Kinetic vacuum treatment devices, systems and methods

Description

1 / 111 KINETIC VACUUM TREATMENT DEVICES, SYSTEMS AND METHODS CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims the benefit of U.S. Provisional Application No. 63 / 498,690, filed April 27, 2023, the full content of which is incorporated herein by this reference. TECHNICAL FIELD

[002] The present invention relates to devices, assemblies and systems for gripping and / or deforming fabric with vacuum pressure and supplying fluid to the fabric. BACKGROUND

[003] Numerous and varied techniques have been developed to improve the delivery of agents in vivo into tissue. Some of these techniques involve modes of injecting the agent into the tissue. These modes include certain types of needle injection (e.g., Mantoux injection, side-port injection) and other types of injection, such as jet injection. Other techniques to improve the delivery of the agent in vivo involve modes of spreading or dispersing an injected agent within the tissue and / or through multiple tissue layers or through the same tissue layer. Iontophoresis, microneedle arrays, suction cup or vacuum cup treatments are examples of such modes. Other techniques to improve the delivery of the agent in vivo involve modes that increase the uptake of the agent directly into target cells within the tissue. Electroporation and sonoporation are examples of this mode.Additional techniques to improve in vivo agent delivery involve methods that include pairing the agent with an adjuvant known to enhance the immune response(s) elicited by the agent. Some techniques accomplish several of the above methods to improve in vivo agent delivery. For example, assisted electroporation treatments... Petition 870250114264, dated 11 / 12 / 2025, page 6 / 175 2 / 111 vacuum (VEP) has been shown to improve in vivo agent delivery by increasing dispersion of the injected substance in the tissue and increasing absorption of the agent directly into target cells within the tissue. SUMMARY

[004] According to one embodiment of the present invention, a method for improving the delivery of an agent into tissue includes placing a housing that defines a chamber adjacent to a tissue surface, thus locating the chamber adjacent to the injection site where the agent was injected into the tissue, applying vacuum pressure to the chamber, thereby pulling a portion of the tissue through an opening in the chamber into the chamber, and moving the housing relative to the tissue while vacuum pressure is applied to improve the delivery of the agent into the tissue.

[005] According to another embodiment of the present invention, a method for improving the administration of an agent into the tissue includes injecting an agent into the individual's tissue, thereby defining an injection site on a tissue surface, placing a housing that defines a chamber at or adjacent to the injection site, and applying vacuum pressure to the chamber, thereby pulling a portion of the tissue through an opening in the chamber into the chamber. While vacuum pressure is applied, the housing is moved relative to the tissue to improve the administration of the agent into the tissue.

[006] According to a further embodiment of the present invention, a system for vacuum-enhanced agent delivery into in vivo tissue includes a housing that defines a chamber and an opening in the chamber, and at least one orifice extending through the housing. The at least one orifice is remote from the at least one opening and can be connected to a vacuum source, such that the at least one orifice is configured to communicate the pressure. Petition 870250114264, dated 11 / 12 / 2025, p. 7 / 175 3 / 111 are vacuum from the vacuum source to the chamber. The housing is configured to communicate a vacuum field to a portion of the tissue and thus pull the tissue portion through the opening and, at least momentarily, hold the tissue portion in the chamber. The housing is further configured to move relative to the tissue while the vacuum field is communicated to the tissue, wherein the relative motion deforms at least part of the tissue. The system includes one or more features that are disposable between a distal end of the housing and a tissue surface to reduce sliding friction between the housing and the tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[007] This patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[008] The preceding summary, as well as the following detailed description of illustrative embodiments of the present application, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the features of the present application, illustrative embodiments are shown in the drawings. It should be understood, however, that the application is not limited to the precise arrangements and instruments shown. In the drawings:

[009] Figure 1A is a diagrammatic view of a kinetic vacuum treatment system employing a vacuum cup, according to an embodiment of the present invention;

[0010] Figure 1B is a perspective view of the vacuum cup illustrated in Figure 1A, showing a vacuum chamber of the cup, according to an embodiment of the present invention;

[0011] Figure 1C is a side view in section of the vacuum cup illustrated in Figure 1B, taken along a sectional plane that Petition 870250114264, dated 11 / 12 / 2025, page 8 / 175 4 / 111 extends along a central axis of the cup;

[0012] Figure 1D is another side view in section of the vacuum cup illustrated in Figure 1B, showing a mound of tissue aspirated into the vacuum chamber in response to the application of vacuum pressure, and also showing the injected fluid dispersed within the skin layer of the tissue;

[0013] Figure 2A is a perspective view of a vacuum cup with a central pin positioned inside the vacuum chamber, according to another embodiment of the present invention;

[0014] Figure 2B is a side view in section of the vacuum cup illustrated in Figure 2A, taken along a sectional plane that extends along a central axis of the cup;

[0015] Figure 2C is another side view in section of the vacuum cup, similar to that shown in Figure 2A, showing a bunch of tissue pulled into the vacuum chamber and in contact with the central pin, responsive to the application of vacuum pressure, and also showing the injected fluid dispersed within the skin layer of the tissue;

[0016] Figure 3A is a side view in section of a vacuum cup, similar to the cup shown in Figures 1A-1D, having an injection channel for an injection needle, according to another embodiment of the present invention;

[0017] Figure 3B is a side view in section of a vacuum cup, similar to the cup shown in Figures 2A-2C, having a jet injector that extends into the vacuum chamber, according to another embodiment of the present invention;

[0018] Figure 4A is a perspective view of a vacuum cup with electrodes for electroporation of aspirated tissue into the vacuum chamber, according to another embodiment of the present invention; Petition 870250114264, dated 11 / 12 / 2025, p. 9 / 175 5 / 111

[0019] Figure 4B is a side view in section of the vacuum cup illustrated in Figure 4A;

[0020] Figure 5A is a perspective view of a handling assembly that includes a vacuum cup, according to another embodiment of the present invention;

[0021] Figure 5B is a partially exploded view of the handling assembly illustrated in Figure 5A, showing the vacuum cup detached from an assembly formation of the handling assembly, the vacuum cup rotated in a bottom view for illustrative purposes, according to an embodiment of the present invention;

[0022] Figures 6A-6G are side views in tissue section showing representative stages of an exemplary kinetic vacuum treatment employing the vacuum cup illustrated in Figures 1A1D, according to an embodiment of the present invention;

[0023] Figure 6H is a cross-sectional side view of the tissue showing representative stages of another exemplary kinetic vacuum treatment;

[0024] Figures 7A-7F are plan views showing exemplary kinetic motions of a vacuum cup for providing kinetic vacuum treatments, according to embodiments of the present invention; Figure 7A shows lateral translational motions of the cup; Figure 7B shows upward and downward or forward and backward translational motions of the cup; Figure 7C shows tortuous translational motions of the cup; Figure 7D shows rotational (e.g., twisting) motions of the cup; Figure 7E shows a particular exemplary sequence of lateral translational motions of the cup; and Figure 7F shows an exemplary unidirectional translational motion of the cup;

[0025] Figure 8 is a diagram of images showing gene expression in guinea pig skin after intradermal injections. Petition 870250114264, dated 11 / 12 / 2025, page 10 / 175 6 / 111 samples of a plasmid encoding the gene for green fluorescent protein (GFP) were then treated with various techniques and devices, each using vacuum treatment;

[0026] Figure 9 is a diagram of images showing gene expression in guinea pig skin after intradermal injections of a plasmid encoding the gene for green fluorescent protein (GFP) and then treatment with various kinetic vacuum (KV) treatments (and one static vacuum (SV) treatment) using various vacuum cup designs;

[0027] Figures 10A and 10B are graphs showing immunogenicity data from linkage ELISA in guinea pigs at two (2) weeks (Figure 10A) and four (4) weeks (Figure 10B) after intradermal injections of a plasmid, followed by various treatments, including kinetic vacuum (KV) treatments, static vacuum (SV) treatments, vacuum electroporation (VEP) treatments and injection-only (INJ) treatments;

[0028] Figures 10C and 10D contain photographs showing the effects of the treatment on the skin tissue at the treatment sites for the study shown in Figures 10A-10B immediately after the treatments (Figure 10C) and seven (7) days after the treatment (Figure 10D).

[0029] Figures 11A and 11B are graphs showing ELISA binding immunogenicity data in guinea pigs at two (2) weeks (Figure 11A) and four (4) weeks (Figure 11B) after intradermal injections of a plasmid, followed by various treatments including kinetic vacuum (KV) treatments, static vacuum (SV) treatments, vacuum electroporation (VEP) treatments and injection-only (INJ) treatments, as a follow-up to the study shown in Figures 10A-10B to confirm the ELISA results;

[0030] Figures 12A and 12B are graphs that show data from Petition 870250114264, dated 11 / 12 / 2025, page 11 / 175 7 / 111 immunogenicity of linkage ELISA in guinea pigs at two (2) weeks (Figure 12A) and four (4) weeks (Figure 12B) after intradermal injections of a plasmid in different volumes, followed by kinetic vacuum (KV) treatments;

[0031] Figure 13 is a graph showing immunogenicity data from binding ELISA in unexposed guinea pigs two (2) weeks after intradermal plasmid injections followed by various treatments, including vacuum electroporation (VEP) treatments, kinetic vacuum (KV) treatments, static vacuum (SV) treatments and injection-only (INJ) treatments;

[0032] Figure 14 is a graph comparing immunogenicity data from linkage ELISA in guinea pigs two (2) weeks after intradermal plasmid injections followed by various kinetic vacuum treatments employing different amounts of cup translations at the injection site, with one of these treatments including hyaluronidase;

[0033] Figure 15 is a graph comparing ELISA immunogenicity data in guinea pigs after intradermal plasmid injections followed by various kinetic vacuum (KV) treatments employing different vacuum pressures;

[0034] Figures 16A and 16B are graphs comparing ELISA immunogenicity data in rabbits on Day 0 (Figure 16A) and Week 2 (Figure 16B) after intradermal plasmid injections followed by various treatments, including kinetic vacuum (KV), static vacuum (SV), vacuum electroporation (VEP), needle electroporation (NEP), and injection-only treatments (INJ);

[0035] Figures 17A and 17B are graphs from a follow-up study to that shown in Figures 16A-16B, again comparing ELISA immunogenicity data in rabbits on Day 0 (Figure 17A) and Week 2 (Figure 17B) after intradermal injections. Petition 870250114264, dated 11 / 12 / 2025, page 12 / 175 8 / 111 plasmid followed by multiple treatments, including kinetic vacuum (KV) treatments;

[0036] Figure 18 is a graph comparing ELISA immunogenicity data in guinea pigs two (2) weeks after intradermal plasmid injections followed by various vacuum treatments, including kinetic vacuum (KV) treatments and static vacuum (SV) treatments with and without hyaluronidase;

[0037] Figure 19 is a graph comparing ELISA immunogenicity data in guinea pigs two (2) weeks after treatment to assess the impact that the number of kinetic vacuum cup (KV) movements and skin thickness at the treatment site have on the immune response;

[0038] Figures 20A and 20B show gene expression in guinea pig skin after intradermal injections of a plasmid encoding the gene for green fluorescent protein (GFP) according to different injection volumes, with and without hyaluronidase, followed by kinetic vacuum (KV) or static vacuum (SV) treatments; Figure 20A is a table of images showing visible GFP expression; Figure 20B is a graph showing quantified values ​​of measured fluorescence;

[0039] Figure 21 is a graph comparing ELISA immunogenicity data in guinea pigs two (2) weeks after treatment to assess the impact that high injection volumes with and without hyaluronidase have on immune responses (ELISA expression) produced by kinetic (KV) and static (SV) vacuum treatments;

[0040] Figure 22 is a graph comparing ELISA immunogenicity data in guinea pigs two (2) weeks after treatment to assess the impact that the combination of kinetic vacuum (KV) treatments with vacuum electroporation (VEP) has on immune responses (ELISA expression); Petition 870250114264, dated 11 / 12 / 2025, page 13 / 175 9 / 111

[0041] Figures 23A-23D are graphs that compare data from Linkage ELISA (Figures 23A and 23B), ELISpot data (Figure 23C), and SARS-CoV-2 pseudovirus neutralization data (Figure 23D) produced by kinetic vacuum (KV) treatments versus mRNA treatments and needle electroporation (NEP) treatments.

[0042] Figure 24 is a table of images showing gene expression in guinea pig skin after various injection partitioning treatments employing multi-bubble injection sites enhanced by a single kinetic vacuum (KV) treatment.

[0043] Figure 25 is a graph from an operator study showing immunogenicity data from binding ELISA in guinea pigs two (2) weeks after intradermal plasmid injections followed by the same kinetic vacuum (KV) treatments performed by four (4) different individuals (operators);

[0044] Figures 26A-26C are graphs from a nearly one (1) year study comparing binding ELISA data (Figure 26A) and SARS-CoV-2 pseudovirus neutralization data (Figures 26B26B) produced by kinetic vacuum (KV) treatments using DNA-launched nanoparticles (DNLP) versus mRNA treatments;

[0045] Figure 27 is a graph showing the comparative scattering effects (measured as a ratio of pre- and post-treatment bubble diameters) for kinetic vacuum (KV) treatments (with and without hyaluronidase added to the injection) and for static vacuum (SV) treatments;

[0046] Figure 28A is a graph showing a vacuum cup device study comparing linkage ELISA data in guinea pigs at two (2) weeks receiving the same kinetic vacuum (KV) treatment (e.g., motion pattern) administered by different devices, including a vacuum cup. Petition 870250114264, dated 11 / 12 / 2025, page 14 / 175 10 / 111 with a central pin and four (4) ready-to-use (OTS) vacuum cup devices;

[0047] Figure 28B is a graph comparing linkage ELISA data in guinea pigs at two (2) weeks produced by performing the same kinetic vacuum (KV) treatment using different vacuum cup devices with different cup sizes and geometries;

[0048] Figure 29 is a graph comparing linkage ELISA data in guinea pigs at two (2) weeks produced by partitioning injection volumes into multiple bubbles, followed by performing the same kinetic vacuum (KV) treatments on the partitioned bubbles in each group collectively;

[0049] Figure 30 is a graph comparing linkage ELISA data in guinea pigs at two (2) weeks produced by injections with reduced dosages of DNA plasmid followed by kinetic vacuum (KV) treatments versus needle electroporation (NEP) treatments;

[0050] Figure 31 is a graph comparing linkage ELISA data in guinea pigs at two (2) weeks produced by reducing the injection volume and DNA dosage in the tested groups, followed by performing the same kinetic vacuum (KV) treatments using the same vacuum cup device for each group;

[0051] Figures 32A-32E are graphs comparing immune responses, particularly ELISA responses (Figures 32A-32C) and T cell responses (Figures 32D and 32E), in rabbits after treatments involving kinetic vacuum (KV) versus needle electroporation (NEP) and intramuscular electroporation (IM-EP);

[0052] Figure 33 is a graph showing a kinetic vacuum (KV) motion pattern study, comparing the effect of patterns Petition 870250114264, dated 11 / 12 / 2025, page 15 / 175 11 / 111 simplified KV movement and repetitions (cycles) in linkage ELISA data in guinea pigs over two (2) weeks;

[0053] Figures 34A and 34B are graphs comparing binding ELISA data (Figure 34A) and neutralization data of SARS-CoV-2 pseudovirus (Figure 34B) produced in pigs by kinetic vacuum (KV) treatments versus needle electroporation (NEP) treatments;

[0054] Figures 35A and 35B illustrate a study exploring the effect of kinetic vacuum (KV) movements outside the bubble on gene expression; Figure 35A is a plan view showing the kinetic movements of a vacuum cup employed in this study; and Figure 35B is a graph showing quantified values ​​of gene expression visible in guinea pig skin after intradermal injections of a plasmid encoding the gene for green fluorescent protein (GFP) following progressively more spaced-out similar KV movements from the injection bubble;

[0055] Figures 36A-36C are graphs that compare data from ELISA linkage at Week 3 (Figure 36A) and Week 4 (Figure 36B) and T cell responses at Week 4 (Figure 36V) in mice resulting from uniform injection volumes (and DNA doses) of a plasmid followed by kinetic vacuum (KV) treatments with hyaluronidase versus injection only (INJ), static vacuum (SV), and needle electroporation (NEP) treatments;

[0056] Figure 37 is a graph comparing Week 2 linkage ELISA data in guinea pigs after different dosages of hyaluronidase added to uniform dosage injections of plasmid DNA followed by uniform kinetic vacuum (KV) treatments;

[0057] Figure 38 is a graph showing the quantified migration of immune cells in guinea pigs after treatment with Petition 870250114264, dated 11 / 12 / 2025, page 16 / 175 12 / 111 kinetic vacuum (KV) versus needle electroporation (NEP) treatment, showing in particular the number of GFP-positive cells in the lymph nodes of subjects 3 days after treatment for the KV versus NEP treatment groups;

[0058] Figure 39 is a graph showing Week 2 linkage ELISA data in rabbits after low-dose injections of plasmid DNA followed by kinetic vacuum (KV) treatments, particularly comparing the immunogenic results provided by the addition of hyaluronidase to the low-dose injections;

[0059] Figure 40 is a graph showing Week 2 linkage ELISA data in rabbits after mid-dose injections of plasmid DNA followed by kinetic vacuum (KV) treatments, particularly comparing the immunogenic results provided by the addition of hyaluronidase to the mid-dose injections;

[0060] Figure 41 is a graph showing the quantified migration of immune cells in rabbits after kinetic vacuum (KV) treatments, particularly showing the number of GFP-positive cells in the individual's lymph nodes on days 1, 2, 3 and 7 post-treatment, thus indicating the period(s) in which the migration of GFP-positive cells occurs;

[0061] Figure 42 is a graph showing Week 2 linkage ELISA data in guinea pigs after low-dose DNA injections and subsequent kinetic vacuum (KV) treatments, particularly comparing the tradeoffs resulting from dilution of low-dose DNA injections with various hyaluronidase dosages;

[0062] Figure 43 is a graph comparing the effect on Week 2 ELISA binding data in guinea pigs produced by adjusting the number of repetitions (cycles) of a kinetic vacuum motion pattern (KV); Petition 870250114264, dated 11 / 12 / 2025, page 17 / 175 13 / 111

[0063] Figure 44 is a graph comparing the effect on Week 2 binding ELISA data in guinea pigs provided by the combination of intradermal (ID) injections of mRNA-1273 (an mRNA vaccine formulated using lipid nanoparticles) at regular and low doses with kinetic vacuum (KV) treatments versus intramuscular (IM) injections of mRNA-1273 at regular and low doses without KV treatments;

[0064] Figure 45 is a graph comparing the effect on Week 2 binding ELISA data in guinea pigs provided by the combination of intradermal (ID) injections of mRNA-1273 (an mRNA vaccine formulated using lipid nanoparticles) at regular, medium, and low doses with kinetic vacuum (KV) treatments versus regular and low dose ID injections of mRNA-1273 without KV treatments; and

[0065] Figure 46 is a graph showing Week 2 linkage ELISA data in guinea pigs after high-dose DNA injections and subsequent kinetic vacuum (KV) treatments, particularly comparing the tradeoffs resulting from dilution of high-dose DNA injections with various hyaluronidase dosages. DETAILED DESCRIPTION OF ILLUSTRATIVE MODALITIES

[0066] The present invention can be more easily understood by reference to the following detailed description taken in connection with the accompanying figures and examples, which form part of this invention. It should be understood that this invention is not limited to the specific devices, methods, applications, conditions or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing specific embodiments only by way of example and is not intended to limit the scope of the present invention.

[0067] In addition, as used in the specification, including the Petition 870250114264, dated 11 / 12 / 2025, page 18 / 175 14 / 111 attached claims, the singular forms a, an, and the singular forms include the plural, and the reference to a specific numerical value includes at least that specific value, unless the context clearly indicates otherwise.

[0068] The term plurality, as used herein, means more than one. When a range of values ​​is expressed, another modality includes from a specific value and / or to another specific value. Similarly, when values ​​are expressed as approximations, by using the antecedent about, it will be understood that the particular value forms another modality. All ranges are inclusive and combinable.

[0069] The terms approximately, about, and substantially, as used herein with respect to dimensions, angles, proportions, and other geometries, take manufacturing tolerances into account. Furthermore, the terms approximately, about, and substantially may include 10% more or less than the stated dimension, proportion, or angle. Additionally, the terms approximately, about, and substantially may be equally applied to the stated specific value.

[0070] The term kinetic vacuum (KV), as used herein, means a moving vacuum pressure field and is caused by the movement of a vacuum applicator (e.g., a vacuum cup) relative to a target tissue surface.

[0071] The term static vacuum (SV), as used herein, means a vacuum pressure field that does not move relative to the target tissue surface. An example of a static vacuum might include a vacuum cup that applies vacuum pressure to the tissue while remaining stationary relative to the tissue.

[0072] The term electroporation (EP), as used herein, means employing an electric field within the tissue that increases time Petition 870250114264, dated 11 / 12 / 2025, page 19 / 175 15 / 111 rapidly and reversibly alters the permeability and / or porosity of cell membranes in the tissue, thus allowing an agent to be introduced into the cells.

[0073] When used here as a postposition to a primary reference character, the character n (e.g., Pn) indicates that the primary reference character (P) may have an open number of counterparts. For example, when referring to various spatial positions P1, P2, P3, etc. shown in the Figures, the combined reference character Pn when used below indicates that there may be additional positions not shown in the respective Figure(s).

[0074] It should be understood that, although terms involving numerical prepositions (e.g., first, second) may be used here to describe various characteristics, such characteristics should not be limited by these terms. These terms are used to distinguish one characteristic from another. For example, a first element could be called a second element in another context and, similarly, a second element could be called a first element in another context, without departing from the scope of the modalities described here.

[0075] The modalities described herein refer to systems and devices that perform kinetic vacuum treatment on tissues, particularly on a target tissue layer, particularly cutaneous tissue, although the treatments described herein may be adapted for adipose tissue and / or muscle tissue. These modalities subject a target area of ​​tissue to a moving vacuum pressure field (i.e., kinetic vacuum) to impose mechanical stress and tension on the target tissue (such as deforming the tissue) in a manner conducive to improving the delivery of the agent in vivo, particularly by ways of increasing the fluid dispersion (spreading) of the injectable within the tissue, increasing the cellular uptake of the agent directly. Petition 870250114264, dated 11 / 12 / 2025, page 20 / 175 16 / 111 te in target tissue cells (e.g., nucleic acid transfection) and enhancing immune responses to injectables (i.e., injected agents). Consequently, it can be said that the modalities described herein provide various ways to enhance agent delivery. The agents delivered by the kinetic vacuum treatments described herein include, by way of non-limiting examples, plasmids (e.g., DNA vaccine plasmids), peptides, small molecules, nucleic acids, DNA-encoded synthetic monoclonal antibodies (DMAbs), DNA-encoded synthetic proteins, cancer antigens, viral antigens associated with chronic infection, bacterial or other microorganism antigens or proteins, and combinations thereof.

[0076] In the embodiments described herein, an open end of a vacuum device, such as a vacuum cup, is placed in contact with an external tissue surface (e.g., skin) overlying a volume of tissue; vacuum pressure is applied to the interior of the cup, thereby pulling a portion of the tissue within the target area into the vacuum cup and momentarily holding the tissue portion within the cup. While the vacuum field is applied to the tissue, the cup is moved relative to the tissue surface, which transmits mechanical stresses and strains (i.e., deformations) to at least part of the tissue that is momentarily and / or reciprocally pulled into the vacuum cup.

[0077] It has been observed that such kinetic vacuum treatments generate a predictable and substantially uniform zone of cell uptake (e.g., transfection zone) within the target tissue area. It has also been observed that the kinetic vacuum treatments provided by the modalities described below provide favorable fluid redistribution within the target tissue area, including favorable in vivo dispersion of an injectable within the tissue volume and also Petition 870250114264, dated 11 / 12 / 2025, page 21 / 175 17 / 111 Favorable in vivo fluid entry and exit into and out of the target zone. For example, the kinetic vacuum treatments described here have been observed to increase the dispersion of the injected fluid throughout the tissue to enlarge the transfection zone and also attract more in vivo fluids to the target tissue area, increasing the number of cells exposed to transfected cells.

[0078] It has been observed that the kinetic vacuum treatments described throughout this invention resulted in increased responses of individuals to the injections. Compared to a static vacuum procedure, in which vacuum pressure is applied and then removed without translating, rotating, or moving the vacuum applicator, kinetic vacuum procedures generate a stronger and more extensive transfection of nucleic acids, a wider dispersion of the injectable throughout the target tissue, and stronger resulting immune responses when the injectable is a vaccine, particularly a nucleic acid vaccine.

[0079] It has also been observed, surprisingly and unexpectedly, that the kinetic vacuum treatments described herein, without the use of electroporation, can cause comparable, and may even surpass, cellular uptake and immune responses resulting from treatments involving electroporation. While not wishing to be limited to any specific theory, it is believed that the fluctuations and movements of the vacuum pressure gradient transmitted by the kinetic use of the vacuum cup (i.e., moving the vacuum pressure field) impose stresses and mechanical strains on the cell membranes within the tissue volume, which increases cell membrane permeability and therefore the observed cellular uptake within the tissue volume. It is further believed that the aforementioned fluid redistribution and mechanical strains likely interact with each other to create a favorable environment within the tissue volume for cellular uptake (e.g., transfection) of external agents directly. Petition 870250114264, dated 11 / 12 / 2025, page 22 / 175 18 / 111 in cells. The use of kinetic vacuum also appears to benefit uniquely from the addition of a spreading agent (e.g., hyaluronidase) that allows the injected substance to flow more freely through the tissue, which is believed to be due to the fluid redistribution capabilities of kinetic vacuum mentioned earlier. For example, adding hyaluronidase to an injectable has been observed to dramatically increase the effect of kinetic vacuum in increasing the dispersion of the injectable throughout the skin tissue.

[0080] Referring to Figures 1A-1B, a vacuum treatment system 100 for treating a patient according to the present invention includes a vacuum applicator 2, which includes a housing body 4 that defines an internal vacuum chamber 6. The vacuum applicator 2 may also be called a vacuum cup or simply a cup. The housing body 4 may be referred to as a cup housing. The cup housing 4 extends from a proximal end 8 to a distal end 9 along a transverse direction Z. The distal end 9 is spaced from the proximal end 8 in a distal direction D along the transverse direction Z, while the proximal end 8 is spaced from the distal end 9 in a proximal direction P that also extends along the transverse direction Z and is opposite to the distal direction D. It should be noted that the proximal and distal directions P, D are unidirectional components of the transverse direction Z, which is bidirectional.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 a central axis of the vacuum chamber 6 and / or a central axis of the vacuum cup 2.

[0081] The vacuum cup 2 is configured so that a user (such as a doctor) can place a distal end surface 10 of the vacuum cup 2 on its distal end 9 over an external tissue surface (e.g., skin) targeted for treatment, such as Petition 870250114264, dated 11 / 12 / 2025, page 23 / 175 19 / 111 a portion of the skin surface overlying a liquid injection (which has been previously injected into the tissue). In this position of the vacuum cup 2 on the skin surface, the user can apply vacuum pressure to the vacuum chamber 6 to pull, aspirate, or otherwise induce tissue (e.g., skin tissue) into the vacuum chamber 6. With the tissue aspirated into the vacuum chamber 6, the user can move the vacuum cup 2 along the tissue surface, thus manipulating the tissue along the path of displacement of the cup, a manipulation that is here referred to as kinetic vacuum treatment. The manipulable target tissue according to the kinetic vacuum treatments described below includes the dermal layers (epidermis and dermis) and may include additional layers such as subcutaneous fat (i.e., the adipose layer), as described in more detail below.

[0082] The cup housing 4 may include a proximal surface 15 at or adjacent to the proximal end 8. In the illustrated embodiment, the cup housing 4 includes a peripheral annular lip 17 extending proximally from the proximal surface 15 to the proximal end 8. In other embodiments, the cup housing 4 need not include the peripheral annular lip 17, such that the proximal surface 15 also defines the proximal end 8 of the cup housing 4. Furthermore, the proximal surface 15 may be substantially flat, as shown, although other surface geometries are within the scope of the present invention.

[0083] Vacuum cup 2 includes one or more couplings, such as orifices, for connection to one or more external components. For example, vacuum cup 2 has an orifice 12 to provide fluid communication between the vacuum chamber 6 and a vacuum source 106, such as a vacuum pump. The orifice 12 may be set along a port coupling 14, such as a rod 14, for connection to tubing 16 that provides fluid communication between the vacuum chamber Petition 870250114264, dated 11 / 12 / 2025, page 24 / 175 20 / 111 and the vacuum source 106. The rod 14 may extend proximally from the proximal surface 15 of the housing body 4. The vacuum source 106 may be in electrical communication with a control unit 114 (also referred to herein as a controller), which may include a processor 116 configured to control the operation of the vacuum treatment system 100, including the operation of the vacuum source 106. The processor 116 may be in electronic communication with the computer memory 118 and may be configured to execute software and / or firmware including one or more algorithms to control the operation of the system 100. The processor 116 may also be in electrical communication with a user interface 120, which may include a display 122 to present information related to the operation of the system 100 and a keyboard 124 allowing an operator, as a user, to input information, such as commands, related to the operation of the system 100.It should be noted that display 122 may be a touch screen, allowing the operator to enter information directly onto display 122. It should also be noted that interface 120 may be a computer interface, such as a desktop or laptop computer, or a portable electronic device, such as a smartphone or similar.

[0084] Referring now to Figures 1B-1C, the distal end 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, although other opening shapes are within the scope of the present invention. The distal end 9 of the vacuum cup 2 (and therefore also the opening 20) 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. Inside the vacuum chamber 6, the housing 4 defines an inner surface 22 extending from the surface of the distal end 10 of the housing 4. Petition 870250114264, dated 11 / 12 / 2025, p. 25 / 175 21 / 111 to a proximal end surface 24 within chamber 6. The proximal end surface 24 may define a proximal end 26 of chamber 6. In the illustrated embodiment, chamber 6 is defined by the inner surface 22 and the proximal end surface 24. Furthermore, the vacuum cup 2 of the present embodiment may be called 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 opposite portions of the inner surface 22 along a radial direction R that is perpendicular to and intersects the central axis Z1. Chamber 6 also defines a chamber depth L1 measured from the distal end 9 to the proximal end 26 of chamber 6 along the transverse direction Z.The diameter of chamber D1 can range from about 1.0 mm to about 50.0 mm, more particularly from about 3.0 mm to about 20.0 mm, and more particularly from about 5.0 mm to about 15.0 mm. The depth of chamber L1 can range from about 1.0 mm to about 50.0 mm, more particularly from about 3 mm to about 20 mm, and more particularly from about 5 mm to about 17 mm.

[0085] Housing 4 also defines an outer surface 28 that is spaced from the inner surface 22 along the radial direction R. Housing 4 also defines a wall 30 that extends from the inner surface 22 to the outer surface 28 of housing 4 along the radial direction R. In the illustrated embodiment, housing 4 has a substantially circular geometry in a reference plane that extends along the first and second X, Y directions, which are perpendicular to each other and also perpendicular to the transverse direction Z. Consequently, the inner and outer surfaces 22, 28 and the wall 30 can rotate uniformly around the central axis Z1 of Petition 870250114264, dated 11 / 12 / 2025, p. 26 / 175 22 / 111 circumferential shape, as shown. It should be noted, however, that other housing geometries are within the scope of the present invention, including the housing (and chamber) geometries described in U.S. Patent Publication No. 2021 / 0290941 A1, published September 23, 2021, entitled VACUUM-ASSISTED ELECTROPORATION DEVICES, AND RELATED SYSTEMS AND METHODS (hereinafter, Reference '941), the entire invention of which is incorporated herein by reference as if set forth herein in its entirety.

[0086] The housing 4 may be made of a preferably transparent or semi-transparent material, thus allowing visualization of the tissue aspirated into the vacuum chamber 6 during use. As shown, the housing 4 may include graduations 32 along the outer surface 28 and be configured to provide a visual indication of the depth to which the tissue is pulled into the chamber 6. The material of the housing 4 may also have a measure of flexibility, particularly by means of elastic deformation, which may reduce patient discomfort during use.The housing material 4 may be a polymeric material, including polyetheretherketones (PEEK), polyphthalamides (PPA), polyethylenes, polycarbonates, polythermides (PEI), polyvinyl chlorides (PVC), polytetrafluoroethylenes (PTFE), polyamides, polyimides, polysiloxanes (silicone), polyethylene terephthalates, polyurethanes, crosslinked or non-crosslinked rubbers (elastomers), polyesters, by way of non-limiting examples. It should be noted that other biocompatible and / or medical-grade materials may be employed for housing 4. Housing 4 may be a monolithic structure that defines the vacuum cup 2, as in the embodiments illustrated herein. In other embodiments, however, housing 4 need not be a monolithic structure and may include two or more body components coupled together. Petition 870250114264, dated 11 / 12 / 2025, p. 27 / 175 23 / 111 define the housing 4. In addition, the surface of the distal end 10 of the vacuum cup 2 may be subjected to one or more finishing processes to reduce the roughness of the surface finish or to smooth and / or polish the surface of the distal end 10, thereby reducing friction with the skin surface during kinetic vacuum treatments and thus improving patient comfort during and after treatment.

[0087] Referring now to Figure 1C, the inner surface 22 preferably has a generally bell-shaped geometry. The inner surface 22 has a primary surface portion 22a that extends between the distal and proximal ends 10, 26 of chamber 6. The primary surface portion 22a may have a linear profile in an axial reference plane, although other profile geometries are within the scope of the present invention. As shown in Figure 1C, the primary surface portion 22a may have a linear profile that is oriented at an acute conical angle A0 with respect to a linear axis oriented along the transverse direction Z in the axial reference plane. In this way, the inner diameter of chamber 6 decreases towards the proximal end 26.The taper angle A0 can be in a range of about 0 degrees to about 60 degrees, and more particularly in a range of about 0.5 degrees to about 15 degrees, and more particularly in a range of about 5 degrees to about 10 degrees.

[0088] The inner surface 22 also preferably includes a distal entry portion 22b that extends from the distal end surface 10 to the primary surface portion 22a. The distal entry portion 22b preferably has a conical and radial contour to reduce or otherwise mitigate tissue damage, such as hematomas, at the periphery of the tissue aspirated into the chamber 6 during the use of the vacuum cup 2. In the embodiments described herein, the Petition 870250114264, dated 11 / 12 / 2025, p. 28 / 175 24 / 111 The diameter of chamber D1 is measured at the interface between the distal inlet portion 22b and the primary surface portion 22a. The diameter of chamber D1 may optionally be measured at other locations along the inner surface 22. The inner surface 22 may include a proximal relief portion 22c extending from the primary surface portion 22a to the proximal end surface 24. The proximal relief surface 22c is preferably rounded or otherwise shaped to reduce stress concentrations within the housing 4 during use. The interfaces between the primary surface portion 22a and the distal inlet portion 22b and the proximal relief surface 22c are indicated in Figure 1C by reference marks 23.

[0089] The distal end 9 of the vacuum cup 2 also preferably has a rounded or chamfered external relief surface 29 that extends between the surface of the distal end 10 and the external surface 28. In this way, the external relief surface 29 is configured to reduce friction and / or abrasion (e.g., scraping) with the skin on the outer edge of the cup 2 during the kinetic movements of the vacuum cup, thus increasing patient comfort. The interfaces between the external relief surface 29 and the surface of the distal end 10 and the external surface 28 of the cup 2 are indicated in Figure 1C by the reference marks 25. The cup wall 30 defines a wall thickness W1 between the inner and outer surfaces 22, 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 inlet portion 22b and the primary surface portion 22a; and (2) the interface 25 between the external relief surface 29 and the external surface 28. The wall thickness W1 may optionally be measured at other locations along the wall 30. It should be noted that the external surface 28 of the vacuum cup 2, in particular. Petition 870250114264, dated 11 / 12 / 2025, page 29 / 175 25 / 111 mentally at the interface 25 with the external relief surface 29, can be used as a visual reference when performing kinetic movements of the vacuum cup. For example, this interface 25 can be used to visually determine when the vacuum cup 2 has been translated through and / or out of the injection site, as described in more detail below.

[0090] Referring now to Figure 1D, the vacuum cup 2 is configured for placement on a tissue surface, such as a skin tissue surface 1, and overlaying an injectable fluid 7 onto the tissue, with the surface of the distal end 10 of the vacuum cup 2 in contact with the tissue surface 3 or with an intermediate substance, such as a gel, oil, lotion, or lubricant to facilitate the smooth movement of the cup along the tissue surface 3. With the vacuum cup 2 positioned in this manner, vacuum pressure can be supplied to chamber 6 through orifice 12 in sufficient magnitude to pull tissue 1 into chamber 6. As shown, tissue 1 can assume a mound-like or dome-like shape within chamber 6 in response to the vacuum pressure.The tissue can be pulled into chamber 6 at various depths L2, measured from the apex of the tissue to the distal end 9 of chamber 6, in response to the magnitude of the vacuum pressure imparted to chamber 6. The vacuum pressure imparted to chamber 6 can be in a range of about -0.1 psi to about -14.7 psi (about -0.70 kPa to about -100 kPa) (about -5 mmHg to about -760 mmHg) and, more particularly, in a range of about -3 psi to about 14.7 psi (about -20 kPa to about -100 kPa) (about -155 mmHg to about -760 mmHg) and, more particularly, in a range of -7.7 psi to about -14.7 psi (about -53 kPa to about -100 kPa) (about -400 mmHg to about -760 mmHg) and, even more particularly, in a range of about -7.7 psi to about -11.6 psi (about -53. Petition 870250114264, dated 11 / 12 / 2025, p. 30 / 175 26 / 111 kPa to about -80 kPA) (about -400 mmHg to about -600 mmHg).

[0091] Referring now to Figures 2A-2C, another embodiment of a vacuum cup 102 is shown having an internal vacuum chamber geometry different from that of vacuum cup 2 described above. In particular, the vacuum cup 102 of the present embodiment includes a protrusion 34 that extends within the chamber 6 from the surface of the proximal end 24 towards the opening 20. The vacuum cup 102 of the present embodiment may otherwise be similar to vacuum cup 2 described above. Consequently, features of vacuum cup 102 that are similar to those of vacuum cup 2 described above may use the same reference characters above. The protrusion 34 preferentially extends centrally along the central axis Z1. Thus, the protrusion 34 may be referred to here as a central pin 34. Furthermore, the vacuum cup 102 of the present embodiment may be called a central pin vacuum cup 102.

[0092] As shown in Figure 2B, the central pin 34 has a length L3 measured from the surface of the proximal end 24 to a distal end 36 of the post 34. The vacuum cup 102 also defines a pin depth L4 measured from the distal end 9 of chamber 6 to the distal end 36 of pin 34. The central pin 34 of the illustrated embodiment has a rounded distal end geometry. For example, as shown, the central pin 34 has a rounded distal end surface with a radius R1. As shown in Figure 2C, the central pin 34 can be configured to contact the surface 3 of the aspirated tissue into chamber 6 and impose additional mechanical stress and tension on the injected tissue, such as through deformation of the tissue that comes into contact with the central pin 34, which can lead to improved tra results. Petition 870250114264, dated 11 / 12 / 2025, page 31 / 175 27 / 111 treatment, as described in more detail below. It should be noted that various other geometric formations may be employed within chamber 6 to increase mechanical stress and tension on the tissue aspirated into chamber 6. For example, the central pin 34 may have a textured outer surface (e.g., protrusions and / or dimples). Additionally or alternatively, a vacuum cup may have a plurality of posts extending distally within chamber 6, which may have various sizes and geometries and may be arranged in various patterns. Additionally or alternatively, a vacuum cup may have one or more posts that can move relative to the cup housing 4, for example, being compressible or extensible, such as along the transverse Z direction.

[0093] Referring now to Figure 3A, a further embodiment of a vacuum cup 202 has an injection channel 38 that extends through the cup housing 4 and into the vacuum chamber 6. These vacuum cups 202 may be called injection channel vacuum cups 202. The injection channel 38 is configured for the passage of an injectable from a proximal side of the cup 202, through the vacuum cup housing and into the vacuum chamber 6. The injection channel 38 may be centrally located along the central axis Z1 of the respective vacuum cup 202, as shown, or it may be offset from the central axis.

[0094] The vacuum cup 202 may be similar to the vacuum cup 2 described above with reference to Figures 1A-1D, with the injection channel 38 configured for the passage of an injection needle 40 into the vacuum chamber 6. In this embodiment, the vacuum cup 202 preferably includes a septum 42 or other device to form a seal with the injection needle 40 as the needle 40 extends into the vacuum chamber 6. In this way, Petition 870250114264, dated 11 / 12 / 2025, page 32 / 175 28 / 111 the septum 42 or other device can maintain the desired vacuum pressure inside chamber 6 while the injection needle 40 extends through the injection channel 38.

[0095] Referring now to Figure 3B, in other embodiments, a vacuum cup 302 can be configured for use with a jet injector 44 and may be similar to the vacuum cups 2, 102 described above. In the present embodiment, the injection channel 38 is wide enough to receive a nozzle 48 from the jet injector 44. As shown, the nozzle 48 can extend into the vacuum chamber 6 and can optionally be positioned to contact the tissue aspirated into the vacuum chamber 6. In this way, the nozzle 48 can deform the tissue aspirated into the chamber 6 in a manner similar to the central pins 34 described above. In the present embodiment, the vacuum cup 302 may include a mounting or other feature for guided coupling with the jet injector 44.

[0096] It should be noted that vacuum cups 202, 302 shown in Figures 3A-3B can be adapted so that their injection channels 38 are configured for use with other types of injection devices. For example, vacuum cup 202 shown in Figure 3A can be adapted so that the injection channel 38 is configured for use with a jet injector or other type of injection device. Similarly, vacuum cup 302 shown in Figure 3B can be adapted so that the injection channel 38 is configured for use with an injection needle or other type of injection device.

[0097] Referring now to Figures 4A-4B, another embodiment of a vacuum cup 402 is shown having one or more electrodes for tissue electroporation during a vacuum treatment, a treatment which may be referred to here as vacuum electroporation (VEP) treatment. Thus, the vacuum cup 402 of the present embodiment Petition 870250114264, dated 11 / 12 / 2025, page 33 / 175 29 / 111 may be referred to herein as a VEP 402 cup. The VEP 402 cup of the illustrated embodiment was primarily used for testing to compare the treatment outcomes resulting from KV and SV treatments with VEP treatments. It should be understood, however, that the VEP 402 cup can be employed to deliver kinetic and / or static VEP treatments within the scope of the present invention.

[0098] The VEP cup 402 of the present embodiment is similar to the vacuum cup 102 described above with reference to Figures 2A-2C, with one difference being that the VEP cup 402 has a central electrode 50 on the central pin 34 and a concentric ring electrode 52 on the distal end 9 of the cup 402. Consequently, the VEP cup 402 of the present embodiment may also be referred to as a central pin electrode VEP cup 402. The central and circular electrodes 50, 52 are configured to deliver one or more electroporative pulses to the aspirated tissue into the vacuum chamber 6 during a vacuum treatment. The electrical parameters of one or more electroporative pulses (e.g., electrical potential (voltage), magnitude of electrical current (amperage), pulse duration, delay between pulses, and number of pulses) may include those described in more detail in Reference '941.

[0099] The ring electrode 52 of the illustrated example extends into a first external electrode end 54, located near an interface with the external surface 28 of the cup housing 4, to a second internal electrode end 56 located on the primary surface portion 22a of the internal surface 22. Thus, the ring electrode 52 can extend along the distal end 9 and the internal surface 22 of the VEP cup 402 and can also define the surface of the distal end 10 of the vacuum cup 402. A transmission element, such as a wire or conducting post 55, extends from the annular electrode 52 to the outside of the cup housing 4 and is Petition 870250114264, dated 11 / 12 / 2025, page 34 / 175 30 / 111 configured to transmit one or more electroporative pulses to the annular electrode 52. As shown, the conductive pin 55 may extend proximally through the cup housing 4 to a contact 57 on the proximal surface 15. The center electrode 38 of the illustrated embodiment defines the center pin 34, although in other embodiments the center electrode 38 may extend along an outer surface of the center pin 34. A proximal end of the center electrode 38 defines a contact 59, which may be located on the proximal surface 15 of the cup housing 4. It should be noted that in other embodiments, a VEP cup may employ several other electrode configurations, including any of those described in Reference '941. It should also be appreciated that the VEP 402 cup can be configured with an injection channel 38 for an injection needle 40 and / or a jet injector 44, similar to the vacuum cups 202, 302 described above with reference to Figures 3A-3B.

[00100] Referring now to Figures 5A-5B, any of the vacuum cups 2, 102, 202, 302, 402 described above can be adapted for attachment to a handling assembly 58 of the vacuum treatment system 100. For illustrative purposes, the handling assembly 58 is shown coupled to the vacuum cup 402 described above with reference to Figures 4A-4B. The handling assembly 58 includes a handling element 60 having a first distal end 62 and a second proximal end 64 opposite the distal end 62. At the distal end 62, the handling element 60 has a mounting formation 66 which is configured to engage in a releasable manner with the proximal end 8 of a vacuum cup. For example, one or more of the vacuum cups 2, 102, 202, 302, 402 described herein may be configured to have a complementary mounting structure 68 that is configured to couple in a releasable manner (i.e., to couple and uncouple repeatedly and non-destructively) to Petition 870250114264, dated 11 / 12 / 2025, page 35 / 175 31 / 111 assembly formation 66 of the handling assembly 58. In this way, the handling assembly 58 can be configured to interchangeably couple with one or more or even all of the vacuum cups 2, 102, 202, 302, 402 described herein. The handling assembly 58 may also include at least one button or trigger 67 that can be configured to control the operation of the vacuum cup 402, such as to control the start and end of the application of vacuum pressure within the vacuum chamber 6, by way of non-limiting example.

[00101] Referring now to Figure 5B, an exemplary handling assembly formation 66 and a complementary cup mounting structure 68 will now be described. For illustrative purposes, the vacuum cup 402 is rotated in a bottom view, while the represented part of the handle element 60 remains in side view. The handle mounting formation 66 may include a base surface 70 and a peripheral bearing surface 72 recessed proximally from the base surface 70. The peripheral bearing surface 72 extends annularly along a periphery of the handle mounting formation 66. The base surface 70 of the handle mounting formation 66 is configured to interact with the proximal surface 15 of the vacuum cup 402 when the cup 402 is attached to the handle mounting formation 66.Furthermore, when cup 402 is attached to the handle mounting formation 66, the peripheral support surface 72 of the handle mounting formation 66 is configured to interact with the proximal end 8 of cup 402, with the peripheral annular lip of the cup 17 extending proximally from the base surface 70 to the peripheral support surface 72. In this way, the coupling engagement between the peripheral annular lip of the cup 17 and the peripheral support surface 72 can provide a centering mechanism for the vacuum cup 402 relative to the handle mounting formation 66.

[00102] The handle mounting formation 66 includes one or more. Petition 870250114264, dated 11 / 12 / 2025, page 36 / 175 32 / 111 fastening elements 74, such as fastening tips 74, extending from the base surface 70 and configured to fasten with one or more complementary fastening formations 76, such as keyed slots 76, of the cup mounting structure 68. The keyed slots 76 are defined in 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 that extends circumferentially from the respective wide slot portion 78. The fastening pins 74 have an extension portion 82 and a locking portion 84 that projects laterally from the extension portion 82.During the coupling of the cup 402 with the handle assembly 58, the locking portions 84 of the fixing pins 74 are configured to advance distally through the wide groove portions 78 of the keyed grooves 76 (while the peripheral annular lip of the cup 17 is advanced to the peripheral support surface 72 of the handle mounting formation 66). Subsequently, the vacuum cup 402 is rotated about the central axis Z1 so that the locking portions 84 protrude over the housing body 4 along the narrow groove portions 80, thus providing mechanical interference between the locking portions 84 and the housing body 4 in the proximal direction P so as to maintain the fixation of the cup 402 to the handle mounting structure 66.To decouple the vacuum cup 402 from the handle mounting structure 66, the vacuum cup 402 is rotated in the opposite direction around the central axis Z1 until the locking portions 84 are aligned with the wide groove portions 78, and the vacuum cup 402 can then be moved in the distal direction D away from the handle mounting structure 66.

[00103] With continuous reference to Figure 5B, handle element 58 includes a vacuum channel 86 configured to couple and provide fluid communication with orifice 12 of vacuum cup 402 for co Petition 870250114264, dated 11 / 12 / 2025, page 37 / 175 33 / 111 transmit vacuum pressure to vacuum chamber 6 when vacuum cup 402 is coupled to handle assembly 58. A distal end of vacuum channel 86 and a proximal end of orifice 12 may have one or more complementary orifice coupling elements to provide a sealed orifice connection between them when vacuum cup 402 is coupled to handle assembly 58.

[00104] Furthermore, as shown, the handle assembly 58 can be adapted for use with VEP cups 402. Consequently, 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 to the handle assembly 58. As shown, the handle mounting structure 66 may include a first contact 88 to contact the contact 59 of the center electrode 50 and a second contact 90 to contact the contact 57 of the annular electrode 52. The contacts 88, 90 of the handle assembly 58 may be in electrical communication with electronic circuits, which may include a printed circuit board (PCB) 92, which may be in electronic communication with an electronic control device to control one or more electroporative pulses, such as the controller 114, which may be located inside or outside the handle assembly. 58.

[00105] Referring again to Figure 5A, the handle assembly 58 may include one or more conduits 94 extending away from the handle element 60. The one or more conduits 94 may include tubing 16 providing fluid communication between the vacuum channel 86 (and therefore also the vacuum chamber 6) and the vacuum source 106. One or more conduits 94 may also include electrical conduits to provide electrical communication between the handle assembly 58 and an external electrical device.

[00106] It should be understood that the set of handle 58 and its for Petition 870250114264, dated 11 / 12 / 2025, p. 38 / 175 34 / 111 handle mounting mechanism 66 and the complementary mounting structure 68 of the vacuum cup described above are offered as non-limiting examples of such components and features, and that various other designs and configurations are within the scope of the present invention.

[00107] Referring now to Figures 6A-6G, a non-limiting example of a kinetic vacuum treatment will be described. In this example, the kinetic vacuum treatment is shown performed by vacuum cup 2 described above with reference to Figures 1A-1D, although it should be noted that similar vacuum treatments may employ any of the other vacuum cups 102, 202, 302, 402 described above. For illustrative purposes, the fabric in Figures 6A-6G has superimposed graduated markings (-4 to +4) to provide a visual reference for the cup movements and distances shown in this non-limiting example of kinetic vacuum treatment. Each interval of these graduated markings is equivalent to the radius R2 of vacuum chamber 6 (R2 = ½ D1).

[00108] As shown in Figure 6A, an injectable fluid containing a drug is administered to the intradermal tissue 1 (i.e., skin tissue) by an injection needle 40, such as by means of Mantoux injection, which creates a bolus of the injected fluid 7 in the intradermal tissue below the skin surface 3. The bolus of injected fluid 7 may cause the formation of a lump or blister 11 on the skin surface 3. As shown in Figure 6B, after the injection, the needle 40 is removed and, optionally, a substance 13 may be applied to the skin surface 3 along the target treatment site around the injected fluid 7 to facilitate the smooth movement of the cup along the skin surface 3 during kinetic vacuum treatment. The substance 13 may be a gel (e.g., ultrasound gel), oil, lotion, or other lubricant. Petition 870250114264, dated 11 / 12 / 2025, page 39 / 175 35 / 111

[00109] As shown in Figure 6C, the vacuum cup 2 can be placed over the substance 13 on the skin surface 3 in a first position P1 adjacent to the injected fluid 7. In this example, the first position P1 is centered over the injected fluid 7 (i.e., the bubble 11). Alternatively, in the first position P1, the cup 2 can be placed at least partially over the bubble 11, but not centered over it, or it can be placed completely offset from the bubble 11.

[00110] As shown in Figure 6D, vacuum pressure can be supplied to vacuum chamber 6, thus applying a vacuum field to the tissue (particularly to the skin surface 3) below vacuum chamber 6, sufficient to draw the tissue into chamber 6. 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 aspirated into 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 aspirated into chamber 6 can be determined by several factors, including the vacuum pressure supplied.

[00111] As shown in Figure 6E, vacuum cup 2 is translated a first distance in a first direction along the skin surface 3 from the first position P1 to a second position P1, thus also effectively pulling the underlying tissue into and out of chamber 6 as chamber 6 passes over. As shown in Figure 6F, vacuum cup 2 is translated a second distance in a second direction opposite to the first direction along the skin surface 3 from the second position P2 to a third position P3, again effectively pulling the underlying tissue into and out of chamber 6 as chamber 6 passes over. It should be noted that vacuum cup 2 may Petition 870250114264, dated 11 / 12 / 2025, page 40 / 175 36 / 111 be moved back and forth across the skin surface 3 between positions P1-P3 shown in Figures 6E and 6F numerous times, including three (3) or more times, by way of non-limiting example. As shown in Figure 6G, the vacuum cup 2 can be removed from the skin surface 3 and the treatment can be completed.

[00112] As shown in the previous example, the translation distance from the first position P1 to the second position P2 is substantially equivalent to the diameter of chamber D1 of cup 2, causing the rear side of the inner surface 18 at P2 to assume the position that the front side of the inner surface 18 occupied at P1. Thus, if the width of bubble 11 is substantially equal to the diameter of chamber D1 and the first position P1 is centered on bubble 11, such movement from P1 to P2 causes the rear side of the inner surface 18 to move from one lateral edge of bubble 11 to the opposite lateral edge of bubble 11. Under these conditions, this movement also causes chamber 6 to go from encompassing substantially the entire bubble 11 at P1 to being substantially displaced from bubble 11 at P2 and P3. Thus, such movements at these distances can effectively cause the edges of chamber 6 to transition between opposite edges of bubble 11.In this way, the user can reference or index the kinetic movements of the vacuum cup by the spatial relationship between the inner surface 18 and the edges of the bubble 11. Thus, during each translation in this example, the user can employ 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 semi-transparent, the inner surface 18 can be used as a visual reference governing the translation.

[00113] Referring now to Figure 6H, in another example of a kinetic vacuum treatment, the outer surface 28 of cup 2 can be used as a visual reference governing the translation. Petition 870250114264, dated 11 / 12 / 2025, page 41 / 175 37 / 111 of the vacuum cup. For illustrative purposes, the fabric in Figure 6H has graduated markings (-6 to +6) superimposed on it to provide a visual reference for the exemplary movements and distances of the cup. In this example, cup 2 is translated from a first position P1 centered on bubble 11 to a second position P2 that is offset from bubble 11, then to a third position P3 offset from bubble 11 on the opposite side of it, then back to a fourth position P4 that is coincident with the first position P1. At the displacement positions P2, P3 of this example, the outer surface 28 of the vacuum cup 28 is spaced from the near edge of bubble 11 by a distance substantially equivalent to one of the graduated intervals (i.e., ½ D1). In other examples, the displacement positions P2, P3 may be such that, at these positions, the near side of the outer surface 28 of cup 2 is substantially aligned with the near edge of bubble 11.In other examples, the user can translate cup 2 from P1 to P2 and from P2 to P3 until the back side of the outer surface 28 reaches the opposite edge of the bubble 11, as guided by visual observation. In these examples discussed with reference to Figure 6H, the translation distances between positions P1-P4 are determined by the width of the bubble 11 and the wall thickness W1 of vacuum cup 2.

[00114] With reference to Figures 7A-7D, examples of cup movements according to various techniques for providing kinetic vacuum treatments will be described. It should be noted that the illustrated examples shown in these Figures are provided for illustrative purposes and for discussion of exemplary types of kinetic vacuum cup movements within the scope of the present invention. It should also be noted that these kinetic vacuum treatments are indicated in Figures 7A-7D by circles labeled with the reference numeral 2z, which generally represent a 2z vacuum cup in Petition 870250114264, dated 11 / 12 / 2025, page 42 / 175 38 / 111 various positions described. In these illustrated examples, the reference circles 2z more accurately represent the position of the vacuum chamber 6; therefore, these exemplary illustrated movements are indexed to the inner surface of the cup 18, which defines the lateral limits 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 circles 2z shown in Figures 7A-7D can analogously represent the outer surface 28 of the cup 2z, depending on how the user decides to index the cup positions. It should also be noted that the vacuum cup 2z shown in these examples represents any of the vacuum cups 2, 102, 202, 302, 402 described herein.

[00115] Referring now to Figures 7A-7B, the example kinetic motions of the vacuum cup include the linear translation of the 2z cup forward and backward, such as laterally from side to side along the first X direction, as shown in Figure 7A, or vertically up and down or horizontally forward and backward along the second Y direction, as shown in Figure 7B. Translations can occur between various positions P1-Pn along a translation axis, such as the linear axis X3 oriented along the first X direction (Figure 7A) or a linear axis Y3 oriented along the second Y direction (Figure 7B). In these illustrated examples, the 2z cup starts at a first position P1, which may be substantially centered on the injection site or bubble 11.From the first position P1, the 2z cup can be translated in a first direction (X1 in Figure 7A; Y1 in Figure 7B) to a second position P2 on one side of bubble 11, and then the 2z cup can be translated in a second opposite direction (X2 in Figure 7A; Y2 in Figure 7B) to a third position P3 on the opposite side of bubble 11. In the illustrated examples, the second and third positions P2,. Petition 870250114264, dated 11 / 12 / 2025, page 43 / 175 39 / 111 P3 represent the lateral or terminal limits of the translational movements and are spaced from the first position P1 so that chamber 6 is completely displaced from bubble 11 in the second and third positions P2, P3. Alternatively, cup 2z may remain partially over bubble 11 in the second and / or third positions P2, P3, as described below. It should be noted that the user may select the first direction of translation based on various factors, such as going with or against the grain of the hair on the skin surface, by way of non-limiting example.

[00116] As shown, a translation distance T1 between the first position P1 to the second and third positions P2, P3 can be substantially equivalent to the chamber diameter D1, and a translation distance T2 between the second and third positions P2, P3 can be twice (x2) the translation distance T1. Thus, in the illustrated examples, the cup 2z starts centered on bubble 11 and moves to the lateral positions P2, P3 that are distant from bubble 11 (positions that can be called outside the bubble). For consistency purposes here, each translational movement in a consistent direction (e.g., X1, X2, Y1 or Y2) can be called a pass or slide, while each movement between the terminal limits P2 and P3 can be called a complete pass or complete slide, and each movement from an intermediate position (e.g., P1) to a final position (e.g., P2, P3) can be called a partial or half pass or slide.

[00117] It should be noted that the translation parameters above can be adjusted as needed. For example, the translation distances T1, T2 can vary as needed. Thus, the translation distance T1 does not need to be equivalent to the chamber diameter D1; for example, the translation distance T1 can be greater or less than the chamber diameter D1. Additionally or Petition 870250114264, dated 11 / 12 / 2025, page 44 / 175 40 / 111 Alternatively, the translation distances T1, T2 may be such that cup 2 remains at least partially over bubble 11 during translation. Additionally or alternatively, the first position P1 does not need to be equidistantly spaced from the second and third positions P2, P3. Alternatively, the translational movement may be limited only between the first and second positions P1, P2 which are located at the terminal ends of the translation (i.e., the first position P1 does not need to be intermediate to the terminal ends). It should also be taken into consideration that the user may perform several numbers of slides and / or partial slides during a kinetic vacuum treatment and may pause the cup movement for various durations between slides.

[00118] Referring now to Figure 7C, further examples of cup movements (kinetics) may include the translation of cup 2 along a tortuous path C1 to various positions P1-Pn. The tortuous path C1 may be repeated or partially repeated. The translation of cup 2 along a complete tortuous path C1 may be called a circuit and along a part of it may be called a partial circuit. In the illustrated example, the tortuous path C1 is circular and rotates (i.e., orbits) around an axis Z1 that intersects the skin surface along a direction orthogonal to it. The axis Z1 may intersect the injection site or the bleb 11, as shown, or it may be displaced from the injection site. It should be understood, however, that several other translational paths are within the scope of the present invention, including eccentric (not centered on the Z1 axis), elliptical, spiral, triangular or other polygonal, zigzag, and virtually countless others.Furthermore, the tortuous translational path C1 may include reversals of direction; for example, with reference to the illustrated example, the tortuous path C1 may include... Petition 870250114264, dated 11 / 12 / 2025, page 45 / 175 41 / 111 include the translation of cup 2 along a circular orbital path in a first rotational direction R1 (e.g., counterclockwise) around the Z1 axis and the reversal of the translation to a second opposite rotational direction R2 (e.g., clockwise) around the Z1 axis. It should be noted that numerous variations in the tortuous translational path C1 may be employed as needed during a kinetic vacuum treatment. Additionally or alternatively, during all or part of any of the preceding orbital motions, cup 2z may also be tilted relative to an axis that is orthogonally oriented to the underlying tissue, so as to cause the cup 6 chamber to remain substantially oriented towards the injection site during the orbital motion(s).For example, during an orbit, the 2z cup can optionally be manipulated to undergo a rolling tilt that causes the central axis of the Z1 cup to remain substantially crossing a geometric center of the injected cake 7.

[00119] Referring now to Figure 7D, additional examples of cup movements (kinetics) may include rotating, pivoting, or twisting cup 2 back and forth between various angular positions P1-Pn around an axis Z1 that intersects the skin surface along a direction orthogonal to it. As shown, the axis Z1 may be centered on the injection site or on the bleb 11, although it may alternatively be offset from there. Each rotational movement between positions may be called a pivot. Cup 2 may be rotated back and forth at various pivot angles A1, A2, A3, An between the angular positions as needed. In the illustrated example, cup 2 is shown rotating from a first position P1 to a second position P2 at a pivot angle A1 of approximately 90 degrees, from the second position P2 to a third position P3 at a pivot angle A2 of approximately 180 degrees, and from the third position P3 back to... Petition 870250114264, dated 11 / 12 / 2025, page 46 / 175 42 / 111 the first position P1 at a pivot angle A3 of approximately 90 degrees. It should be noted, however, that the above items are provided as non-limiting examples and that numerous variations in torsional movements may be employed as needed during a kinetic vacuum treatment.

[00120] It should also be taken into consideration that a user may employ various combinations of the translational (forward and backward, circular) and rotational (twisting or pivoting) movements mentioned above during a kinetic vacuum treatment.

[00121] Referring again to Figures 7A-7D, the various positions of the 2z cup can also be defined by reference to a coordinate system. For example, the linear back-and-forth translations shown in Figures 7A and 7B can be defined by reference to a coordinate system, such as a two-dimensional (x,y) Cartesian coordinate system with x and y axes extending along the X and Y directions, respectively. In the examples illustrated here, the x and y axes intersect at the injection site; therefore, the coordinate system of these examples is centered at the injection site. Thus, in the examples illustrated in Figures 7A and 7B, when the 2z cup is centered over bubble 11, as in the first position P1, this position of the cup can also be denoted as the (0,0) position.

[00122] Referring again to Figure 7A, an exemplary sequence of kinetic back-and-forth motions along the x-axis is shown in Figure 7E and can be characterized as follows (a pattern that is also referred to here as Pattern A). Starting from the position (0,0) centered on bubble 11, cup 2z can undergo a first translation a distance t in the first direction X1 along the x-axis to the second position P2, which can also be denoted as the position (t, 0). From the position (t, 0), cup 2z can undergo a se Petition 870250114264, dated 11 / 12 / 2025, p. 47 / 175 43 / 111 second translation, at a distance t in the second direction X2, back to position (0,0), after which the 2z cup can undergo a third translation, at a distance t in the second direction X2, to the third position P3, which can also be denoted as the position (-t, 0). From the position (-t, 0), the 2z cup can undergo a fourth translation, at a distance t along the first direction X1, back to position (0,0), which can be the final position in the translation sequence. Thus, the previous translation sequence can be characterized as: (0,0)→(t,0)→(0,0)→(t,0)→(0,0). It should be considered that the third translation (0,0)→(-t,0) can occur without discontinuity or deceleration after the second translation (t, 0)→(0,0). In these cases, the second and third translations can be characterized as being two parts of a single translational movement from (t,0) to (-t,0) (that is, from P2 to P3).It should also be noted that the 2z cup can remain in any of the following positions: (0,0), (t,0), (0,0), (-t,0), (0,0) in sequence and / or in any intermediate positions between them.

[00123] Referring again to Figure 7B, an exemplary sequence of kinetic back-and-forth motions along the y-axis can be characterized as follows. Starting from position (0,0), the 2z cup can undergo a first translation, a distance t in a first direction Y1 along the y-axis to the second position P2, which can also be denoted as position (0,t). From position (0,t), the 2z cup can undergo a second translation, a distance t in a second direction Y2, back to position (0,0), after which the 2z cup can undergo a third translation, a distance t in the second direction Y2, to the third position P3, which can also be denoted as position (0,-t). Starting from position (0,-t), the 2z cup can undergo a fourth translation, a distance t along the first direction Y1, back to position (0,0), which can be the final position in the translation sequence. Thus, the previous translation sequence can be characterized Petition 870250114264, dated 11 / 12 / 2025, p. 48 / 175 44 / 111 characterized as: (0,0)à(0,t)à(0,0)à(0,-t)à(0,0). As above, the third translation (0,0)à(0,-t) can occur without discontinuity or deceleration after the second translation (0,t)à(0,0). In these cases, the second and third translations can be characterized as being two parts of a single translational motion from (0,t)à(0,-t) (i.e., from P2 to P3). It should also be appreciated that the 2z cup can remain in any of the positions (0,0), (0,t), (0,0), (0,-t), (0,0) in sequence and / or in any intermediate positions between them.

[00124] The linear back-and-forth translation sequences described above can be repeated numerous times, each of which can be called a cycle. It should be taken into account that the sequence of positions can be maintained or reversed from one cycle to the next. Furthermore, the 2z cup can remain at rest between cycles or, alternatively, a subsequent cycle can begin immediately after the completion of the previous cycle. It should also be noted that the linear back-and-forth translation sequences can be adapted in numerous ways without departing from the scope of the described embodiments.

[00125] Referring again to Figure 7C, the tortuous (orbital) rotating path C1 around the Z1 axis can also be defined by reference to a coordinate system, such as a two-dimensional polar coordinate system (r,θ) centered on the Z1 axis, where r denotes the radial distance from the Z1 axis and θ denotes the polar coordinate (the angle of the zero-angle position).In the illustrated example, the position (0,0) coincides with the Z1 axis. Thus, in the illustrated example, the 2z cup would be at position (0,0) if centered on bubble 11. Using these polar coordinates, an exemplary sequence of a rotating circular translation (orbital) will now be described. The 2z cup can be placed at a first position P1 that is displaced from the Z1 axis by the distance te positioned at the zero-angle polar position, which can be denoted by the position (t,0). From this position, the sequence... Petition 870250114264, dated 11 / 12 / 2025, page 49 / 175 Example 45 / 111 consists of translating the cup 2z along a circular path C1 that makes one (1) complete revolution (orbit) around the Z1 axis. Thus, the revolution example includes the translation of the cup 2z along the circular path C1, in the first rotational direction R1, from the 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 denoted as (t, 2π). Thus, the preceding rotary 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), (t, 3π / 2) are uniformly spaced from each other; however, in other embodiments, the rotary translation sequence may employ unequally spaced angular positions.

[00126] Furthermore, the rotary translation of this sequence can occur at a substantially constant speed (i.e., without discontinuity or deceleration between positions), except perhaps with some acceleration at the beginning (from (t,0)) and deceleration at the end of the sequence (to (t,0)). It should also be appreciated that the 2z cup can remain at any of the positions (t,0), (t, π / 2), (t, π), (t, 3π / 2), (t,0) in the sequence and / or at any intermediate positions between them. The sequence can be repeated numerous times, each of which can be called a cycle. As discussed above, the direction of rotation (and therefore the sequence of positions) can be maintained or reversed from one cycle to another. Furthermore, the 2z cup can remain at rest between cycles or, alternatively, a subsequent cycle can begin immediately after the completion of the previous cycle.It should be understood that rotational (orbital) translations can be adapted in numerous ways without departing from the scope of the modalities described.

[00127] Referring again to Figure 7D, the movements of Petition 870250114264, dated 11 / 12 / 2025, p. 50 / 175 46 / 111 torsion of the 2z cup can also be characterized with reference to a polar coordinate system. Thus, the first, second, and third positions P1, P2, P3 of the illustrated example can also be denoted as (0, π / 2), (0,0), (0, π), respectively. Similarly to what is described above, a sequence of torsion movements can be repeated numerous times, each of which can be called a cycle. Furthermore, the direction of rotation (and therefore the sequence of positions) can be maintained or reversed from one torsion cycle to the next. Additionally, the 2z cup can remain in any position throughout a cycle and / or between cycles, or alternatively, a subsequent cycle can begin immediately after the completion of the previous cycle. It should be noted that torsion movements can be adapted in numerous ways without departing from the scope of the described embodiments.

[00128] Referring now to Figure 7F, an exemplary one-way translational motion of the cup is shown. In this motion example, cup 2 can be placed in a first position P1 which is displaced from a bubble center Z2 by a first displacement distance along a direction. From the first position P1, the cup can be translated through the bubble center Z2 along the direction to a second location P2, which can be spaced from the bubble center Z2 by a second displacement distance measured along the direction. Preferably, the first and second displacement distances are greater than or equal to (i.e., not less than) a maximum internal dimension of the chamber (such as the chamber diameter D1 for circular cup shapes). This displacement spacing ensures that the entire vacuum chamber passes over the bubble center Z2 during one-way sliding. However, other displacement distances may be employed.When the first and second displacement distances are substantial. Petition 870250114264, dated 11 / 12 / 2025, p. 51 / 175 47 / 111 mind-equivalent and both are larger than the maximum internal dimension of the chamber, the unidirectional KV motion through the center of the Z2 bubble can be referred to here as Pattern C, which can also be denoted as: (-t,0)^(t,0).

[00129] Unidirectional kinetic vacuum (KV) motion patterns, such as Pattern C, can be repeated multiple times (cycles). Between these repetitions, the vacuum field does not need to be maintained in the tissue. For example, after performing a unidirectional glide through the bubble, cup 2 can be removed from the tissue (or the vacuum pressure can be discontinued) and cup 2 can be relocated to the first position P1 (or a different position offset from the bubble center Z2), from which another unidirectional glide position can be performed. It should be understood that the above examples represent non-limiting examples of a unidirectional translational kinetic vacuum (KV) motion.

[00130] In any of the previous examples shown in Figures 6A-7D, it should be noted that the cup can optionally lose suction (e.g., by removing the cup from the tissue) between movements. In other words, kinetic vacuum (KV) treatments do not need to apply active vacuum pressure for the entire duration of the treatment. This can increase ease of operation for the user, who can choose to remove or pop the cup from the tissue and reposition it (including in a different position) as needed during a kinetic vacuum (KV) treatment.

[00131] Furthermore, although the previous examples of kinetic vacuum (KV) treatments illustrate each treatment being administered in connection with a single fluid injection 7, any of the KV treatments described herein can be administered to treat multiple fluid injections 7, which can be arranged in various patterns, as described in more detail below with reference to Petition 870250114264, dated 11 / 12 / 2025, page 52 / 175 48 / 111 Figure 24.

[00132] It should also be noted that any of the previous examples of kinetic vacuum (KV) treatments may also employ cup movement in the proximal and / or distal P, D directions (i.e., away from the tissue and / or toward (or into) the tissue, respectively) during, before, or after the application of vacuum pressure. These proximal and / or distal movements may further stress and deform the aspirated tissue to improve agent delivery. TEST RESULTS OVERVIEW

[00133] The results of the tests relating to kinetic vacuum treatments are described below with reference to Figures 8-46. In these tests, several parameters pertaining to kinetic vacuum (KV) treatments were studied to evaluate, among other things, the efficacy and importance of such parameters, including injection volume and dosage, use of adjuvants, addition of hyaluronidase, movements of the kinetic vacuum cup, geometry and diameter of the cup, vacuum pressure and skin thickness at the treatment site, by way of non-limiting examples.Furthermore, in these tests, various kinetic vacuum (KV) treatments were studied to evaluate their performance (e.g., gene expression, immune response, potential skin surface damage) compared to other vaccine-related treatment techniques, including vacuum electroporation (VEP), static vacuum (SV) treatments, injection-only (INJ) treatments, needle electroporation (NEP), intramuscular electroporation (IM-EP), and mRNA vaccine injections. It should be noted that in the description of the following studies, all plasmid injections were performed via Mantoux injection into intradermal (ID) tissue (i.e., skin layers) unless otherwise indicated. Petition 870250114264, dated 11 / 12 / 2025, page 53 / 175 49 / 111 (as for intramuscular electroporation (IM-EP) treatments and intramuscular (IM) mRNA injections).

[00134] The following treatments and their test results include the following: INJECTION ONLY (INJ):

[00135] An intradermal injection. In guinea pigs, the injection is performed on the skin over the flank. In rabbits, the injection is usually applied to the skin over the quadriceps. The injection-only (INJ) treatments discussed below did not involve vacuum treatment or electroporation. STATIC VACUUM (SV):

[00136] An intradermal injection followed by the application of negative pressure (vacuum pressure) via a vacuum cup. The vacuum pressure is used to evacuate the vacuum cup and pull the target tissue (e.g., skin) into the cup. The skin is then subjected to vacuum pressure for a predetermined period of time until the vacuum pressure ceases. After the pressure inside the vacuum cup returns to normal, the cup is removed from the skin. The static vacuum (SV) treatments discussed below did not involve kinetic vacuum movements or electroporation. Unless otherwise indicated below, each static vacuum (SV) treatment applies vacuum pressure for a predetermined period of time of approximately 10 to 20 seconds (duration extending from vacuum start to vacuum shutdown). This 10 to 20 second vacuum period was selected for correlation with certain VEP durations (including vacuum start, EP pulsation, and vacuum shutdown). KINETIC VACUUM (KV):

[00137] An intradermal injection followed by the application of vacuum pressure using a vacuum cup. The vacuum pressure is used to evacuate the vacuum cup and pull the target tissue (e.g. Petition 870250114264, dated 11 / 12 / 2025, page 54 / 175 50 / 111 plo, skin) into the cup. After the skin is pulled into the cup, the vacuum cup is manipulated by an external force, causing it to move (i.e., translate, rotate, or a combination of these factors) relative to the skin. After the treatment is complete, the vacuum pressure ceases and the external force is removed. When the pressure inside the cup returns to normal, the vacuum cup is removed. The kinetic vacuum (KV) treatments discussed below did not involve electroporation. Furthermore, unless otherwise indicated below, the KV treatments evaluated below employed the following side-by-side linear sequence (also referred to below as Pattern A): (0,0)→(t,0)→(0,0)→(-t,0)→(0,0) which sequence was performed three (3) times (i.e., 3 cycles) per treatment. Furthermore, unless otherwise indicated below, the vacuum pressure was applied at approximately -500 mmHg.Furthermore, unless otherwise indicated below, each kinetic vacuum (KV) treatment tested below resulted in the application of vacuum pressure for approximately 10 to 20 seconds (duration that includes vacuum initiation, reaching the desired vacuum pressure, followed by cup translation(s) and vacuum shutdown). As above, this 10 to 20 second vacuum period was selected for correlation with certain VEP durations (including vacuum initiation, EP pulsation, and vacuum shutdown). Vacuum Electroporation (VEP):

[00138] An intradermal injection followed by non-invasive electroporation targeting the dermis and epidermis using a vacuum cup with at least one electrode inside the chamber acting on the skin surface. Vacuum pressure is used to evacuate the vacuum cup and pull the target tissue (e.g., skin) into contact with the electrode(s). All VEP test results discussed below were generated using the VEP 402 cup with the center pin electrode shown in the Petition 870250114264, dated 11 / 12 / 2025, page 55 / 175 51 / 111 Figures 4A-4B. Unless otherwise indicated below, the electroporation component of the VEP treatments below involved three consecutive electroporation pulses applied, each pulse having a pulse duration of 50 milliseconds (ms), an electric current of 0.5 A, a maximum voltage of 200 Volts (V), with a pulse delay of 250 ms between pulses. For each pulse, the ring electrode acts as the delivery electrode and the post electrode acts as the return electrode. Unless otherwise indicated, the total duration of each VEP treatment was approximately 10 to 20 seconds (including vacuum start, EP pulsation, and vacuum interruption). The vacuum electroporation (VEP) treatments discussed below did not involve vacuum kinetic movements. Needle electroporation (NEP):

[00139] An intradermal injection followed by invasive electroporation using three (3) needle electrodes, each with an outer diameter of 0.46 mm. The three (3) needle electrodes are arranged in an isosceles triangle, where a pair of electrodes (i.e., the first and second electrodes) are spaced 3 mm apart, while the third electrode is spaced 5 mm from each of the first and second electrodes. The three (3) needle electrodes are inserted into the dermis and epidermis to a depth of 3 mm from the skin surface. Four (4) consecutive electroporation pulses are applied, with a pulse duration of 52 ms each, an electric current of 0.2 A, a maximum voltage of 200 V, with a 250 ms interval between pulses. During pulses 1 and 3, the first electrode acts as the source electrode, while the second and third electrodes act as return electrodes. During pulses 2 and 4, the second electrode delivers the pulse and electrode 3 acts as a return electrode.The needle electroporation (NEP) treatments discussed below did not involve vacuum treatment (neither KV nor SV) or vacuum electroporation (VEP). Petition 870250114264, dated 11 / 12 / 2025, page 56 / 175 52 / 111 Intramuscular electroporation (IM-EP):

[00140] An intramuscular injection of pDNA at a specific depth in the quadriceps muscle using a bolus needle, followed by invasive electroporation using a 5P array through the skin and into the quadriceps muscle. The 5P array has five (5) needle electrodes arranged in an equilateral pentagon pattern with a pattern diameter of 10 mm. The five (5) needle electrodes are configured for an insertion depth of approximately 19 mm. Three (3) consecutive electroporation pulses are applied, with a pulse duration of 52 ms each, an electric current of 0.5 A, a maximum voltage of 200 V, with a 1-second interval between pulses. The three (3) electroporation pulses are 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); and Pulse 3: from electrode 3 (positive) to electrode 5 (negative).

[00141] The intramuscular electroporation (IM-EP) treatments discussed below did not involve vacuum treatment (neither KV nor SV) or vacuum electroporation (VEP). ADDITIONAL TEST DETAILS:

[00142] Some of the tests below involved off-the-shelf (OTS) vacuum devices. Details about these OTS devices are shown in Table 0 below: TABLE 0: Device Label Cup Shape Cup Size (Chamber Diameter) (mm) Measured Vacuum Pressure (mmHg) Cup 102 Circle 12 -500 OTS-O Circle 14 -420 OTS-1 Circle 10 -508 OTS-2 Circle 9.5 -474 OTS-3 Rounded Rectangle 7.75x6.33 -474 Petition 870250114264, dated 11 / 12 / 2025, page 57 / 175 53 / 111 OTS-4 Interchangeable (e.g., circle, ellipse) Interchangeable (e.g., 3.0 - 11.75) -490 OTS-5 Circle 9.9 -492 OTS-6 Circle 6 489 STUDY 1

[00143] Referring now to Figure 8, gene expression in guinea pig skin is shown after intradermal injections of uniform volumes of a plasmid encoding the gene for green fluorescent protein (GFP) and then treatment according to four (4) treatment groups:

[00144] · Group 1: VEP treatment using a vacuum cup with a D1 diameter chamber of 12 mm;

[00145] · Group 2: SV treatment using a 102 center-pin vacuum cup (see Figures 2A-2C) with a chamber diameter D1 of 12 mm;

[00146] · Group 3: SV treatment using a dome-shaped vacuum cup 2 (see Figures 1A-1D) with a chamber diameter D1 of 12 mm; and

[00147] · Group 4: KV treatment, using the same dome-shaped vacuum cup design as Group 3.

[00148] Each group involved two (2) samples (1a,b; 2a,b; 3a,b; 4a,b), each sample receiving three (3) injections aligned in a column on the skin over the flank. Each injection contained the plasmid 5013 (which encodes the gene for GFP) in a volume of 100 uL with a DNA dose of 0.5 ug. More details on the test parameters for this study are shown in Table 1 below: Petition 870250114264, dated 11 / 12 / 2025, page 58 / 175 54 / 111 TABLE 1: Group number (n / group) Plasmid Treatment Central Pin 1.(n=2) 5013 VEP + 2. (n=2) 5013 SV + 3. (n=2) 5013 SV - 4. (n=2) 5013 KV -

[00149] In this study, SV treatments – both with the central pin cup (2a,b) and the dome-shaped cup (3a,b) – produced a detectable GFP signal, with the dome-shaped cup (3a,b) having a donut-shaped signal with a void in the central area of ​​the injection site, and the central pin cup (2a,b) eliminating the central void (essentially filling the donut, so to speak). In this study, KV treatments using the dome-shaped cup (4a,b) produced a GFP signal comparable to the static vacuum treatments (2a,b), but with larger signal areas. Regarding VEP treatments (1a,b), the GFP signal appears smaller than in the other groups, which likely results from the fact that tissue damage caused by electroporation has been shown to mask the GFP signal. STUDY 2

[00150] Referring now to Figure 9, the expression of the GFP gene in guinea pig skin is shown after treatments involving multiple kinetic vacuum cup movements applied by various vacuum cup designs. Individuals were injected intradermally with uniform volumes of a plasmid encoding the gene for GFP and were then treated according to seven (7) vacuum treatment groups:

[00151] · Group 1: KV treatment using lateral movement (STS) (Standard A, 3 cycles) (see Figure 7A), performed by a 102 center-pin vacuum cup (see Figures 2A-2C) with a chamber diameter Petition 870250114264, dated 11 / 12 / 2025, page 59 / 175 55 / 111 D1 of 12 mm;

[00152] · Group 2: KV treatment using up and down (UD) motion (see Figure 7B) performed by the same 102 center pin vacuum cup design as in Group 1;

[00153] · Group 3: KV treatment using the same movement STS of Group 1, but performed by a dome-shaped vacuum cup 2 (see Figures 1A-1D) with chamber diameter D1 of 12 mm;

[00154] · Group 4: kinetic treatment without vacuum (K, without vacuum) using the same STS motion and cup design 102 as Group 1;

[00155] • Group 5: KV treatment using the same movement STS of Group 1, but performed by a dome-shaped vacuum cup 2 with a chamber diameter D1 of 15 mm;

[00156] · Group 6: KV treatment using a circular orbital motion (ORB) (see Figure 7C), particularly pulling the cup along a circular path around the injection site in two (2) orbits / cycles, counterclockwise and then clockwise (i.e., the first circular orbit in a counterclockwise rotational direction R1, the second along the same circular orbit but in a clockwise rotational direction R2, both cycles performed without twisting or pivoting motion around the central axis Z1), performed by the same central pin vacuum cup design 102 in Group 1; during the specific orbital motions employed for this Group, the cup 102 also effectively underwent a rolling vertical tilt which caused the central axis Z1 of the cup to remain substantially crossing a geometric center of the bubble 11;

[00157] · Group 7: KV treatment using a twisting motion (TWT) (see Figure 7D), particularly rotating the cup 360 degrees clockwise around the central axis Z1, and then rotating the cup 360 degrees counterclockwise around the central axis Z1), performed by the same central pin vacuum cup design 102 in Group Petition 870250114264, dated 11 / 12 / 2025, page 60 / 175 56 / 111 1.

[00158] Groups 1-5 involved two (2) samples (aeb) each, while groups 6-7 involved one (1) sample (a) each, wherein each of the former samples in this study received three (3) flank ID injections, generally shown in columnar form. Each injection site received an independent vacuum treatment. Thus, Groups 1-5 each have six (6) replicates distributed across two (2) flanks, while Groups 6-7 each have three (3) replicates distributed across one (1) 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. For groups 1-3 and 5-7, vacuum pressure for each treatment was applied for 15 seconds to correlate with VEP durations. Group 4 was the only treatment in this study that did not employ vacuum pressure. Further details regarding the test parameters for this study are shown in Table 2 below: TABLE 2: Group number (n / group) Plasmid Treatment Central Pin Movement Pattern 1. (n=2) 5013 KV + STS: (O,O)à(t,O)à(O,O)à(t,O)à(O,O), Repeated 3 times 2. (n=2) 5013 KV + UD: (O,O)à(O,t)à(O,O)à(O,t)à(O,O), Repeated 3 times 3. (n=2) 5013 KV - STS: (O,O)à(t,O)à(O,O)à(t,O)à(O,O), Repeated 3 times 4. (n=2) 5013 K, no vacuum + STS: (O,O)à(t,O)à(O,O)à(t,O)à(O,O), Repeated 3 times 5. (n=2) 5013 KV, 15 mm - STS: (O,O)à(t,O)à(O,O)à(t,O)à(O,O), Repeated 3 times 6. (n=2) 5013 KV + ORB: (0,0) Orbited 360 Petition 870250114264, dated 11 / 12 / 2025, p. 61 / 175 57 / 111 degrees, repeated 3 times. 7. (n=2) 5013 KV + TWT: (0,0), rotated 360 degrees, repeated 3 times

[00159] From this study, it can be observed that the KV treatments (Groups 1-3 and 5-7; images 1a,b, 2a,b, 3a,b, 5a,b, 6a,b, 7a,b) produce similar GFP responses, while the kinetic treatment without vacuum (Group 4; images 4a,b) produced a weaker signal (GFP response) than the KV treatments. This study also demonstrates that, for vacuum cups of the same size, the center-pin cups 102 and the dome-shaped cups 2 produced similar GFP responses (compare the results of the center-pin vacuum cups in Groups 1-2 and 6-7 with the results of the dome-shaped cups in Group 2, each cup having a D1 chamber diameter of 12 mm).This study further suggests that increasing the cup size (diameter) may reduce the GFP signal in some cases (compare results 5a,b from Group 5 (cup 2 in dome shape with D1 = 15 mm) with results 3a,b from Group 3 (cup 2 in dome shape with D1 = 12 mm), both groups 5 and 3 moving the cup from side to side), although more tests are needed regarding the independent effect of cup size / diameter on the GFP signal. This study also shows that circular orbital cup motion (Group 6) and cup twisting motion (Group 7) produced less GFP response than linear translational cup motion (Groups 1-5). STUDY 3.

[00160] Referring now to Figures 10A and 10B, immune responses (ELISA expression) in guinea pigs were tested to evaluate immune responses after kinetic vacuum (KV) treatments compared to immune responses after various static vacuum (SV) treatments, vacuum electroporation (VEP) treatments, and injection-only (INJ) treatments. Petition 870250114264, dated 11 / 12 / 2025, page 62 / 175 58 / 111

[00161] Individuals were injected intradermally with uniform volumes of a plasmid and then treated according to the following treatment groups: • Group 1: VEP treatment using a 402 center-pin electrode VEP cup (see Figures 4A-4B); • Group 2: SV treatment using the same cup design as Group 1; • Group 3: Treatment of SV using a dome-shaped vacuum cup 2 (see Figures 1A-1D); • Group 4: KV treatment using the same central pin cup design as Group 1; and • Group 5: INJ treatment.

[00162] Each group involved six (6) samples, each sample receiving one (1) flank injection of ID. Each injection contained plasmid 2027, which encodes an influenza antigen, in a volume of 100 µL with a DNA dose of 0.05 µg. Group 4 was the only treatment in this study that employed KV. More details on the test parameters for this study are shown in Table 3 below: TABLE 3: Group Number (n / group) Plasmid Delivery Center Pin 1. (n=6) 2027 VEP + 2. (n=6) 2027 SV + 3. (n=6) 2027 SV - 4. (n=6) 2027 KV + 5. (n=6) 2027 INJ N / A

[00163] Combined linkage titer data for all groups are shown in Week 2 (Figure 10A) and Week 4 (after the second vaccination) (Figure 10B). In the results of this study, treatment with KV (Group 4) produced immunogenicity. Petition 870250114264, dated 11 / 12 / 2025, p. 63 / 175 59 / 111 comparable to treatment with VEP (Group 1) and superior to treatments with SV (Groups 2 and 3). Among the SV treatments, the central pin vacuum cup (Group 2) produced superior immunogenicity to that produced by the dome-shaped vacuum cup (Group 3). It was surprising and unexpected that the KV treatments (Group 4) produced immunogenicity substantially equivalent to that produced by the VEP treatments (Group 1). The ability to use KV treatments without electroporation to produce immune responses substantially equivalent to those produced using electroporation (with vacuum or other techniques) could be revolutionary in the field of vaccine delivery.For example, the KV treatments described herein (without electroporation) are believed to provide significant improvements in both immediate pain / irritation and healing time associated with electroporation treatments (particularly invasive electroporation treatments) and, in a more general sense, those associated with methods based on viral vectors, mRNA, proteins, nanoparticles, and other methods that may benefit from the localized and painless administration of the agent provided by KV treatments. The KV treatments described herein are also believed to have the potential to be beneficial in medical fields beyond agent administration.

[00164] Referring now to Figures 10C-10D, the visible effects on the skin imparted by the various treatments discussed above with reference to Figures 10A-10B are shown. In particular, Figure 10C shows the treatment sites immediately after the treatment was applied; and Figure 10D shows the treatment sites seven (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. It can be Petition 870250114264, dated 11 / 12 / 2025, page 64 / 175 60 / 111 observe that treatment with KV (Group 4, images 293L-298L) caused acute redness and irritation on the skin surface immediately after treatment (Figure 10C), although on day 7 there was no visible tissue damage (Figure 10D). In comparison, treatment with VEP did not cause immediate acute visible tissue damage (Figure 10C), but on day 7 some crusting and / or other superficial tissue damage was observed (Figure 10D). STUDY 4

[00165] Referring now to Figures 11A and 11B, the immune responses (ELISA expression) in guinea pigs were tested again to effectively replicate the study shown in Figures 10A-10B to confirm the ELISA results. The same five (5) treatment groups were tested, the main difference being the plasmid used (plasmid 2303 in the present study) and the DNA dose (0.6 ug in the present study). More details on the test parameters for this study are shown in Table 4 below: TABLE 4: Group number (n / group) Plasmid Delivery Central Pin DNA dose (ug) 1.(n=6) 2303 VEP + 60 2. (n=6) 2303 SV + 60 3. (n=6) 2303 SV - 60 4. (n=6) 2303 KV + 60 5. (n=6) 2303 INJ N / A 60

[00166] ELISA data for Groups 1-5 are shown in Figures 11A and 11B. In particular, Figure 11A shows pooled titer data for all Groups at Week 2 and Figure 11B shows pooled titer data for all Groups at Week 4 (after the second vaccination). As shown in the study shown in Figures 10A-10B, the KV treatments (Group 4) produced Petition 870250114264, dated 11 / 12 / 2025, page 65 / 175 61 / 111 showed immunogenicity comparable to treatment with VEP (Group 1). In the present study, however, the KV treatment (Group 4) did not significantly outperform the SV treatments (Groups 2 and 3) at Week 4. The KV treatments (Group 4) showed an increased ELISA response at Week 2 compared to the SV treatments (Groups 2 and 3), but showed similar responses at Week 4 after the second vaccination was administered. Furthermore, at Week 2, the KV treatments (Group 4) showed ELISA responses comparable to those of the VEP treatments (Group 1). Also demonstrated in the present study, the INJ treatment (Group 5) showed strong ELISA responses at Weeks 2 and 4. Typically, seroconversion is not observed at Week 2 for injection-only treatments, such as those shown in the present study.These exceptionally strong results for the INJ treatments (Group 5) may make it difficult to differentiate between the best and worst ELISA responses in this specific study, considering that the lower baseline is exceptionally high. While not wanting to be limited to any specific theory, it is believed that the exceptionally strong results for the INJ treatments suggest that the DNA dosages in this study (0.6 µg for each injection) were higher than necessary for this specific animal (guinea pig), which is supported by the Week 4 ELISA responses, which continue to overlap across all groups, with even the weakest responses being substantially higher than the detection limit. STUDY 5

[00167] Referring now to Figures 12A and 12B, a study attempted to explore the effect of reducing DNA doses (by reducing the injection volume) with kinetic vacuum (KV) treatment on immune responses (binding ELISA responses) in guinea pigs. In this study, individuals in two groups (Group 1 and Group 2) Petition 870250114264, dated 11 / 12 / 2025, page 66 / 175 62 / 111 were injected intradermally with a plasmid and then treated via KV using the same central pin cup design. The only difference between the Groups was the agent volume: Group 1 administered an injection volume of 100 µL, while Group 2 administered a reduced injection volume of 50 µL. The agent in both groups had a DNA concentration of 0.05 mg / mL. Both groups involved five (5) samples, each sample receiving one (1) ID injection of plasmid 2027 in the right flank. ELISA responses for both groups at Week 2 are shown in Figure 12A and at Week 4 are shown in Figure 12B.

[00168] As indicated, the reduced dose (Group 2) had no detectable impact on the ELISA response at Week 2 (Figure 12A), but showed a slight reduction in titers 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 the dose, as the difference between the groups was small and not significant, but this study provides some evidence that the immunogenicity of KV treatments may increase with injection volume. STUDY 6

[00169] Referring now to Figure 13, immune responses (ELISA expression) in unexposed guinea pigs were tested to evaluate immune responses after kinetic vacuum (KV) treatments compared to immune responses after vacuum electroporation (VEP) treatments, static vacuum (SV) treatments, and injection-only treatments.

[00170] Individuals were injected intradermally with uniform volumes of a plasmid and then treated according to the following treatment groups: • Group 1: VEP treatment using a 402 center-pin electrode VEP cup (see Figures 4A-4B); Petition 870250114264, dated 11 / 12 / 2025, page 67 / 175 63 / 111 • Group 2: KV treatment using a 102 center-pin vacuum cup design (see Figures 2A-2C); • Group 3: SV treatment using the same 102 center-pin vacuum cup model used in Group 2; • Group 4: INJ Treatment

[00171] Each group involved eight (8) samples, each sample receiving one (1) ID injection in the left flank. Each injection contained the 2027 plasmid in a volume of 100 uL with a DNA dose of 0.05 ug. Further details on the test parameters for this study are shown in Table 5 below: TABLE 5: Group number (n / group) Plasmid Delivery 1. (n=8) 2027 VEP 2. (n=8) 2027 KV 3. (n=8) 2027 SV 4. (n=8) 2027 INJ

[00172] Title data are shown for Groups 1-4 in Week 2. The results of this study are consistent with the above studies in unexposed subjects, showing that the ELISA responses produced by SV treatments are greater than those produced by INJ treatments, and that KV treatments produce ELISA responses greater than those produced by SV and INJ treatments and that are at the same level as those produced by VEP treatments. One noteworthy item from this study: one subject from Group 2 was removed from the study because anesthesia was interrupted during vaccination, preventing the vacuum procedure from being applied correctly after injection. The removal of this subject does not affect the overall results described above. Petition 870250114264, dated 11 / 12 / 2025, page 68 / 175 64 / 111 STUDY 7

[00173] Referring now to Figure 14, this study evaluates the effects that the number of translations or slides and the use of hyaluronidase have on the immune response (ELISA expression) in guinea pigs that received kinetic vacuum (KV) treatments.

[00174] Individuals were injected intradermally with uniform volumes of a plasmid and then treated according to the following treatment groups: • Group 1: KV Treatment (Standard A, 3 cycles - see Figure 7A); • Group 2: KV treatment employing a single linear translation (one slip), (0,0) to (t,0), also referred to here as Pattern B, 1 cycle, using the same center-pin vacuum cup design as Group 1; and • Group 3: KV treatment equal to Group 1 (Pattern A, 3 cycles), but using a dome-shaped vacuum cup 2 (see Figures 1A-1D); • Group 4: KV treatment, the same as Group 2 (Pattern B, 1 cycle), but using the same dome-shaped vacuum cup design as Group 3; and • Group 5: KV treatment, the same as Group 1 (Pattern A, 3 cycles), but with the injection including hyaluronidase.

[00175] Each group involved six (6) samples, each sample receiving one (1) intradermal (ID) injection in the left flank. Each injection contained plasmid 9517 in a volume of 100 uL with a DNA concentration of 0.25 mg / mL. As mentioned above, the injection for Group 5 included hyaluronidase. The results of the combined titers are shown in Figure 14. More details on the test parameters for this study are shown in Table 6 below: Petition 870250114264, dated 11 / 12 / 2025, page 69 / 175 65 / 111 TABLE 6: Group Number (n / group) Plasmid Treatment Central Pin Motion Cycle Pattern Number of Cycles HYA 1. (n=6) 9517 KV + Pattern A: (0,0)→(t,0)→(0,0)→(-t,0)→(0,0) 3 - 2. (n=6) 9517 KV + Pattern B: (0,0)→(t,0) 1 - 3. (n=6) 9517 KV - Pattern A: (0,0)→(t,0)→(0,0)→(-t,0)→(0,0) 3 - 4. (n=6) 9517 KV - Pattern B: (0,0)→(t,0) 1 - 5. (n=6) 9517 KV + Pattern A: (0,0)→(t,0)→(0,0)→ (-t,O)à(O,O) 3 +

[00176] In the results, Groups 1, 3, and 5, which employed Pattern A, 3 cycles (6 slides in total), produced a greater immune response than the Pattern B groups, 1 cycle (1 slide) (Groups 2 and 4). The results also demonstrate that the addition of hyaluronidase to KV treatment significantly improves immunogenicity. Furthermore, when comparing the results of Group 1 with Group 2 and when comparing Group 3 with Group 4, it is not clear in this study whether the 102 central pin vacuum cup significantly improves immunogenicity compared to the 2 dome cup for KV treatments, although the data slightly point in that direction. Study 8

[00177] Referring now to Figure 15, this study evaluates the effects that vacuum pressure has on the immune response (ELISA expression) in guinea pigs that received similar kinetic vacuum (KV) treatments. Petition 870250114264, dated 11 / 12 / 2025, page 70 / 175 66 / 111

[00178] Individuals in five (5) groups were injected intradermally with uniform volumes of a plasmid and then received KV treatments employing different vacuum pressures but the same kinetic movements of the vacuum cup (Standard A, 3 cycles). The groups employed the following vacuum pressures: 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 for this study); and Group 5 used approximately 420 mmHg. Groups 1 through 4 performed the KV treatments using the same 102 center pin cup design. Group 5 used an off-the-shelf vacuum applicator (OTS), labeled OTS-0 in Table 0, which is the MarvelouSIim applicator, manufactured by Zemits Kosmetik Experte, based in Carlsbad, California, United States.The OTS-O applicator uses a vacuum head with a chamber diameter of approximately 14 mm and distal rollers (ball bearings seated on the distal surface) that facilitate the translation of the vacuum head across the skin surface.

[00179] Each group involved six (6) samples, each sample receiving one (1) intradermal (ID) injection in the left flank. Each injection contained plasmid 2303 in a volume of 100 uL with a DNA concentration of 0.3 mg / mL. For the vacuum cup kinetic movements, each group employed three (3) series of linear translations (six (6) slides in total) at the injection site. Further details on the test parameters for this study are shown in Table 7 below: TABLE 7: Group number (n / group) Plasmid Treatment Vacuum device Vacuum pressure (mmHg) 1. (n=6) 2303 KV cup 102 -100 2. (n=6) 2303 KV cup 102 -300 Petition 870250114264, dated 11 / 12 / 2025, page 71 / 175 67 / 111 3. (n=6) 2303 KV cup 102 -500 4. (n=6) 2303 KV cup 102 -600+ 5. (n=6) 2303 KV OTS-O —420

[00180] The combined ELISA results for each group are shown in Figure 15. These results show a clear correlation between vacuum strength and immunogenicity. In particular, ELISA responses increased with increasing vacuum pressure. Furthermore, in this study, the OTS-O applicator at approximately -420 mmHg (Group 5) produced results generally comparable to the center-pin cups at -500 and -600 mmHg (Groups 3 and 4), although it was observed that the OTS-O applicator tended to inadvertently detach from the skin surface during translation (sliding). The fact that the OTS-O applicator detached from the skin surface during use was not surprising, considering that the literature on the OTS-O applicator mentions the ability to detach to facilitate comfort during use. It was found surprising, however, that the detachment of the OTS-O applicator during use did not appear to significantly decrease the immunogenicity results. STUDY 9

[00181] Referring now to Figures 16A-16B, this study evaluates immune responses (ELISA expression) in rabbits, particularly comparing immune responses produced by kinetic vacuum (KV) treatments in relation to immune responses produced by treatments involving injection only (INJ), static vacuum (SV), vacuum electroporation (VEP) and needle electroporation (NEP).

[00182] Individuals were injected intradermally with uniform volumes of a plasmid and then treated according to the following treatment groups: • Group 1: INJ Treatment; • Group 2: SV treatment using a pi vacuum cup design Petition 870250114264, dated 11 / 12 / 2025, page 72 / 175 68 / 111 in the central 102 (see Figures 2A-2C) with a chamber diameter D1 of 12 mm; • Group 3: KV treatment using the same 102 center-pin vacuum cup design used in Group 2; • Group 4: VEP treatment using a 402 center-pin electrode VEP cup (see Figures 4A-4B); and • Group 5: NEP using the CELLECTRA 2000-3P device, manufactured by Inovio Pharmaceuticals, Inc., located in Plymouth Meeting, Pennsylvania, United States.

[00183] Each group involved five (5) samples, each sample receiving one (1) ID injection into the skin over the left quadriceps. Each injection contained plasmid 9517 in a volume of 100 uL with a DNA dose of 2.5 mg / mL. Further details on the test parameters for this study are shown in Table 8 below: TABLE 8: Group number (n / group) Plasmid Treatment 1. (n=5) 9517 INJ 2. (n=5) 9517 SV 3. (n=5) 9517 KV 4. (n=5) 9517 VEP 5. (n=5) 9517 NEP

[00184] Title 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 responses produced by the KV treatments (Group 3) are greater than those produced by the SV treatments (Group 2), which are greater than those produced by the INJ treatments (Group 1). Surprisingly and unexpectedly, in this study the KV treatments produced greater responses. Petition 870250114264, dated 11 / 12 / 2025, page 73 / 175 69 / 111 ELISA results were higher than treatments with VEP (Group 4) and NEP (Group 5). STUDY 10

[00185] Referring now to Figures 17A-17B, a follow-up study replicates the study described above with reference to Figures 16A-16B, evaluating immune responses in rabbits, but using a different plasmid (this time using plasmid 2303 at a DNA concentration of 3.0 mg / mL). All other parameters in this study were the same as in the study shown in Figures 16A-16B. In particular, Figure 17A shows titer data at Week 0 (and is comparable to Figure 16A), and Figure 17B shows titer data at Week 2 (and is comparable to Figure 16A). As in the previous rabbit study, the present follow-up study again demonstrates that, for immune response, KV treatments (Group 3) outperformed VEP treatments (Group 4), with the latter generally being equivalent to NEP treatments (Group 5).In the present follow-up study, INJ treatments (Group 1) were only slightly less immunogenic than SV treatments (Group 2), which in turn were less immunogenic than VEP and NEP treatments (Groups 4, 5), whereas in the previous study (Figures 16A-16B), SV treatments had similar immunogenicity to VEP and NEP treatments. STUDY 11

[00186] Referring now to Figure 18, this study evaluates the impact that hyaluronidase has on kinetic vacuum (KV) and static vacuum (SV) treatments in guinea pigs, compared to vacuum electroporation (VEP) treatments.

[00187] Individuals were injected intradermally with uniform volumes of a plasmid and then treated according to the following treatment groups: • Group 1: Treatment of SV using a vacuum cup design Petition 870250114264, dated 11 / 12 / 2025, page 74 / 175 70 / 111 center pin 102 (see Figures 2A-2C); • Group 2: VEP treatment using a 402 center-pin electrode VEP cup (see Figures 4A-4B); • Group 3: KV treatment (Standard A, 3 cycles) using the same center-pin vacuum cup design used in Group 1; • Group 4: KV treatment using the cup design and kinetic movements as in Group 3, but with hyaluronidase added to the injection; and • Group 5: SV treatment with hyaluronidase added to the injection, but otherwise using the same SV treatment and cup as Group 1.

[00188] Groups 1-3 involved six (6) samples each, while groups 4-5 involved seven (7) samples each. Each sample received one (1) ID injection over the left flank. Each injection contained plasmid 9517 in a volume of 100 uL with a DNA concentration of 0.25 mg / mL. More details on the test parameters for this study are shown in Table 9 below: TABLE 9: Group number (n / group) Plasmid Treatment HYA 1.(n=6) 9517 SV - 2. (n=6) 9517 VEP - 3. (n=6) 9517 KV - 4. (n=7) 9517 KV + 5. (n=7) 9517 SV +

[00189] Titer data for all Groups are shown in Figure 18. In this study, KV treatments (with and without hyaluronidase) produced higher ELISA responses than VEP treatments. Furthermore, in this study, hyaluronidase increased binding titers for KV treatments but not for SV treatments. In fact, SV treatments produced virtually no response. Petition 870250114264, dated 11 / 12 / 2025, pp. 75 / 175 71 / 111 detectable immune proteins in this study, with or without hyaluronidase. STUDY 12

[00190] Referring now to Figure 19, this study evaluates the impacts that different numbers of kinetic vacuum movements (KV) and, separately, skin thickness have on the immune response (ELISA expression) in guinea pigs.

[00191] Individuals were injected intradermally with uniform volumes of a plasmid and then treated according to the following treatment groups, each administering KV treatments using a 102 center-pin vacuum cup model (see Figures 2A-2C), moving the cup according to Pattern A (linearly back and forth at the injection site), but varying the number of cycles and the injection site: • Group 1: injection in the right flank, followed by Pattern A, 2 cycles (four (4) slides in total) with vacuum cup 102; • Group 2: injection in the right flank, followed by Pattern A, 3 cycles (six (6) slides in total) with vacuum cup 102; • Group 3: injection in the right flank, followed by Pattern A, 4 cycles (eight (8) slides in total) with vacuum cup 102; • Group 4: injection in the back (where the skin of individuals is thicker than on the flank), followed by Pattern A, 3 cycles (six (6) slides in total) with the vacuum cup 102; and • Group 5: injection in the abdomen (where the skin of individuals is thinner than on the flank), followed by Pattern A, 3 cycles (six (6) slides in total) with the vacuum cup 102.

[00192] Groups 1-3 involved six (6) samples each, while groups 4-5 involved seven (7) samples each. Each injection contained plasmid 2303, in a volume of 100 uL, and a DNA concentration of 0.3 mg / mL for each Group. More details on the test parameters for this study are shown in Table 10. Petition 870250114264, dated 11 / 12 / 2025, page 76 / 175 72 / 111 below: TABLE 10: Group Number (n / group) Plasmid Treatment Movement Cycle Pattern Number of Cycles Location Skin Tx 1. (n=6) 2303 KV (O,O)à(t,O)à(O,O)à (-t,O)à(O,O) 2 Flank 2. (n=6) 2303 KV (O,O)à(t,O)à(O,O)à (-t,O)à(O,O) 3 Flank 3. (n=6) 2303 KV (O,O)à(t,O)à(O,O)à (-t,O)à(O,O) 4 Flank 4. (n=7) 2303 KV (O,O)à(t,O)à(O,O)à (-t,O)à(O,O) 3 Back 5. (n=7) 2303 KV (O,O)à(t,O)à(O,O)à (-t,O)à(O,O) 3 Belly

[00193] The combined titer data for all Groups are shown in Figure 19. In this study, the difference in the number of linear translations or slips between 4 and 8 slips at the injection site did not significantly impact the binding titers. Furthermore, the location of the treatment site (thin skin versus thick skin) did not appear to produce a significant difference in immunogenicity. STUDY 13

[00194] Referring now to Figures 20A-20B, this study evaluates the impacts that injection volume and hyaluronidase have on gene expression (GFP) after static vacuum (SV) and kinetic vacuum (KV) treatments administered to guinea pigs.

[00195] Individuals in six (6) groups were injected intradermally into the right flank with a plasmid encoding the gene for GFP and were then treated according to the following treatment groups, each group receiving KV or SV treatments using a 102 center-pin vacuum cup model (see Figures 2A-2C) with a D1 chamber diameter of 12 mm: Petition 870250114264, dated 11 / 12 / 2025, page 77 / 175 73 / 111 • Group 1: SV treatment after injection volume of 100 uL; • Group 2: SV treatment, same as Group 1, but with hyaluronidase; • Group 3: KV treatment after an injection volume of 100 uL, the KV treatment used Standard A, 3 cycles, with a 102 vacuum cup; • Group 4: KV treatment, same as Group 3, but with hyaluronidase; • Group 5: KV treatment, same as Group 4, but using a plasmid injection volume of 400 uL; • Group 6: SV treatment, same as Group 2, but using a plasmid injection volume of 400 uL;

[00196] Groups 1 and 3 involved eight (8) samples each, groups 2 and 4 involved twelve (12) samples each, and groups 5 and 6 involved four (4) samples each. The following groups were paired in test subjects: Groups 1-2, Groups 3-4, so that each pair of groups performed treatments on the same subjects side by side. Groups 5 and 6 were not paired with any other group in a test subject. Each injection contained plasmid 5013 (which encodes the gene for GFP) with a DNA concentration of 0.5 mg / mL. More details on the test parameters for this study are shown in Table 11 below: TABLE 11: Group number (n / group) Plasmid Treatment Injection volume (pL) HYA 1. (n=8) 5013 SV 100 - 2. (n=12) 5013 SV 100 + 3. (n=8) 5013 KV 100 - 4. (n=12) 5013 KV 100 + 5. (n=4) 5013 KV 400 + 6. (n=4) 5013 SV 400 +

[00197] The GFP for the groups is shown in Figure 20A, with all images at the same scale and same image settings. Petition 870250114264, dated 11 / 12 / 2025, p. 78 / 175 74 / 111 gem (e.g., exposure time). GFP quantification for the groups is shown in Figure 20B. In this study, KV treatments (Groups 3-5) significantly increased gene expression compared to SV treatments (Groups 1-2 and 6). Furthermore, within the 100 µL injection volume groups (Groups 1-4), KV treatments benefited dramatically from the addition of hyaluronidase (compare Group 3 with Group 4), while SV treatments that included hyaluronidase produced less gene expression on average than those without hyaluronidase (compare Group 2 with Group 1). In the 400 µL injection volume groups (with hyaluronidase) (Groups 5 and 6), KV treatments (Group 5) produced significantly higher gene expression and in a significantly larger expression area compared to SV treatments (Group 6).In kinetic vacuum (KV) treatments using hyaluronidase, treatments with an injection volume of 400 µL (Group 5) showed a drastic increase in gene expression area compared to treatments with 100 µL (Group 4). In static vacuum treatments with hyaluronidase, treatments with 400 µL (Group 6) showed a slight improvement in gene expression compared to treatments with 100 µL (Group 2). STUDY 14

[00198] Referring now to Figure 21, a further study evaluates the impact that high injection volumes with and without hyaluronidase have on the immune response (ELISA expression) in guinea pigs that received kinetic vacuum (KV) or static vacuum (SV) treatments.

[00199] Individuals in four (4) groups were injected intradermally into the left flank with a plasmid and were then treated according to the following vacuum treatment groups, each using a 102 center-pin vacuum cup model (see Figures 2A-2C) with a D1 chamber diameter of 12 mm: Petition 870250114264, dated 11 / 12 / 2025, page 79 / 175 75 / 111 • Group 1: Treatment with KV after an injection volume of 100 uL without hyaluronidase; • Group 2: KV treatment, same as Group 1 (without hyaluronidase), but with an injection volume of 400 uL; • Group 3: Treatment with KV after injection of a 400 µL volume with hyaluronidase; and • Group 4: SV treatment, otherwise identical to that of Group 3 (400 µL with hyaluronidase).

[00200] All groups in this study involved five (5) samples each, with each injection containing plasmid 2303 with a DNA concentration of 0.3 mg / mL. Further details on the test parameters for this study are shown in Table 12 below: TABLE 12: Group number (n / group) Plasmid Treatment Injection Volume (pL) HYA 1. (n=5) 2303 KV 100 - 2. (n=5) 2303 KV 400 - 3. (n=5) 2303 KV 400 + 4. (n=5) 2303 SV 400 +

[00201] The combined titer data for all groups are shown in Figure 21. In this study, treatments without hyaluronidase (Groups 1-2: KV after injection volumes of 100 µL and 400 µL, respectively) showed similar immune responses. Treatments with KV after injection volumes of 400 µL with hyaluronidase (Group 3) showed the strongest immune response of all groups in this study. Treatments with SV after injection volumes of 400 µL with hyaluronidase (Group 4) had the weakest immune response of all groups in this study, which was unexpected, particularly in light of the results of the study discussed above with reference to Figure 20. The results of the present study shown in Fig. Petition 870250114264, dated 11 / 12 / 2025, page 80 / 175 76 / 111 ra 21 suggest that KV treatments may have an additive and adjuvant effect in addition to the benefit of crude gene expression. STUDY 15

[00202] Referring now to Figure 22, a study was conducted to evaluate the effects that the combination of kinetic vacuum (KV) treatments with vacuum electroporation (VEP) has on the immune response (ELISA expression) in guinea pigs.

[00203] Individuals in four (4) groups were injected intradermally into the left flank with uniform volumes of a plasmid and were then treated according to the following vacuum treatment groups, each group using a 402 center-pin electrode VEP cup (see Figures 4A-4B) with a D1 chamber diameter of 12 mm: • Group 1: KV treatment only, administering three (3) series of slides (six (6) slides in total) with the vacuum cup; • Group 2: VEP treatment followed by the same KV treatment as Group 1; • 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 reverse order); and

[00204] All groups in this study involved five (5) samples each, with each injection containing plasmid 9517 with a DNA concentration of 0.25 mg / mL. The vacuum pressure for each treatment was -500 mmHg. Further details on the test parameters for this study are shown in Table 13 below: TABLE 13: Group number (n / group) Plasmid Treatment step 1 Treatment step 2 1. (n=5) 9517 KV N / A 2. (n=5) 9517 VEP KV 3. (n=5) 9517 KV VEP Petition 870250114264, dated 11 / 12 / 2025, page 81 / 175 77 / 111

[00205] Combined titer data for all Groups are shown in Figure 22. In this study, adding VEP before or after KV treatment did not provide any additional immunological benefit and showed a trend toward decreased immune response. STUDY 16

[00206] Referring now to Figures 23A-23B, a study was conducted in guinea pigs to compare the immune responses (ELISA expression), cellular responses (ELISpot data) and neutralization produced by kinetic vacuum (KV) treatments versus treatments with mRNA / lipid nanoparticle injection only (INJ) and needle electroporation (NEP) treatments.

[00207] Individuals received plasmid injections and were treated according to the following treatment groups: • Group 1: NEP treatment after injection of plasmid 9501, in which NEP was treated using the CELLECTRA™ 2000-3P device; • Group 2: KV treatment after injection of plasmid 9501, in which the KV treatment used a 102 center-pin vacuum cup (see Figures 2A-2C) with a chamber diameter D1 of 12 mm; • Group 3: KV treatment, the same as Group 2, but with hyaluronidase added to the injection; • Group 4: INJ treatment with mRNA-1273 (an mRNA-based vaccine for SARS-CoV-2), produced by Moderna Inc., based in Cambridge, Massachusetts, United States.

[00208] Each group involved five (5) samples, each sample receiving one (1) injection. Groups 1-3 received intradermal (ID) injections of the plasmid (9501) in an injection volume of 100 uL, with a DNA dose of 100 ug and a DNA concentration of 1.0 mg / mL, in the skin over the left flank; Group 4 received intramuscular (IM) injections of the plasmid (mRNA-1273) in a volume of Petition 870250114264, dated 11 / 12 / 2025, page 82 / 175 78 / 111 injection of 50 µL, with a DNA dose of 10 µg and a DNA concentration of 0.2 mg / mL, in the left quadriceps. More details on the test parameters for this study are shown in Table 14 below: TABLE 14: Group Number (n / group) Agent Drug Type Treatment HYA Inj. Volume (pL) DNA Dose (pg) 1. (n=5) 9501 DNA NEP - 100 100 2. (n=5) 9501 DNA KV - 100 100 3. (n=5) 9501 DNA KV + 100 100 4. (n=5) mRNA 1273 mRNA Lipid Nanoparticles - 50 10

[00209] Figures 23A and 23B show peak binding data for all groups at Week 2 (after the first dose / treatment) and Week 5 (after the second dose / treatment), respectively. Figure 23C shows ELISpot data for all groups at Week 5 (after the second dose / treatment). Figure 23D shows SARS-CoV-2 pseudovirus neutralization data for all groups on Day 0 and Day 14 (after the first dose / treatment).

[00210] In this study, in terms of binding titer results, as shown in Figures 23A-23B, the mRNA vaccine (Group 4) outperformed the KV plus hyaluronidase treatments (Group 3), which outperformed the KV without hyaluronidase treatments (Group 2), which outperformed the NEP treatments (Group 1). In terms of cellular responses, as shown in Figure 23C, each of the DNA plasmid treatments (Groups 1-3) outperformed the mRNA vaccine (Group 4). In terms of neutralization, as shown in Figure 23D, the mRNA vaccine (Group 4) outperformed the KV plus hyaluronidase treatments (Group 3), which outperformed the treatments with Petition 870250114264, dated 11 / 12 / 2025, page 83 / 175 79 / 111 KV without hyaluronidase (Group 2), which were generally equivalent to the NEP treatments (Group 1).

[00211] In this study, treatments with KV plus hyaluronidase (Group 3) achieved humoral binding responses closest to the mRNA level (Group 4) (Figures 23A-23B) and pseudovirus neutralization responses (Figure 23D), followed by KV without hyaluronidase (Group 2), with NEP treatments (Group 1) generating the weakest humoral responses (Figure 23C). For neutralization (Figure 23D), only the treatments with KV plus hyaluronidase (Group 3) and the mRNA vaccine (Group 4) generated 100% neutralizing activity after one vaccination. Regarding cellular responses (Figure 23C), DNA treatments (Groups 1-3) generally offered stronger cellular responses than the mRNA vaccine (Group 4), although KV plus hyaluronidase treatments (Group 3) showed a tendency toward slightly higher responses than NEP treatments (Group 1) for the wild-type assay. STUDY 17

[00212] Referring now to Figure 24, a study evaluated whether a single kinetic vacuum (KV) treatment can effectively cause transfection of multiple injections (blisters). Subjects in this study were evaluated after receiving intradermal injections of multiple partitions of a uniform combined total volume of two (2) separate plasmids (one encoding the gene for green fluorescent protein (GFP) and the other encoding the gene for red fluorescent protein (RFP)) and then administering a single KV treatment over the blisters. Subjects were evaluated according to four (4) treatment groups: • Group 1 (Control): KV treatment of one (1) injection of plasmid 5013 (which encodes the gene for GFP) in an injection volume of 100 Petition 870250114264, dated 11 / 12 / 2025, page 84 / 175 80 / 111 uL; • Group 2: KV treatment of two (2) side-by-side injections, one of plasmid 5013 and the other of plasmid 9902 (which encodes the gene for RFP), each with an injection volume of 50 uL; • Group 3: KV treatment of four (4) injections in a square pattern, wherein the top row of injections used plasmid 5013 and the bottom row of injections used plasmid 9902, each injection in a volume of 25 uL; and • Group 4: KV treatment of four (4) injections in a linear line of alternating plasmids 5013 and 9902, each injection in a volume of 25 uL.

[00213] Each group involved five (5) replicates, each replicate receiving its respective skin injection(s) over the flank. Each injection included hyaluronidase (HYA). The KV treatment for each group employed Pattern A, 3 cycles, except for Group 3, which employed a modified version of Pattern A. In particular, for Group 3, the first vacuum chamber position was intermediate between the left bubbles in the top and bottom rows. When vacuum pressure was applied to Group 3, the chamber diameter was large enough that the left bubbles in the top and bottom rows were drawn into the vacuum chamber before performing the vacuum kinetic translations, which were performed side to side in a manner that was otherwise consistent with Pattern A (3 cycles).For the multiple injection groups (Groups 2-4), the use of plasmids encoding GFP and RFP was employed to help visualize differences in expression for each injection site and to help visualize the extent to which different injection sites may or may not mix. More details on the test parameters for this study are shown in Table 15 below. TABLE 15: Petition 870250114264, dated 11 / 12 / 2025, page 85 / 175 81 / 111 Group number (n / group) Plasmid(s) Treatment HYA Number of bubbles Bubble volume (ML) Bubble pattern 1. (n=5) 5013 KV + 1 100 Single bubble 2. (n=5) 5013, 9902 KV + 2 50 Two bubbles in a row - horizontal 3. (n=5) 5013, 9902 KV + 4 25 Four bubbles in a square pattern 4. (n=5) 5013, 9902 KV + 4 25 Four bubbles in a row - horizontal

[00214] Based on the results shown in Figure 24, it can be observed that a single KV treatment was able to transfect multiple injection sites in each group, including individuals in the four-injection groups (Groups 3 and 4). This study demonstrates that the KV treatments described here can be successfully employed with injection partitioning in terms of gene expression. STUDY 18

[00215] Referring now to Figure 25, a study evaluated whether the same kinetic vacuum (KV) treatments performed by different individuals (operators) would produce different immune responses (ELISA expression) in guinea pigs. Individuals were injected intradermally into the flank skin with uniform volumes of plasmid 2027 and then treated according to Groups 1 to 4, where individuals in each group were treated by a separate individual (i.e., individuals in Group 1 were treated by Operator 1, individuals in Group 2 were treated by Operator 2, Petition 870250114264, dated 11 / 12 / 2025, page 86 / 175 82 / 111 etc.). Each group involved five (5) replicates, with each operator performing the KV treatment using pattern A, 3 cycles, with a 102 center-pin vacuum cup (see Figures 2A-2C) with a D1 chamber diameter of 12 mm. The results demonstrate that there was no significant operator-induced difference in ELISA expression at 2 weeks when the same KV treatment pattern was employed. STUDY 19

[00216] Referring now to Figures 26A-26C, a study was conducted in guinea pigs to compare the immune responses (ELISA expression) and neutralization produced by DNA-launched nanoparticles (DLNP) with kinetic vacuum (KV) treatments versus mRNA injection-only (INJ) treatments.

[00217] Individuals received plasmid injections and were treated according to the following treatment groups: • Group 1: KV treatment after injection of plasmid 9528 based on DLNP formulated with hyaluronidase (HYA), where KV treatment employed Pattern A, 3 cycles using a 102 center-pin vacuum cup (see Figures 2A-2C) with a D1 chamber diameter of 12 mm; and • Group 2: INJ treatment of mRNA-1273.

[00218] Each group involved five (5) individuals, each individual receiving one (1) injection. Group 1 received intradermal (ID) injections of the plasmid (9528) in an injection volume of 100 uL, with a DNA dose of 10 ug and a DNA concentration of 0.1 mg / mL, formulated with HYA at a dose of 135 U / mL, in the skin over the flank; Group 2 received intramuscular (IM) injections of the plasmid (mRNA-1273) in an injection volume of 50 uL, with an mRNA dose of 10 ug and an mRNA concentration of 0.2 mg / mL, in the tibialis anterior muscle.

[00219] More details on the test parameters for this study Petition 870250114264, dated 11 / 12 / 2025, page 87 / 175 83 / 111 are shown in Table 16 below: TABLE 16: Group number (n / group) Drug Treatment HYA 1.(n=5) 9528 KV + 2. (n=5) mRNA-1273 Lipid nanoparticles -

[00220] Figure 26A shows peak binding data for both groups on Day 14 (Week 2), Day 28 (Week 4), and Day 46 (approximately Week 6%) after treatment, with the first dose / treatment administered on Day 0 and a 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.

[00221] In this study, in terms of binding titer results, as shown in Figure 26A, the DLNP plus KV plus HYA treatments performed comparably to the mRNA vaccine throughout the study. Furthermore, in terms of neutralization, as shown in Figures 26B-26C, the DLNP plus KV plus HYA treatments exhibited more stable neutralizing titers than the mRNA vaccine and maintained a higher level of neutralization for almost one (1) year. Notably, the magnitude of the mRNA-1273 neutralizing titers fell to less than 5% of their peak during the entire monitoring period, while the DLNP plus KV plus HYA injections maintained 34% of the peak response during the last measured time point.Although mRNA-1273 elicited higher peaks of neutralizing antibodies, the difference in this drop-off response between mRNA and KV-contained DLNP led to equivalent neutralization approximately 100 days after the first vaccination, after which KV-contained DLNP exhibited higher titers than mRNA. Petition 870250114264, dated 11 / 12 / 2025, page 88 / 175 84 / 111 STUDY 20

[00222] Referring now to Figure 27, a study was conducted to compare the scattering effect that kinetic vacuum (KV) treatments have on a bolus injected into skin tissue (a blister) compared to that produced by static vacuum (SV) treatments.

[00223] Individuals in each group were injected with a dye (trypan blue) into the skin of the flank and then treated according to the following treatment groups: Group 1 received SV treatment; Group 2 received KV treatment employing Standard A, 3 cycles, using a 102 center-pin vacuum cup (see Figures 2A2C) with a D1 chamber diameter of 12 mm; and Group 3 injections included hyaluronidase (HYA), but otherwise received the same treatment as Group 2. Each group involved five (5) individuals. Blister diameters were measured in each individual after injection, once before treatment, and again after treatment to compare the physical spreading effect produced by the KV and SV treatments. The spreading effect is quantified as a ratio between the pre-treatment diameter and the post-treatment diameter. The blue dye aided in identifying the extent of spreading during the measurement phase.

[00224] As shown, the SV treatments (Group 1) produced only a marginal scattering effect; the KV treatments (Group 2) produced a positive scattering effect; and the KV plus HYA treatments (Group 3) significantly outperformed Groups 1 and 2. STUDY 21

[00225] Referring now to Figures 28A, a study was conducted to compare immune responses (ELISA expression) in guinea pigs after kinetic vacuum (KV) treatments performed by different devices, particularly a (1) vacuum cup of Petition 870250114264, dated 11 / 12 / 2025, page 89 / 175 85 / 111 center pin 102 (see Figures 2A-2C) and four (4) ready-to-use (OTS) vacuum devices, all with similar chamber sizes and applying similar vacuum pressures.

[00226] All individuals in this study received injections of the same plasmid (2303) at the same dosage into the skin of the flank. Individuals were grouped according to the vacuum cup: (1) cup 102 - a center-pin vacuum cup 102 (see Figures 2A-2C) with a chamber diameter of 12 mm; (2) OTS-1 device; (3) OTS-2 device; (4) OTS-3 device; and (5) OTS-4 device. Further details on the test parameters for this study are shown in Table 17 below: TABLE 17: Device Cup Shape Chamber Diameter (mm) Measured Vacuum Pressure (mmHg) Cup 102 Circle 12 -500 OTS-1 Circle 10 -508 OTS-2 Circle 9.5 -474 OTS-3 Rounded Rectangle 7.75x6.33 -474 OTS-4 Circle 8.8 -490

[00227] Each group involved five (5) individuals, and each KV treatment employed Pattern A, 3 cycles. Title data are shown for the groups at Week 2. Results show that the five (5) vacuum devices produced similar immunogenic responses when using similar chamber sizes and applying similar vacuum pressures. STUDY 22

[00228] Referring now to Figure 28B, a study similar to Study 21 (Fig. 28A) was conducted to determine the effect of cup size on immune responses (ELISA titers) in guinea pigs after kinetic vacuum (KV) treatments. All tests Petition 870250114264, dated 11 / 12 / 2025, page 90 / 175 86 / 111 tests in this study were performed using the OTS-4 device (see Table 0), with five (5) different vacuum cups interchangeably attached to it. The use of the same OTS-4 device with different interchangeable cups helped to maintain uniform vacuum pressure when using different chamber sizes and geometries, which are found in Table 18 below: TABLE 18: Group Number (n / group) Plasmid Delivery Cup Format Chamber Diameter (mm) 1. (n=5) 9501 KV Circle 11.75 2. (n=5) 9501 KV Circle 5.8 3. (n=5) 9501 KV Circle 3.8 4. (n=5) 9501 KV Ellipse 8.3 x 3.0 5. (n=5) 9501 KV Circle 8.8

[00229] All individuals in this study received injections of the same plasmid (9501) at the same dosage into the skin of the flank. Individuals were grouped according to the vacuum cup used. Each group involved five (5) individuals, and each KV treatment employed Pattern A, 3 cycles. Title data are shown for the groups at Week 2. For the cup sizes tested, the results show a strong correlation where the immune response increased with cup size. At the lower end of cup size, the smallest cup tested (Group 3: chamber diameter of 3.8 mm) failed to produce a significant immune response. These results demonstrate that, in KV treatments using similar vacuum pressures and motion patterns, cup size is a significant factor in the immune response. STUDY 23

[00230] Referring now to Figure 29, a study was conducted Petition 870250114264, dated 11 / 12 / 2025, page 91 / 175 87 / 111 to determine whether dividing a given injection volume into smaller volumes produced an immunogenic effect in guinea pigs after kinetic vacuum (KV) treatment over those injection sites.

[00231] Individuals received injections of plasmid 2027 and were treated according to the following treatment group: • Group 1: total injection volume of 100 pL in one (1) injection (i.e., 1 bubble partition); • Group 2: total injection volume of 100 pL divided into three (3) injections (i.e., 3 bubble partitions of 33.3 pL each); • Group 3: total injection volume of 300 pL in one (1) injection (i.e., 1 bubble partition); and • Group 4: total injection volume of 300 pL divided into nine (9) injections (i.e., 9 bubble partitions of 33.3 pL each).

[00232] Each group involved five (5) individuals, each individual being injected into the skin of the flank and then administered KV treatment using the OTS-3 device (see Table 0), employing Pattern A, 3 cycles. For the groups with multiple bubble partitions (i.e., groups 2 and 4), the KV treatment involved a single treatment performed on all bubble partitions in the group (i.e., in each Pattern A cycle, the vacuum cup was moved over all bubble partitions in the group). Further details on the test parameters for this study are shown in Table 19 below: TABLE 19: Group number (n / group) Plasmid Treatment Total volume (ML) Bubble partitions Volume per bubble (pL) 1. (n=5) 2027 KV 100 1 100 2. (n=5) 2027 KV 100 3 33.3 3. (n=5) 2027 KV 300 1 300 4. (n=5) 2027 KV 300 3 33.3 Petition 870250114264, dated 11 / 12 / 2025, page 92 / 175 88 / 111

[00233] The title data are shown for the groups in Week 2. For Groups 1-2, the results show no immunological effect when splitting the 100 pL injection volume into three (3) injections, although this may be due to immune responses that were below detectable levels and not necessarily due to a lack of immunological difference between Groups 1 and 2. For the higher total volume groups (Groups 3-4), splitting the 300 pL injection volume into nine (9) injections may have caused a greater trend in the immune response. These results show a trend that splitting the injection volume with KV treatments into high-volume bubbles may increase immune responses. These results also corroborate other test results and data showing that KV treatments are effective in increasing the immune response, provided the treatment involves passing the vacuum cup over the entire injection zone. STUDY 24

[00234] Referring now to Figure 30, another study explores the effect of reducing DNA dosage (by reducing injection volume) with kinetic vacuum (KV) treatment on immune responses (binding ELISA responses) in guinea pigs. Compare with Study 5 (Figures 12A-12B). In the present study, however, needle electroporation (NEP) treatments were also tested for comparison with KV treatments in reduced dosage scenarios.

[00235] In this study, individuals in two groups (Group 1 and Group 2, each group with five (5) individuals) were injected intradermally into the flank with the same plasmid (9501) in uniform volumes (100 uL), uniform doses of DNA (2.5 ug) and formulated with hyaluronidase (HYA), and then administered with KV treatment (Group 1) or NEP treatment (Group 2). The KV treatment was administered using Petition 870250114264, dated 11 / 12 / 2025, page 93 / 175 89 / 111 of a central pin vacuum cup 102 (see Figures 2A-2C, with a chamber diameter D1 of 12 mm, using Standard A, 3 cycles.

[00236] ELISA responses at Week 2 are shown for both groups in Figure 30 (with the dashed line indicating the limit of detectable titers). The results show that, at the low doses tested, the KV treatment group produced a measurable immune response, while the NEP treatment group did not. These results also provide evidence that KV treatments may have dose-saving benefits compared to NEP. STUDY 25

[00237] Referring now to Figure 31, another study was conducted to explore the effect of reducing DNA dosage (by reducing injection volume) with kinetic vacuum (KV) treatment on immune responses (binding ELISA responses) in guinea pigs. Compare with Study 24 (Figure 30). In the present study, individuals in four (4) groups (five (5) individuals per group) received intradermal injections of plasmid 2303 into the skin of the flank, where each successive group received half the dosage of the previous group. Group 1 received an injection volume of 100 uL with a DNA dose of 30 ug; Group 2 received an injection volume of 50 uL with a DNA dose of 15 ug; Group 3 received an injection volume of 25 uL with a DNA dose of 7.5 ug; Group 4 received an injection volume of 12.5 µL with a DNA dose of 3.75 µg.All other factors were constant for each group: KV treatments were administered using the OTS-3 device (see Table 17), employing Standard A, 3 cycles.

[00238] ELISA responses in Week 2 are shown for all Groups. Interestingly, no measurable decrease in ELISA immunogenicity was observed in the reduced dosage groups. Petition 870250114264, dated 11 / 12 / 2025, page 94 / 175 90 / 111 of which provides further evidence that KV treatments have potential dose-saving benefits. STUDY 26

[00239] Referring now to Figures 32A-32E, a study was conducted to compare immune responses, particularly ELISA responses (Figures 32A-32C) and T-cell responses (Figures 32D and 32E), in rabbits after treatments involving kinetic vacuum (KV), needle electroporation (NEP), and intramuscular electroporation (IM-EP).

[00240] In this study, subjects received injections of plasmid 9501 (which encodes the entire length of the SARS-CoV-2 Spike glycoprotein) and were treated according to the following treatment groups: • Group 1: KV treatment (using OTS-3 device, Standard A, 3 cycles) after intradermal injection of a 100 pL volume with a 100 µg dose of DNA, with hyaluronidase (HYA); • Group 2: Treatment with NEP after intradermal injection of a 100 pL volume with a DNA dose of 100 µg; and • Group 3: IM-EP treatment after intramuscular injection of a 1000 pL volume with a DNA dose of 1000 µg.

[00241] Further details on the test parameters for this study are shown in Table 20 below: TABLE 20: Group number (n / group) Plasmid Treatment HYA Total volume (pL) DNA dose (pg) 1. (n=7) 9501 KV + 100 100 2. (n=6) 9501 NEP - 100 100 3. (n=6) 9501 IM-EP - 1000 1000

[00242] ELISA responses are shown for all groups Petition 870250114264, dated 11 / 12 / 2025, page 95 / 175 91 / 111 in Week 2 (Figure 32A), Week 4 (Figure 32B), and Week 5 (Figure 32C). These results show that the KV with HYA treatment outperformed the NEP and IM-EP treatment groups at all time points in terms of ELISA immunogenicity. Although the IM-EP treatments matched the NEP treatments over time, they were unable to match the results of the KV plus HYA treatments.

[00243] T cell responses are shown for all groups at week 2 (Figure 32D) and week 5 (Figure 32E). These results show similar T cell immunogenicity for IM-EP, NEP, and KV plus HYA treatments. STUDY 27

[00244] Referring now to Figure 33, a study was conducted to compare the effects on immune responses (ELISA responses) in guinea pigs provided by different movement patterns and cycles of kinetic vacuum (KV) treatment. The KV movement patterns in this study were all performed using the same vacuum cup device, specifically the OTS-2 device found in Table 17. Individuals were intradermally injected with uniform volumes of plasmid 2303 into the skin over the flank and then received KV treatments according to four (4) movement-specific treatment groups (each group with six (6) individuals): • Group 1: KV treatment using lateral movement (STS) (Standard A, 3 cycles); • Group 2: KV treatment using a unidirectional (UNI) movement, starting outside the bubble at position (-t, 0), employing a linear translation (a glide) through the bubble: (-t,0) to (t,0), also referred to here as Pattern C (see Figure 7F), repeated six (6) times (i.e., 6 cycles); Petition 870250114264, dated 11 / 12 / 2025, page 96 / 175 92 / 111 • Group 3: KV treatment using the same UNI movement as Group 2, but only 4 cycles; and • Group 4: KV treatment using the same UNI movement as Group 2, but only 2 cycles;

[00245] Details regarding the test parameters for this study are summarized in Table 21 below: TABLE 21: Group Number (n / group) Plasmid Treatment Standard Movement KV Standard Abbreviation Number of Cycles 1. (n=6) 2303 KV Standard A: (O,O)à(t,O)à(O,O)à(t,O)à(O,O) STS 3 2. (n=6) 2303 KV Standard C: (-t,O)à(t,O) UNI 6 3. (n=6) 2303 KV Standard C: (-t,O)à(t,O) UNI 4 4. (n=6) 2303 KV Standard C: (-t,O)à(t,O) UNI 2

[00246] ELISA responses are shown for all groups at week 2. The results demonstrate that unidirectional (UNI) movement patterns performed similarly to the lateral movement pattern (STS). Surprisingly and unexpectedly, this remained true even when reducing the number of cycles in unidirectional (UNI) movements to 2 cycles. These results provide evidence that the immunogenic response benefits provided by KV treatment are applicable even when employing only one (1) unidirectional slide on the bubble. Furthermore, when viewed in connection with the operator study results discussed above (Study 18, Figure 25), these results support the conclusion that the KV treatments described here are substantially operator-friendly, i.e., easy to perform and require only one (1) precise slide on the bubble. Petition 870250114264, dated 11 / 12 / 2025, page 97 / 175 93 / 111 STUDY 28

[00247] Referring now to Figures 34A-34B, a study compares the immune responses (ELISA responses) and neutralizing activity in pigs resulting from kinetic vacuum (KV) treatments versus needle electroporation (NEP). In this study, pig subjects in two (2) groups (with three (3) subjects per group) were injected intradermally with uniform volumes of plasmid 9501 (encoding the SARS-CoV-2 Spike glycoprotein) and uniform doses of DNA. Each subject received two (2) injections at separate skin sites over the quadriceps, with KV subjects (Group 2) also receiving hyaluronidase (HYA) with the injection. After injection, subjects in Group 1 received NEP treatment and subjects in Group 2 received KV treatment with the OTS-2 device (see Table 0), using Pattern A, 3 cycles. For both groups, the ELISA responses in Week 2 are shown in Figure 34A and the neutralizing titers are shown in Fig.Figure 34B (with dashed lines in both Figures indicating the limit of detectable titers). In terms of ELISA response (Figure 34A), responses to NEP treatment bordered on the detectability threshold, while KV treatments were comfortably detectable in all individuals. In terms of pseudovirus neutralization (Figure 34B), the KV treatment group showed superior neutralizing activity compared to the NEP treatment group. Despite the fact that swine individuals present challenges in demonstrating ELISA responses after intradermal injections (including with electroporation treatments), the results of this study are consistent with those showing positive ELISA responses in guinea pigs and rabbits resulting from KV treatments. STUDY 29

[00248] Referring now to Figures 35A and 35B, a study was Petition 870250114264, dated 11 / 12 / 2025, page 98 / 175 94 / 111 conducted to evaluate the impacts that off-target kinetic vacuum (KV) treatments (i.e., where the vacuum cup does not contact the center of the injection bubble) have on gene expression (GFP) in guinea pigs. Individuals in four (4) groups (with six (6) individuals per group) were injected intradermally into the flank skin with a plasmid encoding the gene for GFP and then received KV treatment employing the same motion pattern, but at different displacements from the center of the Z2 bubble along the second Y direction perpendicular to the translation direction of the X cup. The KV motions employed in this study are shown in Figure 35A. The KV motion patterns are effectively the same as Pattern C (Figure 7F), but at different displacement distances d along the second Y direction.For quantification purposes, displacement distances d are measured along the second Y direction from the bubble center Z2 to the translation axis X3 of the vacuum cup. The KV treatments for each group were performed using the same OTS-2 device (see Table 0) and grouped according to the bubble displacement distance d, as follows and as shown in Figure 35A:. • Group 1: displacement distance d=0 (i.e., on target, meaning that the translation axis of cup X3 intersects the center of bubble Z2); • Group 2: displacement distance d=t / 2, where t is equivalent to the diameter of the cup chamber in this study; • Group 3: displacement distance d=1, which means that the translation axis of cup X3 is spaced from the center of bubble Z2 by a distance equivalent to the diameter of the cup chamber t; and • Group 4: initial position at d=2, that is, the translation axis of cup X3 is spaced from the center of bubble Z2 by a distance equivalent to two (2) chamber diameters (2t). Petition 870250114264, dated 11 / 12 / 2025, page 99 / 175 95 / 111

[00249] The quantification of GFP for the groups is shown in Figure 35B. The results show that KV movements on the target (i.e., without displacement of the Z2 bubble center) (Group 1) provide strong expression, with expression decreasing at a displacement of d=t / 2 (Group 2) and expression dropping completely when the displacement is d=1 or greater (i.e., the vacuum cup is fully spaced from the bubble during KV movements) (Groups 3 and 4). The results for Groups 3 and 4 are only marginally better (if at all) than the GFP expression after injection (INJ) of GFP alone. Notably, losing the bubble by a small distance (Group 3) performed similarly poorly to losing the bubble by a large distance (Group 4) in terms of gene expression. These results demonstrate that simply stretching the skin around the injection bubble (Group 3) is not sufficient to increase gene expression.Instead, the results suggest that gene expression is increased if the injected tissue is pulled into the vacuum chamber, and increases with the percentage of injected tissue pulled into the vacuum chamber. STUDY 30

[00250] Referring now to Figure 36A-36C, a study was conducted to compare immune responses (ELISA responses) and T cell responses in mice after kinetic vacuum (KV) treatments with hyaluronidase (HYA) versus injection-only (INJ) treatments, static vacuum (SV) treatments, and study-specific intramuscular (IM) needle electroporation (NEP) treatments, labeled below as NEP-IM. Subjects in this study received injections of plasmid 9517 in uniform volumes (30 µL) and uniform doses of DNA (5 µg) and were treated according to the following treatment groups (with five (5) subjects per group): • Group 1: INJ Treatment; Petition 870250114264, dated 11 / 12 / 2025, pages 100 / 175 96 / 111 • Group 2: KV treatment after intradermal injection of plasmid with HYA into the flank skin, KV treatment employed Standard C, 4 cycles, performed using the OTS-6 device with a chamber diameter of 6 mm at a pressure of 489 mmHg (see Table 0); • Group 3: SV Treatment; and • Group 4: NEP-IM Treatment after intramuscular injection, NEP-IM was applied using three (3) needle electrodes, each with a diameter of 0.46 mm and arranged in an isosceles triangle; four (4) consecutive pulses were applied, each with a pulse duration of 52 ms, a defined current of 0.2 A, a maximum voltage of 200 V and a pulse delay of 250 ms.

[00251] ELISA responses are shown for all groups at Week 3 (Figure 36A) and Week 4 (Figure 36B). These results show that the KV plus HYA treatments outperformed the NEP-IM treatment at all time points in terms of ELISA immunogenicity. In turn, the NEP-IM treatments showed immunogenicity at weeks 3 and 4, although less than the KV plus HYA group. The INJ and SV treatments were not immunogenic in this study. T cell responses are shown in Figure 36C for all Groups at Week 4. In terms of T cell immunogenicity, the NEP-IM and KV plus HYA treatments performed similarly, which is consistent with studies in other species, and both treatments significantly outperformed the INJ and SV treatments. Interestingly, the NEP-IM treatments provide good T cell responses comparable to the KV plus HYA treatments, but weaker antibody responses. STUDY 31

[00252] Referring now to Figure 37, a study was conducted to evaluate the effect of hyaluronidase (HYA) dosage in kinetic vacuum (KV) treatments in terms of immune responses (responses Petition 870250114264, dated 11 / 12 / 2025, pp. 101 / 175 97 / 111 ELISA) in guinea pigs. Individuals in five (5) groups received the same KV treatments after the same injections, with the only difference between the groups being 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; and • Group 5: 0 U / mL (i.e., no HYA).

[00253] Each group involved five (5) individuals, who received injections of plasmid 9501 into the skin of the flank in uniform volumes (100 uL) and doses of DNA (25 ug) and subsequently treated with the same KV treatment (Pattern C, 4 cycles), using the same vacuum cup, particularly the OTS-5 vacuum cup (see Table 0).

[00254] The results show that even small doses of HYA added to the plasmid injection provide significant benefits in terms of ELISA immunogenicity, even at a dose of 10 U / ml (Group 4), with a sharp drop in immunogenicity from the 10 U / ml dose (Group 4) to the 0 U / ml dose (Group 5). These results demonstrate the benefit of combining KV treatments with HYA, even at reduced dosages, such as 10% of the typical HYA dosages in KV treatments discussed throughout this invention. Reducing HYA dosages can provide significant cost savings for KV treatments and limits the dilutive impact of HYA on the DNA dose administered to patients. STUDY 32

[00255] Referring now to Figure 38, a study was conducted to compare the effects that kinetic vacuum (KV) treatments and needle electroporation (NEP) treatments have on immune cell migration in guinea pigs. Individuals in two (2) groups Petition 870250114264, dated 11 / 12 / 2025, page 102 / 175 98 / 111 (with four (4) individuals per group) were injected with plasmid 5013 (which encodes the gene for GFP) in uniform volumes and doses of DNA into the skin over the flank. For the first group, the injections included hyaluronidase (HYA) and this group subsequently received KV treatment, using the OTS-5 vacuum cup (see Table 0), employing Pattern C, 4 cycles. The second group received NEP treatment after the injection (which did not include hyaluronidase). Three days after treatment, the lymph nodes of both groups were analyzed to measure the number of GFP-positive cells and to assess the extent of immune cell migration along the immunogenic pathway from the skin, where transfection occurred, to the lymph nodes.

[00256] The results show that treatments with KV plus HYA resulted in higher numbers of cells expressing reporter genes in lymph nodes compared to treatments with NEP. These results are consistent with other studies described here regarding gene expression and immune responses. Furthermore, these results provide evidence that treatments with KV plus HYA successfully increase the migration of immune cells along immunogenic pathways (in this case, GFP trafficking from lymph nodes) in a process subsequent to the initial transfection events. STUDY 33

[00257] Referring now to Figure 39, a study was conducted to evaluate the effect of hyaluronidase (HYA) in kinetic vacuum (KV) treatments with low pDNA dosage in terms of immune responses (ELISA responses) in rabbits. See also Study 31 (Figure 37). The pDNA dosage (1.5 µg) in this study was intentionally set low enough that the treatments were not expected to produce a detectable ELISA response. In essence, this study evaluates whether the use of HYA can salvage a KV treatment. Petition 870250114264, dated 11 / 12 / 2025, pp. 103 / 175 99 / 111 with low success rate. In this study, individuals in two (2) groups (with nine (9) individuals per group) were injected with plasmid 9501 in uniform volumes and doses of DNA (1.5 µg) into the skin over the flank. The injections in the second group included HYA (135 U / mL), while those in the first group did not. After injection, both groups received KV treatment using the OTS-5 vacuum cup (see Table 0), employing Pattern C, 4 cycles. The results show that even at low doses of pDNA, HYA is able to improve detectable ELISA titer responses, with most individuals in the second group producing detectable titer levels, while only two (2) individuals in the first group (without HYA) showed detectable levels. These results show trends that adding HYA to KV treatments may improve or rescue immunogenicity conditions that might otherwise be poorly or non-immunogenic. STUDY 34

[00258] Referring now to Figure 40, a study similar to Study 33 (Figure 39) was conducted in rabbits, but at an average pDNA dosage, specifically at 15 µg (which is ten times (10x) the pDNA dosage used in Study 33). In the present study, a different DNA plasmid was used: plasmid 2303. All other conditions (besides the pDNA plasmid and dosage) in this study were the same as in Study 33. Again, individuals in two groups (with nine (9) individuals per group) were injected with plasmid 2303 in uniform volumes and average doses of DNA (15 µg) into the skin over the flank. The injections in the second group included hyaluronidase (HYA) at 135 U / mL, while those in the first group did not. After the injection, both groups received KV treatment using the OTS-5 vacuum cup (see Table 0), employing Pattern C, 4 cycles. The results are consistent with those of Study 33, showing here that Petition 870250114264, dated 11 / 12 / 2025, pp. 104 / 175 At 100 / 111 medium doses of pDNA, HYA improves ELISA titer responses in KV treatments.

[00259] When combined, the results of Study 33 (Figure 39) and Study 34 (Figure 40) suggest that adding HYA to KV treatments not only improves immunogenicity (and can be seen as dose economy), but does so to the extent that KV treatments involving HYA can improve treatment outcomes for individuals who would otherwise hardly achieve significant immune responses. STUDY 35

[00260] Referring now to Figure 41, another study was conducted to evaluate the effects of kinetic vacuum (KV) treatments on immune cell migration, this time in rabbits. In this sense, this study presents similarities with Study 32 (Figure 38), which was performed in guinea pigs. In the present study, individuals in four (4) groups (with six (6) individuals per group) were injected with plasmid 5013 (which encodes the gene for GFP) in uniform volumes and doses of DNA into the skin over the flank. After injection, each group received KV treatment, using the OTS-5 vacuum cup, employing Pattern C, 4 cycles. Lymph nodes were analyzed on different days after treatment to measure the number of GFP-positive cells for each group, as follows: Group 1 on Day 1; Group 2 on Day 2; Group 3 on Day 3; Group 4 on Day 7. This analysis aims to identify the timing of the migration of immune cells from the skin to the lymph nodes.

[00261] The results show a strong migration of immune cells occurring on days 1 and 2, with a drop on day 3, and then the migration remaining stable until day 7. These results suggest that most immune cell migration (in this case, GFP trafficking from lymph nodes) occurs in the first 48 hours. These results Petition 870250114264, dated 11 / 12 / 2025, pages 105 / 175 101 / 111 data, together with those from Study 32 (Figure 38), provide evidence that the immunogenic response involves the trafficking of antigens along immunogenic pathways (e.g., lymph node trafficking) concentrated in the first 2 days after transfection. STUDY 36

[00262] Referring now to Figure 42, a study was conducted to evaluate the trade-off relationship between DNA dosage and hyaluronidase (HYA) dosage in a given total injection volume for kinetic vacuum (KV) treatments in guinea pigs. Individuals in four (4) groups (with five (5) individuals per group) received the same KV treatments after injections of plasmid 2303 of varying percentages of DNA dose to HYA dose. For Groups 1-3, the DNA and HYA doses were limited to a uniform total injection volume (therefore, HYA dosage was performed at the expense of DNA dosage and vice versa). For Group 4, the total injection volume was not restricted and involved a high dose for both DNA and HYA dosage. The dosages in Groups 1-4 were as follows: • Group 1: DNA dose of 5ug; HYA dose of 75U / mL (i.e., a 50 / 50 mixture, meaning half the dose of DNA and half the dose of HYA); • Group 2: DNA dose of 9 µg; HYA dose of 12.5 U / mL (i.e., a DNA dose diluted by 10% due to the presence of a low dose of HYA); and • Group 3: DNA dose of 10 µg; HYA dose of 0 U / mL (i.e., 100% DNA dosage, without HYA); • Group 4: DNA dose of 10 ug; HYA dose of 135 U / mL (i.e., high HYA dosage without sacrificing DNA dosage).

[00263] Details regarding the test parameters for this study are summarized in Table 22 below: TABLE 22: Petition 870250114264, dated 11 / 12 / 2025, pp. 106 / 175 102 / 111 Group number (n / group) Plasmid Treatment DNA dose (ug) HYA dose (U / mL) 1. (n=5) 2303 KV 5 75 2. (n=5) 2303 KV 9 12.5 3. (n=5) 2303 KV 10 0 4. (n=5) 2303 KV 10 135

[00264] For all Groups, injections were performed intradermally in the skin of the flank. All KV treatments in this study were performed using the OTS-5 vacuum cup (see Table 0), employing Pattern C, 4 cycles.

[00265] The results demonstrate that the presence of HYA in each of the dosages tested (Groups 1, 2, and 4) increased the immune response measured by the ELISA titer. Comparatively, individuals without HYA (Group 3) produced almost no measurable ELISA response, if any at all. Interestingly, the formulation with high DNA content and high HYA content (Group 4) performed similarly to the 50 / 50 formulation (Group 1), where both Groups 1 and 4 demonstrated measurable ELISA responses, with Group 2 (90% DNA and low HYA formulation) outperforming Groups 1 and 4. These results suggest that DNA dosage does not need to be sacrificed to increase HYA dosage in order to produce enhanced immunogenicity, provided some HYA is added. Furthermore, these results demonstrate that a low HYA dosage (Group 2) can outperform a high HYA dosage (Group 4), and interestingly, even a high HYA dosage without sacrificing DNA dosage (again, Group 4).Another interesting observation from these results is that adding HYA to plasmid injections produced beneficial immunogenicity, even if the DNA dosage is... Petition 870250114264, dated 11 / 12 / 2025, pp. 107 / 175 103 / 111 sacrificed in the process (e.g., compare Group 1 (50 / 50 mixture) with Group 3 (100% DNA dose, no HYA). Considering the potential clinical use of KV+HYA administration, these results support the inclusion of HYA at low doses (such as, but not limited to, 10% of the total injection volume) in plasmid injections for KV treatments. Overall, these results provide interesting implications regarding the potential for significant cost savings to be achieved by reducing HYA to even a small fraction of historical dosages in plasmid injections for KV treatments, without sacrificing its beneficial effects. STUDY 37

[00266] Referring now to Figure 43, a study was conducted to compare the effect on the ELISA immune response provided by adjusting the number of kinetic vacuum (KV) translational movements (cycles, in this case glides) in similar KV treatments. Individuals in four groups (with five (5) individuals per group) were intradermally injected with plasmid 2027 in volumes and dosages of unformed DNA in the skin over the flank and subsequently received KV treatments using the same OTS vacuum cup, particularly the OTS-5 vacuum cup (see Table 0), employing different numbers of cycles of the same KV movement pattern, particularly Pattern C in the injection bubble (see Figure 7F). The cycles administered to each group were as follows: • Group 1 received one (1) cycle (i.e., one slip); • Group 2 received two (2) cycles; • Group 3 received three (3) cycles; and • Group 4 received four (4) cycles.

[00267] The results show substantially equivalent immune responses in terms of ELISA responses, regardless of the number of slips in the injection bubble. Furthermore, when Petition 870250114264, dated 11 / 12 / 2025, pp. 108 / 175 104 / 111 seen in connection with the results of the operator study described above (Study 18, Figure 25), the unidirectional motion (UNI) study (Study 27, Figures 33A and 33B) and the off-target KV study (Study 29, Figures 35A and 35B), these results support the conclusion that the KV treatments described herein are substantially operator-friendly, i.e., easy to perform and require only one (1) precise slide on the injection bubble to achieve a beneficial immune response. In simpler terms, these studies provide evidence that one slide on the bubble is as effective as multiple slides on the bubble for delivering an immunogenic treatment against KV. STUDY 38

[00268] Referring now to Figure 44, a study was conducted to explore the effects provided by kinetic vacuum (KV) treatments at reduced dosages of mRNA-1273 (an mRNA-based vaccine for SARS-CoV-2) on immune responses (binding ELISA responses) in guinea pigs. In this study, individuals in four (4) groups (with five (5) individuals per group) received mRNA-1273 injections at a low mRNA dose (0.1 µg) or at a high mRNA dose (1 µg). Furthermore, for each dosage in this study, the mRNA-1273 plasmid was injected by intramuscular (IM) injection (which is the Moderna injection protocol) or by intradermal (ID) injection, with intradermal injections followed by KV treatments using the same vacuum cup (the OTS-5 vacuum cup, see Table 0) employing the same motion pattern (Pattern C), 4 cycles. The dosages in Groups 1-4 were as follows: • Group 1: mRNA dose of 1ug (high dose, for the purposes of this study), via intramuscular (IM) injection; • Group 2: mRNA dose of 0.1 ug (low dose, for the purposes of this study), via intramuscular (IM) injection; Petition 870250114264, dated 11 / 12 / 2025, pp. 109 / 175 105 / 111 • Group 3: mRNA dose of 1 µg (high dose), via intradermal injection (ID) followed by treatment with KV; and • Group 4: mRNA dose of 0.1 µg (low dose), via intradermal injection (ID) followed by treatment with KV.

[00269] Details regarding the test parameters for this study are summarized in Table 23 below: TABLE 23: Group number (n / group) Agent Injection type Additional treatment mRNA dose (µg) 1. (n=5) mRNA-1273 IM - 1 2. (n=5) mRNA-1273 IM - 0.1 3. (n=5) mRNA-1273 ID KV 1 4. (n=5) mRNA-1273 ID KV 0.1

[00270] Week 2 ELISA responses are shown for all Groups (with the dashed line indicating the limit of detectable titers). Results show that at the high dose level (1 µg), both IM administration (Group 1) and ID administration plus KV treatment (Group 3) produced similar immunogenicity in terms of ELISA titers. At the low dose level (0.1 µg), IM administration (Group 1) had completely undetectable ELISA responses, while ID administration plus KV treatment (Group 4) remained immunogenic, although it experienced a drop compared to its high-dose counterpart (Group 3). These results demonstrate that at low mRNA doses, intradermal (ID) administration of mRNA-1273 with supplemental KV treatment is far superior to intramuscular (IM) administration in terms of ELISA response. STUDY 39

[00271] Referring now to Figure 45, a follow-up study Petition 870250114264, dated 11 / 12 / 2025, p. 110 / 175 106 / 111 The follow-up to Study 38 (Figure 44) was conducted to further explore the effects provided by kinetic vacuum (KV) treatments at reduced dosages of mRNA-1273 on immune responses (binding ELISA responses) in guinea pigs. In this follow-up study, KV treatments after intradermal (ID) injections of mRNA-1273 at, for the purposes of this study, high, medium, and low doses (1 µg, 0.5 µg, and 0.1 µg, respectively) were compared to intradermal injection (ID) only (INJ) treatments of high and low doses of mRNA-1273 (as opposed to intramuscular (IM) injection treatments of mRNA only, as in Study 38). Thus, in the present study, all injections were intradermal (ID). The KV treatments were administered using the same vacuum cup and the same motion pattern and cycles as in Study 38, namely the OTS-5 vacuum cup (see Table 0), using Pattern C, 4 cycles. The dosages in Groups 1-5 were as follows: • Group 1: mRNA dose of 1 ug (high dose); • Group 2: mRNA dose of 0.1 ug (low dose); • Group 3: mRNA dose of 1ug (high dose), followed by treatment with KV; • Group 4: mRNA dose of 0.1 ug (low dose), followed by treatment with KV; and • Group 5: mRNA dose of 0.5 ug (medium dose), followed by treatment with KV.

[00272] Details regarding the test parameters for this study are summarized in Table 24 below: TABLE 24: Group number (n / group) Agent Injection type Additional treatment mRNA dose (µg) 1. (n=5) mRNA-1273 ID - 1 2. (n=5) mRNA-1273 ID - 0.1 Petition 870250114264, dated 11 / 12 / 2025, p. 111 / 175 107 / 111 3. (n=5) mRNA-1273 ID KV 1 4. (n=5) mRNA-1273 ID KV 0.1 5. (n=5) mRNA-1273 ID KV 0.5

[00273] Week 2 ELISA responses are shown for all Groups (with the dashed line indicating the limit of detectable titers). Results show that, in both high-dose (1 µg) and low-dose (0.1 µg) mRNA injection treatments, KV treatments (Groups 3 and 4) outperformed ID injection-only treatments (Groups 1 and 2) in terms of ELISA titers, although high-dose mRNA injection-only treatments (Group 1) were more immunogenic than low-dose mRNA injection-only treatments (Group 4). Medium-dose mRNA injection-only treatments (0.5 µg) (Group 5) modestly outperformed high-dose mRNA injection-only treatments (Group 1). Interestingly, at low mRNA dose levels (0.1 µg), ID injection-only treatments (Group 2) were more immunogenic than intramuscular (IM) injection-only treatments from Study 38 (Group 2).

[00274] When the results of the present study are combined with those of Study 38, the data demonstrate that, at low mRNA doses, intradermal (ID) administration of mRNA-1273 with supplemental KV treatment is far superior to intramuscular (IM) and intradermal (ID) injection treatments in terms of ELISA response alone. The combined results also show that medium-dose mRNA KV treatments provided immunogenicity comparable to high-dose mRNA-1273 ID and IM injection treatments. Thus, the combined results suggest that intradermal injection of mRNA-1273 followed by KV treatment may provide dose-saving effects for mRNA-1273 injections, whether administered intramuscularly (IM) or intradermally (ID), without sacrificing immunogenicity. Additionally, the ad Petition 870250114264, dated 11 / 12 / 2025, page 112 / 175 108 / 111 Administration of mRNA from lipid nanoparticles into the skin using KV has been shown to be more immunogenic than administration into the muscle or skin without the use of KV, suggesting that KV generally increases cellular uptake of foreign cargo, not just plasmid DNA STUDY 40

[00275] Referring now to Figure 46, another hyaluronidase dosage study was conducted, specifically to evaluate the trade-off relationship between DNA dosage and hyaluronidase (HYA) dosage in high DNA dosage scenarios for kinetic vacuum (KV) treatments in guinea pigs. Individuals in two (2) groups (with five (5) individuals per group) received the same KV treatments after high-dose plasmid 2303 injections, as follows: • Group 1: DNA dose of 100 µg; HYA dose of 0 U / mL (i.e., 100% DNA dosage, without HYA); and • Group 2: DNA dose of 75 µg; HYA dose of 33.75 U / mL (i.e., a DNA dose diluted 25% due to the presence of HYA).

[00276] The results show that, at high doses of DNA, the addition of HYA improved immunogenicity, even at the cost of reducing the DNA dosage by 25%. NOTES

[00277] Based on the data and results of the studies described above with reference to Figures 8-46, the following observations were made. Kinetic vacuum treatments provide immune responses comparable to vacuum electroporation (VEP) or needle electroporation (NEP) in several studies, and greater responses in guinea pigs or rabbits in several studies (see Figures 17-18, 23A-23B, 30, 32A-32E, 34A-34B). Furthermore, kinetic vacuum treatments improved immune responses compared to static vacuum treatments in all studies described herein, both in Petition 870250114264, dated 11 / 12 / 2025, page 113 / 175 109 / 111 rabbits as well as guinea pigs. Furthermore, kinetic vacuum treatments provided superior gene expression compared to vacuum electroporation (VEP) treatments, and in the study shown in Figure 8, kinetic vacuum treatments produced a larger transfection zone. Although kinetic vacuum treatments were observed to cause acute redness at the treatment site, it disappeared quickly and did not cause any crusting, burning, or other tissue damage, unlike electroporation treatments in the studies described here, which caused more tissue damage and required longer healing times to recover to normal compared to kinetic vacuum treatments.KV also led to superior antigen trafficking to lymph nodes than NEP (Figure 38) and proved to be dose-sparing, generating stronger immune responses compared to NEP when suboptimal (poorly immunogenic or non-immunogenic) DNA vaccine doses were used.

[00278] Furthermore, for kinetic vacuum treatments, six (6) slips (three round-trip cycles) outperformed half a slip, although even a single slip was subsequently shown to be immunogenic in a manner similar to multiple slip patterns. Additionally, the magnitude of gene expression was associated with the percentage of the injection site touched by the slip; the absence of a percentage of the injection site correspondingly reduced gene expression.It was also observed that the addition of hyaluronidase (HYA) significantly improved gene expression and immune responses to kinetic vacuum treatments, but did not similarly benefit static vacuum or electroporation treatments. HYA concentrations ranging from 12.5 U / mL to 135 U / mL were all equally effective in increasing the immunogenicity of KV DNA vaccine administration, suggest. Petition 870250114264, dated 11 / 12 / 2025, p. 114 / 175 110 / 111 laughing that even a small amount of HYA can trigger this benefit. The magnitude of the immune response after KV administration increased with increasing vacuum strength and increasing cup diameter, until the cup diameter became approximately similar to the diameter of the injection site on the skin. These studies also demonstrated that decreasing the injection volume in kinetic vacuum treatments can slightly reduce immunogenicity. It was also observed that supplementing kinetic vacuum treatments with vacuum electroporation (VEP) did not produce beneficial results compared to kinetic vacuum treatments.Furthermore, it was observed that kinetic vacuum treatments including hyaluronidase approach parity with mRNA-level immune responses to lipid nanoparticles, with superior cellular responses, although DNA-based kinetic vacuum treatments still require approximately ten times (10x) the mRNA dose to achieve near parity. The use of a DNA-released nanoparticle plasmid, rather than a monomeric antigen-encoding plasmid, administered using KV + HYA, was able to elicit more durable immune responses than lipid nanoparticle mRNA using the same nucleic acid dose in each group. Finally, administration of lipid nanoparticle mRNA to the skin using KV proved to be more immunogenic than administration to muscle or skin without the use of KV, suggesting that KV generally enhances cellular uptake of foreign cargo, not just plasmid DNA. MODIFICATIONS

[00279] It should be understood that the various parameters of vacuum cups, systems, and kinetic vacuum treatment techniques described above are provided as exemplary characteristics for providing vacuum-enhanced tissue transfection. These parameters may Petition 870250114264, dated 11 / 12 / 2025, pages 115 / 175 111 / 111 may be adjusted as necessary without departing from the scope of the present invention. For example, although the kinetic vacuum treatments and vacuum cups described above are primarily described in relation to enhancing skin tissue transfection, the kinetic vacuum treatments and vacuum cups described herein may be adapted to treat adipose tissue and / or muscle tissue to enhance transfection therein.

[00280] It should also be noted that in other embodiments, the various vacuum cups 2, 102, 302, 402 and support components (see Figures 1A, 3A-3B and 5A-5B) may be supplied in a kit that includes a plurality of vacuum cups 2, 102, 302, 402 that a user may use interchangeably.

[00281] Although the invention has been described in detail, it should be understood that various alterations, substitutions, and modifications may be made herein without departing from the spirit and scope of the invention, as defined by the appended claims. Furthermore, the scope of the present invention is not intended to be limited to the particular embodiments described in the descriptive report. In particular, one or more of the features of the preceding embodiments may be employed in other embodiments described herein. As one with ordinary skill in the art will readily perceive, processes, machines, manufacturing, material composition, means, methods, or steps, now existing or hereafter developed, which substantially perform the same function or achieve substantially the same result as the corresponding embodiments described herein may be used in accordance with the present invention. Petition 870250114264, dated 11 / 12 / 2025, pp. 116 / 175

Claims

1 / 6 CLAIMS 1. A method for improving the delivery of an agent into tissue, characterized in that it comprises: placing a housing that defines a chamber adjacent to a tissue surface, wherein the placement step locates the chamber adjacent to an injection site where the agent has been injected into the tissue; applying vacuum pressure to the chamber, thereby pulling a portion of the tissue through an opening in the chamber and into the chamber; and moving the housing relative to the tissue while vacuum pressure is applied, thereby subjecting the tissue to a vacuum pressure motion field that imposes mechanical stress and tension on the tissue to improve the delivery of the agent into the tissue, increasing cellular uptake of the agent directly into the target cells of the tissue.

2. Method according to claim 1, characterized in that the motion step comprises translation of the housing along a displacement path that causes at least a portion of the chamber to pass through a center of the injection site.

3. Method, according to claim 1, characterized in that the placement step positions the chamber in a first position that is spaced from the center of the injection site at a first displacement distance measured from the center of the injection site along a direction, the translation step comprises translating the housing from the first position and through the center of the injection site along the direction to a second position, wherein the second position is spaced from the center of the injection site at a second displacement distance measured along the direction.

4. Method according to claim 3, characterized in that at least one of the first and second displacement distances is not less than a maximum internal dimension of the chamber measured along the direction.

5. Method according to claim 4, characterized in that the first and second displacement distances are not less than the maximum internal dimension of the chamber.

6. Method, according to claim 3, characterized in that the translation step of the housing from the first position to the second position is performed at least twice.

7. Method according to claim 2, characterized in that the translation step comprises a first translation step of the housing along a straight path, at least partially, through the injection site in a first direction and, subsequently, a second translation step of the housing along a straight path, at least partially, through the injection site in a second direction opposite to the first direction.

8. Method according to claim 7, characterized in that the first and second translation steps are repeated a plurality of times.

9. Method according to claim 1, characterized in that the motion step comprises at least one of the following: translating the housing along a substantially circular path around the injection site, such that at least a portion of the chamber overlaps the center of the injection site while the housing translates along at least a portion of the substantially circular path; and rotating the housing around a central axis of the vacuum chamber, the central axis oriented substantially orthogonally to the tissue surface on which the chamber is placed.

10. Method according to claim 1, characterized in that the applied vacuum pressure is in a range of about -300 mmHg to about -760 mmHg during the motion step.

11. Method according to claim 10, characterized in that the applied vacuum pressure is in a range of about -400 mmHg to about -600 mmHg during the motion step.

12. Method according to claim 1, characterized in that the portion of the tissue pulled into the chamber is pulled into contact with at least one protrusion of the housing positioned inside the chamber, thereby deforming the tissue that comes into contact with the protrusion.

13. Method for improving the delivery of an agent into tissue, characterized in that it comprises: injecting an agent into the individual's tissue, thereby defining an injection site on a tissue surface; placing a housing that defines a chamber at or adjacent to the injection site; applying vacuum pressure to the chamber; after the injection step, pulling a portion of tissue through an opening in the chamber and into the chamber in response to the application of vacuum pressure in the chamber; and moving the housing relative to the tissue while vacuum pressure is applied, thereby subjecting the tissue to a vacuum pressure motion field that imposes mechanical stress and tension on the tissue to improve the delivery of the agent into the tissue, increasing cellular uptake of the agent directly into the target cells of the tissue.

14. Method according to claim 13, characterized in that the motion step comprises translation of the housing along a displacement path that causes at least a portion of the chamber to traverse a center of the injection site one or more times.

15. Method according to claim 13, characterized in that the applied vacuum pressure is in a range of about -300 mmHg to about -760 mmHg during the motion step.

16. Method according to claim 13, characterized in that the tissue is skin tissue, the tissue surface is a skin surface, and the injection step comprises performing a Mantoux injection into the skin tissue.

17. Method according to claim 16, characterized in that the injected agent includes a spreading agent.

18. Method according to claim 17, characterized in that the spreading agent is hyaluronidase.

19. Method according to claim 18, characterized in that hyaluronidase comprises from about 5% to about 20% of the total injection volume of the agent.

20. Method according to claim 13, characterized in that the injected agent comprises a plasmid containing mRNA and lipid nanoparticles.

21. Method according to claim 13, characterized in that the injected agent comprises a plasmid containing DNA-launched nanoparticles. Petition 870250114277, dated 11 / 12 / 2025, page 8 / 17 5 / 6 22. Method according to claim 13, characterized in that: the injection step comprises injecting a total volume of the agent into the tissue, wherein the total volume is divided into a plurality of subvolumes, such that the injection step comprises performing a plurality of injections into the tissue at a plurality of respective injection sites, each of the plurality of injections injecting a respective subvolume of the plurality of subvolumes into the tissue; and the movement step comprises translating the housing along a displacement path that causes the chamber to traverse at least most of the plurality of injection sites.

23. System for vacuum-enhanced agent administration in vivo, characterized in that it comprises: a housing that defines a chamber and an opening to the chamber; at least one orifice extending through the housing, wherein the at least one orifice is remote from the at least one opening and is connectable to a vacuum source, such that the at least one orifice is configured to communicate vacuum pressure from the vacuum source to the chamber, wherein the housing is configured to communicate a vacuum field to a portion of the tissue and thus pull the portion of tissue through the opening and at least momentarily hold the portion of tissue in the chamber, and the housing is further configured to move relative to the tissue while the vacuum field is communicated to the tissue, wherein the relative movement deforms at least part of the tissue for increased cellular uptake of the agent directly into the target cells of the tissue; and Petition 870250114277, dated 11 / 12 / 2025, p.9 / 17 6 / 6 one or more disposable features between a distal end of the housing and a tissue surface to reduce sliding friction between the housing and the tissue.

24. System according to claim 23, characterized in that one or more features are selected from the group comprising: a lubricant; and rollers coupled to the distal end of the housing.

25. System according to claim 23, characterized in that it further comprises a handling member, wherein the housing can be attached to the handling member.

26. System according to claim 23, characterized in that the housing is configured to communicate the vacuum field at a vacuum pressure in a range of about -300 mmHg to about -760 mmHg during movement of the housing relative to the tissue.

27. System according to claim 26, characterized in that the chamber has a circular cross-sectional shape in a plane orthogonal to a central axis of the chamber, and an inner surface of the housing within the chamber defines a chamber diameter in a range of about 5 mm to about 15 mm. Petition 870250114277, dated 11 / 12 / 2025, p. 10 / 17

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