.

[0075] Additionally, while the aforementioned examples of dynamic vacuum (KV) treatments illustrate each treatment administered in relation to a single fluid injection 7, any of the KV treatments herein can be administered to treat multiple fluid injections 7, which can be arranged in various patterns, as will be described in more detail below with reference to Figure 24.

[0076] Furthermore, it should be recognized that in any of the aforementioned examples of dynamic vacuum (KV) procedures, cups can be used that move proximal P and / or distal D (i.e., away from and / or toward (or into) the tissue, respectively) during, before, or after the application of vacuum pressure. Such proximal and / or distal movement can further stress and deform the aspirated tissue, thereby enhancing drug delivery.

[0077] Test results overview The test results related to dynamic vacuum treatment are described below with reference to Figures 8 to 46. In these studies, various parameters related to dynamic vacuum (KV) treatment were studied to evaluate the effectiveness and importance of such parameters, including, but not limited to, injection volume and administration, use of immunosuppressants, addition of hyaluronidase, movement of the dynamic vacuum cup, geometric shape and diameter of the cup, vacuum pressure, and skin thickness at the treatment site. In addition, these studies studied various dynamic vacuum (KV) treatments in comparison to other vaccine-related treatment techniques, including vacuum electroporation (VEP), static vacuum (SV) treatment, injection-only (INJ) treatment, needle electroporation (NEP), intramuscular electroporation (IM-EP), and mRNA vaccine infusion, to evaluate their performance (e.g., gene expression, immune response, potential damage to the skin surface). In the following description of this study, unless otherwise specified (regarding intramuscular electroporation (IM-EP) procedures and intramuscular (IM) mRNA injection, it should be recognized that all plasmid injections were performed via mantle injection into intradermal (ID) tissue (i.e., layers of skin).

[0078] The following procedures and their test results are as follows:

[0079] Injection only (INJ): Intradermal injection. In the guinea pigs studied, injections were performed in the skin above the flank. In the rabbits studied, injections were typically performed in the skin above the quadriceps femoris muscle. The injection-only (INJ) procedures discussed below did not involve vacuum treatment or electroporation.

[0080] Static vacuum (SV): Intradermal injection after applying negative pressure (vacuum pressure) via a vacuum cup. The vacuum pressure is used to evacuate the vacuum cup and draw the target tissue (e.g., skin) into the cup. The skin is then subjected to the vacuum pressure for a predetermined amount of time until the vacuum pressure is released. After the pressure in the vacuum cup returns to normal, the cup is removed from the skin. The static vacuum (SV) procedures discussed below did not involve dynamic vacuum transfer or electroporation. Unless otherwise specified below, each static vacuum (SV) procedure applied vacuum pressure for a predetermined amount of time of approximately 10–20 seconds (this duration is extended from vacuum activation to deactivation). This 10–20 second vacuum period was selected for its correlation with the specific VEP duration (including vacuum activation, EP pulse generation, and vacuum deactivation).

[0081] Dynamic vacuum (kV): Intradermal injection after applying vacuum pressure via a vacuum cup. The vacuum pressure is used to evacuate the vacuum cup and draw the target tissue (e.g., skin) into the cup. After the skin is drawn into the cup, the vacuum cup is manipulated by an external force that moves the vacuum cup relative to the skin (i.e., translation, rotation, or a combination thereof). After the procedure is complete, the vacuum pressure is stopped and the external force is removed. Once the pressure in the cup returns to normal, the vacuum cup is removed. The dynamic vacuum (KV) procedures discussed below did not involve electroporation. Additionally, unless otherwise noted below, the KV procedures evaluated below used the following linear left-right sequence (hereinafter also referred to as "Pattern A"): (0,0)→(t,0)→(0,0)→(-t,0)→(0,0), performed three times (i.e., three cycles) per procedure. Also, unless otherwise noted below, the vacuum pressure was applied at approximately -500 mmHg. Furthermore, unless otherwise noted below, each of the dynamic vacuum (KV) procedures tested below resulted in the application of vacuum pressure for approximately 10–20 seconds (this duration includes vacuum initiation, reaching the desired vacuum pressure, followed by cup translation(s) and vacuum shutdown). As described above, this 10–20 second vacuum period was selected for its correlation with specific VEP durations (including vacuum initiation, EP pulse generation, and vacuum shutdown).

[0082] Vacuum electroporation (VEP): This is an intradermal injection following non-invasive electroporation targeting the dermis and epidermis using a vacuum cup, with at least one electrode in the chamber acting on the surface of the skin. Vacuum pressure is used to evacuate the vacuum cup and bring the target tissue (e.g., skin) into contact with the electrode(s). All VEP test results discussed below were generated using a central support electrode VEP cup 402 shown in Figures 4A-4B. Unless otherwise noted below, the electroporation components of the following VEP procedures consisted of three consecutive electroporation pulses applied, each pulse having a pulse duration of 50 milliseconds (ms), a current of 0.5 Amp, a maximum voltage of 200 volts (V), and an inter-pulse delay of 250 ms between pulses. For each pulse, the ring electrode acted as the delivery electrode and the support electrode acted as the return electrode. Unless otherwise noted, the total duration of each VEP procedure was approximately 10-20 seconds (including vacuum initiation, EP pulse generation, and vacuum shutdown). The vacuum electroporation (VEP) procedure discussed below did not involve dynamic vacuum motion.

[0083] Needle electropermeabilization (NEP): This procedure involves intradermal injection following invasive electropermeabilization using three needle electrodes, each with an outer diameter of 0.46 mm. The three needle electrodes are arranged in an isosceles triangle, with a pair of electrodes (i.e., the first and second electrodes) spaced 3 mm apart from each other, while the third electrode is spaced 5 mm apart from each of the first and second electrodes. The three needle electrodes are inserted into the dermis and epidermis to a depth of 3 mm from the skin surface. Four consecutive electropermeabilization pulses are applied, each pulse having a pulse duration of 52 ms, a current of 0.2 Amp, and a maximum voltage of 200 V, with an interpulse delay of 250 ms between pulses. During pulses 1 and 3, the first electrode functions as the source electrode, while the second and third electrodes function as the return electrodes. During pulses 2 and 4, the second electrode delivers the pulse, and electrode 3 functions as the return electrode. The needle electroporation (NEP) procedures discussed below did not involve vacuum procedures (neither KV nor SV) or vacuum electroporation (VEP).

[0084] Intramuscular electropermeability (IM-EP): Using a bolus needle, pDNA was intramuscularly injected into the quadriceps femoris muscle at a target depth, followed by invasive electroporation into the quadriceps femoris muscle through the skin using a 5P array. The 5P array has five needle electrodes arranged in an equilateral pentagonal pattern with a pattern diameter of 10 mm. The five needle electrodes are arranged at an insertion depth of approximately 19 mm. Three consecutive electroporation pulses were applied, each pulse having a pulse duration of 52 ms, a current of 0.5 Amp, and a maximum voltage of 200 V, with an interpulse delay of 1 second between pulses. The three electroporation pulses were delivered by the electrodes as follows: Pulse 1: From electrode 1 (positive) to electrodes 3 and 4 (negative), Pulse 2: From electrode 2 (positive) to electrodes 4 and 5 (negative), Pulse 3: From electrode 3 (positive) to electrode 5 (negative). The intramuscular electroporation (IM-EP) procedure discussed below was not involved in vacuum treatment (neither KV nor SV) nor vacuum electroporation (VEP).

[0085] Additional test details: Some of the following tests involved commercially available (OTS) vacuum devices. Details of these OTS devices are shown in Table 0 below. [Table 1]

[0086] Research 1 Referring to Figure 8, we see the gene expression in guinea pig skin after intradermal injection of a uniform volume plasmid encoding the gene for green fluorescent protein (GFP), followed by treatment according to the following four treatment groups. Group 1: VEP procedure using a vacuum cup with a chamber diameter D1 of 12 mm. Group 2: SV treatment using a central support vacuum cup 102 having a chamber diameter D1 of 12 mm (see Figures 2A to 2C). Group 3: SV procedure using a dome-shaped vacuum cup 2 with a chamber diameter D1 of 12 mm (see Figures 1A to 1D). Group 4: KV treatment using the same dome-shaped vacuum cup design as Group 3.

[0087] Each group consisted of two samples (1a, b; 2a, b; 3a, b; 4a, b), each sample receiving three aligned injections in a column within the skin on the flank. Each injection contained plasmid 5013 (encoding the gene for GFP) in a volume of 100 μL with a DNA dose of 0.5 μg. Details of the test parameters for this study are shown in Table 1 below. [Table 2]

[0088] In this study, SV treatment using both centrally oriented cups (2a, b) and dome-shaped cups (3a, b) produced a detectable GFP signal. The dome-shaped cups (3a, b) had some donut-shaped signal with a void in the central region of the injection site, while the centrally oriented cups (2a, b) eliminated the central void (essentially "filling" the donut). In this study, KV treatment using dome-shaped cups (4a, b) produced a GFP signal comparable to static vacuum treatment (2a, b), but with a larger signal region. With respect to VEP treatment (1a, b), the GFP signal appeared lower than in the other groups, which is likely due to the fact that tissue damage caused by electroporation has been shown to mask the GFP signal.

[0089] Research 2 Referring to Figure 9, GFP gene expression in guinea pig skin is shown after treatment involving various dynamic vacuum cup transfers applied by different vacuum cup designs. Subjects were intradermally injected with a uniform volume plasmid encoding the gene for GFP and then treated according to the following seven vacuum treatment groups. Group 1: KV treatment using left-right (STS) movement (pattern A, 3 cycles) (see Figure 7A) performed by a central support vacuum cup 102 (see Figures 2A-2C) having a chamber diameter D1 of 12 mm. Group 2: KV treatment using vertical (UD) movement (see Figure 7B) implemented by the same central support vacuum cup 102 design as in Group 1. Group 3: KV treatment using the same STS movement as Group 1, but performed by a dome-shaped vacuum cup 2 with a chamber diameter D1 of 12 mm (see Figures 1A-1D). Group 4: Dynamic treatment without vacuum (K, no vacuum), using the same STS transfer and cup 102 design as Group 1. Group 5: The same STS movement as in Group 1, but the KV treatment is performed by a dome-shaped vacuum cup 2 having a chamber diameter D1 of 15 mm. Group 6: In particular, the KV treatment using a detour trajectory (ORB) movement (see Figure 7C) is performed by pulling the cup counterclockwise and then clockwise along a circular path around the injection site in two trajectories / cycles (i.e., a first circular trajectory in a counterclockwise rotation direction R1, and a second trajectory along the same circular trajectory but in a clockwise rotation direction R2, both cycles performed without twisting or pivoting motion around the central axis Z1), using the same central support vacuum cup 102 design as in Group 1. During the specific trajectory movement used in this group, the cup 102 also effectively underwent a rotating, vertical inclination, leaving the cup central axis Z1 substantially intersecting the geometric center of the blister 11. Group 7: In particular, the KV treatment using torsional (TWT) movement (see Figure 7D) by twisting the cup 360 degrees clockwise around the central axis Z1 and then twisting the cup 360 degrees counterclockwise around the central axis Z1 was carried out by the same central support vacuum cup 102 design of Group 1.

[0090] Groups 1–5 each contained two samples ("a" and "b"), while groups 6–7 each contained one sample ("a"). Each of the aforementioned samples in this study received three ID injections on the flanks, generally shown in a columnar pattern. Each injection site underwent independent vacuum treatment. Thus, groups 1–5 each have six repeats spread across two flanks, and groups 6–7 each have three repeats spread across one flank. Each injection contained plasmid 5013 (encoding the gene for GFP) in a 100 μL volume with a DNA dose of 0.5 μg. For groups 1–3 and 5–7, vacuum pressure was applied for each treatment for 15 seconds to correlate with the VEP duration. Group 4 was the only treatment in this study that did not use vacuum pressure. Details of the test parameters for this study are shown in Table 2 below. [Table 3]

[0091] This study shows that KV treatment (groups 1-3 and 5-7; images 1a, b, 2a, b, 3a, b, 5a, b, 6a, b, 7a, b) produced similar GFP responses, while dynamic treatment without vacuum (group 4; images 4a, b) produced weaker signals (GFP responses) than KV treatment. This study also demonstrates that for vacuum cups of the same size, centrally strutted cup 102 and dome-shaped cup 2 produced similar GFP responses (the results for centrally strutted vacuum cups in groups 1-2 and 6-7 were compared with the results for the dome-shaped cup in group 2, each cup having a chamber diameter D1 of 12 mm). This study further suggests that increasing the cup size (diameter) may reduce the GFP signal in some cases (comparing results 5a and 5b of group 5 (dome-shaped cup 2 with D1=15mm) with results 3a and 3b of group 3 (dome-shaped cup 2 with D1=12mm), where both groups 5 and 3 involved moving the cups from side to side), however, further testing is needed regarding the standalone effect of cup size / diameter on the GFP signal. This study also further shows that the movement of circular orbital cups (group 6) and torsional cups (group 7) produced less GFP response than the movement of linear translational cups (groups 1-5).

[0092] Research 3 Referring to Figures 10A and 10B, the immune response (ELISA expression) in guinea pigs was tested to evaluate the immune response after dynamic vacuum (KV) treatment compared to the immune responses after various static vacuum (SV), vacuum electroporation (VEP), and injection-only (INJ) treatments.

[0093] The subjects were intradermally injected with a uniform volume of plasmid, and then treated according to the following treatment groups. Group 1: VEP treatment using the central support electrode VEP cup 402 (see Figures 4A to 4B). Group 2: SV treatment using the same cup design as Group 1. Group 3: SV treatment using dome-shaped vacuum cup 2 (see Figures 1A to 1D). Group 4: KV treatment using the same central support cup design as Group 1. Group 5: INJ treatment.

[0094] Each group consisted of six samples, each receiving one ID injection on the flank. Each injection contained plasmid 2027 encoding the influenza antigen in a 100 μL volume with a DNA dose of 0.05 μg. Group 4 was the only group in this study to be treated with KV. Further details regarding the test parameters for this study are shown in Table 3 below. [Table 4]

[0095] Combination titer data for all groups are shown at week 2 (Figure 10A) and week 4 (after the second vaccination) (Figure 10B). The results of this study show that KV treatment (group 4) produced immunogenicity comparable to VEP treatment (group 1) and superior to SV treatment (groups 2 and 3). Among the SV treatments, the central-pillar vacuum cup (group 2) produced superior immunogenicity than that produced by the dome-shaped vacuum cup (group 3). The inventors found it surprising and unexpected that KV treatment (group 4) produced immunogenicity substantially equivalent to that produced by VEP treatment (group 1). The ability to use KV treatment without electroporation to produce an immune response substantially equivalent to that produced using electroporation (vacuum or other techniques) may be revolutionary in the field of vaccine administration. For example, the inventors believe that the KV treatment (without electroporation) disclosed herein brings about significant improvements in both instantaneous pain / irritation and healing time associated with electroporation treatment (particularly invasive electroporation treatment), and, in a more general sense, significant improvements in viral vector-based, mRNA-based, protein-based, nanoparticle-based, and other methods that can benefit from the painless, localized drug delivery provided by KV treatment. The KV treatment disclosed herein also is thought to have potential benefits in the medical field beyond drug delivery.

[0096] Referring to Figures 10C to 10D, the visible effects on the skin from the various treatments discussed above with reference to Figures 10A to 10B are shown. In particular, Figure 10C shows the treatment site immediately after the treatment was applied, and Figure 10D shows the treatment site 7 days after the treatment. In these figures, the images showing the treatment sites of the various groups are labeled as follows: Group 1-275L-280L, Group 2-281L-286L, Group 3-287L-292L, Group 4-293L-298L, and Group 5-299L-304L. The KV treatment (Group 4, images 293L-298L) caused acute redness and irritation on the skin surface immediately after treatment (Figure 10C), but there was no visible tissue damage by 7 days (Figure 10D). In comparison, VEP treatment did not cause immediate acute visible tissue damage (Figure 10C), but some scabbing and / or other superficial tissue damage was observed by day 7 (Figure 10D).

[0097] Research 4 Here, referring to Figures 11A and 11B, the immune response (ELISA expression) in guinea pigs was re-examined to effectively replicate the studies shown in Figures 10A-10B and confirm the ELISA results. The same five treatment groups were tested, with the main differences being the plasmid used (plasmid 2303 in this study) and the DNA dose (0.6 μg in this study). Further details regarding the test parameters for this study are shown in Table 4 below. [Table 5]

[0098] ELISA data for groups 1-5 are shown in Figures 11A and 11B. Specifically, Figure 11A shows the total potency data for all groups at week 2, and Figure 11B shows the total potency data for all groups at week 4 (after the second vaccination). As shown, KV treatment (group 4) generated immunogenicity comparable to VEP treatment (group 1), as in the studies shown in Figures 10A-10B. However, in this study, KV treatment (group 4) did not significantly outperform SV treatment (groups 2 and 3) at week 4. KV treatment (group 4) showed an increased ELISA response at week 2 compared to SV treatment (groups 2 and 3), and showed a similar response at week 4 after the second vaccination. Furthermore, at week 2, KV treatment (group 4) showed an ELISA response comparable to that of VEP treatment (group 1). Furthermore, as shown in this study, the INJ treatment (group 5) showed a strong ELISA response at weeks 2 and 4. Typically, with infusion-only treatments like the one shown in this study, serum conversion is not observed at week 2. These unusually strong results for the INJ treatment (group 5), considering the unusually high low-end baseline, may have made it more difficult to distinguish between the best and worst ELISA responses in this particular study. While we do not wish to be bound by any particular theory, we believe that the unusually strong results for the INJ treatment suggest that the DNA dose in this study (0.6 μg per infusion) was higher than necessary for this particular animal (guinea pig), which is supported by the week 4 ELISA response, which continues to overlap across all groups, even when the weakest response is substantially above the detection limit.

[0099] Research 5 Referring here to Figures 12A and 12B, there is a study that attempted to investigate the effect of reducing DNA dose (via reduction of injection volume) using dynamic vacuum (KV) treatment on the immune response (binding ELISA response) in guinea pigs. In this study, subjects in two groups (Group 1 and Group 2) were intradermally injected with plasmids and then treated via KV using the same central stratification cup design. The only difference between the groups was the drug volume. Group 1 was administered an injection volume of 100 μL, while Group 2 was administered an injection volume reduced to 50 μL. The drug in both groups had a DNA concentration of 0.05 mg / mL. Both groups contained five samples, each sample receiving a single ID injection of plasmid 2027 on the right flank. The ELISA response for both groups at week 2 is shown in Figure 12A, and the ELISA response at week 4 is shown in Figure 12B.

[0100] As shown, the reduced dose (group 2) did not show a detectable effect on the ELISA response at week 2 (Figure 12A), but showed a slight reduction in titer at week 4 (after the second vaccine dose) (Figure 12B). Based on this study, it is not entirely clear whether KV is sensitive to half doses, as the difference between groups is small and not statistically significant, but this study provides some evidence that the immunogenicity of KV treatment may be proportional to the infusion volume.

[0101] research 6 Referring to Figure 13, the immune response (ELISA expression) in naive guinea pigs was tested to evaluate the immune response after dynamic vacuum (KV) treatment compared to the immune responses after vacuum electroporation (VEP), static vacuum (SV) treatment, and injection-only treatment.

[0102] The subjects were intradermally injected with a uniform volume of plasmid, and then treated according to the following treatment groups. Group 1: VEP treatment using the central support electrode VEP cup 402 (see Figures 4A to 4B). Group 2: KV treatment using a central support vacuum cup 102 design (see Figures 2A-2C). Group 3: SV treatment using the same central support vacuum cup 102 design as used in Group 2. Group 4: INJ treatment.

[0103] Each group consisted of eight samples, each receiving a single ID injection in the left flank. Each injection contained plasmid 2027 in a volume of 100 μL with a DNA dose of 0.05 μg. Further details regarding the test parameters for this study are shown in Table 5 below. [Table 6]

[0104] The titer data for groups 1-4 at week 2 are shown. The results of this study are consistent with the above study in non-naive subjects, showing that the ELISA response generated by SV treatment was greater than that generated by INJ treatment, and that KV treatment produced an ELISA response that was greater than that generated by SV and INJ treatments and comparable to that generated by VEP treatment. One notable finding from this study is that one subject in group 2 was excluded from the study because anesthesia was interrupted during vaccination and vacuum treatment was not properly applied after injection. This exclusion of the subject does not affect the overall results described above.

[0105] Research 7 Referring to Figure 14, this study evaluates the effects of the number of translational or "swiping" steps and the use of hyaluronidase on the immune response (ELISA expression) in guinea pigs treated with dynamic vacuum (KV).

[0106] The subjects were intradermally injected with a uniform volume of plasmid, and then treated according to the following treatment groups. Group 1: KV treatment (Pattern A, 3 cycles - see Figure 7A). Group 2: KV treatment using one single linear translation (one swipe), (0,0)→(t,0), also referred to herein as "Pattern B", one cycle, uses the same central support vacuum cup design as Group 1. Group 3: The KV treatment is the same as in Group 1 (Pattern A, 3 cycles), but a dome-shaped vacuum cup 2 is used (see Figures 1A to 1D). Group 4: The KV treatment is the same as Group 2 (Pattern B, 1 cycle), but uses the same dome-shaped vacuum cup design from Group 3. Group 5: The KV treatment is the same as in Group 1 (Pattern A, 3 cycles), but an infusion containing hyaluronidase is used.

[0107] Each group consisted of six samples, each receiving a single intradermal (ID) injection on the left flank. Each injection contained plasmid 9517 in a 100 μL volume with a DNA concentration of 0.25 mg / mL. As mentioned above, the injection for group 5 included hyaluronidase. The overall titer results are shown in Figure 14. Further details regarding the test parameters for this study are shown in Table 6 below. [Table 7]

[0108] As a result, groups 1, 3, and 5, using pattern A, 3 cycles (6 swipes in total), generated a greater immune response than the pattern B, 1 cycle (1 swipe) groups (groups 2 and 4). The results also demonstrate that the addition of hyaluronidase to KV treatment significantly improves immunogenicity. Additionally, by comparing the results of group 1 to group 2 and group 3 to group 4, it is unclear from this study whether the central support vacuum cup 102 significantly improves immunogenicity compared to the dome-shaped cup 2 in KV treatment, although the data slightly suggest this trend.

[0109] research 8 Referring to Figure 15, this study evaluated the effect of vacuum pressure on the immune response (ELISA expression) in guinea pigs that otherwise underwent similar dynamic vacuum (KV) treatment.

[0110] Five groups of subjects received intradermal injection of a uniform volume of plasmid, followed by KV treatment using different vacuum pressures but the same dynamic vacuum cup transfer (Pattern A, 3 cycles). The vacuum pressures used by the groups were as follows: Group 1 used -100 mmHg, Group 2 used -300 mmHg, Group 3 used -500 mmHg, Group 4 used -600 mmHg (the maximum vacuum pressure of the pump used in this study), and Group 5 used approximately -420 mmHg. Groups 1-4 underwent KV treatment using the same central support cup 102 design. Group 5 used a ready-made (OTS) vacuum applicator labeled "OTS-0" in Table 0, which is a MarvelouSlim applicator manufactured by Zemits Kosmetik Experte, headquartered in Carlsbad, California, USA. The OTS-0 applicator uses a vacuum head with a chamber diameter of approximately 14 mm and a distal roller (a ball bearing seated on the distal surface) that facilitates the translation of the vacuum head across the skin surface.

[0111] Each group consisted of six samples, each receiving a single intradermal (ID) injection on the left flank. Each injection contained plasmid 2303 in a 100 μL volume with a DNA concentration of 0.3 mg / mL. For dynamic vacuum cup transfer, each group used three sets of linear translation (a total of six swipes) across the injection site. Further details regarding the test parameters for this study are shown in Table 7 below. [Table 8]

[0112] The combined ELISA results for each group are shown in Figure 15. These results demonstrate a clear correlation between vacuum strength and immunogenicity. In particular, the ELISA response increased with increasing vacuum pressure. Additionally, in this study, the OTS-0 applicator at approximately -420 mmHg (group 5) produced results roughly equivalent to the central support cups at -500 and -600 mmHg (groups 3 and 4), although the inventors observed that the OTS-0 applicator tended to detach inadvertently from the skin surface during translation (swiping). The removal of the OTS-0 applicator from the skin surface during use was not surprising, given that the OTS-0 applicator literature mentions its ability to be removed for ease of use and comfort. However, the inventors were surprised that removal of the OTS-0 applicator during use did not appear to significantly reduce immunogenicity results.

[0113] Research 9 Referring to Figures 16A and 16B, this study evaluates the immune response (ELISA expression) in rabbits, and in particular compares the immune response generated by dynamic vacuum (KV) treatment with the immune response generated by treatments including infusion only (INJ), static vacuum (SV), vacuum electroporation (VEP), and needle electroporation (NEP).

[0114] The subjects were intradermally injected with a uniform volume of plasmid, and then treated according to the following treatment groups. Group 1: INJ treatment. Group 2: SV treatment using a central support vacuum cup 102 design with a chamber diameter D1 of 12 mm (see Figures 2A-2C). Group 3: KV treatment using the same central support vacuum cup 102 design as used in Group 2. Group 4: VEP treatment using the central support electrode VEP cup 402 (see Figures 4A to 4B). Group 5: NEP using the CELLECTRA2000-3P device manufactured by Inovio Pharmaceuticals, headquartered in Plymouth Meeting, Pennsylvania, USA.

[0115] Each group consisted of five samples, each receiving a single ID injection intracutaneously above the left quadriceps femoris muscle. Each injection contained plasmid 9517 in a 100 μL volume with a DNA dose of 2.5 mg / mL. Further details regarding the test parameters for this study are shown in Table 8 below. [Table 9]

[0116] Titer data for all groups are shown on day 0 (Figure 16A) and week 2 (Figure 16B). The results of this study are consistent with the guinea pig studies described above, showing that the ELISA response produced by KV treatment (group 3) was greater than that produced by SV treatment (group 2) and greater than that produced by INJ treatment (group 1). Surprisingly and unexpectedly, in this study, KV treatment produced a greater ELISA response than both VEP treatment (group 4) and NEP treatment (group 5).

[0117] research 10 Referring here to Figures 17A–17B, the follow-up study evaluates the immune response in rabbits, but uses a different plasmid (this time, plasmid 2303 at a DNA concentration of 3.0 mg / mL) and replicates the study disclosed above, referring to Figures 16A–16B. All other parameters of this study were the same as those of the study shown in Figures 16A–16B. In particular, Figure 17A shows the titer data at week 0 (equivalent to Figure 16A), and Figure 17B shows the titer data at week 2 (equivalent to Figure 16A). As with previous studies in rabbits, this follow-up study again demonstrates that the KV treatment (group 3) outperforms the VEP treatment (group 4) in terms of immune response, and the latter is generally equivalent to the NEP treatment (group 5). In this follow-up study, INJ treatment (group 1) was only slightly less immunogenic than SV treatment (group 2), and was itself less immunogenic than VEP and NEP treatments (groups 4 and 5). However, in previous studies (Figures 16A-16B), SV treatment had similar immunogenicity to VEP and NEP treatments.

[0118] Research 11 Referring to Figure 18, this study evaluates the effects of hyaluronidase on dynamic vacuum (KV) and static vacuum (SV) treatments in guinea pigs, in comparison to vacuum electroporation (VEP) treatment.

[0119] The subjects were intradermally injected with a uniform volume of plasmid, and then treated according to the following treatment groups. Group 1: SV treatment using a central support vacuum cup 102 design (see Figures 2A-2C). Group 2: VEP treatment using the central support electrode VEP cup 402 (see Figures 4A to 4B). Group 3: KV treatment using the same central support vacuum cup design as used in Group 1 (Pattern A, 3 cycles). Group 4: KV treatment using a cup design and dynamic movement as in Group 3, but with hyaluronidase added to the injection. Group 5: SV treatment using the same SV treatment and cup as Group 1, but with the addition of hyaluronidase to the injection.

[0120] Groups 1-3 each contained six samples, and groups 4-5 each contained seven samples. Each sample was administered with a single ID infusion to the left flank. Each infusion contained plasmid 9517 in a volume of 100 μL with a DNA concentration of 0.25 mg / mL. Further details regarding the test parameters for this study are shown in Table 9 below. [Table 10]

[0121] Titer data for all groups are shown in Figure 18. In this study, KV treatment (with or without hyaluronidase) produced a larger ELISA response than VEP treatment. Additionally, in this study, hyaluronidase enhanced binding titer for KV treatment but not for SV treatment. In fact, SV treatment, with or without hyaluronidase, did not produce a substantially detectable immune response in this study.

[0122] research 12 Referring to Figure 19, this study evaluates the effects of different numbers of dynamic vacuum (KV) transfers and distinct skin thicknesses on the immune response (ELISA expression) in guinea pigs.

[0123] Subjects were intradermally injected with a uniform volume of plasmid, and then treated according to the following treatment groups. Each treatment group received KV treatment using a central support vacuum cup 102 design (see Figures 2A-2C) in which the cup was moved linearly back and forth across the injection site according to pattern A, but the number of cycles and the injection site were varied. Group 1: Injection into the right flank, followed by Pattern A using vacuum cup 102, 2 cycles (4 swipes in total). Group 2: Injection into the right flank, followed by Pattern A using vacuum cup 102, 3 cycles (6 swipes in total). Group 3: Injection into the right flank, followed by Pattern A using vacuum cup 102, 4 cycles (8 swipes in total). Group 4: Injection into the back (where the target skin is thicker than the flank), followed by Pattern A using vacuum cup 102, 3 cycles (a total of 6 swipes). Group 5: Injection into the abdomen (where the target skin is thinner than the flank), followed by Pattern A using vacuum cup 102, 3 cycles (a total of 6 swipes).

[0124] Groups 1-3 each contained six samples, and groups 4-5 each contained seven samples. Each injection contained plasmid 2303 in a volume of 100 μL, with a DNA concentration of 0.3 mg / mL for each group. Further details regarding the test parameters for this study are shown in Table 10 below. [Table 11]

[0125] The overall titer data for all groups are shown in Figure 19. In this study, differences in linear translation or the number of swipes between 4 and 8 swipes across the injection site did not have a significant effect on binding titer. Furthermore, the location of the treatment site (thin skin vs. thick skin) did not appear to produce a significant difference in immunogenicity.

[0126] Research 13 Referring to Figures 20A and 20B, this study evaluates the effects of injection volume and hyaluronidase on gene (GFP) expression after static vacuum (SV) and dynamic vacuum (KV) treatments administered to guinea pigs.

[0127] Six groups of subjects received intradermal injection of plasmids encoding the gene for GFP into the right flank, followed by treatment according to the following treatment groups, each group receiving either KV treatment or SV treatment using a central support vacuum cup 102 design with a chamber diameter D1 of 12 mm (see Figures 2A-2C). Group 1: SV treatment after injecting a volume of 100 μL. Group 2: Similar to Group 1, but with SV treatment using hyaluronidase. Group 3: KV treatment after injection of 100 μL volume. The KV treatment was performed using pattern A, 3 cycles, along with vacuum cup 102. Group 4: Similar to Group 3, but with KV treatment using hyaluronidase. Group 5: Similar to Group 4, but with KV treatment using plasmid injection in a volume of 400 μL. Group 6: Similar to Group 2, but SV treatment using plasmid injection in a volume of 400 μL.

[0128] Groups 1 and 3 each contained 8 samples, groups 2 and 4 each contained 12 (12) samples, and groups 5 and 6 each contained 4 samples. The following groups were paired together for the test subjects: Groups 1-2 and 3-4 were treated side-by-side on the same subject. Groups 5 and 6 were not paired with any other groups on the test subjects. Each injection contained plasmid 5013 (encoding the gene for GFP) with a DNA concentration of 0.5 mg / mL. Further details regarding the test parameters for this study are shown in Table 11 below. [Table 12]

[0129] GFP for each group is shown in Figure 20A, with all images shown at the same scale and image settings (e.g., exposure time). GFP quantification for each group is shown in Figure 20B. In this study, KV treatment (groups 3-5) significantly increased gene expression compared to SV treatment (groups 1-2 and 6). Additionally, within the 100 μL injection groups (groups 1-4), KV treatment dramatically benefited from the addition of hyaluronidase (group 3 compared to group 4), while SV treatment with hyaluronidase produced less gene expression on average than SV treatment without hyaluronidase (group 2 compared to group 1). Within the 400 μL injection (with hyaluronidase) groups (groups 5 and 6), KV treatment (group 5) produced significantly greater gene expression compared to SV treatment (group 6), and this expression was generated across a significantly larger expression region. In KV dynamic vacuum treatment using hyaluronidase, the 400 μL injection treatment (group 5) dramatically increased the gene expression region compared to the 100 μL treatment (group 4). In static vacuum treatment using hyaluronidase, the 400 μL treatment (group 6) showed a milder improvement in gene expression than the 100 μL treatment (group 2).

[0130] research 14 Referring to Figure 21, further studies will evaluate the effect of high infusion volumes, with or without hyaluronidase, on the immune response (ELISA expression) in guinea pigs administered dynamic vacuum (KV) or static vacuum (SV) treatment.

[0131] For subjects in the four groups, the plasmid was intradermally injected into the left flank, and then a central support vacuum cup 102 design with a chamber diameter D1 of 12 mm (see Figures 2A-2C) was treated according to the following vacuum treatment groups used for each group. Group 1: KV treatment after infusion of 100 μL of hyaluronidase-free solution. Group 2: Same as Group 1 (without hyaluronidase), but with KV treatment using a 400 μL injection. Group 3: KV treatment after infusion of 400 μL of hyaluronidase. Group 4: SV treatment; otherwise, same as Group 3 (400 μL including hyaluronidase).

[0132] Each group in this study contained five samples, with each injection containing plasmid 2303 at a DNA concentration of 0.3 mg / mL. Further details regarding the test parameters for this study are shown in Table 12 below. [Table 13]

[0133] The overall titer data for all groups are shown in Figure 21. In this study, treatments without hyaluronidase (groups 1-2: KV after infusion of 100 μL and 400 μL volumes, respectively) showed similar immune responses. KV treatment after infusion of 400 μL volume containing hyaluronidase (group 3) showed the strongest immune response among all groups in this study. SV treatment after infusion of 400 μL volume containing hyaluronidase (group 4) showed the weakest immune response among all groups in this study, which was unexpected, especially considering the results from the studies discussed above with reference to Figure 20. The results of this study, shown in Figure 21, suggest that KV treatment may have an additional immunosuppressant effect on top of the benefits of raw gene expression.

[0134] research 15 Referring to Figure 22, a study was conducted to evaluate the effect of combining dynamic vacuum (KV) treatment with vacuum electroporation (VEP) on the immune response (ELISA expression) in guinea pigs.

[0135] For the four groups of subjects, a uniform volume of plasmid was intradermally injected into the left flank, and then treated according to the following vacuum treatment groups, each group using a central support electrode VEP cup 402 with a chamber diameter D1 of 12 mm (see Figures 4A-4B). Group 1: KV treatment only, administering 3 sets of swipes (6 sets of swipes in total) using a vacuum cup. Group 2: Following VEP treatment, the same KV treatment as Group 1 is administered. Group 3: The same KV treatment as Group 1, followed by the same VEP treatment as Group 2 (i.e., Group 3 used the same KV and VEP treatments as Group 2, but in a reserve order).

[0136] Each group in this study contained five samples, with each injection containing plasmid 9517 having a DNA concentration of 0.25 mg / mL. The vacuum pressure for each procedure was -500 mmHg. Further details regarding the test parameters for this study are shown in Table 13 below. [Table 14]

[0137] The total potency data for all groups are shown in Figure 22. In this study, adding VEP before or after KV treatment did not result in any additional immune benefit and tended to lead to a decrease in the immune response.

[0138] research 16 Referring to Figures 23A and 23B, studies were conducted in guinea pigs to compare the immune response (ELISA expression), cellular response (ELISpot data), and neutralization generated by dynamic vacuum (KV) treatment with mRNA / lipid nanoparticle injection (INJ) treatment and needle electroporation (NEP) treatment.

[0139] The subjects received plasmid injection and were treated according to the following treatment groups. Group 1: NEP treatment after plasmid 9501 injection, wherein the NEP treatment is performed using a CELLECTRA® 2000-3P device. Group 2: KV treatment after injection of plasmid 9501, wherein the KV treatment is performed using a central support vacuum cup 102 having a chamber diameter D1 of 12 mm (see Figures 2A-2C). Group 3: Same as Group 2, but with the addition of hyaluronidase during the injection (KV treatment). Group 4: INJ treatment of mRNA-1273 (an mRNA-based vaccine for SARS-CoV-2) manufactured by Moderna, Inc., based in Cambridge, Massachusetts, USA.

[0140] Each group consisted of five samples, each receiving one injection. Groups 1-3 received intradermal (ID) injections of plasmid (9501) into the skin on the left flank with an injection volume of 100 μL, a DNA dose of 100 μg, and a DNA concentration of 1.0 mg / mL. Group 4 received intramuscular (IM) injections of plasmid (mRNA-1273) into the left quadriceps femoris muscle with an injection volume of 50 μL, a DNA dose of 10 μg, and a DNA concentration of 0.2 mg / mL. Further details regarding the test parameters for this study are shown in Table 14 below. [Table 15]

[0141] Figures 23A and 23B show spike-coupled data for all groups at week 2 (after first dose / treatment) and week 5 (after second dose / treatment), respectively. Figure 23C shows ELISpot data for all groups at week 5 (after second dose / treatment). Figure 23D shows SARS-CoV-2 pseudovirus neutralization data for all groups at day 0 and day 14 (after initial dose / treatment).

[0142] In this study, regarding binding titer results, as shown in Figures 23A-23B, the mRNA vaccine (group 4) outperformed KV + hyaluronidase treatment (group 3), KV treatment without hyaluronidase (group 2), and NEP treatment (group 1). Regarding cellular response, as shown in Figure 23C, each of the DNA plasmid treatments (groups 1-3) outperformed the mRNA vaccine (group 4). Regarding neutralization, as shown in Figure 23D, the mRNA vaccine (group 4) outperformed KV + hyaluronidase treatment (group 3), KV treatment without hyaluronidase (group 2), and was generally equivalent to NEP treatment (group 1).

[0143] In this study, KV + hyaluronidase treatment (group 3) achieved the closest results to the mRNA level (group 4) in humoral binding response (Figures 23A-23B) and pseudoviral neutralization response (Figure 23D), followed by hyaluronidase-free KV (group 2), while NEP treatment (group 1) produced the weakest humoral response (Figure 23C). For neutralization (Figure 23D), only KV + hyaluronidase treatment (group 3) and mRNA vaccine (group 4) produced 100% neutralizing activity after a single vaccination. Regarding cellular responses (Figure 23C), DNA treatments (groups 1-3) generally provided stronger cellular responses than mRNA vaccines (group 4), although KV + hyaluronidase treatment (group 3) tended to produce a slightly higher response than NEP treatment (group 1) for the wild-type assay.

[0144] research 17 Referring here to Figure 24, there is a study that evaluated whether a single dynamic vacuum (KV) treatment can effectively induce transfection of multiple injections (blisters). In this study, subjects were evaluated after receiving intradermal injections of various fractions of a uniformly combined total volume of two distinct plasmids (one encoding the gene for green fluorescent protein (GFP) and the other encoding the gene for red fluorescent protein (RFP)), and then a single KV treatment was administered over the blisters. Subjects were evaluated according to four treatment groups. Group 1 (control): KV treatment with a single infusion of plasmid 5013 (encoding the GFP gene) in an injection volume of 100 μL. Group 2: KV treatment with two parallel injections, each with an injection volume of 50 μL, one containing plasmid 5013 and the other plasmid 9902 (encoding the gene for RFP). Group 3: KV treatment with four injections in a square pattern, with the top row injection using plasmid 5013 and the bottom row injection using plasmid 9902, each injection being 25 μL in volume. Group 4: KV treatment of four injections in alternating linear rows of plasmids 5013 and 9902, with each injection being 25 μL in volume.

[0145] Each group included five repetitions, each repetition receiving its respective injection(s) in the skin on the flank. Each injection contained hyaluronidase (HYA). KV treatment for each group, except for group 3, used pattern A, 3 cycles, or a modified version of pattern A. Specifically for group 3, the first position of the vacuum chamber was midway between the topmost and bottommost left blisters. When vacuum pressure was applied for group 3, the chamber diameter was large enough that both the topmost and bottommost left blisters were drawn into the vacuum chamber before dynamic vacuum translation was performed, and the dynamic vacuum translation was performed laterally in a manner consistent with pattern A (3 cycles) otherwise. For the multiple injection groups (groups 2-4), the use of plasmids encoding GFP and RFP helped to visualize differences in expression at each injection site and to visualize the extent to which different injection sites might blend together. Further details regarding the test parameters for this study are shown in Table 15 below. [Table 16]

[0146] The results shown in Figure 24 demonstrate that a single KV treatment was able to transfect multiple injection sites in each group, including subjects in the four injection groups (groups 3 and 4). This study demonstrates that the KV treatment described herein can be successfully used in conjunction with injection splitting in terms of gene expression.

[0147] research 18 Referring to Figure 25, there is a study that evaluated whether the same dynamic vacuum (KV) treatment performed by different individuals (operators) would produce different immune responses (ELISA expression) in guinea pigs. Subjects were intradermally injected into the flank skin with a uniform volume of plasmid 2027 and then treated according to groups 1-4, with each group treated by a different individual (i.e., subjects in group 1 were treated by operator 1, subjects in group 2 were treated by operator 2, etc.). Each group included five replicates, with each operator performing the KV treatment using pattern A, 3 cycles, and the central pillar vacuum cup 102 (see Figures 2A-2C) having a chamber diameter D1 of 12 mm. The results demonstrate that there was no significant operator-dependent difference in ELISA expression at 2 weeks when the same KV treatment pattern was used.

[0148] research 19 Referring to Figures 26A to 26C, guinea pig studies were conducted to compare the immune response (ELISA expression) and neutralization generated by DNA firing nanoparticles (DLNPs) with dynamic vacuum (KV) treatment versus mRNA injection-only (INJ) treatment.

[0149] The subjects received plasmid injection and were treated according to the following treatment groups. Group 1: KV treatment following injection of DLNP-based plasmid 9528 formulated with hyaluronidase (HYA), wherein the KV treatment is performed using a central support vacuum cup 102 (see Figures 2A-2C) with a chamber diameter D1 of 12 mm, using pattern A, 3 cycles. Group 2: INJ treatment of mRNA-1273.

[0150] Each group consisted of five subjects, each receiving one injection. Group 1 received an intradermal (ID) injection of plasmid (9528) formulated with a 10 μg DNA dose and a DNA concentration of 0.1 mg / mL in the skin of the flank, with a dose of 135 U / mL HYA. Group 2 received an intramuscular (IM) injection of plasmid (mRNA-1273) in the pretibial muscle, with a 50 μL injection volume, a 10 μg mRNA dose and a mRNA concentration of 0.2 mg / mL.

[0151] Further details regarding the test parameters for this study are shown in Table 16 below. [Table 17]

[0152] Figure 26A shows spike-coupled data for both groups after treatment on day 14 (week 2), day 28 (week 4), and day 46 (approximately week 6 and 1 / 2), with the first dose / treatment administered on day 0 and the second dose / treatment administered on day 21. Figures 26B–26C show SARS-CoV-2 pseudovirus neutralization data for both groups over a period exceeding 300 days.

[0153] In this study, regarding binding titer results, as shown in Figure 26A, DLNP+KV+HYA treatment was performed compared to mRNA vaccine over the long course of this study. Furthermore, regarding neutralization, as shown in Figures 26B-26C, DLNP+KV+HYA treatment showed more stable neutralizing titers than mRNA vaccine and maintained a high level of neutralization for almost one year. In particular, the magnitude of the neutralizing titer for mRNA-1273 decreased to less than 5% of the peak throughout the entire monitoring period, while DLNP+KV+HYA infusion maintained 34% of the peak response until the last time point measured. Although mRNA-1273 induced a higher peak neutralizing antibody, this difference in response reduction between mRNA and DLNP with KV resulted in comparable neutralization approximately 100 days after initial vaccination, after which DLNP with KV exhibited superior titers compared to mRNA.

[0154] research 20 Referring to Figure 27, a study was conducted to compare the spreading effect of dynamic vacuum (KV) treatment on boluses injected into skin tissue (blisters) with the spreading effect generated by static vacuum (SV) treatment.

[0155] Subjects in each group were injected with a dye (trypan blue) into the flank skin and then treated according to the following treatment groups: Group 1 received SV treatment; Group 2 received KV treatment using pattern A, 3 cycles with a central support vacuum cup 102 (see Figures 2A-2C) having a chamber diameter D1 of 12 mm; and Group 3 received the same treatment as Group 2, except that the injection contained hyaluronidase (HYA). Each group consisted of 5 subjects. Blister diameter was measured in each subject once before treatment and again after treatment to compare the physical spreading effect produced by KV and SV treatments. The spreading effect was quantified as the ratio of the diameter before and after treatment. The blue dye helped to identify the degree of spreading during the measurement phase.

[0156] As shown, the SV treatment (group 1) produced only a minimal spreading effect, the KV treatment (group 2) produced a positive spreading effect, and the KV + HYA treatment (group 3) significantly outperformed groups 1 and 2.

[0157] research 21 Here, referring to Figure 28A, we conducted a study to compare the immune response (ELISA expression) in guinea pigs after dynamic vacuum (KV) treatment performed with different devices, in particular, one central-support vacuum cup 102 (see Figures 2A-2C) and four off-the-shelf (OTS) vacuum devices, all having similar chamber sizes and applying similar vacuum pressures.

[0158] All subjects in this study received the same dose of the same plasmid (2303) injected into the skin on the flank. Subjects were grouped according to the vacuum cup as follows: (1) "Cup 102" - a central support vacuum cup 102 with a chamber diameter of 12 mm (see Figures 2A-2C), (2) OTS-1 device, (3) OTS-2 device, (4) OTS-3 device, and (5) OTS-4 device. Further details regarding the test parameters for this study are shown in Table 17 below. [Table 18]

[0159] Each group consisted of five subjects, and each KV treatment used pattern A, 3 cycles. Titer data for the groups at week 2 are shown. The results indicate that the five vacuum devices produced similar immunogenic responses when using similar chamber sizes and applying similar vacuum pressures.

[0160] research 22 Referring here to Figure 28B, a study similar to Study 21 (Figure 28A) was conducted to determine the effect of cup size on the immune response (ELISA titer) in guinea pigs after dynamic vacuum (KV) treatment. All tests in this study were performed using an OTS-4 device (see Table 0), with five different vacuum cups interchangeably attached to the device. Using the same OTS-4 device with different interchangeable cups helped maintain a uniform vacuum pressure while using different chamber sizes and geometric shapes. This is found in Table 18 below. [Table 19]

[0161] All subjects in this study received the same dose of the same plasmid (9501) injections into the skin on the flank. Subjects were grouped according to the vacuum cup used. Each group consisted of five subjects, and each KV treatment used pattern A, 3 cycles. Titer data for the groups at week 2 are shown. For the cup sizes tested, the results showed a strong correlation in which the immune response increased with cup size. At the lower end of the cup size range, the smallest cup tested (group 3: 3.8 mm chamber diameter) did not produce a meaningful immune response. These results demonstrate that cup size is a significant factor in the immune response in KV treatments using similar vacuum pressure and migration patterns.

[0162] research 23 Referring to Figure 29, studies were conducted to determine whether dividing a given injection volume into smaller volumes produced an immunogenic effect in guinea pigs after dynamic vacuum (KV) treatment across those injection sites.

[0163] The subjects received plasmid 2027 injection and were treated according to the following treatment groups. Group 1: Total injection volume of 100 μL in one injection (i.e., one blister compartment). Group 2: A total injection volume of 100 μL divided over three injections (i.e., three blister compartments of 33.3 μL each). Group 3: Total injection volume of 300 μL in one injection (i.e., one blister compartment). Group 4: A total injection volume of 300 μL divided over 9 injections (i.e., 9 blister compartments of 33.3 μL each).

[0164] Each group consisted of five subjects, each of whom was injected into the skin of their flank and then administered the KV treatment using the OTS-3 device (see Table 0) in three cycles of Pattern A. For those groups with multiple blister compartments (i.e., groups 2 and 4), the KV treatment involved a single treatment performed on all blister compartments within the group (i.e., the vacuum cup was moved across all blister compartments within the group in each cycle of Pattern A). Further details regarding the test parameters for this study are shown in Table 19 below. [Table 20]

[0165] The titer data for the groups at week 2 are shown. For groups 1 and 2, the results showed no immunological effect by dividing the 100 μL infusion volume into three infusions, which may be due to an immune response below detectable levels and not necessarily to the lack of immunological difference between groups 1 and 2. For the higher total volume groups (groups 3 and 4), dividing the 300 μL infusion volume into nine infusions can be seen as eliciting a higher trend in the immune response. These results suggest a trend that infusion volume division using KV treatment in high-volume blisters may increase the immune response. These results also support other study results and data showing that KV treatment is effective in enhancing the immune response, insofar as the treatment involves passing the vacuum cup through the entire infusion zone.

[0166] research 24 Referring to Figure 30, another study investigated the effect of reducing DNA dosage (by reducing injection volume) associated with dynamic vacuum (KV) treatment on the immune response (binding ELISA response) in guinea pigs. Compare this with Study 5 (Figures 12A-12B). However, in this study, needle electroporation (NEP) treatment was also tested in a reduced dosage scenario to compare with KV treatment.

[0167] In this study, subjects in two groups (Group 1 and Group 2, 5 subjects each) were intradermally injected with the same plasmid (9501) in a uniform volume (100 μL) and uniform DNA dose (2.5 μg) into the flank. After formulation with hyaluronidase (HYA), they were administered either KV treatment (Group 1) or NEP treatment (Group 2). KV treatment was administered using pattern A, 3 cycles, with a central support vacuum cup 102 having a chamber diameter D1 of 12 mm (see Figures 2A-2C).

[0168] The ELISA response at week 2 is shown in Figure 30 for both groups (the dashed line indicates the limit of detectable titer). The results show that, at the low doses tested, the KV-treated group produced a measurable immune response, while the NEP-treated group did not. These results also provide evidence that KV treatment may have a dose-saving advantage compared to NEP.

[0169] research 25 Referring now to FIG. 31, yet another study was conducted to investigate the effect of reducing the DNA dosage (by reducing the injection volume) using dynamic vacuum (KV) treatment on the immune response (binding ELISA response) in guinea pigs. Compare with Study 24 (FIG. 30). In this study, four groups of subjects (five subjects per group) received intradermal injection of plasmid 2303 into the skin of the flank, and each successive group received half the dosage of the previous group. Group 1 received an injection volume of 100 μL with a DNA dosage of 30 μg, Group 2 received an injection volume of 50 μL with a DNA dosage of 15 μg, Group 3 received an injection volume of 25 μL with a DNA dosage of 7.5 μg, and Group 4 received an injection volume of 12.5 μL with a DNA dosage of 3.75 μg. All other factors were constant for each group. The KV treatment was administered by an OTS-3 device (see Table 17) using Pattern A, 3 cycles.

[0170] The ELISA responses at week 2 for all groups are shown. Interestingly, no measurable decrease in ELISA immunogenicity was observed in the reduced dosage groups, providing further evidence that the KV treatment has a potential dosage-saving effect.

[0171] Study 26 Referring now to FIGS. 32A - 32E, a study was conducted to compare the immune responses, particularly the ELISA responses (FIGS. 32A - 32C) and T cell responses (FIGS. 32D and 32E), in rabbits after treatments including dynamic vacuum (KV), needle electropermeabilization (NEP), and intramuscular electropermeabilization (IM-EP).

[0172] In this study, the subjects received an injection of plasmid 9501 (encoding the full-length spike glycoprotein of SARS-CoV-2) and were treated according to the following treatment groups. · Group 1: KV treatment (using an OTS-3 device, Pattern A, 3 cycles) after intradermal injection of a volume of 100 μL with a DNA dosage of 100 μg with hyaluronidase (HYA). Group 2: NEP treatment after intradermal injection of 100 μL of DNA with a dose of 100 μg. Group 3: IM-EP treatment following intramuscular injection of 1000 μL of DNA containing a 1000 μg dose. Further details regarding the test parameters for this study are shown in Table 20 below. [Table 21]

[0173] The ELISA response is shown for all groups at week 2 (Figure 32A), week 4 (Figure 32B), and week 5 (Figure 32C). These results indicate that KV treatment with HYA outperformed the NEP-treated and IM-EP-treated groups in terms of ELISA immunogenicity at all time points. IM-EP treatment became equivalent to NEP treatment over time, but the results were not consistent with those of KV+HYA treatment.

[0174] T cell responses are shown for all groups at week 2 (Figure 32D) and week 5 (Figure 32E). These results demonstrate similar T cell immunogenicity for IM-EP treatment, NEP treatment, and KV+HYA treatment.

[0175] research 27 Referring to Figure 33, an investigation was conducted to compare the effects of different migration patterns and cycles of dynamic vacuum (KV) treatment on the immune response (ELISA response) in guinea pigs. All KV migration patterns in this study were performed using the same vacuum cup device, particularly the OTS-2 device shown in Table 17. Subjects were intradermally injected with a uniform volume of plasmid 2303 into the skin on their flank, and then administered KV treatment according to four migration-specific treatment groups (each group having six subjects). Group 1: KV treatment using left-right (STS) movement (Pattern A, 3 cycles). Group 2: KV treatment using unidirectional (UNI) movement begins away from the blister at position (-t,0) and uses one linear translation (one swipe) across the blister. This (-t,0)→(t,0) sequence, also referred to as "Pattern C" (see Figure 7F), is repeated six times (i.e., six cycles). Group 3: The same UNI movement as Group 2 was used, but with KV treatment applied only to those four cycles. Group 4: The same UNI movement as Group 2 was used, but KV treatment was applied only for those two cycles. Details regarding the test parameters for this study are summarized in Table 21 below. [Table 22]

[0176] ELISA responses are shown for all groups at week 2. The results demonstrate that the unidirectional (UNI) movement pattern was performed similarly to the lateral (STS) movement pattern. Surprisingly and unexpectedly, this was also true when the number of cycles in the unidirectional (UNI) movement was reduced to two cycles. These results provide evidence that the immunogenicity benefits provided by KV treatment are applicable even when using only one unidirectional swipe across the blister. Furthermore, viewed in conjunction with the results of the operator study discussed above (Study 18, Figure 25), these results support the conclusion that the KV treatment disclosed herein is substantially operator-friendly, i.e., easy to perform and requiring only one precise swipe across the blister.

[0177] research 28 Referring here to Figures 34A and 34B, there is a study comparing the immune response (ELISA response) and neutralizing activity in pigs resulting from dynamic vacuum (KV) treatment versus needle electroporation (NEP) treatment. In this study, pig subjects in two groups (each group having 3 subjects) were intradermally injected with a uniform volume of plasmid 9501 (encoding the SARS-CoV-2 spike glycoprotein) and a uniform DNA dose. Each subject received two injections at separate sites on the skin above the quadriceps femoris muscle, and KV subjects (group 2) also received hyaluronidase (HYA) along with the injection. After injection, subjects in group 1 were administered NEP treatment, and subjects in group 2 were administered KV treatment using the OTS-2 device with pattern A, 3 cycles (see Table 0). For both groups, the ELISA response at week 2 is shown in Figure 34A, and the neutralizing titer is shown in Figure 34B (dashed lines in both figures indicate the limit of detectable titer). Regarding the ELISA response (Figure 34A), the NEP-treated response approached the detection threshold, while the KV-treated response was easily detectable in all subjects. Regarding pseudovirus neutralization (Figure 34B), the KV-treated group showed superior neutralizing activity compared to the NEP-treated group. Despite the fact that demonstrating an ELISA response after intradermal injection (including electroporation) in porcine subjects is known to present challenges, the results of this study are not inconsistent with positive ELISA responses observed in guinea pigs and rabbits treated with KV.

[0178] research 29 Referring here to Figures 35A and 35B, a study was conducted to evaluate the effect of improper dynamic vacuum (KV) treatment (i.e., when the vacuum cup does not contact the center of the injected blister) on gene (GFP) expression in guinea pigs. Four groups of subjects (each group having 6(6) subjects) were intradermally injected into the flank skin using a plasmid encoding the GFP gene, and then KV treatment was administered using the same movement pattern, but at a different offset from the blister center Z2 along a second direction Y which is perpendicular to the cup translation direction X. The KV movement used in this study is shown in Figure 35A. The KV movement pattern is substantially the same as pattern C (Figure 7F), but at a different offset distance "d" along the second direction Y. For quantification, the offset distance d is measured along the second direction Y from the blister center Z2 to the translation axis X3 of the vacuum cup. KV treatment was performed for each group using the same OTS-2 device (see Table 0), and the groups were then grouped according to the blister offset distance d, as shown below and in Figure 35A. Group 1: Offset distance d=0 (i.e., accurate, meaning the cup translation axis X3 intersects the blister center Z2). Group 2: The offset distance d = t / 2, where t is equivalent to the cup chamber diameter in this study. Group 3: An offset distance d=1 means that the cup translation axis X3 is separated from the blister center Z2 by a distance equivalent to the cup chamber diameter t. Group 4: The starting position at d=2 means that the cup translation axis X3 is separated from the blister center Z2 by a distance equivalent to the two chamber diameters (2t).

[0179] GFP quantification for each group is shown in Figure 35B. The results indicate that precise KV movement (i.e., no offset from the blister center Z2) (group 1) provides robust expression, with decreasing expression at an offset of d=t / 2 (group 2) and completely decreased expression when the offset is d=1 or greater (i.e., the vacuum cup is completely separated from the blister during KV movement) (groups 3 and 4). The results for groups 3 and 4 are slightly better (if present) than GFP expression after GFP injection-only (INJ) globulinization. In particular, blister deletion at short distances (group 3) performed as poorly as blister deletion at long distances (group 4) in terms of gene expression. These results demonstrate that simply stretching the skin around the injected blister (group 3) is not sufficient to enhance gene expression. Instead, the results suggest that gene expression is enhanced when the injected tissue is drawn into the vacuum chamber, and increases with the proportion of injected tissue drawn into the vacuum chamber.

[0180] research 30 Referring to Figures 36A-36C, studies were conducted to compare the immune response (ELISA response) and T cell response in mice after dynamic vacuum (KV) treatment with hyaluronidase (HYA), injection-only (INJ) treatment, static vacuum (SV) treatment, and study-specific intramuscular (IM) needle electroporation (NEP) treatment (hereinafter labeled "NEP-IM"). The subjects in this study received injection of plasmid 9517 in a uniform volume (30 μL) and uniform DNA dose (5 μg) and were treated according to the following treatment groups (using five subjects per group). Group 1: INJ treatment. Group 2: KV treatment following intradermal plasmid injection of HYA into the flank skin, using pattern C, 4 cycles, performed with an OTS-6 device having a chamber diameter of 6 mm at a pressure of 489 mmHg (see Table 0). Group 3: SV treatment. ·Group 4: NEP-IM treatment after intramuscular injection, where NEP-IM was applied using three needle electrodes each having a diameter of 0.46 mm and arranged in an isosceles triangle. Four consecutive pulses were applied, each having a pulse duration of 52 ms, a set current of 0.2 A, a maximum voltage of 200 V, and a pulse delay of 250 ms.

[0181] ELISA responses are shown for all groups at week 3 (Figure 36A) and week 4 (Figure 36B). These results indicate that the KV+HYA treatment outperformed the NEP-IM treatment at all time points in terms of ELISA immunogenicity. In that regard, the NEP-IM treatment showed immunogenicity at week 3 and week 4, but less than the KV+HYA group. In this study, the INJ treatment and the SV treatment were not immunogenic. T cell responses are shown for all groups at week 4 in Figure 36C. In terms of T cell immunogenicity, the NEP-IM treatment and the KV+HYA treatment were comparable, which is consistent with studies in other species, and both of these treatments significantly outperformed the INJ treatment and the SV treatment. Interestingly, the NEP-IM treatment provides a good T cell response comparable to the KV+HYA treatment, but a weaker antibody response.

[0182] Study 31 Here, referring to Figure 37, a study was conducted to evaluate the effect of hyaluronidase (HYA) dosage on the dynamic vacuum (KV) treatment in terms of the immune response (ELISA response) in guinea pigs. Five groups of subjects were administered the same KV treatment after the same injection, but the only difference between the groups was the HYA dosage, which was administered as follows. ·Group 1: 135 U / mL; ·Group 2: 75 U / mL; ·Group 3: 25 U / mL; ·Group 4: 10 U / mL; ·Group 5: 0 U / mL (i.e., without HYA). Each group consisted of five subjects, all of whom received a uniform volume (100 μL) and DNA dose (25 μg) of plasmid 9501 injections into the flank skin, and were subsequently treated with the same KV treatment (pattern C, 4 cycles) using the same vacuum cups, particularly OTS-5 vacuum cups (see Table 0).

[0183] The results show that even small amounts of HYA added to plasmid injection, up to a dose of 10 U / mL (group 4), provide a significant benefit in terms of ELISA immunogenicity, with a sharp decrease in immunogenicity from 10 U / mL (group 4) to 0 U / mL (group 5). These results demonstrate the merits of pairing KV treatment with HYA, even at reduced doses such as 10% of the typical HYA dose in KV treatment discussed throughout this disclosure. Reducing the HYA dose can result in significant cost savings for KV treatment and limits the dilution effect of HYA on the amount of DNA delivered to the patient.

[0184] research 32 Referring to Figure 38, a study was conducted to compare the effects of dynamic vacuum (KV) treatment and needle electroporation (NEP) treatment on immune cell migration in guinea pigs. Plasmid 5013 (encoding the gene for GFP) was injected into the skin on the flank of two groups (four subjects per group) with a uniform volume and DNA dose. For the first group, the injection included hyaluronidase (HYA), and this group was subsequently administered KV treatment using an OTS-5 vacuum cup (see Table 0) with pattern C, 4 cycles. The second group was administered NEP treatment after the injection (without hyaluronidase). Three days after treatment, lymph nodes from both groups were analyzed to measure the number of GFP-positive cells and assess the degree of immune cell migration along the immunogenic pathway from transfection-induced skin to lymph nodes.

[0185] The results indicate that KV+HYA treatment resulted in more reporter gene-expressing cells in the lymph nodes compared to NEP treatment. These results are consistent with other studies described herein regarding gene expression and immune responses. Additionally, these results provide evidence that KV+HYA treatment successfully enhances immune cell migration along the immunogenic pathway (in this case, lymph node GFP transport) in downstream processes of the initial transfection event.

[0186] research 33 Referring here to Figure 39, a study was conducted to evaluate the effect of hyaluronidase (HYA) in low-dose pDNA dynamic vacuum (KV) treatment in rabbits in terms of immune response (ELISA response). See also Study 31 (Figure 37). The pDNA dose (1.5 μg) in this study was intentionally set low enough that the treatment was expected to fail to produce a detectable ELISA response. Essentially, this study evaluates whether the use of HYA can “rescue” KV treatment that would otherwise have a low success rate. In this study, subjects in two groups (with 9 subjects per group) were injected with plasmid 9501 at a uniform volume and DNA dose (1.5 μg) into the skin on the flank. The injection in the second group included HYA (135 U / mL), while the injection in the first group did not. After injection, both groups underwent KV treatment using OTS-5 vacuum cups (see Table 0) with pattern C, 4 cycles. The results show that even at low pDNA doses, HYA can improve the detectable ELISA titer response, with the majority of subjects in the second group producing detectable titer levels, while only two subjects in the first group (without HYA) had detectable levels. These results suggest a tendency for adding HYA treatment to KV treatment to enhance or rescue immunogenic conditions that might otherwise be insufficient or non-immunogenic.

[0187] research 34 Referring to Figure 40, a study similar to Study 33 (Figure 39) was conducted in rabbits, but with a moderate pDNA dose, specifically 15 μg (10 times the pDNA dose used in Study 33). This study used a different DNA plasmid, namely plasmid 2303. All other conditions in this study (except for the pDNA plasmid and dose) were the same as in Study 33. Again, two groups (with 9 subjects per group) were injected into the skin on their flanks with a uniform volume of plasmid 2303 and a "moderate" DNA dose (15 μg). The injection in the second group contained 135 U / mL of hyaluronidase (HYA), while the injection in the first group did not. After injection, both groups underwent KV treatment using OTS-5 vacuum cups (see Table 0) with pattern C, 4 cycles. The results are consistent with those of Study 33, which demonstrates that HYA improves the ELISA titer response in KV treatment at moderate pDNA doses.

[0188] Combining the results of Study 33 (Figure 39) and Study 34 (Figure 40), the findings suggest that adding HYA to KV treatment not only improves immunogenicity (which can be considered dose saving), but also, to some extent, that KV treatment with HYA may improve treatment outcomes in subjects who are unlikely to achieve a meaningful immune response.

[0189] research 35 Referring to Figure 41, this study conducted another research project to evaluate the effect of dynamic vacuum (KV) treatment on immune cell migration in rabbits. In this respect, this study is similar to study 32 (Figure 38) conducted in guinea pigs. In this study, four groups of subjects (each group having 6 subjects) were injected with plasmid 5013 (encoding the GFP gene) into the skin on the flank at a uniform volume and DNA dose. After injection, each group underwent KV treatment using an OTS-5 vacuum cup with pattern C, 4 cycles. Lymph nodes were analyzed on different days after treatment, and the number of GFP-positive cells in each group was measured as follows: Group 1 on day 1, Group 2 on day 2, Group 3 on day 3, and Group 4 on day 7. This analysis aimed to identify the timing of immune cell migration from the skin to the lymph nodes.

[0190] The results indicate that strong immune cell migration occurs on days 1 and 2, decreases on day 3, and then migration reaches a level on day 7. These results suggest that the majority of immune cell migration (in this case, lymph node GFP transport) occurs within the first 48 hours. These results, along with those of Study 32 (Figure 38), provide evidence that the immunogenic response involves antigen transport along the immunogenic pathway (e.g., lymph node transport) that is concentrated in the first two days after transfection.

[0191] research 36 Referring here to Figure 42, a study was conducted to evaluate the "trade-off" relationship between DNA dose and hyaluronidase (HYA) dose in a given total injection volume for dynamic vacuum (KV) treatment in guinea pigs. Four groups (each with five subjects) received the same KV treatment after injecting plasmid 2303 at varying ratios of DNA dose to HYA dose. For groups 1-3, the DNA and HYA doses were limited within a uniform total injection volume (thus, the HYA dose was made at the expense of the DNA dose, and vice versa). For group 4, the total injection volume was unlimited, and high doses were given for both DNA and HYA. The doses for groups 1-4 were as follows: Group 1: 5 μg DNA dose; 75 U / mL HYA dose (i.e., 50 / 50 mixture, i.e., half dose of DNA and half dose of HYA). Group 2: 9 μg DNA dose; 12.5 U / mL HYA dose (i.e., DNA dose diluted by 10% in the presence of a low dose of HYA). Group 3: 10 μg DNA dose; 0 U / mL HYA dose (i.e., 100% DNA dose, no HYA). Group 4: 10 μg DNA dose; 135 U / mL HYA dose (i.e., a high HYA dose without sacrificing DNA dose). Details regarding the test parameters for this study are summarized in Table 22 below. [Table 23]

[0192] For all groups, the injection was performed intradermally within the skin across the flank. All KV treatments in this study were performed using Pattern C, 4 cycles, with an OTS-5 vacuum cup (see Table 0).

[0193] The results demonstrate that the presence of HYA at each tested dose (groups 1, 2, and 4) enhanced the immune response measured by ELISA titer. In comparison, the non-HYA control (group 3) produced no measurable ELISA response at all. Interestingly, the high-DNA, high-HYA formulation (group 4), performed similarly to the 50 / 50 formulation (group 1), showed measurable ELISA responses in both groups 1 and 4, while group 2 (90% DNA, low-HYA formulation) outperformed both groups 1 and 4. These results suggest that, as long as some HYA is added, it is not necessary to sacrifice the DNA dose to increase the HYA dose in order to produce enhanced immunogenicity. Furthermore, these results demonstrate that the low-HYA dose (group 2) can outperform the high-HYA dose (group 4) without sacrificing the DNA dose, which is interesting. Another interesting observation from these results is that adding HYA to plasmid infusion generated beneficial immunogenicity, even at the expense of DNA dosage in the process (e.g., group 1 (50 / 50 mixture) compared to group 3 (100% DNA dose, no HYA)). Considering the potential clinical use of KV+HYA delivery, these results support the inclusion of HYA in plasmid infusions for KV treatment at low doses (but not limited to, e.g., 10% of the total infusion volume). Overall, these results offer groundbreaking insights into the potential for significant cost savings that could be obtained by even a slight reduction in the historical dosage of HYA in plasmid infusions for KV treatment, without sacrificing beneficial effects.

[0194] research 37 Referring here to Figure 43, we conducted a study to compare the effects on the ELISA immune response provided by varying the number of dynamic vacuum (KV) translational transfers (cycles, in this case "swipes") in other similar KV treatments. Four groups of subjects (five subjects per group) received intradermal injection of plasmid 2027 in a uniform volume and DNA dose on the skin over the flank, followed by KV treatment with the same OTS vacuum cup, particularly the OTS-5 vacuum cup (see Table 0), using different numbers of cycles of the same KV transfer pattern, particularly pattern C across the injection blister (see Figure 7F). The cycles administered to each group were as follows: Group 1 received one cycle (i.e., one swipe). Group 2 underwent two cycles. Group 3 underwent three cycles. Group 4 underwent four cycles.

[0195] The results show substantially equivalent immune responses in terms of ELISA response, regardless of the number of swipes used across the infused blister. In addition, viewed in relation to the results of the operator studies (Study 18, Figure 25), unidirectional (UNI) transfer studies (Study 27, Figures 33A and 33B), and inappropriate KV studies (Study 29, Figures 35A and 35B) described above, these results support the conclusion that the KV treatment disclosed herein is substantially operator-friendly, i.e., easy to administer and requires only one precise swipe across the infused blister to achieve a beneficial immune response. In short, these studies provide evidence that a single swipe across a blister is as effective as multiple swipes across the blister for administering immunogenic KV treatment.

[0196] research 38 Referring to Figure 44, a study was conducted to investigate the effect of dynamic vacuum (KV) treatment on reduced doses of mRNA-1273 (an mRNA-based vaccine for SARS-CoV-2) on the immune response (binding ELISA response) in guinea pigs. In this study, subjects in four groups (five subjects per group) received mRNA-1273 injections at either a low mRNA dose (0.1 μg) or a high mRNA dose (1 μg). Additionally, for each dose in this study, the mRNA-1273 plasmid was injected either intramuscularly (IM) (this is the Moderna protocol injection) or intradermally (ID), and the intradermal injections were followed by KV treatment using the same migration pattern (pattern C) and the same vacuum cup (OTS-5 vacuum cup, see Table 0) for four cycles. The doses for groups 1-4 were as follows: Group 1: 1 μg mRNA dose ("high dose" for the purposes of this study) via intramuscular (IM) injection. Group 2: 0.1 μg mRNA dose ("low dose" for the purposes of this study) via intramuscular (IM) injection. Group 3: Intradermal (ID) injection followed by KV treatment, delivering 1 μg (high dose) of mRNA. Group 4: Intradermal (ID) injection followed by KV treatment, delivering a low dose of 0.1 μg of mRNA. Details regarding the test parameters for this study are summarized in Table 23 below. [Table 24]

[0197] ELISA responses at week 2 are shown for all groups (dashed lines indicate the limits of detectable titer). The results show that at high dose levels (1 μg), both IM delivery (group 1) and ID delivery + KV treatment (group 3) produced similar immunogenicity in terms of ELISA titer. At low dose levels (0.1 μg), IM delivery (group 1) had a completely undetectable ELISA response, while ID delivery + KV treatment (group 4) remained immunogenic but experienced a decrease from its high-dose counterpart (group 3). These results demonstrate that at low mRNA doses, intradermal (ID) delivery of mRNA-1273 with additional KV treatment is far superior to intramuscular (IM) delivery in terms of ELISA response.

[0198] research 39 Referring to Figure 45, a follow-up study to Study 38 (Figure 44) was conducted to further investigate the effect of dynamic vacuum (KV) treatment on reduced doses of mRNA-1273 on the immune response (binding ELISA response) in guinea pigs. In this follow-up study, KV treatment after intradermal (ID) injection of mRNA-1273 at “high,” “medium,” and “low” doses (1 μg, 0.5 μg, and 0.1 μg, respectively) was compared to dedicated intradermal (ID) injection (INJ) treatment of high and low doses of mRNA-1273 (in contrast to dedicated intramuscular (IM) injection of mRNA, as in Study 38). Therefore, all injections in this study were intradermal (ID). KV treatment was administered using the same vacuum cup, as well as the same transfer pattern and cycle as in Study 38, i.e., using the OTS-5 vacuum cup (see Table 0), with pattern C, 4 cycles. The doses for groups 1-5 were as follows: Group 1: 1 μg mRNA dose (high dose). Group 2: mRNA dose of 0.1 μg (low dose). Group 3: 1 μg mRNA dose (high dose), followed by KV treatment. Group 4: 0.1 μg mRNA dose (low dose), followed by KV treatment. Group 5: 0.5 μg mRNA dose (medium dose), followed by KV treatment. Details regarding the test parameters for this study are summarized in Table 24 below. [Table 25]

[0199] ELISA responses at week 2 are shown for all groups (dashed lines indicate the limits of detectable titers). The results show that, in both high-dose (1 μg) and low-dose (0.1 μg) mRNA ID infusions, KV treatment (groups 3 and 4) closely outperformed ID infusion-only treatments (groups 1 and 2) in terms of ELISA titer, but high-mRNA-dose ID infusion (group 1) was more immunogenic than low-mRNA-dose KV treatment (group 4). Moderate mRNA-dose (0.5 μg) KV treatment (group 5) slightly outperformed high-mRNA-dose ID infusion-only treatment (group 1). Interestingly, at low mRNA-dose levels (0.1 μg), ID infusion-only treatment (group 2) was more immunogenic than intramuscular (IM) infusion-only treatment from Study 38 (group 2 in that study).

[0200] Combining the results of this study with those of Study 38, the data demonstrate that, at low mRNA doses, intradermal (ID) delivery of mRNA-1273 with additional KV treatment is far superior in terms of ELISA response to both intramuscular (IM) and intradermal (ID) injection-only treatments. The combined results also show that KV treatment of moderate mRNA doses provided comparable immunogenicity to both ID and IM injection-only treatments of high-dose mRNA-1273. Therefore, the combined results suggest that intradermal injection of mRNA-1273 followed by KV treatment, whether injected intramuscular (IM) or intradermal (ID), can provide a dose-saving effect for mRNA-1273 injection without sacrificing immunogenicity. Additionally, the use of KV to deliver lipid nanoparticle mRNA to the skin has been shown to be more immunogenic than delivery to muscle or skin without KV, suggesting that KV generally enhances cellular uptake of foreign cargo as well as plasmid DNA.

[0201] research 40 Referring here to Figure 46, another hyaluronidase administration study was conducted to evaluate the "trade-off" relationship between DNA dose and hyaluronidase (HYA) dose, particularly in guinea pigs, in high-DNA dose scenarios for dynamic vacuum (KV) treatment. Two groups of subjects (five subjects per group) were administered the same KV treatment after infusion with a high dose of plasmid 2303, as follows: Group 1: 100 μg DNA dose; 0 U / mL HYA dose (i.e., 100% DNA dose, no HYA). Group 2: 75 μg DNA dose; 33.75 U / mL HYA dose (i.e., 25% diluted DNA dose due to the presence of HYA). The results indicate that, at high DNA doses, the addition of HYA improved immunogenicity, even at the cost of a 25% reduction in DNA dose.

[0202] observation Based on the data and results from the studies described above, with reference to Figures 8–46, the inventors made the following observations: Dynamic vacuum treatment provides a comparable immune response to vacuum electroporation (VEP) or needle electroporation (NEP) across multiple studies, and a larger response in guinea pigs or rabbits across multiple studies (see Figures 17–18, 23A–23B, 30, 32A–32E, 34A–34B). Additionally, dynamic vacuum treatment improved the immune response in both rabbits and guinea pigs compared to static vacuum treatment in all studies herein. Furthermore, dynamic vacuum treatment provides superior gene expression compared to vacuum electroporation (VEP) treatment, and in the study shown in Figure 8, dynamic vacuum treatment generated a larger transfection zone. Dynamic vacuum treatment was observed to cause acute redness at the treatment site, but unlike electroporation treatment in this study, which caused more tissue damage and required a longer healing time to recover normally, it disappeared rapidly and did not cause any scabbing, burning, or other tissue damage compared to dynamic vacuum treatment. KV also resulted in better antigen transport to lymph nodes than NEP (Figure 38), was shown to be dose-saving, and generated a stronger immune response compared to NEP when using suboptimal (low or non-immunogenic) DNA vaccine doses.

[0203] Furthermore, for dynamic vacuum treatment, six swipes (three forward and backward cycles) outperformed half-swipes, but even a single swipe was subsequently shown to be equally immunogenic as multiple swipe patterns. Additionally, the magnitude of gene expression was correlated with the percentage of injection site contacted by the swipe, with a corresponding decrease in gene expression when the percentage of injection site contact was absent. It was also observed that the addition of hyaluronidase (HYA) significantly improved both gene expression and immune response for dynamic vacuum treatment, but similarly, it was not beneficial for static vacuum treatment or electroporation. All HYA concentrations in the range of 12.5 U / mL to 135 U / mL were equally effective in enhancing the immunogenicity of KV delivery of DNA vaccines, suggesting that even small amounts of HYA can produce this benefit. The magnitude of the immune response after KV delivery increased with increasing vacuum intensity and cup diameter until the cup diameter was approximately equal to the diameter of the injection site in the skin. These studies also demonstrated that reducing the injection volume in dynamic vacuum treatment may slightly reduce immunogenicity. Furthermore, it was observed that supplementing dynamic vacuum treatment with vacuum electroporation (VEP) did not yield more beneficial results than dynamic vacuum treatment alone. Additionally, dynamic vacuum treatment containing hyaluronidase was observed to approach the level of immune responses at the lipid nanoparticle mRNA level, which exhibited superior cellular responses, while DNA-based dynamic vacuum treatment still required approximately 10-fold (10-fold) doses to achieve nearly equivalent mRNA levels. The use of DNA-launching nanoparticle plasmids, rather than plasmids encoding monomeric antigens delivered using KV+HYA, was able to induce more durable immune responses than lipid nanoparticle mRNA using the same dose of nucleic acid in each group. Finally, delivery of lipid nanoparticle mRNA to the skin using KV was shown to be more immunogenic than delivery to muscle or skin without KV, suggesting that KV generally enhances cellular uptake of foreign cargo as well as plasmid DNA.

[0204] qualification It should be recognized that the various parameters of the vacuum cups, systems, and dynamic vacuum treatment techniques described above are provided as exemplary features for providing vacuum-enhanced transfection of tissue. These parameters can be adjusted as needed without departing from the scope of this disclosure. For example, while the dynamic vacuum treatment and vacuum cups described above are primarily described in relation to enhancing transfection of skin tissue, the dynamic vacuum treatment and vacuum cups herein can be adapted to treat adipose tissue and / or muscle tissue to enhance transfection therein.

[0205] It should also be noted that in additional embodiments, various vacuum cups 2, 102, 302, 402 and support components (see Figures 1A, 3A-3B, and 5A-5B) may be provided in a kit containing multiple vacuum cups 2, 102, 302, 402 that can be used interchangeably by the user.

[0206] Although this disclosure has been described in detail, it should be understood that various modifications, substitutions, and alternatives can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Furthermore, the scope of this disclosure is not intended to be limited to the specific embodiments described herein. In particular, one or more features from the embodiments described above can be used in other embodiments herein. As will be readily apparent to those skilled in the art, existing or subsequently developed processes, machines, manufactures, compositions of materials, means, methods, or steps that perform substantially the same functions or achieve substantially the same results as the corresponding embodiments described herein can be utilized in accordance with this disclosure.

Claims

1. A method for enhancing drug delivery into tissues, The step of arranging a housing that defines a chamber adjacent to the surface of the tissue, wherein the arranging step involves positioning the chamber adjacent to the injection site where the drug is injected into the tissue. By applying vacuum pressure to the chamber, a portion of the tissue is drawn through the opening of the chamber and into the chamber. A method comprising moving the housing relative to the tissue while the vacuum pressure is applied, in order to enhance drug delivery within the tissue.

2. The method according to claim 1, wherein the moving step includes translating the housing in at least a portion of the chamber along a path that crosses the center of the injection site.

3. The method according to claim 1, wherein the positioning step involves positioning the chamber at a first location which is spaced apart from the center of the injection portion by a first offset distance measured from the center of the injection portion along a certain direction, and the translation step involves translating the housing from the first location and along the direction to a second location which is spaced apart from the center of the injection portion by a second offset distance measured along the direction.

4. The method according to claim 3, wherein at least one of the first offset distance and the second offset distance is greater than or equal to the maximum internal dimension of the chamber measured along the direction.

5. The method according to claim 4, wherein the first offset distance and the second offset distance are each greater than or equal to the maximum internal dimension of the chamber.

6. The method according to claim 3, wherein the step of translating the housing from the first location to the second location is performed at least twice.

7. The method according to claim 2, wherein the translation step includes a first step of translating the housing in a first direction along a linear path at least partially over the injection site, and a second step of translating the housing in a second direction opposite to the first direction along a linear path at least partially over the injection site.

8. The method according to claim 3, wherein the first translation step and the second translation step are repeated a plurality of times.

9. The aforementioned step of moving, The housing is translated along at least a portion of a substantially circular path, while the housing is translated along the substantially circular path around the injection site such that at least a portion of the chamber overlaps the center of the injection site. The method according to claim 1, comprising at least one of: pivoting the housing about a central axis of the vacuum chamber, such that the central axis is oriented substantially orthogonal to the surface of the tissue on which the chamber is located.

10. The method according to claim 1, wherein the applied vacuum pressure is in the range of about -300 mmHg to about -760 mmHg during the moving step.

11. The method according to claim 11, wherein the applied vacuum pressure is in the range of about -400 mmHg to about -600 mmHg during the moving step.

12. The method according to claim 1, wherein the portion of the tissue drawn into the chamber is drawn in such a way that it comes into contact with at least one projection of the housing located within the chamber, thereby deforming the tissue in contact with the projection.

13. A method for enhancing drug delivery into tissues, The process involves injecting a drug into the target tissue, thereby defining the injection site on the surface of the tissue, A housing is provided that defines a chamber at or adjacent to the injection site, By applying vacuum pressure to the chamber, a portion of the tissue is drawn through the opening of the chamber and into the chamber. A method comprising moving the housing relative to the tissue while the vacuum pressure is applied, in order to enhance drug delivery within the tissue.

14. The method according to claim 13, wherein the moving step includes translating the housing along a path that crosses the center of the injection site once or more times in at least a portion of the chamber.

15. The method according to claim 13, wherein the applied vacuum pressure is in the range of about -300 mmHg to about -760 mmHg during the moving step.

16. The method according to claim 13, wherein the tissue is skin tissue, the surface of the tissue is a skin surface, and the injecting step comprises performing a Mantou injection within the skin tissue.

17. The method according to claim 16, wherein the injected agent includes a diffusing agent.

18. The method according to claim 17, wherein the diffusing agent is hyaluronidase.

19. The method according to claim 18, wherein the hyaluronidase constitutes about 5% to about 20% of the total injection volume of the drug.

20. The method according to claim 13, wherein the injected drug comprises a plasmid containing mRNA and lipid nanoparticles.

21. The method according to claim 13, wherein the injected drug comprises a plasmid containing DNA firing nanoparticles.

22. The injection step includes injecting the total volume of the drug into the tissue, and the injection step includes performing multiple injections into the tissue at each of the multiple injection sites, wherein the total volume is divided into multiple sub-volumes, and each of the multiple injections injects one of the multiple sub-volumes into the tissue. The method according to claim 13, wherein the moving step includes translating the housing along a path that traverses at least a large portion of the plurality of injection sites in the chamber.

23. A system for vacuum-enhanced drug delivery into tissues within a living organism, A housing that defines the chamber and the opening into the chamber, At least one port extending through the housing, wherein the at least one port is separated from the at least one opening and is connectable to a vacuum source, thereby configuring the at least one port to communicate vacuum pressure from the vacuum source to the chamber. The housing is configured to communicate a vacuum field to a portion of the tissue, thereby drawing the portion of the tissue through the opening and holding the portion of the tissue within the chamber at least momentarily, and the housing is further configured to move relative to the tissue while the vacuum field is communicating with the tissue, and the relative motion deforms at least a portion of the tissue, with at least one port, A system comprising one or more features that can be disposed between the distal end of the housing and the surface of the tissue in order to reduce sliding friction between the housing and the tissue.

24. The one or more of the above features, Lubricant and The system according to claim 23, selected from the group, comprising a roller coupled to the distal end of the housing.

25. The system according to claim 23, further comprising a handle member, wherein the housing is attachable to the handle member.

26. The system according to claim 23, wherein the housing is configured to communicate the vacuum field at a vacuum pressure in the range of approximately -300 mmHg to approximately -760 mmHg during the movement of the housing relative to the tissue.

27. The system according to claim 26, wherein the chamber has a circular cross-sectional shape in a plane perpendicular to the central axis of the chamber, and the inner surface of the housing within the chamber defines a chamber diameter in the range of about 5 mm to about 15 mm.

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