EA043519B1 - METHOD AND DEVICE FOR DELIVERY OF VACCINES AGAINST HEPATITIS B VIRUS (HBV) - Google Patents

Description

Cross reference to related applications

This application claims priority to the International Patent Application

No. PCT/US2017/067269, filed December 19, 2017, and US provisional patent application

No. 62/607430, filed December 19, 2017, the entire disclosure of which is incorporated herein by reference.

Link to sequence list submitted electronically

This application contains a sequence listing, which is presented in electronic form via EFS-Web as a sequence list in ASCII format under the file name 688097-331WO Sequence Listing, created on December 10, 2018 and having a size of 46.6 kilobytes. This sequence listing provided on EFS-Web is part of this application and is incorporated herein by reference in its entirety.

Field of invention

The present invention relates to the administration of prophylactic and/or therapeutic vaccines against hepatitis B virus (HBV) to individuals in need thereof, and in particular to the reproducible, consistent and effective delivery of prophylactic and/or therapeutic vaccines against HBV, such as nucleic acids encoding HBV antigens, to specific areas in a selected tissue region of interest, where said delivery is facilitated by local application of electrical fields in a safe, effective and consistent manner for heterogeneous groups of recipients with minimal user training.

Prerequisites for creating the invention

Hepatitis B virus (HBV) is a small 3.2-kb hepatotropic DNA virus that encodes four open reading frames and seven proteins. About two billion people are infected with HBV, and approximately 240 million people have chronic hepatitis B infection (chronic HBV infection), characterized by the persistent presence of viral and subviral particles in the blood for more than 6 months (Cohen et al. J Viral Hepat. (2011) 18 (6), 377-83). Persistent HBV infection results in depletion of T cells in the circulation and depletion of intrahepatic HBV-specific CD4 + and CD8 ⁺ T cells through chronic stimulation of HBV-specific T cell receptors by viral peptides and circulating antigens. As a result, the multifunctionality of T cells is reduced (i.e., the levels of IL-2, tumor necrosis factor (TNF)-α, IFN-γ are reduced, and there is a lack of proliferation and degranulation). Therapeutic vaccination eliminates HBV from chronically infected individuals (Michel et al. J. Hepatol. (2011) 54 (6), 1286-1296). Immunogenic compositions containing one or more nucleic acid molecules encoding one or more HBV antigens can be used to produce therapeutic immunity in an individual in need thereof, such as a person with chronic HBV infection.

Although naked DNA can be delivered to cells in vivo and result in gene expression, the efficiency of gene transfer is relatively low and variable. Local application of electrical signals has been shown to improve the distribution and absorption of macromolecules in living tissue. Delivery of such electrical signals into tissue in combination with administration of a prophylactic or therapeutic HBV vaccine may produce the desired effect on the tissue and/or the agent being delivered. In particular, techniques such as electroporation and iontophoresis have been applied to significantly improve the distribution and/or uptake of nucleic acids, including DNA and RNA sequences. Potential clinical applications of such methods include the delivery of nucleic acid sequences for prophylactic and therapeutic immunization, and the delivery of nucleic acid sequences encoding therapeutic proteins or peptides.

Procedures for such delivery involve administering the agent of interest to a target tissue site in combination with the application of electrical fields of sufficient magnitude and duration to achieve the desired effects on delivery, distribution and/or activity of the agent. Electric fields are propagated by two or more electrodes through an electrically conductive connection to the tissue. Electrodes with configurations suitable for use in these methods are tissue penetrating electrodes, surface contact electrodes, and air gap electrodes. Specific electrode configurations include, but are not limited to, elongated needle or rod electrodes, point electrodes, tortuous electrodes (ie, wire-shaped electrodes), flat electrodes, and combinations thereof. The specific type and location of electrodes is selected depending on the type of target tissue and the purpose of the procedure. The desired result is best achieved when electric fields are propagated into the target tissue in the presence of the agent of interest.

A wide variety of methods and devices have been described for delivering electrical fields to tissue in the presence of an agent of interest to improve delivery of the agent to skin and muscle tissue. These devices use both surface- and tissue-penetrating electrode systems, as well as combinations of both. Despite the promising prospects associated with electrically mediated delivery of agents and the possible clinical application of these methods, there is still

- 1 043519 there is a need for reliable and consistent delivery of nucleic acids of interest to individuals. Significant reasons for this variability are differences in technique and operator skill level. Other causes of variability that are not relevant to current systems are differences in the physiological characteristics of individuals, which may influence the use of a given procedure. Other considerations when designing suitable devices are ease of use and implementation of designs that reduce the frequency and severity of potential user errors.

Given that safe, reliable, accurate, and consistent delivery of clinical therapies is highly desirable, the development of improved delivery systems is warranted. Such design should include means to minimize operator-related variability while providing a means to account for inter-individual differences that may occur during widespread clinical use of electrically mediated agent delivery. In other words, specific areas for such improvement include the ability to consistently maintain the effectiveness of such a system for heterogeneous groups of recipients and reducing the training and skill requirements required for its effective use by the user. In addition, the device, system or method must be designed to help prevent user or device errors and minimize their impact should they occur.

This Background section is provided to summarize the material presented below in the Summary and Detailed Description of the Invention sections. This section is not intended to assist in defining the scope of the claimed subject matter and should not be construed as limiting the claimed subject matter to methods of carrying out the invention that overcome any or all of the disadvantages or solve any or all of the problems described above.

The essence of the invention

The present invention relates to devices, kits and methods for reproducibly, consistently and effectively delivering an HBV vaccine containing a nucleic acid molecule encoding an HBV antigen to an individual in need thereof using electrically mediated therapeutic agent delivery (EMTAD). In embodiments described herein, the therapeutic agent is an HBV vaccine.

In one aspect, the present invention relates to a device for delivering an HBV vaccine to a predetermined site in an individual, comprising a unit for controlled administration of an HBV vaccine to the individual, including a reservoir containing the HBV vaccine, at least one opening through which the agent is administered , and a controlled energy source sufficient to transport a predetermined amount of HBV vaccine at a predetermined rate from the reservoir through the opening to a predetermined site in the individual. In addition, the device may include a plurality of penetrating electrodes positioned in a predetermined spatial relationship with respect to the opening, and an electrical signal generator operably coupled to the electrodes.

In another aspect, the present invention relates to a kit for delivering an HBV vaccine to a predetermined site on an individual, wherein the kit includes an HBV vaccine and a device containing a unit for controlled administration of an HBV vaccine to the individual, wherein the device includes a reservoir for the HBV vaccine. HBV, at least one orifice through which HBV vaccine is administered, and a controlled energy source sufficient to transport a predetermined amount of HBV vaccine at a predetermined rate from the reservoir through the orifice to a predetermined site in the individual. In addition, the device may include a plurality of penetrating electrodes arranged in a predetermined spatial relationship with respect to the opening, and an electrical signal generator operably coupled to the electrodes.

The HBV vaccine included in the device or kit according to the invention may contain:

a first nucleic acid molecule comprising a first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity;

a second nucleic acid molecule containing a second polynucleotide encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14; and a pharmaceutically acceptable carrier, wherein the first nucleic acid molecule and the second nucleic acid molecule are present in the same nucleic acid molecule or in two different nucleic acid molecules.

Other aspects of the invention include methods including administering a vaccine against

- 2 043519

HBV in controlled spatial and temporal relationship with electrical signal application (ESA).

The beneficial effects and advantages of certain methods of carrying out the invention in accordance with the principles according to the invention are quite varied. Some methods of carrying out the invention allow you to select the depth of insertion of the needle and electrode and introduce them into the desired tissues of different types (eg, dermis, muscle, etc.) for all heterogeneous groups of individuals with different body weights and physiques. Such an implementation also facilitates the adaptation of methods for use in specific target groups, such as, but not limited to, patients with chronic HBV infection and/or HBV disease. The present invention relates to systems and methods including design features that make the systems and methods resistant to accidental discharge or potential misuse of the device, such as due to drop, vibration and/or breakage. In some embodiments, the present invention relates to devices configured with multiple injection holes. Systems and methods according to the principles of the present invention allow for multiple safety interlocks to reduce the frequency and/or negative impact of user errors. These measures facilitate the correct preparation and calculation of the administered dose, which will ensure that the required force is applied to the recipient tissue and will ensure the removal of the protective cap, etc. Systems, devices, kits and methods according to the principles of the present invention allow for highly consistent therapy regardless of the skill of the administerer or the type of recipient. The systems, devices, kits and methods described herein can provide, for example, a consistent force profile that can be achieved before and during administration of a therapeutic agent so that the doses can be administered to recipients with varying skin and muscle characteristics.

This summary of the invention is presented to select a concept in a simplified form. These concepts are also described in the Detailed Description of the Invention section. It is possible to use elements or steps other than those described in the Summary of the Invention section, but these elements or steps are not necessary. This summary is not intended to identify key features or important features of the claimed subject matter, nor is it intended to assist in defining the scope of the claimed subject matter. The claimed subject matter of the invention is not limited to methods of carrying out the invention that eliminate any or all of the disadvantages described in any part of this application.

Other aspects, features and advantages of the present invention will be apparent from the following disclosure, including the detailed description of the invention and preferred embodiments thereof and the accompanying claims.

Brief description of drawings

The specifics of the disclosure of the invention are precisely set forth in the accompanying claims. To better understand the features and advantages of the disclosure, reference is made to the following detailed description, which provides illustrative embodiments of the invention in accordance with the principles of the disclosure of the present invention and the accompanying drawings, wherein:

in fig. Figure 1 illustrates potential sources of spatial variability associated with injection with a standard syringe and needle;

in fig. 2 is a general view of a system in accordance with the principles of the present invention, including a cassette unit 100, an applicator 400, and a control system 700;

in fig. 3A-3B show various views of the device described herein. In fig. 3A is a side view of a cassette unit 100 in accordance with the principles of the present invention. In fig. 3B is a side view of an HBV 101 vaccine reservoir manufactured into a syringe according to the principles of the present invention;

in fig. 4 shows various representative components of a cassette unit 100 in accordance with the principles of the present invention;

in fig. 5A-5E show views of the inner cassette and the rear of the cassette in the apparatus described herein. In fig. 5A is a top view of the inner cassette 103 in accordance with the principles of the present invention. In fig. 5B shows a bottom view of the inner cassette 103 and a rear portion of the cassette according to the principles of the present invention. In fig. 5C is a side perspective view of a detailed view of the inner cassette 103 in accordance with the principles of the present invention. In fig. 5D is a side view of the rear of a cassette 112 in accordance with the principles of the present invention. In fig. 5E shows a reservoir lock 120 with improved locking features to prevent the rear portion of the cassette 112 from inadvertently moving forward;

in fig. 6 is a detailed view of a cassette unit showing rack 154, initiation signal 172, and continuation signal 174 in accordance with principles of the present invention;

in fig. 7A-7B show various views of the electrodes and/or electrode contacts of the device described herein. In fig. 7A shows in detail the electrode contacts 130 and various

- 3 043519 parts of electrode contacts according to the principles of the present invention. In fig. 7B shows in detail the electrodes 122 and various parts of the electrode contacts in accordance with the principles of the present invention;

in fig. 8A-8B show the power contact locking systems in the device described herein in various forms. In fig. 8A is a top view of a power contact locking system in accordance with the principles of the present invention. In fig. 8B shows details of a power contact interlocking system in accordance with the principles of the present invention;

in fig. 9A-9D show various views of the device described herein. 9A shows a needle 105 and distal inner cassette electrodes 137 for insertion into tissue according to the principles of the present invention. In fig. 9B shows details of a protective shield 134 in accordance with the principles of the present invention. In fig. 9C illustrates the alignment panel 108 and side screen 168 in accordance with the principles of the present invention. In fig. 9D shows shield supports integrated into the outer cover of the cassette 106;

in fig. 10A-10D show various views of the outer cassette cover of the apparatus described herein. 10A shows an outer cassette cover 110 in accordance with the principles of the present invention. 10B shows a side view of an outer cassette cover 110 in accordance with the principles of the present invention. In fig. 10C shows an outer cassette cover 110 used in the leveling panel 108 and side screen in accordance with the principles of the present invention. In fig. 10D shows the outer cassette cover 110 with extension members for holding the inner cassette 103 in position during transport and loading;

in fig. 11A-11C show details of the protective screens of the device described here in various forms. In fig. 11A shows a hook 182 for holding the protective shield 134 in accordance with the principles of the present invention. In fig. 11B shows details of a protective shield 134 in accordance with the principles of the present invention. In fig. 11C shows shield supports 132 holding shield 134 in position in accordance with the principles of the present invention;

in fig. 12 shows details of an electrode support 124 in accordance with the principles of the present invention;

in fig. 13A-13B show images of an applicator in the apparatus described herein. In fig. 13A is a side view of an applicator 400 in accordance with the principles of the present invention. In fig. 13B is a top view of an applicator 400 in accordance with the principles of the present invention;

in fig. 14 is a three-dimensional view of an applicator 400 in accordance with the principles of the present invention;

in fig. 15 shows details of the side housing and electrode connection for electroporation 496 of the applicator 400 in accordance with the principles of the present invention;

in fig. 16 is a three-dimensional view of an applicator in accordance with the principles of the present invention, illustrating the cassette loading subunit 456;

in fig. 17A-17B show images of an applicator in the apparatus described herein. In fig. 17A is a three-dimensional view of an applicator in accordance with the principles of the present invention, illustrating the loading drive subassembly 454. FIG. 17B shows a rack 154 of a drive subassembly for loading 454 in accordance with the principles of the present invention;

in fig. 18A-18C show various views of the applicator in the apparatus described herein. In fig. 18A shows details of a cassette loading sub-unit 456 in accordance with the principles of the present invention, wherein the insertion/injection applicator drive unit is coupled to the cassette unit. In fig. 18B shows a cross-section of a cassette unit in accordance with the principles of the present invention, wherein the insertion/injection applicator drive unit is coupled to the cassette unit. In fig. 18C shows details of the cartridge loading, electrode insert, and injection subassemblies 452 of the applicator 400 in accordance with the principles of the present invention;

in fig. 19 illustrates various components of a control system in accordance with the principles of the present invention;

in fig. 20A-20D show an image of the device described herein. In fig. 20A illustrates various components of a control system in accordance with the principles of the present invention. In fig. 20B shows details of an applicator connection port 708 and a regulator system tray 710 in accordance with the principles of the present invention. In fig. 20C shows details of a stimulator display screen of a controller system in accordance with the principles of the present invention. In fig. 20D is a detailed rear view of a control system in accordance with the principles of the present invention;

in fig. 21 is a flowchart illustrating a method of operation according to the principles of the present invention;

in fig. 22 shows a TriGrid electrode array (in cross section) for intramuscular (i.m.) delivery, which consists of four electrodes arranged in two equilateral triangles in a diamond shape surrounding a central injection needle;

in fig. 23A-23B depict TriGrid TDS-IM v1.0 devices adapted for use in a mouse model (FIG. 23A) and a non-human primate (NHP) model (FIG. 23B);

in fig. 24 shows the TDS-IM v2.0 TriGrid device adapted for use in a non-human primate (NHP) model;

in fig. 25A-25H show the design and optimization of expression clusters and DNA plasmids encoding HBV pol antigens and core antigens as described in Example 1. FIG. 25A schematically illustrates an expression strategy where the coding sequences of the core antigens and HBV pol antigens are linked to each other in frame conservation. In fig. 25B schematically illustrates an expression strategy where the coding sequences for the HBV core and pol antigens are expressed from a single plasmid via an FA2 ribosomal excision site. In fig. 25C schematically illustrates an expression strategy where core and pol antigens are expressed from two separate plasmids. In fig. 25D illustrates Western blot analysis of core antigen expression in HEK293T cells transfected with a plasmid expressing core antigen in the presence and absence of the post-transcriptional regulatory element WPRE; expression was tested in cell lysate (left) and supernatant (sup; right) using an antibody against a-core antigen. In fig. 25E shows a Western blot analysis illustrating a comparison of core antigen expression in HEK293T cells transfected with a plasmid expressing core antigen and including an intron/exon sequence derived from the human apolipoprotein A1 precursor (intron AI) from the R-U5 long terminal repeat untranslated domain. (LTR) human T-cell leukemia virus type 1 (HTLV-1), (HTLVR) or from the triple sequence of hybrid enhancers HTLV-1 LTR, synthetic rabbit intron - globin and splicing enhancer (triple); the unlabeled lane represents purified core protein used as a size marker; expression was tested in lysate (left) and supernatant (sup; right); core antigen expression was highest in the case of triple sequence hybrid enhancers. In fig. 25F shows Western blot analysis of core antigen secretion using various signal peptides attached to the N-terminus of HBV core antigen; and the most efficient protein secretion was observed in the case of cystatin S signal peptide. In FIG. 25G schematically shows optimized HBV core antigen/pol antigen expression clusters for each of the three expression strategies illustrated in FIG. 25A25S. CMVpr: human CMV-IE promoter. TRE: Triple enhancer sequence. SP: cystatin S signal peptide. FA2: FMDV ribosome excision site. pA: BGH polyadenylation signal;

in fig. 25H shows Western blot analysis for the expression of HBV core antigen and pol antigen of pDK vectors containing each of the expression clusters shown in FIG. 25G. Lanes 1 and 2: pDK core antigen. Lanes 3 and 4: pDK-pol. Lanes 5 and 6: pDK-coreFA2Pol. Lanes 7 and 8: pDK-core antigen-pol antigen hybrid. The most favorable expression profile for cellular and secreted core and pol antigens was observed when the antigens were encoded by separate vectors;

in fig. 26A-26B are schematic representations of DNA plasmids according to embodiments of the invention. In fig. 26A shows a DNA plasmid encoding an HBV polymerase (pol) antigen according to an embodiment of the invention. In fig. 26B shows a DNA plasmid encoding HBV core antigen, according to an embodiment of the invention. HBV core and pol antigens are expressed under the control of the CMV promoter via the N-terminal cystatin S signal peptide, which is cleaved from the expressed antigen after secretion from the cell. The transcriptional regulatory elements of the plasmid include an enhancer sequence located between the CMV promoter and the polynucleotide sequence encoding the HBV antigen, and a bGH polyadenylation sequence located downstream of the polynucleotide sequence encoding the HBV antigen. A second expression cluster is included in the plasmid in reverse orientation, including the kanamycin resistance gene under the control of the Amp ^r (bla) promoter, and the origin of replication (pUC) is also included in reverse orientation.

Like reference numerals denote like elements throughout the specification. Elements have not been scaled unless otherwise noted.

Detailed Description of the Invention

The following description and examples illustrate in detail embodiments of the invention. It should be noted that the present invention is not limited to the specific embodiments described herein, and such embodiments may vary. It will be apparent to those skilled in the art that there are many variations and modifications of the invention that fall within the scope of the invention.

All terms are used in the generally accepted meanings known to those skilled in the art. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the invention relates.

The section headings used herein are for organizational purposes only and should not be construed as limiting the scope of the invention described.

Although various features of the invention may be described in the context of a single embodiment of the invention, these features may also be presented separately or in any suitable combination. Conversely, for clarity, although the present invention may be described herein in the context of individual embodiments of the invention, however, the present invention may also be implemented in one embodiment of the invention.

In an attempt to assist the reader in understanding the present specification, this specification has been divided into various paragraphs or sections or subdivided into various versions of this application. These sections should not be construed as a departure from the spirit of a paragraph or section or variations of the invention from the spirit of another paragraph or section or variations. Conversely, one skilled in the art will understand that this description has a broad application and covers all combinations of various sections, paragraphs and sentences that may be considered. The discussion of any embodiment of the invention is provided for illustrative purposes only and it is not intended that the scope of the disclosure, including the claims, be limited to these examples. Thus, for example, although variants of the HBV vectors that may be used in the application described herein (e.g., plasmid DNA or viral vectors) may contain specific components, including, but not limited to, certain promoter sequences, enhancer sequences or regulatory sequences, signal peptides, HBV antigen coding sequence, polyadenylation signal sequences, etc., arranged in a particular order, however, it will be apparent to those skilled in the art that the concepts disclosed herein may equally apply to other components arranged in a different order, which can be used in HBV vectors suitable for use. Such use involves the use of any of the components used in any combination having any sequence that can be used in HBV vectors suitable for their use, whether such specific combination is specified or not.

The following definitions supplement those found in the literature and apply to this application, but they should not be construed as applicable to any related or unrelated documents, such as any patents or applications having a common owner. Although any methods and materials similar or equivalent to those described herein may be used to practice the present invention, the materials and methods described herein are preferred. Accordingly, the terminology used herein is intended to describe specific embodiments of the invention only, and is not intended to limit the scope of the invention.

In this application, a word used in the singular includes that word in the plural unless otherwise indicated. It should be noted that singular nouns used in the present invention may also refer to plural nouns unless the context of the description indicates otherwise. In this application, the conjunction used or means and/or, unless otherwise specified. Moreover, as used herein, the term including, as well as its other forms such as include and included, is not limiting.

As used herein, references to certain embodiments of the invention, an embodiment of the invention, a variant of the invention, or other embodiments of the invention mean that the particular features, structures, or characteristics described in combination with the embodiments of the invention are included in at least some of the embodiments and, although not required, in all embodiments of the invention.

As used in this specification and in the claims, the words containing (and any form thereof, such as contain and contains), having (and any form thereof, such as have and has), including (and any form thereof, such as include and include) or containing (and any form thereof such as contains and contain) have an inclusive or non-exhaustive meaning and do not exclude additional unspecified elements or method steps. It is intended that any embodiment of the invention discussed herein can be implemented in any method or composition of the invention, and vice versa. In addition, the compositions of the invention can be used to carry out the methods of the invention.

As used herein, the term approximately refers to a reference numeric value used herein, and its grammatical equivalents may include the numeric value itself and a range of values plus or minus 10% of that numeric value. Thus, for example, the term approximately 10 includes 10 and any values from 9 to 11. Thus, for example, the term approximately, when used with respect to a reference numerical value, may also include the range of values plus or minus 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1% of this value.

The present invention discloses improved systems, kits, methods and devices for the reproducible, consistent and effective delivery of HBV vaccines, such as nucleic acids encoding HBV antigens and combinations thereof, administered by electrically mediated delivery of a therapeutic agent (e.g., HBV vaccine). (EMTAD).

In one aspect, the present invention relates to a device for delivering an HBV vaccine to a predetermined site in an individual, where the device includes a cassette

- 6 043519 a unit comprising an outer cassette, an inner cassette, a reservoir containing an HBV vaccine, wherein the reservoir receptacle is located in the outer cassette and is configured to be supplied to the reservoir; an applicator comprising a cassette unit receiver of the required volume, a needle hub and an injection detector, where the injection detector detects the loading of the reservoir through its receiver; at least one lock, where such lock facilitates the correct execution of the procedure for administering the HBV vaccine; at least one injection port through which the HBV vaccine is administered; a plurality of penetrating electrodes with a given spatial arrangement relative to the hole; an electric field generator for generating an electrical signal operably connected to the electrodes; and a controlled energy source sufficient to transport a predetermined amount of HBV vaccine at a predetermined rate from the reservoir through the opening to a predetermined site in the individual.

In another aspect, the present invention relates to a kit for delivering an HBV vaccine to a predetermined site in an individual, wherein the kit contains an HBV vaccine and a device including a cassette unit comprising an outer cassette; internal cassette; a reservoir containing the HBV vaccine, wherein the reservoir receiver is located in an external cassette and is configured to be delivered to the reservoir; an applicator comprising a cassette unit receptacle, a needle hub, and an insertion detector, wherein the insertion detector detects loading of the reservoir through its receptacle; at least one lock, where the lock facilitates proper execution of the HBV vaccine administration procedure; at least one injection port through which the HBV vaccine is administered; a plurality of penetrating electrodes with a given spatial relationship with respect to the hole; an electric field generator for generating an electrical signal operably connected to the electrodes; and a controlled energy source sufficient to transport a predetermined amount of HBV vaccine at a predetermined rate from the reservoir through the opening to a predetermined site in the individual.

The HBV vaccine included in a device or kit according to the invention may contain:

a first nucleic acid molecule comprising a first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity;

a second nucleic acid molecule containing a second polynucleotide encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14; and a pharmaceutically acceptable carrier, wherein the first nucleic acid molecule and the second nucleic acid molecule are present in the same nucleic acid molecule or in two different nucleic acid molecules.

In some aspects of the disclosure, EMTAD may mean administering an HBV vaccine to a biological tissue of interest, and pre-, simultaneously, or sequentially applying electrical signals to the biological tissue to enhance the penetration of the HBV vaccine into the tissue and/or the uptake of the vaccine into the tissue. The EMTAD method consists of two steps: 1) administration of a therapeutic agent (ie, HBV vaccine) (TAA) and 2) application of an electrical signal (ESA) sufficient to induce the desired EMTAD effect. In the present invention, TAA can be carried out, for example, in a controlled manner called controlled administration of a therapeutic agent (eg, HBV vaccine) (CTAA). As used herein, the term CTAA may refer to methods or devices that provide spatial and/or temporal control of HBV vaccine administration to induce an EMTAD effect. Regulated administration methods may use variations of a conventional syringe and needle (eg, an auto-injector) and/or various needle-free techniques (eg, jet injector, transdermal/transdermal patch, oral, gel, cream, or inhalation). As used herein, the term ESA may mean the application of electrical signals to facilitate or improve the delivery of active agents, for example, HBV vaccines, by enhancing the penetration of said agents into said tissue and/or the uptake of said agents by said tissue, and thereby inducing an EMTAD effect. ESA methods, when used to facilitate or improve the delivery of HBV vaccine, such as electroporation, iontophoresis, electroosmosis, electropenetration, electrical stimulation, electromigration and electroconvection, are various variants of EMTAD, where one or more of these methods may be included in those described herein ways.

Specific applications of the devices, kits and systems described herein include, but are not limited to, the delivery of HBV vaccines containing one or more nucleic acid molecules. Typically in such applications, EMTAD is initiated by injecting HBV vaccine using a standard syringe and needle. After administration of the agent, a device suitable for ESA is administered to the individual at a specific site. Finally, the appropriate ESA protocol is used to provide the desired ease or improvement in HBV vaccine delivery. However, despite the traditional use of EMTAD, the desired spatial and temporal relationship between agent administration and ESA cannot yet be realized.

Spatial parameters.

In some embodiments of the systems, kits, methods, and devices described herein, HBV vaccine is administered using a conventional syringe and needle. The need to deliver certain agents via EMTAD adds additional complexity to the implementation of TAA. As shown in FIG. 1, with any injection using a conventional syringe with a needle, when the needle 5 is inserted into the tissue, the insertion depth 1 and angle 2 relative to the surface of the tissue 3 may be difficult to regulate. In addition, the point of insertion of the needle 4 on the surface of the tissue 3 may not correspond to the location of the hole 6 and the area of introduction of the agent 7 into the target tissue. As a representative example, a transdermal intramuscular injection may not correspond to the skin injection site since the two tissues can often move relative to each other.

Although this traditional approach is generally adequate for the delivery of many different therapeutics that do not require EMTAD, however, these changes result in post-injection HBV vaccine distribution that is often inappropriate and/or uncertain and may hinder effective delivery of EMTAD. In some embodiments described herein, the most effective use of EMTAD involves the use of a predetermined relationship between the HBV vaccine and the ESA in an individual. As a result, in the absence of spatial control of TAA in the target tissue, the use of a conventional syringe and needle may result in reduced effectiveness of EMTAD compared to a device, method or system that provides spatial and temporal control. One illustrative example of this concept is the use of electroporation to facilitate HBV vaccine delivery. Electroporation is generally most effective at improving HBV vaccine delivery when TAA and ESA colocalize to the target tissue. In many cases, if the delivered agent and the induced electroporation effect together are not localized to the target tissue, then the delivery of the agent is suboptimal.

Another example of the need for adequate spatial regulation of TAA in EMTAD is iontophoresis. This EMTAD method uses electric fields to initiate the movement of charged molecules. To achieve the desired movement of the agent, proper spatial relationship between the electrodes and the HBV vaccine must be implemented. If a negatively charged agent is placed in close proximity to the location of the positive electrode, little or no movement of the agent through the tissue will be observed. In contrast, localization of said negatively charged agent near the negative electrode will result in significant movement of the agent through the tissue towards the positive electrode.

As shown in the previous examples, it is important to adjust the exact location of the TAA relative to the ESA to achieve the desired effect. Thus, the embodiments of devices and methods described herein provide regulation of the precise location of the TAA relative to the ESA site and are suitable for achieving reproducible, consistent and well-characterized distribution of one or more HBV vaccines.

Temporary parameters.

In the case of a standard injection using a syringe with a needle, the injection rate for TAA may vary depending on the operator, which will result in inappropriate distribution of the agent into the tissue. Additional time variability occurs when multiple devices are required to complete the EMTAD method. For example, one application of EMTAD requires the introduction of plasmid DNA encoding a therapeutic protein, followed by the generation of an electroporation-induced electric field. Using the traditional EMTAD method, the HBV vaccine is administered using a syringe with a needle, and then the electroporation device is introduced and activated. If placement of two separate devices (first the syringe and needle and then the ESA device) is required, the procedure may have different effects in different individuals due to the timing of each device being used inappropriately by the operator. Additionally, placing two separate devices in use will inevitably result in a time lag between the physician's placement of the device and the activation of each device. This is further complicated when multiple injection sites are required to achieve adequate delivery of the agent to a specific location in the target tissue.

These problems are particularly relevant when using agents such as nucleic acids that can be degraded or inactivated in the extracellular environment. Degradation of nucleic acids in the HBV vaccine may result in decreased efficacy and consistency of therapy. In addition, the degree of degradation of the nucleic acids in the HBV vaccine is not consistent between individuals, which contributes to the general therapeutic incompatibility of injection with a standard syringe and needle in combination with ESA and, more specifically, electroporation therapy.

Because of the complexity of spatial and temporal variability consistently observed when injection with a conventional syringe and needle in combination with ESA, the exact location and timing of TAA relative to ESA is often unknown. As a result, effective administration and dosing of HBV vaccines using EMTAD may be inconsistent and reproducible. Although injection with a conventional syringe and needle is sometimes sufficient to administer HBV vaccine, reproducible and consistent delivery of HBV vaccines is greatly improved by regulating the spatial and temporal relationship between HBV vaccine administration and induction of the desired EMTAD effect.

Thus, although the traditional EMTAD procedure may be adequate for certain purposes, however, temporal and spatial regulation is highly desirable for clinical applications where a high degree of consistency and reproducibility is typically required. In contrast to the traditional EMTAD method, variations of the methods, systems and devices described herein facilitate CTAA and ESA and are more effective methods and devices for clinical use of EMTAD. The present invention utilizes various aspects of CTAA in combination with ESA to provide reproducible, consistent and effective delivery of an HBV vaccine. The present invention describes methods and devices for providing spatial and temporal control in the administration of an HBV vaccine along with electrical signals, thereby improving the movement of the vaccine into a target tissue and/or its uptake by that tissue.

In some embodiments, the invention describes methods and devices that provide a controlled spatial relationship between the administration of an HBV vaccine and the delivery of electrical signals. Before treatment, the optimal location for the TAA relative to the ESA is determined. This spatial relationship between TAA and ESA depends on treatment parameters, including the nature of the agent administered and the properties of the target tissue into which the agent is administered. In a representative embodiment of the invention, the electrical signals are preferably provided at a significant distance from the site of administration of the HBV vaccine. In some other embodiments of the invention, the spatial relationship involves applying electrical signals via the EMTAD to a site located in the vicinity of the site of administration of the agent. In some cases, colocalization of TAA and ESA is preferable. This is often observed when electroporation and/or iontophoresis are used to achieve the desired EMTAD effect.

In another aspect of the invention, the device described herein provides an adjustable timing relationship between the sequence and timing of the TAA and ESA. Before treatment, the optimal sequence and timing of the combination of TAA and ESA is determined. As with the spatial relationship, the desired temporal relationship between TAA and ESA is determined by parameters such as the nature of the agent administered and the properties of the target tissue into which the agent is administered. In some cases, exposure to electrical fields associated with ESA may adversely affect the HBV vaccine. In such a practical application, after generating such electric fields, CTAA is carried out. However, a typical timing relationship is to conduct a CTAA and then an ESA.

The present invention relates to improved methods and devices for reproducible, consistent and effective delivery of HBV vaccines containing nucleic acid-based constructs together with EMTAD. This goal is achieved by regulating the spatial and temporal administration of the HBV vaccine relative to the delivery of electrical signals. In a certain embodiment of the invention, EMTAD is initiated by injecting the HBV vaccine using a conventional syringe and needle. After administration of the agent, a device suitable for ESA is administered to the individual at a specific site. An appropriate ESA protocol provides the desired facilitation or improvement in HBV vaccine delivery. A representative ESA method that has proven effective for almost all cell types is electroporation. Other representative electrically mediated delivery methods include, but are not limited to, iontophoresis, electroosmosis, electropenetration, electrical stimulation, electromigration, and electroconvection. These terms are used for illustrative purposes only and should not be construed as limiting the scope of the invention.

Electroporation uses electric fields to induce a transient increase in cell membrane permeability and the movement of charged particles. By permeabilizing cell membranes in the target tissue, electroporation significantly improves the intracellular uptake of exogenous substances that have been introduced into the target tissue. Increasing cell membrane permeability and molecular transport through electroporation is a method of delivering an HBV vaccine by passing it through a barrier such as a cell membrane. The use of electroporation as a method of inducing EMTAD has the advantage that the physical nature of this method allows electroporation to be used in tissues of almost all types. Accordingly, in various aspects and embodiments of the invention, electroporation as a method of inducing EMTAD is discussed, but not limited to.

HBV vaccines.

As used herein, the term HBV vaccine is used herein in its broadest sense to include any agent capable of providing a desired or beneficial immune response, therapeutic and/or prophylactic effect against HBV in living tissue. Thus, the term includes both prophylactic and therapeutic vaccines against HBV, as well as any other category of agents that have such desired effects. The scope of the present invention is quite broad and includes controlled delivery of any HBV vaccines, regardless of their classification. HBV vaccines include, but are not limited to, pharmaceutical drugs and vaccines, and nucleic acid sequences (such as supercoiled, sterically relaxed and linear plasmid DNA, RNA, antisense constructs, artificial chromosomes or any other nucleic acid-based therapeutics) and any of their compositions. Such agent formulations include, but are not limited to, cationic lipids, cationic and/or nonionic polymers, liposomes, saline, nuclease inhibitors, anesthetics, poloxamers, preservatives, sodium phosphate solutions or other compounds that may improve administration, stability and/or or the effect of the HBV vaccine. Additional formulations include agents and additives that impart the ability to regulate the viscosity and electrical resistance of the administered agent.

In a preferred aspect of the invention, the HBV vaccine for use in the present invention may include:

a first nucleic acid molecule, and preferably a first plasmid DNA vector, comprising a first polynucleotide encoding an HBV polymerase antigen, and preferably an HBV polymerase antigen, having an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, for example, by at least 98, 98.5, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity;

a second nucleic acid molecule, and preferably a second plasmid DNA vector, comprising a second polynucleotide encoding a truncated HBV core antigen, and preferably a truncated HBV core antigen, consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14; and a pharmaceutically acceptable carrier; and wherein the first nucleic acid molecule and the second nucleic acid molecule are present in the same nucleic acid molecule, such as the same plasmid DNA vector, or in two different nucleic acid molecules, such as two separate plasmid DNA vectors.

As used herein, each of the terms HBV core antigen, HBcAg and core antigen means an HBV antigen capable of inducing an immune response, such as a humoral and/or cell-mediated response against the HBV core protein in an individual. The terms core, core polypeptide, and core protein each refer to the HBV viral core protein. A full-length core antigen is typically 183 amino acids long and includes an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain (amino acids 150 to 183). The 34-residue nucleic acid binding domain is required for encapsidation of pregenomic RNA. This domain also functions as a nuclear import signal. It contains 17 arginine residues and is highly basic, consistent with its function. The cellular HBV protein is dimeric in solution, and the dimers are capable of self-assembly into icosahedral capsids. Each core protein dimer has four α-helical bundles flanked by an aspirated domain on both sides. Truncated HBV core proteins that do not contain a nucleic acid binding domain are also capable of forming capsids.

In one aspect of the present application, the HBV antigen used in the present invention is a truncated HBV core antigen. As used herein, the term truncated HBV core antigen means an HBV antigen that does not contain the full-length HBV core protein but is capable of inducing an immune response against the HBV core protein in an individual. For example, an HBV core antigen can be modified to remove one or more amino acids from the C-terminal nucleic acid binding domain of the core antigen, which has a high positive charge (arginine-rich), and such antigen typically contains seventeen arginine residues (R). The truncated HBV core antigen of the invention is preferably a C-terminally truncated HBV core protein that does not contain the HBV core nuclear import signal and/or a truncated HBV core protein from which the C-terminal HBV core nuclear import signal has been removed. In one embodiment of the invention, the truncated HBV core antigen has a deletion in the C-terminal nucleic acid binding domain, such as deletion of amino acid residues 1-34 of the C-terminal nucleic acid binding domain, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33

- 10 043519 or 34 amino acid residues, and preferably, deletion of all 34 amino acid residues. In a preferred embodiment of the invention, the truncated HBV core antigen has a deletion in

The C-terminal nucleic acid binding domain, preferably all 34 amino acid residues.

The HBV core antigen used in the present invention may be a consensus sequence derived from multiple HBV genotypes (eg, genotypes A, B, C, D, E, F, G and H). As used herein, the term consensus sequence means an artificial amino acid sequence derived from an alignment of the amino acid sequences of homologous proteins, for example, as determined by alignment (eg, using Clustal Omega) of the amino acid sequences of homologous proteins. The order of the most frequently occurring amino acid residues found at each position in a sequence alignment can be calculated based on the sequences of HBV antigens (eg, core, pol, etc.) from at least 100 natural HBV isolates. The consensus sequence may be a non-natural sequence and a sequence different from native viral sequences. Consensus sequences can be constructed by aligning multiple HBV antigen sequences from different sources using multiple sequence alignment programs and at different alignment positions, selecting the most frequently occurring amino acids. Preferably, the HBV consensus antigen sequence is derived from HBV genotypes B, C and D. The term consensus antigen is used to refer to an antigen having a consensus sequence.

A typical truncated HBV core antigen used in the present invention does not have a nucleic acid binding function and can induce an immune response in a mammal against at least two HBV genotypes. Preferably, the truncated HBV core antigen can induce a T cell response in a mammal against at least HBV genotypes B, C and D. More preferably, the truncated HBV core antigen can induce a CD8 T cell response in a human against at least HBV genotypes A , B, C and D.

Preferably, the HBV core antigen used in the present invention is a consensus antigen, and preferably a consensus antigen derived from HBV genotypes B, C and D, and more preferably a truncated consensus antigen derived from HBV genotypes B, C and D. A representative truncated HBV core consensus antigen of the invention consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, for example at least 90, 91, 92, 93, 94, 95 , 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99 .7, 99.8, 99.9 or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14. SEQ ID NO: 2 and SEQ ID NO: 4 are core consensus antigens derived from HBV genotypes B, C and D. SEQ ID NO: 2 and SEQ ID NO: 14 contain a native core antigen domain that binds to a nucleic acid and has a high positive charge (arginine rich) with a C-terminal deletion of 34 amino acids.

In a specific embodiment of the invention, the HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence SEQ ID NO: 2. In another specific embodiment of the invention, the HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence SEQ ID NO: 14 .

Examples of polynucleotide sequences encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 include, but are not limited to, a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1, e.g. 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least by 96%, by at least 96.5%, by at least 97%, by at least 97.5%, by at least 98%, by at least 98.5%, by at least 99 %, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to SEQ ID NO: 1, and preferably about 98%, approximately 99% or 100% identical to SEQ ID NO: 1.

Examples of polynucleotide sequences encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 14 include, but are not limited to, a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 15, e.g. 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least by 96%, by at least 96.5%, by at least 97%, by at least 97.5%, by at least 98%, by at least 98.5%, by at least 99 %, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to SEQ ID NO: 15, and preferably about 98%, 11 043519 is approximately 99% or 100% identical to SEQ ID NO: 15.

In specific embodiments, the HBV vaccine used in the present invention comprises a non-naturally occurring nucleic acid molecule encoding a truncated HBV core antigen, and such non-naturally occurring nucleic acid molecule contains the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

As used herein, the term HBV polymerase antigen, HBV Pol antigen, or HBV pol antigen means an HBV antigen capable of inducing an immune response, for example, a humoral and/or cell-mediated response against HBV polymerase in an individual. The terms polymerase, polymerase polypeptide, Pol, and pol each refer to HBV viral DNA polymerase. The HBV viral DNA polymerase has four domains, including, from N-terminus to C-terminus, a protein terminal domain (TP), which acts as a primer for minus-strand DNA synthesis; a spacer that does not play a significant role in the functioning of the polymerase; reverse transcriptase (RT) domain for transcription; and RNase H domain.

In one embodiment of the present application, the HBV antigen comprises an HBV Pol antigen or any immunogenic fragment or combination thereof. The HBV Pol antigen may contain additional modifications to enhance the immunogenicity of the antigen, for example by introducing mutations into the active sites of the polymerase and/or RNase H domains to reduce or significantly eliminate certain enzymatic activities.

Preferably, the HBV Pol antigen suitable for use in the present invention does not have reverse transcriptase activity and RNase H activity and is capable of inducing an immune response in a mammal against at least two HBV genotypes. Preferably, the HBV Pol antigen can induce a T cell response in a mammal against at least HBV genotypes B, C and D. More preferably, the HBV Pol antigen can induce a CD8 T cell response in a human against at least HBV genotypes A, B , C and D.

Thus, in some embodiments, the HBV Pol antigen used in the present invention is an inactivated Pol antigen. In one embodiment of the invention, the inactivated HBV Pol antigen contains one or more amino acid mutations in the active site of the polymerase domain. In another embodiment of the invention, the inactivated HBV Pol antigen contains one or more amino acid mutations in the active site of the RNase H domain. In a preferred embodiment of the invention, the inactivated HBV Pol antigen contains one or more amino acid mutations in the active site of the polymerase domain and the RNase H domain. Thus for example, the YXDD motif in the polymerase domain of the HBV pol antigen, which may be required for nucleotide/metal ion binding, can be mutated, for example, by replacing one or more aspartate residues (D) with aspartic residues (N) to eliminate or reduce the coordination function with metal, which will thereby reduce or significantly eliminate the function of reverse transcriptase. Alternatively, or in addition to mutation of the YXDD motif, the DEDD motif in the RNase H domain of the HBV pol antigen required for coordination with Mg ²⁺ can be mutated, for example, by replacing one or more aspartate residues (D) with aspartic residues (N) and/or replacing glutamate residue (E) with glutamine (Q), which will thereby reduce or substantially eliminate the function of RNase H. In a specific embodiment of the invention, the HBV pol antigen is modified by (1) replacing aspartate residues (D) with aspartic residues (N) in polymerase domain YXDD motif; and (2) replacing the first aspartate residue (D) with an aspartic residue (N) and the first glutamate residue (E) with a glutamine residue (N) in the DEDD motif of the RNase H domain, thereby reducing or substantially eliminating reverse transcriptase and RNase function H antigen pol.

In a preferred embodiment of the present application, the HBV pol antigen is a consensus antigen, and preferably a consensus antigen derived from HBV genotypes B, C and D, and more preferably an inactivated consensus antigen derived from HBV genotypes B, C and D. Representative Consensus The HBV pol antigen of the invention contains an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 4, for example at least 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5 , 97, 97.5, 98, 98.5, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to SEQ ID NO: 4, and preferably at least 98% identical to SEQ ID NO: 4, for example, at least 98, 98.5, 99, 99.1, 99.2, 99, 3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to SeQ ID NO: 4. SEQ ID NO: 4 is a consensus HBV pol antigen derived from HBV genotypes B, C and D, and containing four mutations in the active sites of the polymerase and RNase H domains. Specifically, the four mutations include the replacement of aspartic acid residues (D) with aspartic residues (N) in the YXDD motif of the polymerase domain; and replacement of the first aspartate residue (D) with an aspartic residue (N) and replacement of the first glutamate residue (E) with a glutamine residue (Q) in the DEDD motif of the RNase H domain.

In a specific embodiment of the invention, the HBV pol antigen used in the present invention contains the amino acid sequence of SEQ ID NO: 4. In other embodiments of the present application, the HBV core antigen used in the present invention consists of the amino acid

- 12 043519 sequence SEQ ID NO: 4.

Examples of polynucleotide sequences encoding an HBV pol antigen containing the amino acid sequence of SEQ ID NO: 4 include, but are not limited to, a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16, for example, at least 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.1, 99.2, 99, 3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16, and preferably 98, 99 or 100% identical is identical to SEQ ID NO: 3 or SEQ ID NO: 16. In specific embodiments of the present application, the HBV vaccine used in the present invention contains a non-naturally occurring nucleic acid molecule encoding an HBV pol antigen, and the non-naturally occurring nucleic acid molecule acid contains the polynucleotide sequence SEQ ID NO: 3 or SEQ ID NO: 16.

As used herein, the term fusion protein or hybrid means a single polypeptide chain having at least two polypeptide domains that are not typically present in a single naturally occurring polypeptide.

In an aspect of the present invention, the HBV antigen used in the present invention may include a fusion protein comprising a truncated HBV core antigen operably linked to an HBV Pol antigen, or a HBV Pol antigen operably linked to a truncated HBV core antigen, preferably via a linker. As used herein, the term linker means a compound or portion thereof that acts as a molecular bridge to operably link two different molecules, where one portion of the linker is operably attached to a first molecule and the other portion of the linker is operably attached to a second molecule. As used herein, the term operably attached refers to a relationship or position where the described components are in a relationship that allows them to function in a certain manner. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of regulating transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is capable of secreting or translocating the amino acid sequence of interest to a desired site.

Thus, for example, in a fusion protein comprising a first polypeptide and a second heterologous polypeptide, the linker serves primarily as a spacer between the first and second polypeptides. In one embodiment of the invention, the linker consists of amino acids linked to each other by peptide bonds, preferably 1-20 amino acids linked by peptide bonds, where the amino acids are selected from 20 naturally occurring amino acids. In one embodiment of the invention, 1-20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine. Preferably, the linker consists of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Representative linkers are polyglycines, and in particular (Gly)5, (Gly)8; poly(O1y-A1a) and polyalanines. One representative suitable linker is (AlaGly) _n , where n is an integer from 2 to 5.

Preferably, the fusion protein used in the present invention can induce an immune response in a mammal against HBV core antigen and HBV Pol antigen of at least two genotypes. Preferably, the fusion protein can induce a T cell response in a mammal against at least genotypes B, C and D of HBV. More preferably, the fusion protein can induce a CD8 T cell response in humans against at least HBV genotypes A, B, C and D.

In one aspect of the present application, the fusion protein used in the present invention may comprise a truncated HBV core antigen having an amino acid sequence that is at least 90, such as at least 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99, 7, 99.8, 99.9 or 100% identical to SEQ ID NO: 2 or 14; HBV Pol linker and antigen having an amino acid sequence that is at least 90, for example at least 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5 , 98, 98.5, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to SEQ ID NO: 4.

Preferably, the fusion protein used in the present invention contains a truncated HBV core antigen consisting of the amino acid sequence SEQ ID NO: 2 or 14; a linker comprising (AlaGly)n, where n is an integer from 2 to 5, and an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 4. More preferably, the fusion protein used in the present invention contains the amino acid sequence of SEQ ID NO: : 20.

In some embodiments of the present application, the fusion protein used in the present invention also contains a signal sequence. Preferably, the signal sequence has the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19. More preferably, the fusion protein contains the amino acid sequence of SEQ ID NO: 21.

Examples of polynucleotide sequences encoding a fusion protein used in the present invention include, but are not limited to, the polynucleotide sequence

- 13 043519 identity that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15, for example at least 90, 91, 92, 93, 94, 95, 95.5, 96, 96, 5, 97, 97.5, 98, 98.5, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99, 9 or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 15, and preferably 98, 99 or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 15, which is operably linked to a coding linker sequence that is at least at least 90% identical to SEQ ID NO: 22, for example, at least 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5 , 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to SEQ ID NO: 22, and preferably is 98, 99 or 100% identical to SEQ ID NO: 22, which is also operably linked to a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16, for example, at least 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16, and preferably 98, 99 or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16. In specific embodiments of the present application, the HBV vaccine used in the present invention contains a non-naturally occurring nucleic acid molecule encoding a fusion protein, and the non-naturally occurring nucleic acid molecule contains SEQ ID NO: 1 operably linked to SEQ ID NO: 22, which is also operably linked to SEQ ID NO: 3.

In an aspect of the present application, in the HBV vaccine used in the present invention, the first and second nucleic acid molecules are first and second plasmid DNA vectors, respectively, and each first and second plasmid DNA vectors contain an origin of replication, an antibiotic resistance gene, and a 5' to 3', a promoter sequence, an enhancer sequence, a signal peptide coding sequence, a first polynucleotide sequence or a second polynucleotide sequence, and a polyadenylation signal sequence. In a preferred embodiment of the invention, the plasmid DNA is an expression vector suitable for expression of the protein in mammalian host cells. Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNATM, pcDNA3TM, pVAX, pVAX-1, and the like. Preferably, the expression vector is based on pVAX-1, which can be further modified to optimize protein expression in mammalian cells. pVAX-1 is the most commonly used plasmid in DNA vaccines and contains the strong human cytomegalovirus immediate early promoter (CMV-IE) followed by a polyadenylation (pA) sequence derived from bovine growth hormone (bGH). In addition, pVAX-1 contains a pETS origin of replication and a kanamycin resistance gene induced by a small prokaryotic promoter that allows propagation of the bacterial plasmid. Preferably, the plasmid DNA vector contains a codon optimized kanamycin resistance gene having a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 12, and preferably 100% identical to SEQ ID NO: 12.

In a specific embodiment, the HBV vaccine used in the present invention includes:

a) a first plasmid DNA vector comprising from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7, an enhancer sequence containing the polynucleotide sequence of SEQ ID NO: 8, a signal peptide coding sequence and containing the polynucleotide sequence of SEQ ID NO: 5, a first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 3, and a polyadenylation signal sequence containing the polynucleotide sequence of SEQ ID NO: 11;

b) a second plasmid DNA vector comprising from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7, an enhancer sequence containing the polynucleotide sequence of SEQ ID NO: 8, a signal peptide coding sequence and containing the polynucleotide sequence of SEQ ID NO: 5, a second polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 1, and a polyadenylation signal sequence containing the polynucleotide sequence of SEQ ID NO: 11; And

c) a pharmaceutically acceptable carrier, wherein the first plasmid DNA vector and the second plasmid DNA vector also contain a kanamycin resistance gene having the polynucleotide sequence SEQ ID NO: 12, and an origin of replication having the polynucleotide sequence SEQ ID NO: 10, and where the first the plasmid DNA vector and the second plasmid DNA vector are in the same composition or in two different compositions.

In those embodiments in which the HBV vaccine includes a first vector, such as a first DNA plasmid, and a second vector, such as a second DNA plasmid, the amount of each of the first and second vectors is not particularly limited. Thus, for example, the first DNA plasmid and the second DNA plasmid may be present in a ratio of 10:1 to 1:10 by weight, for example, 10:1, 9:1, 8:1, 7:1,

- 14 043519

6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1: 8, 1:9 or 1:10 by weight. Preferably, the first and second DNA plasmids are present in a 1:1 ratio by weight.

The compositions and immunogenic combinations of the invention may contain additional polynucleotides or vectors encoding additional HBV antigens and/or additional HBV antigens or immunogenic fragments thereof. However, in certain embodiments of the invention, the compositions and immunogenic combinations of the invention do not contain certain antigens. In preferred embodiments of the invention, the HBV vaccine used in the present invention does not contain a nucleic acid molecule encoding an HBV antigen selected from the group consisting of hepatitis B virus surface antigen (HBsAg), HBV envelope antigen (Env) and L protein antigen HBV and also does not contain an HBV antigen selected from the group consisting of hepatitis B virus surface antigen (HBsAg), HBV envelope antigen (Env) and HBV L protein antigen.

The HBV vaccine used in the present invention may also contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is non-toxic and should not adversely affect the effectiveness of the active ingredient. Pharmaceutically acceptable carriers may include one or more components such as water, glycols, sugars, oils, amino acids, alcohols, preservatives, emollients, stabilizers, coloring agents, and the like.

Other examples of HBV vaccines used in the present invention are described in the International Patent Application entitled Hepatitis B Virus (HBV) Vaccines and their Use, filed on the same date as the present application under Attorney Document Number 688097-403WO1, Contents which is incorporated herein by reference in its entirety.

Sets/Systems.

In its general aspect, the present invention relates to a kit or system for controlled delivery of an HBV vaccine to a predetermined tissue site of an individual in need thereof, wherein said kit comprises an HBV vaccine and a device for administering the HBV vaccine to a predetermined tissue site by means of electroporation. For example, a kit or system may contain a pre-packaged container, such as a syringe, containing a pre-measured amount of HBV vaccine that can be loaded into the device described herein for subsequent administration of HBV vaccine.

Methods for inducing an immune response.

In another general aspect, the present invention relates to a method of inducing an immune response against hepatitis B virus (HBV) in an individual in need thereof, the method comprising administering to the individual an immunogenically effective amount of an HBV vaccine using a device, kit or system of the invention. Any of the devices, kits or systems of the invention described herein may be used in the methods of the invention. As used herein, the term infection means invasion of a host by a disease-causing agent. A disease agent is considered infectious if it is capable of infecting a host and replicating or multiplying within the host. Examples of infectious agents are viruses, such as HBV and some types of adenoviruses, prions, bacteria, fungi, protozoa, etc. The term HBV infection specifically means the invasion of a host organism, such as host cells and tissues, by the HBV virus.

As used herein, the term inducing an immune response, as it relates to the methods described herein, encompasses producing a desired immune response or effect in an individual in need thereof against an infection, for example, an HBV infection. The term inducing an immune response also encompasses the provision of therapeutic immunity to treat a disease caused by a pathogenic agent, such as HBV. As used herein, the term therapeutic immunity or therapeutic immune response means that the body of a vaccinated individual is capable of fighting infection with the pathogenic agent against which the vaccination was given, for example, producing immunity against HBV infection conferred by an HBV vaccine. In an embodiment of the invention, the term inducing an immune response means producing immunity in an individual in need thereof, for example, to provide a therapeutic effect against a disease such as HBV infection. In some embodiments, the term inducing an immune response means producing or enhancing cellular immunity, such as a T cell response against HBV. In some embodiments, the term inducing an immune response means producing or enhancing a humoral immune response against HBV. In some embodiments, the term inducing an immune response means producing or enhancing a cellular and humoral immune response against HBV.

As used herein, the term protective immunity or protective immune response means that the body of a vaccinated individual is capable of fighting off infection by the pathogenic agent against which the vaccination was given. Typically, an individual who has developed a protective immune response will have only mild to moderate clinical symptoms, or

- 15 043519 these symptoms are completely absent. Typically, an individual who has a 'protective immune response' or immunity against a particular agent does not die from infection with said agent.

Typically, administration of an HBV vaccine in accordance with embodiments of the present invention is carried out for therapeutic purposes to generate an immune response against HBV after infection with HBV or the development of symptoms characteristic of HBV infection, for example, for therapeutic vaccination.

As used herein, the term immunogenically effective amount or immunologically effective amount means an amount of a composition, polynucleotide, vector or antigen sufficient to induce the desired immune effect or immune response in an individual in need thereof. In one embodiment of the invention, an immunogenically effective amount means an amount sufficient to induce an immune response in an individual in need thereof. In another embodiment, an immunogenically effective amount means an amount sufficient to produce immunity in an individual in need thereof, for example, to provide a therapeutic effect against a disease such as HBV infection. The immunogenically effective amount may vary depending on a number of factors, such as the individual's physical condition, age, body weight, health status, and the like; from the specific use, for example, providing protective immunity or therapeutic immunity; and from a specific disease, for example, a viral infection, against which it is desirable to develop immunity. An immunogenically effective amount can be readily determined by one skilled in the art from the description of the invention.

In specific embodiments of the invention, an immunogenically effective amount means an amount of a composition or immunogenic combination (such as an HBV vaccine) that is sufficient to achieve one, two, three, four or more of the following effects, such as: (i) reducing or ameliorating severity HBV infection or associated symptom; (ii) reducing the duration of HBV infection or associated symptoms; (iii) preventing progression of HBV infection or associated symptoms; (iv) inducing regression of HBV infection or associated symptoms; (v) preventing the development or occurrence of HBV infection or associated symptoms; (vi) preventing recurrence of HBV infection or associated symptoms; (vii) reducing the number of hospitalizations of individuals with HBV infection; (viii) reducing the length of hospitalization of an individual having HBV infection; (ix) improving survival of individuals with HBV infection; (x) eliminating HBV infection in the individual; (xi) inhibiting or reducing HBV replication in an individual; and/or (xii) enhancing or enhancing the prophylactic or therapeutic effect(s) of another therapy.

In other specific embodiments of the invention, an immunogenically effective amount is an amount sufficient to reduce HBsAg levels until they are completely eliminated, as indicated by clinical seroconversion; achieving sustained clearance of HBsAg associated with a decrease in the number of infected hepatocytes under the influence of the individual's immune system; inducing activated T cell populations specific for HBV antigen; and/or for persistent loss of HBsAg for 12 months. Examples of a target index are a lower HBsAg value, that is, a value below the International Units (IU) HBsAg cutoff of 500 copies, and/or a higher CD8 count.

In accordance with general guidance, an immunogenically effective amount, when used with respect to plasmid DNA, may range from about 0.1 mg/ml to 10 mg/ml of the total amount of plasmid DNA, for example 0.1, 0.25 , 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/ml. Preferably, the immunogenically effective amount of DNA plasmid is less than 8 mg/ml, more preferably less than 6 mg/ml, and even more preferably 3-4 mg/ml. The immunogenically effective amount can be calculated from a single vector or plasmid or from a plurality of vectors or plasmids. An immunogenically effective amount may be administered in a single composition or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions (e.g., tablets, capsules, or injections), wherein multiple administrations capsules or injections provides an immunogenically effective amount to an individual. It is also possible to administer an immunogenically effective amount to an individual followed by another dose of an immunogenically effective amount to the same individual in a so-called prime-boost regimen. This general concept of a prime-boost regimen is well known to those in the vaccine field. If necessary, in addition to this regimen, additional booster injections can be carried out, but not necessarily.

According to embodiments of the invention, an immunogenic combination comprising two DNA plasmids, for example, a first DNA plasmid encoding HBV core antigen and a second DNA plasmid encoding HBV pol antigen, can be administered to an individual by mixing both plasmids and delivering the mixture to one anatomical site. plot. Alternatively, two separate immunizations can be performed, each delivering one expression plasmid. In such embodiments, regardless of whether both plasmids are administered in one immunization as a mixture or in two separate immunizations, the first DNA plasmid and the second DNA plasmid can be administered in a ratio of 10:1 to 1: 10 by weight, for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1,

1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 by weight. Preferably, the first and second DNA plasmids are administered in a 1:1 weight ratio.

In some embodiments of the invention, the individual to be treated in accordance with the methods of the invention is an HBV-infected individual, and in particular, an individual who has a chronic HBV infection. Acute HBV infection is characterized by effective activation of the innate immune system, which is complemented by a subsequent broad adaptive response (eg, production of HBV-specific T cell neutralizing antibodies), usually resulting in successful inhibition of replication or elimination of infected hepatocytes. In contrast, such responses are impaired or reduced due to high viral and antigenic load, for example, HBV envelope proteins are produced in abundance and can be released in subviral particles in 1000-fold excess relative to the infectious virus.

Chronic HBV infection is described in phases characterized by viral load, liver enzyme levels (necroinflammatory activity), HBeAg or HBsAg load, or the presence of antibodies to these antigens. ssDNA levels remain relatively constant at approximately 10 to 50 copies per cell, even though viremia can vary significantly. The persistent presence of ssDNA species leads to chronic infection. More specifically, the phases of chronic HBV infection include: (i) the immunotolerant phase, characterized by a high viral load and normal or minimally elevated liver enzyme levels; (ii) an HBeAg-positive phase of immune activation, in which lower or declining levels of viral replication are observed with significantly elevated liver enzyme levels; (iii) the inactive phase of HBsAg maintenance, which represents a low replicative state with low viral loads and normal serum liver enzyme levels, which may then undergo HBeAg seroconversion; and (iv) an HBeAg-negative phase, in which periodic viral replication (reactivation) occurs with accompanying fluctuations in liver enzyme levels, where mutations in the precore and/or basal core promoter are most common and therefore HBeAg is not produced by the infected cell.

As used herein, the term chronic HBV infection refers to an individual in whom the presence of HBV has been detected for more than 6 months. An individual with chronic HBV infection may be in any phase of chronic HBV infection. The term chronic HBV infection has its generally accepted meaning known to those skilled in the art. Chronic HBV infection may, for example, be characterized by the persistent presence of HBsAg for 6 months or more after acute HBV infection. For example, the term chronic HBV infection used here corresponds to the definition given by the Centers for Disease Control and Prevention (CDC), according to which chronic HBV infection can be characterized by laboratory criteria such as: (i) a negative IgM antibody test against hepatitis B core antigen (anti-HBc IgM) and a positive test for hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg), or a test for the presence of a nucleic acid, namely, hepatitis B virus DNA, or ( ii) a positive HBsAg or HBV DNA nucleic acid test, or a positive HBeAg test performed twice at least 6 months apart.

In accordance with specific embodiments of the invention, an immunogenically effective amount means an amount of a composition or immunogenic combination that is sufficient to treat chronic HBV infection.

In some embodiments of the invention, an individual having a chronic HBV infection is treated with a nucleoside analogue (NUC), and the infection is suppressed by NUC. As used herein, the term NUC-suppressed refers to an individual having undetectable levels of HBV virus and stable alanine aminotransferase (ALT) levels for at least six months. Examples of nucleoside/nucleotide analogue treatment are treatment with HBV polymerase inhibitors such as entacavir and tenofovir. Preferably, the individual having chronic HBV infection does not have advanced fibrosis or cirrhosis of the liver. Such an individual typically has a METAVIR score of less than 3 for fibrosis and a fibroscan result of less than 9 kPa. The METAVIR score is a scoring system that is commonly used to assess the degree of inflammation and fibrosis by histopathological evaluation of liver biopsies from patients with hepatitis B. The scoring system includes two standardized scores: one score indicating the degree of inflammation and one score indicating the degree of fibrosis .

Elimination or reduction of chronic HBV infection is believed to allow early detection of severe liver disease, including viral cirrhosis and hepatocellular carcinoma. Thus, the methods according to the invention can also be used as therapy for the treatment of diseases caused by HBV. Examples of HBV-induced diseases include, but are not limited to, cirrhosis, cancer (eg, hepatocellular carcinoma) and fibrosis, and in particular,

- 17 043519 advanced fibrosis, characterized by a METAVIR score of 3 or higher for fibrosis. In such embodiments, an immunogenically effective amount means an amount sufficient to achieve persistent loss of HBsAg for 12 months and significant attenuation of the clinical disease (eg, cirrhosis, hepatocellular carcinoma, etc.).

Methods according to embodiments of the invention further comprise administering to the individual in need thereof another immunogenic agent (such as another HBV antigen or another antigen) or another anti-HBV agent (such as a nucleoside analogue or other anti-HBV agent) in combination with a composition of the invention.

The ability to induce or stimulate an immune response against HBV after administration to an animal or human can be assessed either in vitro or in vivo using a number of assays that are standard in the art. A general description of methods for assessing the initiation and activation of an immune response can be found, for example, in Coligan et al. (1992 and 1994, Current Protocols in Immunology; ed. J Wiley & Sons Inc., National Institute of Health). Measuring cellular immunity can be accomplished by measuring the profiles of cytokines secreted by activated effector cells, including cells derived from CD4+ and CD8 ⁺ T cells (for example, by quantifying cells producing IL-10 or IFN-gamma using ELISPOT) , by determining the activation status of immune effector cells (eg, using classical [3H]-thymidine uptake T-cell proliferation assays), using an antigen-specific T-lymphocyte assay in a sensitized individual (eg, peptide-specific lysis in a cytotoxicity assay and so on.).

The ability to stimulate a cellular and/or humoral response can be determined by antibody binding and/or competitive binding (see, eg, Harlow, 1989, Antibodies, Cold Spring Harbor Press). For example, titers of antibodies produced in response to administration of a composition providing immunogenicity can be assessed using an enzyme-linked immunosorbent assay (ELISA). Immune responses can also be determined using a neutralizing antibody assay, where virus neutralization is defined as loss of infectivity through reaction of the virus with a specific antibody/inhibition/neutralization of the virus by that antibody. The immune response can be further assessed using an antibody-dependent cellular phagocytosis (ADCP) assay.

Target tissues.

Target tissues most suitable for EMTAD using the methods, devices and systems described herein are both healthy and pathological cells present, for example, in epidermal, dermal, hypodermal, connective and muscle tissue. This method can also be applied to healthy or diseased organs that must be accessed by minimally invasive or other surgical methods. Such target tissues include the liver, lungs, heart, blood vessels, lymphatic system, brain, kidneys, pancreas, stomach, intestines, colon, bladder and reproductive organs. In some embodiments of the invention, the desired therapeutic effect can be achieved using a method or device described herein for delivering a specified amount of agent to certain cell types typically present in the target tissues, as well as to other cell types that are abnormal in those tissues ( for example, during chemotherapy treatment of tumors).

As discussed previously and as shown in FIG. 1, the disadvantages of traditional EMTAD are the lack of an accurate and reproducible spatial and temporal relationship between HBV vaccine administration and electrical signal delivery. In contrast to the traditional EMTAD method, the present disclosure discloses methods and devices for combined CTAA and ESA performed to provide a more advantageous clinical use of EMTAD. The present invention discloses various aspects of CTAA in combination with ESA to achieve reproducible, consistent and effective delivery of an HBV vaccine. The methods and devices described herein provide spatial and temporal control of HBV vaccine administration relative to the delivery of electrical signals, and thereby improve the transport of the agent to a target tissue and/or its uptake by that tissue.

Methods.

In one aspect, the present invention relates to systems, kits and devices for use in methods of controlled administration of an HBV vaccine to an individual in need thereof, followed by ESA. As used herein, the term individual means any animal, preferably a mammal, and most preferably a human, that can be or has been treated with a method in accordance with an embodiment of the invention. As used herein, the term mammal includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc. etc., and more preferably, a person.

- 18 043519

In another aspect, the present invention relates to systems, kits and devices for use in methods of controlled administration of an HBV vaccine, which is preceded by an ESA. In another aspect, the invention described herein relates to systems and devices for use in methods of controlled administration of an HBV vaccine that is accompanied by an ESA. These methods include, but are not limited to, spatial or coordinated relationship with the determination of treatment parameters, subject preparation procedures, CTAA, ESA and additional measures.

Determination of treatment parameters.

In some embodiments of the invention, treatment parameters are based on the desired amounts and/or duration of administration of HBV vaccine doses. HBV vaccine dosage may depend, for example, on the specific indication or treatment application (such as the type and location of the target tissue), as well as on various characteristics of the individual (such as age and body weight). Dosing of the HBV vaccine can be controlled by parameters related to the administration of the HBV vaccine and ESA. Representative control parameters related to CTAA include, but are not limited to, agent volume, agent viscosity, and injection rate. Representative control parameters related to ESA include, but are not limited to, electrical signal characteristics, tissue volume exposed to electrical signals, and electrode array format. The relative timing and location of CTAA and ESA are parameters that provide additional regulation of HBV vaccine dosing.

Preparation of the individual.

In the embodiments described herein, the methods described herein may include the step of preparing the individual. Preparation of the individual may include, but is not limited to, antiseptic cleansing and administration of an anesthetic, including local or regional anesthesia, regional anesthesia, spinal anesthesia, epidural anesthesia, or general anesthesia. In a representative case of intramuscular (IM) ESA, protocols to minimize the effects of electrical muscle stimulation may be included in the method described herein, for example, including thermoregulation (eg, muscle cooling), administration of anesthetics, and/or alternative stimulation patterns sufficient to alleviate discomfort. It should be noted that the selected means of preparing an individual should not adversely affect therapeutic efficacy if acceptable alternatives exist. For example, it has been shown that in some cases, intramuscular administration of amide-based anesthetics may have undesirable effects on therapy based on intramuscular delivery of plasmid DNA, which is likely due to the weak myotoxicity of these agents, which can inhibit the ability of muscle cells to expression of the protein encoded by the input DNA sequence.

CTAA and ESA.

In certain embodiments described herein, a method is provided in which CTAA and ESA are combined to provide consistent and reproducible delivery of an HBV vaccine. In some cases, the present invention relates to devices or kits suitable for CTAA, including, for example, a device containing at least one of automatic injection devices and jet injectors.

The present invention relates to methods, kits and devices that allow the transdermal placement of multiple elongated electrodes to the desired depth in a safe and consistent manner relative to the desired site of agent distribution in recipients with heterogeneous skin thickness and composition to maintain the delivery of electrical fields into the tissue to facilitate intramuscular, intradermal and/or subcutaneous administration of therapeutic or prophylactic agents, such as nucleic acids, pharmaceuticals, antibodies, peptides, proteins, or combinations thereof.

Systems and methods in accordance with the principles of the present invention enable the coordinated transcutaneous placement of multiple elongated tissue penetrating electrodes at a predetermined target tissue site to propagate electrical fields in the skin, subcutaneous tissue and/or skeletal muscle. The invention disclosed herein enables the user, with minimal training, to place electrodes to the desired depth while maintaining appropriate spatial relationships between multiple electrodes, even when such procedure is performed on areas with different skin characteristics. Such variations may be due to variability in skin characteristics in different areas within one individual or among different groups of recipients. In other words, the systems and methods of the invention must provide a consistent profile regardless of the administerer or recipient. In some embodiments of the invention, placement of the electrodes is accompanied by the introduction of one or more injection needles that are configured to be suitable for administering HBV vaccines to the target tissue area and are located in a predetermined spatial relationship with the electrodes used for ESA. In a representative embodiment of the invention, the electrodes are arranged such that any electrical signals from said electrodes are preferably applied to

- 19 043519 a certain distance from the site of administration of the HBV vaccine using one or more injection needles. In another embodiment of the invention, the electrodes are positioned such that any electrical signals are preferably applied to a site proximal to the site of administration of the HBV vaccine by one or more injection needles.

Aspects of the disclosure may be used alone or in combination to provide transdermal delivery of electrodes for the purpose of delivering electric fields in vivo to facilitate intramuscular, intradermal and/or subcutaneous administration of nucleic acids, small molecule drugs, antibodies, peptides, proteins, and combinations thereof. In some embodiments of the invention, the placement of the electrodes and the subsequent propagation of the electric field is carried out in a coordinated manner with the distribution of the agent of interest over the target tissue site. In a representative embodiment of the invention, the administration of the agent and the application of one or more electric fields are carried out in a controlled and controlled manner so as to maximize the likelihood of achieving spatial and temporal co-localization of the distribution of the agent at the site of the electric field application.

In general, the present invention discloses methods and devices for transdermal placement of electrodes at a predetermined site in the skin, subcutaneous tissue and/or skeletal muscle of a recipient in combination with the administration of an agent of interest and local application of electrical fields to enhance delivery, absorption and /or biological action of the agent. In some embodiments, the present invention has been implemented so that installation and use of the device can be performed efficiently and reliably by users with minimal training. In another embodiment, the present invention also includes the installation of a variety of interlocks, sensors and feedback loops to reduce the frequency and/or potential impact of possible user errors made during setup and use of the device. In fig. 2, an embodiment of the device described herein includes a cassette unit 100, which is a removable device interfaced to an applicator 400 that is configured to be connected to a controller 700 that acts as a source of electrical energy to activate the electrode and subsequently generate an electrical field. as well as for diagnostics and other routine regulatory procedures. The controller 700 also has a user interface, a tray, a holder for an applicator 400, and various other elements described. As can be seen in FIG. 2, in the application method, a reservoir 101 of a suitable size and shape can be inserted into the cassette unit 100.

Details of the cassette unit 100 provided in some embodiments of the device described herein are described in detail, for example, in FIG. 3A-12, together with corresponding interacting parts of the applicator 400 in FIG. 13A-18C, followed, where appropriate, by parts of the controller 700 in FIG. 19-20D. The remaining parts of the applicator 400 are presented next, followed by the remaining parts of the regulator 700.

Moreover, as shown in FIG. 3A-4, in some embodiments described herein, the cassette unit 100 may have a support structure configured for connection to the applicator 400 and comprising two or more elongate electrodes 122 mounted on the structure to form the array. To avoid unwanted propagation of electrical currents within the device, the design and materials of the electrode mounting structure must be specified so that there is a sufficient dielectric barrier between electrodes of opposite polarity within the device. The distal region 137 of the elongated electrodes is connected to the mounting structure through standard mechanical elements and/or adhesives appropriate to the material composition of the electrode mounting structure and the electrodes.

In a representative embodiment of the invention, the outer structure of the cassette body 102 is configured for connection to a fluid reservoir 101 containing an HBV vaccine, wherein the reservoir 101 and the cassette body structure 102 are configured to be operatively coupled to at least one aperture for injection (105 needle) through which the HBV vaccine is injected into the target tissue. In some embodiments of the invention, this configuration facilitates co-localization of the distribution of the agent of interest at the site of the electric field application. In another embodiment of the invention, this configuration facilitates the implementation of a predetermined spatial relationship between the device for ESA and CTAA. In another exemplary embodiment of the invention, a syringe 101 is inserted into a cartridge 100 where, after loading the cartridge into the applicator 400, the syringe 101 is advanced toward the needle hub 152 and connects the cartridge to the needle.

Some embodiments of the invention may include the use of syringes, vials, ampoules, cassettes, or equivalent structures for storing one or more HBV vaccines. In some embodiments of the invention, the reservoirs may be made of at least glass or plastic, the material being selected to be compatible with the agent of interest. The reservoir used may be coated to provide the desired lubricating or protective properties. As described above, electrodes 122 may be hollow and

- 20 043519 in some cases, they may have injection ports that can be operatively connected to fluid reservoirs. Alternatively, the injection port may include one or more hypodermic needles and/or needle-free injection ports suitably positioned relative to the electrodes. The choice of injection port type and size may depend on the desired route of administration, tissue distribution, and physical properties of the agent of interest. In a certain embodiment of the invention, the design of the cassette ensures a predetermined spatial relationship between the injection port and the electrodes at their location such that the distribution of the agent of interest occurs substantially within the tissue defined by the conductive regions of the plurality of electrodes. To minimize the need for the user to handle sharp objects, in a certain embodiment of the invention, the cassette 100 for use with hypodermic needles is configured such that the hypodermic needle must be connected to the cassette at the time of manufacture rather than the more common case of connecting a needle to a syringe during use. In certain embodiments of the invention, an aspect of the invention may include features in the cartridge and/or needle to ensure that the needle is retained during manufacture, dispensing, processing and use, as well as features to ensure proper connection of the reservoir to the needle prior to use. In some embodiments of the invention, such features may minimize the risks of leakage of agent from the reservoir or reservoir connection opening due to breakage or improper connection.

In some embodiments of the invention, the cartridge may include a tissue contact surface located at the distal end of the subunit. In some embodiments of the invention, the tissue contact surface has a substantially planar structure positioned perpendicular to the elongated electrodes and having one or more openings that are configured to allow the electrodes to pass through the tissue contact surface. For embodiments of the invention including an integral reservoir and an injection port, such contact surface also has an aperture for accommodating an injection port or needleless injection. To minimize the risk of contamination of the electrodes and injection needle, as well as inadvertent contact of the user or recipient with sharp objects, in a certain embodiment of the invention, the openings for the placement of electrodes and injection needles should be sized to prevent accidental contact with the electrodes and injection needle. . Most often, the tissue contact surface consists of one or more plastics suitable for at least short-term contact with the tissue.

To avoid potential cross-contamination of biological material by recipients, cassette unit 100 may be configured for single use. In some cases, the cassette contains one or more mechanical, electrical and/or identification elements that limit the use of the cassette to a single administration. Examples of this type of mechanical elements include, for example, but are not limited to, interlocks and/or latches that secure the electrode mounting structure and/or shield (see below) in the on state after use. Examples of electrical elements are, for example, fuses or fuses located in series with one or more electrodes, which are turned off by the electrical power source after the first use of the cassette. Examples of identification elements are, for example, serial radio frequency identification devices, bar codes or quick response codes designed to be read by an applicator, and/or an electrical power source. The identification information for a particular cassette can then be used to prevent accidental or intentional reuse of that cassette by the applicator and/or power source. In one embodiment of the invention, one or more redundant elements are included to minimize the possibility of reusing the cartridges.

In one embodiment of the device described herein, as shown in FIG. 2, applicator 400 includes a support structure configured for connection to cassette unit 100, user interface 410-418 (FIG. 13B), electrically conductive electrical connections configured to provide an operative connection between a conductive contact area located distal areas of elongated electrodes, and a source of electrical energy when introducing electrodes into the target tissue of the recipient. In some cases, the user interface includes a handle, one or more display elements configured to convey information to the user, and one or more elements capable of receiving input from the user. In some embodiments of the invention, the pixels are configured to convey information about the operating status of the device during setup and use, as well as associated warning/error messages. Such displays may include mechanical elements, light sources, alphanumeric displays, and/or electronic display screens. In some cases, elements capable of receiving user input are configured to allow the user, at an appropriate point in the procedure, to disable safety elements in the device preventing accidental discharge and to make a selection of specific procedure parameters (for example, the intended injection depth ) and initiate the insertion procedure, and such elements may include buttons, triggers, mechanical rails and/or levers.

In some embodiments of the invention, the applicator 400 also includes drive mechanisms that interact with the cassette unit, and which are configured to transdermally place electrodes, position the injection port with respect to target tissues, release the agent of interest from the reservoir through the port to the tissue site. target and/or transmitting electrical signals from an electric field generator, such as a controller 700, to a cassette 100. The applicator 400 may be designed such that the energy to operate the mechanisms is supplied by the user or, more preferably, the device may contain one or more inactive energy sources functionally connected to the drive mechanisms in the applicator. Such inactive energy sources include, for example, electromechanical devices (solenoids, motors, lead screws), mechanical components (springs and related devices) and liquefied gases.

A representative implementation of cassette unit 100 is described in FIG. 3A, where the cassette unit 100 includes an opening for loading a reservoir 140 and a reservoir receptacle 142 for receiving and storing medication in the reservoir 101. The cassette unit 100 is necessary because for a device in which an electric field is to be generated and used during therapy, an electric generator fields, such as controller 700, are required to electrically interact with a device containing electrodes designed to contact target tissue. Since the regulator may be reusable and the reservoir 101 may be disposable, the cassette unit 100 may serve to hold the electrodes 122 and the reservoir 101 may be coupled to the reusable device and the disposable cassette unit 100. use. Thus, the applicator 400 may be a reusable component, and the cassette unit 100 may be intended for single use. The cassette unit 100 may also be placed in a state that prevents its subsequent use if errors or distortions occur when inserting the reservoir 101 or if defects are present.

The term reservoir 101 may refer to a syringe, vial, or any other device that may contain an HBV drug or vaccine and which may be connected to a device having an opening, such as the needle shown in FIG. 4, namely, a needle 105 having a sleeve for a needle 152. The reservoir 101 for a cassette unit 100 of this type has a generally conventional shape and size. Various components in the cassette unit 100 are subject to variation from precise dimensions and/or manufacturing tolerances, but are generally of the usual shapes and sizes necessary to reduce the risk that drugs not labeled for their use in the cassette 100 will be erroneously shipped. this device. If a suitably sized reservoir 101 is not available to the user, then one or more interlocks in the cassette unit 100 will not be able to disable it and the system will be non-functional until a suitably sized reservoir 101 is inserted.

As shown in FIG. 3B, the reservoir 101 may be provided with a piston and a drug outlet opening 156. A removable cap 158 may also be provided to maintain the sterility and integrity of the agent until the reservoir is inserted and used. The drug release hole may be adjacent to the needle hub 152 being used, and the piston may be located opposite the drug release hole. As an alternative to an open drug outlet, in some embodiments of the invention, the reservoir may be designed to include a baffle that covers and seals the end of the container opposite the piston. The baffle may be made of elastomeric compounds such as silicone or butyl rubber, with a specific composition and coating of material selected to provide stability and compatibility with the agent contained in the reservoir. A component such as a baffle is usually held in place by a crimp seal or other fastening mechanism. This septum seal configuration eliminates the need for a removable cap, but requires that the needle 105 be provided with a suitable piercing element, such as a needle, spike, or other elements, to access the liquid contained in the reservoir. Specific implementations for this configuration include the use of double-ended needles and spiked ampoule adapters.

The cassette unit 100 is designed not only to receive the reservoir 101, but also to be received by the applicator 400 through the receiving opening 401 of the applicator cassette unit (FIG. 2). Thus, the cassette unit 100 includes a device that allows the applicator 400 to be drawn into and retained at least partially within the internal volume. In some embodiments, the device comprises one or more racks on the surface(s) of the cassette unit 100 that engage a corresponding moving gear assembly in the applicator 400. In other embodiments, the applicator 400 may be coupled to the cassette unit 100 without having to pull it in.

In other embodiments of the invention, other technologies may be used to allow the cassette unit 100 to be coupled to the applicator 400, such as moving tracks or brackets, etc., to which the cassette unit 100 may be coupled.

The applicator 400 is further provided with interface elements allowing it to control certain actions within the cassette unit 100. In particular, the applicator 400 may be configured to control needle insertion, drug delivery, electrode insertion, and electrode activation using various subsystems. In some cases, these stages are interconnected, such that a single action of the applicator 400 initiates a plurality of these stages. In some embodiments, all of these steps except drug delivery and electrode activation are triggered by a single action, as described below in a representative embodiment of the invention.

After appropriate activation, such as through the use of electrical or optical signals applied to the mechanical, electrical, or optical elements of the cassette unit 100, the applicator 400 can provide testing of the subsystems in the cassette unit 100 to ensure that they operate as intended and are correctly configured for drug delivery. means by applying an electric field. Thus, for example, such subsystems are subsystems that can be tested using the applicator 400 to ensure that the cassette unit 100 has not previously been used; that the reservoir has been correctly placed in the cassette unit; that the corresponding force applied to the individual's body was effected by the leveling panel/side screen 108; that the depth was correctly selected by the user; and that the outer cassette cover 110 has been removed. In addition, the applicator 400 may be designed to allow monitoring of the status of the cassette functions during a procedure. Thus, for example, such subsystems are subsystems that can be tested using the applicator 400 to ensure that the electrodes 122 are located in the desired area on the subject before the drug is administered; that the piston in the reservoir was properly actuated before the electrical fields were applied; and that the user maintains an appropriate force applied to the individual's body during the insertion procedure and the like.

In addition to the subsystems that are controlled by the applicator 400, the cassette unit 100 may include corresponding subsystems, including subsystems that interact with the applicator 400 and subsystems that do not interact with it, so as to allow drug delivery and therapy by applying an electric field. . Such subsystems are a subsystem for inserting a needle and electrode, a subsystem for protecting users from contact with sharp objects after therapy; subsystem for providing different depths of needle/electrode insertion; a subsystem for ensuring adequate force is applied to the recipient tissue prior to the commencement of the procedure and subsequently during the insertion procedure, etc. Although this is often described in relation to placeable needles and electrodes, it should be noted that this is not strictly necessary, and that systems with fixed or fixed needles and electrodes also have advantages when using systems and methods according to the principles of the present invention, including the described subsystems.

In one representative embodiment of the invention, as shown in FIG. 4, the cassette unit 100 includes an outer cassette 102, in some cases referred to as a housing. The outer cassette 102 is terminated at the distal end by an outer cassette cap 106. The outer cassette 102 includes an inner cassette receptacle 150 for feeding into an inner cassette 103, which is received and slidably moved relative to the outer cassette 102. The inner cassette 103 includes a reservoir receptacle 142, and therein there may be a reservoir 101. The inner cassette 103 engages the inner cassette cover 104 at the distal end. The inner cassette cover 104 performs a number of functions, including locking the electrodes 122 in place (the inner cassette 103 itself has recesses into which the electrodes 122 are placed) and providing a support surface for the protective shield 134. The inner cassette cover 104 is secured to the inner cassette 103.

The cassette rear portion 112 engages the reservoir receptacle portion 142 of the inner cassette 103, that is, the portion opposite the inner cassette cover 104. The reservoir detection cover 118 engages the cartridge rear portion 112 by a reservoir detection spring 116. The cassette retaining ring 114 locks the system to a specific location, including the rear cassette portion 112 engaged with the inner cassette 103. The reservoir detection spring 116 also serves to push the reservoir 101 into engagement with the needle hub 152 and also serves to create the allowable dimensions of the reservoir 101.

The reservoir lock 120 provides a mechanical lock to prevent inadvertent or unwanted activation of the cartridge elements. Specifically, the reservoir lock 120, also referred to as the first reservoir insertion trigger, is located below the inner cassette 103 and has pins that extend through slots or holes provided in the inner cassette 103 (see FIG. 5B). The fingers prevent the rear cassette portion 112 from sliding relative to the inner cassette 103, and in particular from moving within the inner cassette 103 toward the inner cassette cover 104 before the reservoir is inserted into the reservoir receptacle 142.

When the reservoir 101 is correctly inserted into the reservoir receptacle 142, the reservoir lock 120 is pushed down and the fingers also move down and no longer advance into the reservoir receptacle 142. Such lowering or pressing of the reservoir lock 120 may also be accomplished with an audible, tactile, or tactile click that may inform the user of correct insertion. Once pressed, the rear cassette portion 112, which is no longer locked by the reservoir lock fingers 120, can then be moved and, in particular, can be moved towards the inner cassette cover 104.

Movement of the rear cassette portion 112 is caused by the action of the spring/cassette cover joint 470 when the cassette unit 100 is inserted into the applicator 400 in the manner described below. When the rear portion 112 of the cartridge is moved far enough forward, it locks into place, securing the reservoir 101 in the reservoir receiver 142, and ensuring its correct position relative to the needle hub 152, which will ensure the movement of intact fluid from the reservoir 101 to the opening of the needle 105.

In those embodiments of the device in which the injection needle is built into the cartridge 100, the use of standard commercially available disposable hypodermic needles can be accomplished using such a device. However, the performance and reliability characteristics of the device can be improved by incorporating specially designed features that are not found in hypodermic needles intended for conventional parenteral administration procedures. Particular features of the needle hub 152 may include the material of which it is composed, retention features to prevent displacement of the needle hub 152 from the inner cartridge 103 during dispensing and use, and the orientation of any beveled features in the needle relative to the hub.

Standard disposable injection needles typically consist of injection molded polypropylene thermoplastic. However, for many applications, the toughness, tensile strength and flexural strength of polypropylene may be insufficient to ensure the integrity of the sleeve when subjected to the forces inherent in needle insertion and injection with this device. Specific drawbacks of concern are failure of the sleeve wall due to impact or injection forces, and failure of the sleeve-to-needle connection due to these reasons. Although adjustments to the bushing design, including geometry and wall thickness, can be made to correct these deficiencies, it is not always possible to change the design sufficiently to prevent bushing failure while still ensuring that the bushing will retain its properties. the size required to properly mate with tapered luer slip connectors as described in the related standards document published by the International Standards Organization (ISO) ISO 80369-7:2016, entitled Small diameter connectors for liquids and gases used in healthcare - part 7. Connectors for intravascular or subcutaneous use. In particular, given the forces that the syringe needle and hub are subjected to during administration, some embodiments of the invention utilize a material with improved toughness, tensile strength, and flexural strength. One example is the use of injection molded polycarbonate plastics (such as ZELUX® GS, Makrolon or Lexan) or copolyesters (such as Eastman Tritan Copolyester MX731, MX711 and MX 730). When assessed according to ISO 180:2000 Plastics - Determination of Izod impact strength, a notched impact strength suitable for this purpose is considered to be at least 70 kJ/m ² . When assessed in accordance with ISO 527-1:2012. Plastics - Determination of tensile properties. Part 1: General principles, the tensile strength suitable for this purpose is at least 30 MPa. When assessed in accordance with ISO 178:2010. Plastics - Determination of flexural properties, a flexural strength suitable for this purpose is considered to be at least 50 MPa. In some embodiments of the invention, the specifically selected resin is compatible with the intended sterilization method (eg, gamma radiation) and does not exhibit any undesirable changes in physical properties that may impair its function.

Those embodiments that use a custom-made injection needle include one or more mechanical features not typically found in standard syringe hubs that allow the device to be inserted into the internal cassette 103. Such features may include hinges, latches, or projections. with corresponding mechanical elements located in the inner cassette 103. In some cases, the elements are configured such that the sleeve mates with the inner cassette 103 in a consistent orientation. The combination with a needle manufacturing method that provides a consistent orientation of the bevel or other feature of the needle hole ensures that variations in the injection site or drug distribution due to the location and design of the hole can be taken into account in the design of the device. For example, needles with asymmetrical penetrating tips (eg, beveled) may cause directional displacement during insertion into tissue due to the interaction between the tissue and the asymmetrical penetrating element on the needle. If the electrodes have a symmetrical tip element for penetration (for example, a trocar needle tip), then the electrodes will not have a corresponding displacement when they are inserted. Therefore, the fastener for the needle hub 152 on the inner cassette 103 may provide an offset in the position of the injection hole of the needle 105 relative to the electrodes 122 prior to their placement, taking into account the expected asymmetric bevel properties of the needle during placement. The exact amount of displacement may depend on the nature of the target tissue and the expected range of penetration depth, but in some embodiments of the invention, the needle moves 0.5-1 mm for every 10 mm of penetration depth. When using electrodes and injection needles with different tip profiles, or when the tip profiles must be sequentially oriented with respect to each other, such features are advantageous for providing co-localization during drug distribution by applying electric fields.

The inclusion of a syringe detection cap 118 mounted on a syringe detection spring 116 ensures that the cassette unit 100 can receive and correctly position the syringe 101 relative to the needle hub 152 within the range of manufacturing tolerances expected for the syringe 101. The applicator 400 allows the back of the cassette 112 to move forward during the loading procedure when the spring cap/cassette joint 470 in the applicator 400 moves distally relative to the cassette unit 100. This action occurs when the cassette unit 100 is loaded into the applicator 400 and the cassette unit 100 is retracted into the applicator 400, for example, by the action of a mechanism loading, such as the rack and pinion mechanism described below.

Movement of the rear cassette portion 112 may act as a second lock. In particular, in one embodiment of the invention, as seen in FIG. 5C-5D, the line of sight is visible and detected by a suitably designed sensor in the cassette unit, in the cassette unit receiver 403, through a series of reservoir blocking holes 144' (FIG. 5D). This line of sight is visible when the cassette unit 100 is loaded into the applicator 400. This light line of sight, or occlusion thereof, may act as part of a second interlock that must be disabled in order for the controller 700 to activate and operate the device, including insertion of the needle and electrode, drug delivery and electrode activation.

Thus, for example, in one embodiment of the invention, the reservoir blocking openings 144' (FIG. 5D) must be closed for the device to operate. If a visible line of sight is present, for example, as detected by an IR emitter or a visible light emitter and detector coupled to the applicator cassette unit receiver 403, then the device may be rendered inoperative and may generate and display an error message on the applicator display. 404 and/or controller 700, including controller display 712, to notify the user of the status of the device, as well as recommended steps to take to resolve the error.

Thus, in this embodiment of the invention, one error condition may be that the reservoir 101 was not loaded into the cassette unit 100, or that the syringe was not properly installed in the internal cassette 103. In this case, the lockout 120 the reservoir cannot be pressed because the reservoir 101 does not perform this action. In this case, the rear cassette portion 112 cannot be moved forward in a distal direction, that is, toward the inner cassette cover 104. The design of these components may be such that the open state of the rear portion provides an open line of sight 146 through the first reservoir blocking holes 144' (Fig. 5D). An additional check is that the rear portion 112 of the cassette may not be closed, and this may manifest as the inability of the cassette to move the required distance back or distally into the receptacle 403 of the cassette unit. Since these conditions are defined by the system as an error condition, the same can be identified and used when an error message is received, for example, a corresponding message to the user on the user interface on the display 404 of the applicator and/or controller 700, including the controller display 712. A similar error condition may occur if the tank 101 is not loaded correctly, or if the tank lock 120 is broken. Typically, in this case, the associated error message may be accompanied by instructions for the user to remove the cassette, reinstall a new reservoir, and attempt to reinsert the cassette unit 100 into the applicator 400.

Another error condition may be that the rear portion 112 of the cartridge is moved forward manually by the user without the reservoir 101, which occurs when the user manually pushes the lock 120 out of the reservoir receptacle 142. This situation can also be defined as an error condition, and the same situation can be detected because another (second) series of reservoir blocking holes 144 (FIG. 5C) is placed on a portion of the outer cassette 102. If the reservoir is not installed, but the rear portion 112 the cassette moves forward under the action of the 120 reserve lock

- 25 043519 voir is in the pressed state, the blocking holes 144' of the reservoir (Fig. 5D) are aligned with the blocking holes 144 of the reservoir (Fig. 5C), which again leads to the creation of an open line of sight 146 and a subsequent error condition. This error condition can also occur if the reservoir lock 120 is damaged and its fingers are no longer in the reservoir receiver 142. In a certain embodiment of the invention, this error condition cannot be corrected by attempting to reset the reservoir because the reservoir may not fit into the reservoir receiver 142 with the rear cassette portion 112 locked. In a certain embodiment of the invention, a new cassette unit 100 is required.

In contrast, if the appropriately sized reservoir 101 is located in the desired location, the reservoir detection cover 118 is folded back toward the reservoir detection spring 116, and the movement of the reservoir detection cover 118 closes the reservoir blocking holes 144 (FIG. 5C) and the reservoir blocking holes 144' (Fig. 5C). Fig. 5D). In this case, there is no error condition, allowing the device to operate. Occlusion and occlusion detection occur within the applicator body 400 after the cassette unit 100 is inserted, and thus, attempts by the user to remove this lock will be unsuccessful, regardless of whether such removal is intentional, accidental, or caused by some defect. It should be noted that this error-free condition still occurs even if the user intentionally or inadvertently closes the cassette back 112 while operating the cassette 100.

Depending on the state in which the error occurs, the cassette unit 100 may remain usable or not. If the cassette back 112 is locked in place, the cassette unit 100 becomes unusable. However, if the cassette back 112 has not been locked in place, the cassette unit 100 can be removed from the applicator 400 and a new reservoir 101 inserted.

While the above set of two interlocks (one mechanical with reservoir interlock 120 and one with light emitter and collector and reservoir interlock openings 144 and 144') have been found to be particularly suitable in some embodiments of the invention, it should be understood that there may be Other types of locks were also used (Figs. 5C-5D). Thus, for example, instead of an error condition if the reservoir blocking holes 144 were not closed, an error condition may occur (as a result of changing the location of the reservoir blocking hole and program logic) if the reservoir blocking holes 144 were closed (and a clear line of sight 146 will correspond to a state without errors). The reservoir lock 120 may include additional mechanical signaling elements that are not visible in the cassette unit when the lock is engaged, but become visible to a suitably configured sensor when the syringe 101 has been correctly inserted and the reservoir lock 120 is in the disengaged position. In other embodiments of the invention, to determine the presence of a line of sight, such as optical, acoustic, electrical, or the like. Other methods may be used, provided that a suitable emitter and collector are located in the applicator 400. In order to determine whether the reservoir 101 is properly loaded, other methods may also be used, such as mechanical methods known to those skilled in the art. this area. Depending on implementation goals, if an error condition is detected, the applicator 400 may be disabled from handling the cassette unit 100 at a particular location, or the applicator 400 may be disabled from even accepting the cassette unit 100 in the first place. Thus, for example, the cartridge is designed such that the reservoir lock 120 includes mechanical tabs or locking elements that extend from one or more surfaces of the cartridge until the syringe 101 is properly inserted into the cartridge 100. The mechanical tabs are designed to engage with a corresponding lock. located in the applicator 400 such that loading of the cassette 100 into the applicator 400 is physically blocked until the reservoir lock 120 is released. This mechanical interaction will provide feedback to the user or system and indicate that an error condition must be resolved before the cartridge 100 is loaded into the applicator 400. In other embodiments of the invention, more or less than two interlocks may be used, although both locks can be appropriately associated with different security profiles.

In some embodiments, other elements may also be used in the above determination methods or to improve upon these methods. Thus, for example, when a motor is used to push the cassette unit 100 into the applicator 400, the sensors described below may be used to detect the spatial position of the cassette unit 100 during insertion. In other words, the applicator 400 can determine where the cassette unit 100 is located in the cassette unit receiver 403. In some cases, this makes it possible to detect other error conditions either directly or by triggering the activation of additional sensors to evaluate the status of the device. Thus, for example, in a cassette used, the rear portion 112 of the cassette is fixed in a certain position. If you attempt to reuse the cassette unit 100, the optical detector will detect the cassette unit 100 in another

- 26 043519 position different from the position of the unused cassette unit 100. Using the electric motor for one or more system functions also provides the ability to monitor its operating status, including the amount of voltage and current supplied to the motor, as well as the number of revolutions the motor performs during a particular operations. Measurement of these quantities while the system is operating can be made using a primary or secondary method for detecting potential or actual fault conditions. Thus, for example, the use of mechanical interlocks designed to block the loading procedure if the cassette 100 was improperly designed may be implemented in combination with sensors and logic to ensure that the cassette 100 is loaded correctly into the applicator 400. Thus, for example, the interaction of the mechanical elements described above designed to prevent loading of the cassette 100 without a properly installed reservoir 102 will result in increased load on the motor drive mechanism, which will result in greater current consumption. Detection of an increase in current drawn by the motor will cause the boot procedure to stop and notify the user of the stoppage and fault condition, for example, on the stimulator display 712 and/or the applicator display 404.

Because it is possible that drugs not intended for administration by this method may be contained in reservoirs having the same size and configuration as the reservoirs intended for use by this delivery method. Therefore, an additional aspect of the system is the use of one or more methods to ensure that the reservoir 101 inserted by the user into the cartridge 100 will be specifically designed for use with the device. The introduction of such elements will reduce the risk of inappropriate administration of the drug to a given individual. Typically, specific information in the instructions for use and labeling of a drug includes the schedule and route of administration. However, to further reduce the likelihood of user error, it may be desirable to include mechanical, optical and/or electrical elements in the reservoir and device. In one embodiment of the invention, the syringe may be designed to include one or more unique mechanical elements not found in other reservoirs, which may be similar to reservoirs intended for use in this delivery method. Thus, for example, the reservoir may be specially manufactured to include a fin or other elongated element on the flange or body of the reservoir. In this embodiment of the invention, a corresponding interface must be included in the reservoir lock 120 such that the reservoir lock can only be released if the reservoir with the corresponding interface has been correctly inserted into the device. If it is not possible to directly use this element in the design of the tank, then, as an alternative, a second mechanical component that will be unique to tanks intended for use with this device may be included in the tank. Thus, for example, a ring or other suitably designed element designed to slide along the body of the reservoir may be used to select the reservoir for use with this device by mating with a corresponding element in the outer cassette 102, the inner cassette 103, in the reservoir lock 120, in the detectable reservoir cap 118 or other suitable element in the cassette 100. An additional embodiment of the invention includes a specially manufactured mark of suitable size, color and/or electrical conductivity applied to a predetermined location on the outer surface of reservoirs that are intended for use with this device. Appropriate optical or electrical sensors in the applicator 400 may be designed to detect the presence or absence of a mark on the surface of the reservoir to confirm that the drug introduced into the cartridge will be intended for use with the device. The detection method involves the use of optical or electrical signals applied to a surface to determine the presence or absence of a tag. In this way, reservoirs containing medications not intended for use with the device (and therefore mislabeled) can be detected and removed to avoid possible misuse.

The configuration of the sensors for detecting cartridge loading and syringe loading described above is described below, where such sensors also form part of the cartridge loading subunit in the applicator 400. In more detail and with reference to FIG. 6, 17B and 18C, a representative method of sensing the location of the cassette unit 100 is through the use of a cassette loading sensor 436 and a cassette end of loading sensor 438, which form part of a loading drive sub-unit 454, where the sub-unit 454 further includes cassette guide rails 442 and a loading motor 444. which is coupled to a gear assembly 448 that pushes the cassette unit 100 into the cassette unit receptacle 403 via rack and pinion gears 154 on the base of the outer cassette 102. In more detail, if the cassette load sensor 436 detects an initiation signal 172 on the cassette unit 100 (see FIG. 6 ), then the engine can be ready to start loading. When the cassette end of load sensor 438 detects the same signal, loading can be terminated. A continuation signal may be used

- 27 043519

174, which should indicate that the download will continue.

The first teeth of the rack 154 may be configured to provide a tactile (or audible or tactile) sensation to the user when the cassette unit 100 is inserted into the cassette unit receptacle 403 . Such a configuration may have the shape and/or size of the teeth of the rack 154, as well as the degree of bending allowed by their location in the outer cassette. By adapting the implementation of the rack teeth, the desired degree of haptic feedback can be achieved while ensuring that it does not significantly impact the loading motor 444 once the cassette unit 100 has been received and loaded.

Moreover, as shown in FIG. 17A, and as noted above, the cassette unit 100 is inserted into the cassette unit receptacle 403 in the applicator 400. Although various methods can be used for this insertion, one particularly suitable method has been found, namely, by engaging the gear assembly 448 with racks 154 on the outer cassette 102. The use of more than one rack provides additional stability, and in particular resistance to vortex vibrations during the loading phase. As shown in FIG. 18A, where the insertion/injection drive assembly 456 is shown, in addition to retracting the cassette unit 100 into the cassette unit receptacle 403, the insertion process further compresses the electrode/needle insertion spring 472 through the spring cap/cassette joint 470. The electrode/needle insertion spring 472 is used as the primary driving force for insertion of the needle and electrode during drug delivery.

This hybrid motor-spring action offers many benefits. The motor drive is advantageous because it is highly controllable and allows the cartridge 100 to be loaded into the applicator 400 in a semi-automatic manner with minimal mechanical force required by the user. As described above, the implementation of mechanisms based on the motor drive provides monitoring of the operating state of the system. For example, transferring current consumption and speed through logic and control circuits in the system provides an additional method for detecting and diagnosing possible fault conditions. Despite these advantages, in some cases, electric motors may be poorly adapted to apply the required linear force over sufficiently short time intervals that are most desirable for efficient transdermal delivery of matrices containing multiple elongated electrodes, and in some embodiments, hypodermic needles. injections. In particular, penetration into the dermal tissue is best achieved by applying a large linear force for a short period of time. In some embodiments of the invention, the most favorable injection characteristics are achieved when the penetrating electrodes and the injection needle, if present, contact the skin at a higher speed. This is because sharp objects moving at higher speeds at the point of contact with the skin cause less deformation of the tissue as it cuts or penetrates the tissue. Therefore, in some embodiments of the invention, it is desirable to quickly accelerate the movement of sharp objects before contacting the skin. In some embodiments, a plurality of electrodes and injection needles are often used. In another embodiment of the invention, the speed of the electrodes is at least 50 mm/second before contact with the skin. In yet another embodiment of the invention, the speed of the electrode is at least 500 mm/second before contact with the skin. This method of insertion minimizes the discomfort felt by the individual during electrode penetration and is most beneficial for maintaining a consistent spatial relationship between multiple electrodes. In contrast to electromechanical motors, spring mechanisms exhibit a more favorable discharge profile that is capable of imparting rapid pulse force to the electrodes and injection needle, which is desirable for percutaneous electrode implantation. In particular, the force acting by compressing the spring reaches its peak during the initial discharge. This may be advantageous for transdermal administration where high velocity at the point of contact with the skin is favorable and, due to the viscoelasticity of skin tissue, the greatest force is required to penetrate the skin, especially when the skin is in contact with multiple electrodes and/or injection needles. Moreover, the spring mechanism is able to generate this force from a simple, durable and compact device that can be easily incorporated into a portable device format. However, a disadvantage of spring mechanisms is that they typically require significant mechanical force from the user to operate them, especially for springs with high force constants and/or large displacements. The use of a hybrid motor-spring mechanism as described in the present invention provides the desired force characteristics for placement while being easy to operate for the user. Although a hybrid motor-spring mechanism is the preferred embodiment of the invention, however, depending on the application, other hybrid mechanisms comprising two or more drive mechanisms, where one is capable of generating a fast impulse force and the other is capable of triggering, may also be used. a pulsed force mechanism, such as a pump capable of creating a liquefied gas in the chamber and then injecting the liquefied gas to provide a pulse force for placement of electrodes and, if necessary, hypodermic needle(s)

- 28 043519 tions.

In either case, once loaded, the desired electrode depth and/or agent delivery is selected by the physician or other drug administration specialist and input into the applicator 400. As shown in FIG. 13B, the depth can be selected using the depth selection buttons 409 (or other equivalent interface such as a toggle switch or slide switch), and the result is displayed on the injection depth selection indicators 408 (or, again, another equivalent interface). The available injection depth is communicated to the user by appropriate markings on the applicator 400 and/or cassette 100. In some embodiments of the invention, any markings regarding injection depth are located on the cassette 100 and remain visible to the user once installed in the applicator 400. For example, the available depth injection may be marked on the top surface of the alignment panel/side screen 108. To avoid instances where the user forgets or neglects to select the injection depth, it is preferable that such a device does not allow the user to continue the injection procedure until the correct selection is made. This can be achieved by introducing appropriate control logic into the system so that subsequent adjustments or use of the device are not available until the user makes the correct depth selection. In some embodiments of the invention, when the user is asked to finalize an injection depth, the controller display may provide information to the user regarding the appropriate means for the individual to evaluate and determine an appropriate injection depth for the selected injection site.

Moreover, as shown in FIG. 18A-18B, when the cassette unit is properly installed, the spring cap/cassette joint 470 engages and is also pressed against the spring cap opening 471 and tabs 491. Although the large spring force pushes against the inner cassette 103, however, this force prevents forward movement due to engagement of the plurality of support legs 488 with the walls 494 of the outer cassette 102. However, the force exerted on 470 increases the distance between the tabs 491 (which prevents accidental rotation of the lock ring during processing) and thereby allows rotation of the cassette lock ring 114 by the motor drive mechanism. Rotation of the cassette lock ring 114 initiates rotation of the support legs 488. The support legs 488 can be rotated toward the first depth channels 490 or the second depth channels 492. The length of the first depth channels 490 corresponds to one of the depth options, and the length of the second depth channels 492 corresponds to another option, and one or the other depth is selected by the user using buttons 409. So, for example, the length of the channels 490 can be in the range of 20-30 mm, and the length of the channels 492 can be in the range of 12-20 mm. The need for the user to select the correct depth is ensured by another lock. Without the correct selection, the applicator cannot allow activation/insertion of the needle. Thus, the rotation of the cassette locking ring 114 is carried out by clockwise or counterclockwise rotation, adjustable by the applicator 400 according to the user's requirements. Requiring rotational movement of a series of posts into such channels to accommodate the electrodes and injection needle, if present, will significantly reduce the risk of accidental discharge even after dangerous interference or shutdowns.

In more detail, the rotation of the cassette lock ring 114 is transmitted to the cassette unit 100 by means of support posts 488, which are installed after the cassette unit 100 is correctly inserted into the slots on the drive ring of the insertion mechanism 478. FIG. 18A, the slots on the insertion drive ring 478 are at the 3 and 9 o'clock clockwise positions. The insertion drive ring 478 is mounted on the partial insertion drive ring 479, which is driven by the insertion drive motor 482. Triggering the partial insertion mechanism ring 479 activates the insertion mechanism drive ring 478, causing either clockwise or counterclockwise rotation. The signal 481 on the ring 480 and the corresponding insertion mechanism position sensor 483 are used to determine the position of the insertion mechanism drive ring 478 and are also used to accurately return it to the 3 and 9 o'clock position when the applicator 400 is to be reused with another cassette.

The above implementation provides various advantages. For example, the user must select the depth before continuing with the insertion procedure. In doing so, the user must evaluate the correct injection depth for the selected injection site and prepare the site as indicated in the manual or instructions in the document for use. As noted, the insertion mechanism, which requires a rotational motion for placement, is largely protected from accidental misplacement due to drop, vibration, breakage, or the like.

Various options will be known to those skilled in the art. Thus, for example, although two channels and two support posts are used for each depth, one channel and one support post may also be used. Various types of motors and mechanisms can be used to transmit the rotational motion required to rotate the posts in the channels. Other options are also known, including the use of solenoids and the like. Instead of using channels to select depth, a motor-controlled insertion device may be used and such

- 29 043519 the depth can be adjusted simply by determining how much this motor is capable of adjusting the initiation of placement. Preferably, in this context, the described hybrid drive mechanism should be designed such that a pulse discharge mechanism (eg, a spring mechanism) is used for initial insertion into the dermis, and then a motor drive mechanism is used to insert the electrodes to the desired depth.

One or more interlocks may be set and must be released before the insert mechanism drive ring 478 begins to rotate, rotating the support posts 488 in the channels.

First, a force detection override subsystem may be installed that requires the device to apply a force greater than a predetermined amount to the individual before beginning the insertion procedure. This force may be measured by a suitable mechanical or electromechanical system, and the result of such measurement is fed back to the controller 700 and used as an interlock to prevent the device from starting if the force is insufficient. In some embodiments of the invention, controller 700 is capable of communicating a force lock condition to the user via visual, tactile, or audible cues so that the inadequate force condition can be corrected and the user can begin insertion. In the event that the user attempts to continue insertion in a state of inadequate force contact (eg, by pressing the trigger button 407 or other trigger buttons), additional visual, tactile, or audible cues may be provided to the applicator 400 or controller 700 to notify the user that that the lock must be disabled before administration begins.

Determining the force acting on an individual can be done in several ways. According to a specific implementation as shown in FIG. 4 and 8A-8B, the alignment panel/side screen 108 is provided with a force contact transducer 128. The alignment panel/side screen 108 can be mechanically displaced distally (towards the individual) by the action of one or more force contact springs 126, four of which are shown in Fig. 4. The power contact converter 128 changes its position due to the force applied to the alignment panel/side shield 108. In doing so, it also changes the state of the electrical circuit formed by the power contact converter 128, the series of first pads 162, the series of second pads 164 and the flexible circuit 160. Specifically, by checking the continuity between one or more pads 162 and one or more corresponding pads 164, it is possible to determine how far or close the alignment panel/side screen 108 moves under the applied force, and thus determine , to what extent this force is sufficient for correct delivery. The state of the circuit is read by the applicator 400 under the action of the contacts of the sensor 434 (see Fig. 16). If the sensor indicates sufficient force, the power contact lock is released, allowing the user to operate the device.

In one embodiment of this implementation, at the first point of contact, the system may not register any specific force applied. At the second point of contact, the system can register partial (but insufficient) pressure. At the third contact point, the system can register that the preset pressure level required to continue the insertion procedure has been reached, and the lock can be released. Preferably, the state of the power contact circuit is communicated to the user on the applicator display 404.

The options should be understandable to one skilled in the art. Thus, for example, the power contact interlock may form an electrical lock that is released either in the controller 700 or in the applicator 400 itself. Alternatively, the power contact circuitry may be designed to provide information about the state of the device throughout the insertion procedure. In particular, the system may include a feedback loop between the force contact circuit and the controller 700, wherein reducing the force applied by the user accelerates the generation of a visual, tactile, or audible signal by the controller 700 and/or the applicator 400 so that the reduced force condition can be corrected. In some embodiments of the invention, a feedback loop is present between the force contact circuit and the controller 700 such that detection of a change in applied force causes the system to initiate a check to see if the electrodes are correctly placed in the tissue of the subject, for example, by checking the impedance or resistance between the electrodes. If the check is passed, the procedure continues as usual. In the event that the position of the electrodes is no longer acceptable, the procedure can be interrupted and the user is notified of the status of the device through the generation of a visual, tactile or audible signal by the controller 700 and/or applicator 400. This feedback loop is of particular importance during drug administration. facilities. By monitoring the position of the device and the state of the electrodes, the feedback loop between the power contact circuit and the resistance/impedance of the electrodes, the system can detect that if the electrodes (and therefore the injection needle) are no longer in the individual's possession, it allows the mechanism system to be terminated. 456 of the injection drive, and thereby stop pressing the reservoir piston 484 and stop

- 30 043519 injection of a medicinal product. Although this embodiment of the invention is most easily implemented using a motor driven injection actuator 456, other embodiments may be implemented with manual or spring driven mechanisms where triggering mechanical interlocks can be used to stop the action of the reservoir piston upon detection of a fault condition. . This feature may be particularly suitable for preventing accidental release of HBV vaccine into the environment when the applicator 400 is removed from the subject's tissue before delivery of the drug is completed. For drugs or therapeutic agents that are potentially hazardous to the user or the environment, this design avoids unintentional releases/exposures.

Because the amount of dose delivered to an individual in a partial dose situation can play an important role in informing the clinician's decision regarding further treatment, it is preferred that the injection drive mechanism 456 include appropriate sensing and control functions to determine the position of the drive piston 484 for injection at a time when the injection procedure has been stopped due to the detection of a malfunction or other circumstances under which it is necessary to stop the injection procedure. Preferably, this is accomplished by monitoring the RPM of the engine used to actuate the injection drive piston 484, but other methods of monitoring the position of the injection drive piston 484 may be used, including optical or electrical sensors. Based on the known dimensions of the injection drive piston 484, the injection depth, and the reservoir 101, the use of appropriate logic and control circuits allows the position of the injection drive piston to be used to judge the volume of drug remaining in the syringe at the time the injection procedure is stopped. Such information may be communicated to the user at display 712.

In addition to the function of ending the injection procedure if a fault condition is detected, use of the injection motor 456 drive may also provide information regarding the detection of a fault condition or other operating problem. In particular, the inclusion of appropriate measurement and logic circuits to monitor the operation of the circuit via the motor during the injection procedure can be carried out to confirm that the injection has been administered in accordance with certain requirements. Expected ranges for the current drawn by the motor can be set at various stages of the injection procedure, including: the initial startup of the injection drive piston 484 before it contacts the piston blocker 159, the initial interface between the injection drive piston 484 and the piston blocker 159; actuating the piston blocker 159 forward in the reservoir cylinder 101; and completing the injection procedure once the piston blocker 159 contacts the end of the reservoir cylinder 101. By correlating the position of the injection drive piston 484 with the measured current draw of the motor to expected values during each agent injection phase, the system is able to detect possible fault conditions and transmit to their user. Thus, for example, if a user accidentally inserted reservoir 101, in which piston blocker 159 was partially actuated (and therefore did not contain the full intended dose of drug), into cartridge 100, then the system may indicate that the expected increase in current, consumed by the injection motor drive 456 does not occur if the injection drive piston 484 reaches the external piston positioning tolerance. In this case, the system may terminate the insertion procedure and notify the user of the detected malfunction. Additional fault conditions could potentially be detected using this method, including (but not limited to) faulty components in the applicator 400, a broken reservoir 101, and/or a faulty cartridge 100.

Another interlock to facilitate correct insertion is provided by the alignment panel/side screen 108, which is shown in FIG. 9C. In this figure, the leveling panel/side shield 108 is shown with beveled members 168 and an opening 170 therein for sliding movement of the shield 134. The shield 134 is shown in FIG. 9B, and also shows electrode openings 167. For transdermal insertion of electrodes and, if necessary, injection needles, the presence of an appropriate skin contact surface facilitates placement of the electrodes into the target tissue while maintaining the desired spatial relationship between the plurality of elements. In particular, correct placement is best achieved if the skin surface is perpendicular to the direction of insertion. Additionally, misalignment of electrodes and injection needles can be corrected by tensioning the skin in an orientation perpendicular to the direction of placement. As can be seen, the protective shield 108 includes mechanical elements, including ribs and edges for adhesion to the skin and for tensioning it perpendicular to the direction of placement of the electrodes. In combination with the force contact circuit conversion system described above, which ensures a constant force is applied to the skin, the alignment panel/side screen 108 will ensure that the skin and its tension are perpendicular to the direction of electrode placement. Although this embodiment uses mechanical ribbed elements to secure the skin to the surface of the device, however, many other devices can be used for such fixation.

- 31 043519 designs and elements, including the use of alternative materials with a high coefficient of friction in contact with the skin (for example, the use of rubber inserts, adhesive patches and

etc.) or alternative mechanical elements, including molded structures, cuts and saw tooth elements capable of creating tension on the skin.

As can be seen, the alignment panel/side screen 108 has a certain preferred orientation. As described above, spatial and temporal co-localization of drug distribution and electrical field application are desirable. It is noteworthy that the structural properties inherent in skeletal muscle determine the characteristic pattern of ellipsoidal distribution of intramuscular injections, where the main axis of the ellipsoid coincides with the striated structure of muscle fibers. Therefore, when used for intramuscular injections, it is desirable to place the electrodes so that they give an ellipsoidal electric field profile. To ensure proper orientation of the electrode array and the resulting electric field profile relative to the striated structure of the target skeletal muscle, the use of an alignment panel is particularly suitable for intramuscular administration. The alignment panel is intended to facilitate placement of the device so that the orientation of the electrode array is most favorable to the striated muscles and to facilitate the final distribution of the drug after injection. For injection into the arm, it is desirable for the alignment panel/side shield 108 to wrap horizontally around the arm, similar to an arm band. A similar orientation would be desirable for the legs, with the shield wrapped horizontally around the leg. The alignment panel/side screen 108 has been found to be configured to allow drug delivery to the user with >98% accuracy even in the absence of verbal instructions. A preferred orientation has been shown, and this final placement on the skin is particularly suitable for intramuscular injections, since the diamond-shaped electrode array (see the array of distal ends of the electrodes 137 in Fig. 9A, which approximately matches the shape of the protective shield 134 and associated holes 167) then is oriented properly to ensure drug delivery and initiate drug electroporation, preferably along the direction of the striated structure of the muscle. The main feature is that when properly aligned, the skin should be flush with the skin interface where the hole is located, for example where the needle appears. This creates a consistent skin interface.

When alignment with the target muscle is incorrect, the archwire typically causes a visually visible gap, for example, 2-5 mm, between the alignment panel/side shield 108 and the skin. The visually observed gap may be used to remind the user to reorient the applicator 400. The applicator 400 may be less resistant to the individual's skin. Leveling panel elements where the distance between the edges of the element wings is at least 1.3 times the vertical height of the element are preferred to ensure proper placement. Additionally, in some cases, the element is designed such that the fabric contact surface can be flush with the skin in the desired orientation while still having an air gap of at least 2 mm when positioned at a 90° angle from the desired orientation.

Those skilled in the art will appreciate the various design options for a leveling panel. For example, instead of an arcuate leveling panel/side screen 108, V-shapes may be used. Other options are also known, the key feature of which is that the device can be placed directly against the skin in the desired orientation, while there is a visually visible gap of 2 mm or more between the interface of the tissue and the recipient's skin if the device is offset relative to striated target muscles.

As discussed above, in some embodiments of such an implementation, the alignment panel/side shield 108 is oriented to the shape of the electrode array because the diamond symmetry of the electrode array has a specific distribution associated with it, and such distribution must be in a predetermined relationship with the shape of the alignment panel/side shield 108 In Fig. 9A9B shows the electrodes, and in particular the distal parts 137, the number of which is four. The electrodes are arranged in a diamond-shaped array, which could mean that they have second-order symmetry, meaning they have double symmetry, meaning they can rotate and look the same in two different positions. The electrodes extend in this array from the holes 167 present in the protective shield 134. The use of a diamond-shaped array is particularly useful as it is generally desired for intramuscular injections, as discussed above. It should be recalled that a symmetric second-order matrix produces a symmetrically applied second-order electric field. This type of applied electric field may have a preferred direction along the transverse muscle bands if the alignment panel/side shield 108 is oriented properly. Thus, the alignment panel/side shield 108 and the second order electric field have a synergistic orientation that promotes drug distribution toward the striated muscle.

Generally speaking, a diamond shape or other second-order symmetrical shape usually has a major (long) axis and a minor (short) axis. The axes may have a predetermined relationship to the preferred axis of the leveling panel/side screen 108. Thus, for example, if the leveling panel/side screen 108 is intended to have arcuate wings surrounding a lever, then a straight line connecting the center of the wings may be perpendicular to the major axis diamond-shaped matrix.

The changes should be understandable to one skilled in the art. For example, although a rhombus is one possible shape for an electrode array, another representative shape is a rectangle, which also has second order symmetry. The electrodes may be placed at appropriate positions, but they may be moved within a predetermined or desired tolerance, for example within 5%, 10%, or the like. The electrodes can have various other shapes, provided that they create a rotationally symmetric second-order electric field.

Alternatively, symmetries of a different order may be used for non-intramuscular injections. For example, for the skin there is usually no preferred direction of drug distribution, and therefore even a ring electrode array can be used for intradermal injections.

Other considerations regarding electrode arrays are as follows. The simplest array configuration contains two electrodes connected to opposite poles of an electrical energy source. As disclosed in US Pat. Nos. 5,873,549 and 6,041,252 (which are incorporated by reference in their entirety), three or more simultaneously active electrodes arranged in a multi-element array can be used to increase target tissue volume and increase uniformity in electric field propagation. in target tissue. A wide range of geometric electrode arrangements and activation schemes have been developed to deliver an electric field into tissue. Such electrodes are electrodes arranged in a linear, rectangular, circular or triangular configuration and capable of propagating electrical fields throughout a tissue volume approximately in the shape of an ellipsoid, cuboid, cylinder or spheroid. Most commonly, the electrodes are positioned parallel to each other and are configured for percutaneous insertion in an orientation substantially perpendicular to the skin surface. To ensure that the electric field will act on the target tissue of a certain volume and shape, it is desirable that the intended spatial relationship between each of the electrodes in the array is achieved after percutaneous administration. In particular, inadvertent changes in electrode spacing should be avoided as they may cause changes in the magnitude of electrical fields propagating into the target tissue, which may have negative consequences for the safety and/or effectiveness of the procedure.

Another lock includes the use of an outer cassette cover 110. In particular, the alignment panel/side screen 108 is covered by an outer cassette cover 110 during storage. The outer cassette cover 110 is configured to not only generally protect the distal end of the device until use, but It also performed the blocking function itself. Although the protection of the distal end of the cassette unit 100 serves to protect the needle and electrodes from the environment, a common problem is that users often forget to remove such covers. One solution to this problem is to make the lid a bright color that is different from the color(s) of the other components in the cassette 100, thereby reminding the user of its presence and thus the need to remove it. Another part of this solution is to include an extension or reminder signal 190, as shown by the arcuate portion adjacent to the rectangular portion, where the arcuate portion is visible even when the cassette unit 100 is positioned opposite the object. Since this reminder signal 190 is visible, it can serve as a reminder to remove the outer cassette cover 110 even if the reminder of the outer cassette cover 110 was not visible.

As shown in more detail in FIG. 10A-10B, the outer cassette cover 110 includes an outer surface distal to the object and an inner surface facing the alignment panel/side screen 108. The inner surface includes a plurality of hooks 176 that engage a corresponding alignment panel wall 180. side screen 108. These hooks hold the outer cassette cover 110 in place.

However, if the outer cassette cover 110 is accidentally left in place and the applicator 400 is pressed against the insertion site with a certain amount of force, then the force of the alignment panel/side screen 108 against the outer cassette cover 110 may cause the outer cassette cover 110 to open due to the repulsion from the engagement position of the leveling panel/side screen 108. However, the hooks 176 have chamfered surfaces 178 that tend to act on the protective screen 134 when pressure is applied, which will cause the hooks 176 to expand outward and increase their holding effect on the leveling panel/side screen 108 and pre

- 33 043519 prevent the outer cassette cover 110 from detaching. In addition, the beveled surface acts further on the protective screen to prevent the leveling panel/side screen 108 from moving relative to the protective screen 134. If the leveling panel/side screen 108 cannot move relative to the protective screen 134, then the force detection interlock cannot be disabled because the force contact transducer 128 cannot move to the third force contact point described above where total pressure is detected (or, in this case, even to the second force contact point). The regulator can provide information about what is happening with the regulator. So, for example, a message may appear on the user interface display, such as You need to remove the outer cassette cover, and the same is caused by pressing the start button without adequate force.

Although some locks have been described above, however, more or fewer locks may be used in any given embodiment of the invention. Thus, for example, other elements may be used to detect force and serve as interlock trigger signals. Other types of locks may also be used, including trigger locks, safety switches, etc. Other types of interlocks will also be apparent to those skilled in the art from the description of the invention.

Once the insertion depth has been correctly selected by the user and the outer cassette cover 110 has been removed and the self-force has been detected through the force lock, activation of the trigger will cause the cassette retaining ring 114 to rotate clockwise or counterclockwise, where the direction of rotation will depend from the insertion depth selected by the user. The rotation causes the support posts 488 to move into the channels of the selected depth, which in turn allows the placement of needles and electrodes. Specifically, the inner cassette 103, the cassette back 112, the reservoir 101, the inner cassette cover 104, the needle hub 152, the needle 105, the electrodes 122 and associated members are moved forward by the spring 472 for inserting the electrodes/needles. In an embodiment of the invention, these elements are rigidly connected to each other and thus can move forward as a separate element.

To further minimize the risk of injury to the user from sharp objects, it is preferred that the cassette unit also include a mechanism for coating the electrodes and any injection needles after they are removed from the recipient tissue. This can be achieved by including a protective shield that will serve as a shroud for the electrodes and injection needle (if present) before they are used, and then this extends to the electrodes and injection needle (if present) after they have been placed and removed from individual tissue. Typically, the tissue contacting surface includes the distal surface of the security element. Although the use of manually constructed security features may be contemplated, it is preferred that the device include a mechanism to automatically expand the shield onto the electrodes and further to engage a locking function to maintain the shield feature in the expanded state after it has been removed from the recipient tissue. An example is (are) a screen element that is slidable and engages with the outer cassette body and is operatively coupled to a stored energy source, such as a spring, that moves the screen forward as the electrodes are withdrawn from recipient tissue. Alternatively, the mechanism may be contained within the applicator and may be configured to vary the pitch of the electrode, thereby retracting the electrodes and any associated injection needles away from the tissue contacting surface. This can be achieved using simple electromechanical translational mechanisms such as motors or solenoids.

In a particular embodiment of such an implementation and as shown in FIG. 4, and also in FIG. 11A and 11C, the shield 134 is configured to remain in the drug delivery position but move the inner cassette 103 and other associated components forward during drug delivery, which will cause the shield spring 138 to compress. screen. This compressed spring of the protective shield 138 will then be released as the applicator is withdrawn from the subject, since the protective shield 134 is forced to move forward if the applicator 400 is removed from the subject, covering the needle and electrodes and protecting the subject, etc. from contact with uncovered sharp objects. Notably, this feature also prevents the visualization of sharp objects during their removal, which may prevent individuals from experiencing anxiety associated with these needles.

Otherwise, although the weakened spring is capable of being recompressed, and in particular if the user were to move the shield 134 in a proximal direction, this may, however, be prevented by the action of the shield support arm 132, which is mounted into the interior of the outer cassette 102 and act as a ratchet when the protective screen 134 moves forward. In particular, as shown in FIG. 11A and 11C, the shield support arms 132 move across various successive support walls and can easily do so as the shield 134 moves distally. However, in the proximal direction, movement of the shield 134 is prevented by the shield support arms 132, which

- 34 043519 rest against the supporting walls in this direction using a ratchet mechanism and do not allow passage.

An initial series of support walls 188 prevent proximal movement of the shield 134 prior to use of the cassette unit 100. A series of first support walls 184 for the first selected depth prevent proximal movement of the shield 134 after discharge at the first selected depth. A series of second support walls 184 for a second selected depth prevent proximal movement of the shield 134 following a discharge at the second selected depth. The shield 134 is prevented from being completely removed from the cassette unit 100 due to the action of the shield support hooks 182 acting on the inner cassette 103.

These options should be understood by one skilled in the art. Thus, for example, in one embodiment, the shield support arms 132 may be provided with stamped metal support arms that engage with the outer cassette 102. In some embodiments, the support arm members are directly incorporated as injection molded plastic members, into the outer cassette cover 106 and/or into the alignment panel/side shield 108. The specific material must be selected to provide sufficient rigidity and support for the elongated electrodes while maintaining sufficient elasticity to prevent failure of the component under load. Material selection should also consider the intended method of sterilization of the electrode array (e.g., by gamma radiation, steam sterilization, ethylene oxide treatment, or electron radiation) to ensure that such elements will retain adequate material properties after sterilization. In an exemplary embodiment of the invention, appropriately designed members should retain the protective shield 134 from proximal movement when a force of at least 5 N, and more preferably at least 15 N, is applied. Other methods of providing ratchet function may also be used. to prevent proximal movement of the shield, including a rack inserted into the shield 134 and a corresponding ratchet member inserted into the outer cassette cover 106.

As shown in FIG. 4, each elongate electrode 122 in the array includes a distal portion 137 and a proximal portion 135 that are in conductive communication. The distal portion is designed to penetrate tissue and propagate an electrical field into the tissue, and the proximal portion is designed to have a conductive contact region capable of reliably conducting conductive communication with electrical connections contained in the applicator with a suitable source of electrical energy. The source of electrical energy can, due to a temporary change in the power applied to the individual electrodes, produce a spatially and temporally varying electric field in the body of the individual, which is usually limited by the volume around the area of drug distribution, and the same can be used in electroporation therapy .

Relevant electrode characteristics include shape, diameter, tip profile, length, material composition and conductivity, the technical parameters of which are selected depending on the intended application. Most often, electrodes contain electrically conductive elongated rods with a curved cross-sectional diameter of 0.1-1.5 mm. Electrodes can be solid or hollow. Most commonly, hollow electrodes contain one or more openings and an operative connection to a fluid reservoir that allows the agent of interest or other concomitant drugs, including anesthetics, surfactants, proteins, adjuvants, or stimulants, to be administered from the electrode itself. Depending on the application, suitable metal electrode materials include, but are not limited to: titanium, gold, silver, aluminum, copper, tantalum, tungsten, molybdenum, stainless steel, MP35N and their alloys. Electrodes may also consist of electrically conductive ceramics or plastics. To minimize unwanted electrochemical reactions at the electrode-tissue interface, it is often desirable that one or more electrodes be coated with a conductive material that provides improved electrochemical stability compared to the electrode material itself. Such plating materials include, but are not limited to: platinum, iridium, palladium, osmium, gold, silver, titanium and their alloys.

As described above, the tip of the distal portion is configured to penetrate tissue. Common tip profiles include, but are not limited to: trocar, bevel, cone, blade, peak and beveled cone. For percutaneous electrode placement, tip profiles with one or more cutting edges are preferred. In some cases where the profile of the penetrating tip may not be desirable for generating the required electric fields, the tip may consist of a non-conductive material located at the distal tip of the electrode, or the penetrating tip may be coated with an adhesive coating or spacer layer consisting of a biocompatible electrical insulating material such as p-xylylene polymer, polyolefin, polyvinyl chloride, polyurethane, polyester, polyimide, silicone rubber, thermoplastic elastomer/rubber, ethylene tetrafluoroethylene, fluorinated ethylene propylene and/or perfluoroalkoxy plastic.

Adjacent to the penetrating tip are one or more conductive areas having

- 35 043519 configuration suitable for the propagation of electric fields in tissue. To limit the propagation of electric fields into the target tissue region, typically, and especially for subcutaneous and intramuscular administration, at least a portion of the length of the electrode, having a configuration suitable for penetration into the tissue, is coated with an adhesive coating or spacer layer consisting of a biocompatible electrical insulating material, such as p-xylylene polymer, polyolefin, polyvinyl chloride, polyurethane, polyester, polyimide, silicone rubber, thermoplastic elastomer/rubber, ethylene tetrafluoroethylene, fluorinated ethylene propylene and/or perfluoroalkoxy plastic. In this way, electrodes can be designed to only activate at a certain depth in the tissue.

In general, the number of electrodes and their interelectrode distance, as well as the penetration depth of the electrodes and the length of their conductive region, determine the volume of tissue to which electric fields are applied. These parameters are selected based on the specific objectives of the administration procedure, including the target tissue site, volume, dose and viscosity of the agent delivered, and changes in skin and subcutaneous tissue thickness in the intended recipient population. Typically, matrices for intradermal administration to a human recipient contain 2-16 electrodes with a diameter of 0.2-0.7 mm, with a distance between electrodes of 2-8 mm, an electrode penetration depth of 0.5-4 mm and a conductive length of 0.5- 4 mm. Typically, matrices for subcutaneous administration to a human recipient contain 2-8 electrodes with a diameter of 0.3-0.8 mm, with a distance between electrodes of 4-10 mm, an electrode penetration depth of 5-15 mm and a conductive length of 2-8 mm. Typically, matrices for intramuscular administration to a human recipient contain 2-8 electrodes with a diameter of 0.3-1.2 mm, with a distance between electrodes of 4-12 mm, an electrode penetration depth of 10-60 mm and a conductive length of 2-20 mm.

To avoid unwanted visualization and interference with the electrodes during use of the device, the cassette unit 100 is preferably configured such that the electrodes 122 are recessed into the device prior to insertion into the recipient tissue. Most commonly, this is achieved by allowing the outer housing structure 102 to slide and engage the electrode mounting support, which, in one embodiment of the invention, includes an inner cassette 103 having recesses formed therein in which the electrodes are located. The electrodes are, prior to use, recessed in the outer housing 102. During use, the sliding engagement of the electrode mounting structure, such as the inner cassette 103, with the outer housing allows the electrodes 122 to slide forward relative to the distal tip of the outer housing 102, and position the electrodes 122 in the outer housing 102. The length of the sliding engagement between the electrode mounting structure and the outer housing corresponds to the desired maximum electrode penetration depth for a given application.

As can be seen in FIG. 4, the electrodes 122 have a proximal portion 135 and a distal portion 137. The proximal portion is separated from the distal portion by an arm or bend 139. The arm or bend 139 is secured to each other and between the inner cassette cover 104 and the inner cassette 103. The arm or bend 139 performs a number of functions. First, although the electrodes at the distal end of the cassette unit 100 are necessary to form a matrix of a certain size and shape, as described above, however, installing the electrodes 122 in the form of a matrix of the desired size and shape at the proximal end of the cassette unit 100 is impractical due to the presence of the reservoir 101 In other words, the electrodes 122 must bend over a distance to make room for the reservoir 101. In addition, the presence of the bend, especially when the bend is fixed at the friction point between the inner cassette 103 and the inner cassette cover 104, provides a surface for the electrodes 122 to meet, if the insertion force of the needle and electrodes moves distally as the electrodes 122 interact with the tissue during their placement. The bending may be resistant to axial rotation, which is advantageous in that the electrodes do not exhibit significant torsional motion and rotate away from their electrical pads 130 (as described below). Additionally, unlike prior art methods where multiple electrodes are connected to axially separated coaxial rings requiring a different configuration for each electrode, the present system allows the use of a common electrode type for all four electrodes.

Systems and methods according to the principles of the present invention provide the propagation of an electric field in the skin, subcutaneous tissue and/or skeletal muscle of the recipient, which facilitates intracellular delivery of the HBV vaccine to the specified tissue of the recipient. In this aspect, the device includes two or more elongate electrodes 122 arranged in a predetermined spatial relationship and configured to locate the electrodes in the target tissue into which the agent of interest is to be administered or distributed. The desired enhancement of agent activity depends on achieving co-localization of the agent's distribution site with the propagation of electric fields of sufficient magnitude to induce the desired physiological effects. Therefore, it is desirable that the device achieves consistent placement of the electrode at a given depth and with a certain interelectrode distance. In particular, this will ensure that the electric fields propagate in the desired area of the tissue, since the magnitude of the electric fields propagating in the tissue depends

- 36 043519 on the distance between the electrodes, and on the safe and effective electric field strength. However, variations in the thickness, density, and composition of skin and subcutaneous tissue between sites within a given recipient and between groups of recipients can result in significant changes in electrode placement characteristics. For example, in recipients with increased skin density, there will be increased sensitivity to electrode distortion and/or penetration depth will be insufficient, which may negatively affect co-localization at the site of agent distribution.

Systems and methods according to the principles of the present invention also facilitate the coordinated transdermal administration of electrode arrays containing 2-16 electrodes with a diameter of 0.2-1.3 mm with an interelectrode distance of 2-12 mm and an electrode penetration depth of 0.5-60 mm, to recipients with skin of different thickness, composition and condition. To enable percutaneous administration, tissue penetrating electrodes are most often elongated and configured with a tissue penetrating tip, a tissue contact area at the distal portion, and an electrical contact area at the proximal portion. Given the elongated configuration of the electrodes, they are susceptible to bending, bending and/or corrugation (collectively referred to as distortion) when subjected to compressive forces encountered during percutaneous administration to the recipient. Such distortions during electrode insertion are undesirable as they can lead to incorrect electrode insertion characterized by insufficient penetration depth and/or excessive variation in the interelectrode distance. It should be noted that humans and animals exhibit significant intra- and inter-species variation in skin thickness, composition, and condition, any of which can have a significant impact on the forces that electrodes experience during percutaneous insertion. In some embodiments, devices for transdermal insertion of extended electrodes are designed to withstand the forces encountered under the most severe electrode insertion conditions encountered in the recipient population receiving the electrodes. The occurrence of electrode distortion can be partially eliminated by choosing the electrode material, as well as by reducing the length and increasing the diameter of the electrodes in the matrix. However, material selection may be limited due to performance, cost, processability, and biocompatibility issues. In addition, electrodes of reduced length may prevent adequate coverage of the target population of individuals with varying tissue thicknesses, while electrodes of larger diameter are associated with increased discomfort and tissue trauma. Thus, the present invention relates to methods and devices for facilitating percutaneous insertion of electrodes regardless of these parameters.

In a first embodiment of the invention, the cassette subassembly containing the electrode array includes one or more dynamic support elements that are in physical contact with one of the electrodes during insertion into tissue, and is configured to limit the movement of the electrodes perpendicular to the direction of insertion and maintain the desired spatial relationship between electrodes. So, for example, in FIG. 12 shows an electrode support member 124 that includes holes 192 for electrodes and a hole 194 for needle passage. The tendency of elongated electrodes to warp or bend under load is exacerbated by any bending, eccentricity, or bending of the electrodes that may occur during manufacturing or assembly. Thus, it is preferable to use a device such as the electrode support member 124 to fix the electrode in order to prevent or eliminate any distortion, eccentricity or bending of the electrodes that may occur at rest, and in order to prevent unwanted perpendicular movement of the electrode after loading that occurs at rest. the process of its embedding into tissue.

In the context of the disclosure of the invention, a dynamic support element is defined as a structural element that provides sliding engagement with elongated electrodes during insertion into recipient tissue, and which is configured to change position, size and/or conformation during electrode insertion, to maintain the desired spatial the relationship of the electrodes as they are placed into the target tissue to a certain depth. Since the electrodes are subject to the greatest stress upon initial contact with the skin, the adhesion of the dynamic support element to the electrodes making up the matrix is preferably addressed prior to initial contact with the recipient tissue and continues to support these electrodes as they are positioned to their full penetration depth. It is also desirable that the dynamic support member be designed to support these electrodes and the injection needle with minimal loss of electrode penetration force due to friction.

Although the primary function of the dynamic support element is to maintain the desired spatial relationship of the plurality of electrodes in the array with respect to each other and to the injection needle, it is also desirable that the design of the dynamic support element include elements capable of stabilizing the array as a whole. This is preferably achieved by providing additional structural support elements that limit the lateral movement of the support link. In some embodiments of the invention, these support elements

- 37 043519 are built into a sharps guard which also serves to protect the user from accidental contact with electrodes and/or injection needles. Various embodiments of the dynamic support elements according to the invention are disclosed below.

One embodiment of the invention described above as an electrode support member 124 includes the use of a flat support structure 124 positioned perpendicular to the elongated electrodes. A planar support structure was created with one or more holes 192 that correspond to the positions of said electrodes in their predetermined spatial relationship. The size, shape and position of the holes allow the support structure to slide smoothly along the extended electrodes within the array while limiting unwanted movement perpendicular to the direction of electrode placement. Typically, the openings may be holes or slits 192 in a planar structure with sufficient clearance for the electrode (at least 10% larger than the largest cross-section of the electrode, including any coatings or other adhesive materials). However, if more substantial support is required for particular electrodes, then one or more of the openings may comprise tubular structures 196 positioned perpendicular to the planar support structure. Holes containing such tubular structures increase the surface area in contact with the electrode and thus improve support for the elongated electrodes. The planar support structure may be made of any material with suitable structural characteristics, including metal, polymer, ceramic, or composite materials, and may be manufactured by molding, machining, injection molding, or other methods. To avoid unwanted electrical interactions with the electrodes, it is preferable that the interface between the electrodes and the planar support structure be non-conductive. The material and method of manufacture should also be chosen so that the amount of friction at the interface between the electrodes and the dynamic support element can be minimized. Given a number of factors, including low cost, ease of fabrication, and favorable electrical properties, the electrode support structure is typically made from a thermoplastic such as polycarbonate, polystyrene, polypropylene, acrylic, or polyethylene. The specific material must be selected to have sufficient rigidity to support the extended electrodes while maintaining sufficient elasticity to prevent failure of the component under load. Material selection must also consider the intended method of sterilization of the electrode array (eg, gamma irradiation, steam sterilization, ethylene oxide treatment, or electron irradiation) to ensure that the dynamic support element is compatible. The specific dimensions and design of the support element depend on the properties of the selected material. However, it is desirable to minimize the size of the support structure so that it does not unduly limit the distance between the electrodes to be placed or negatively affect other functional properties of the device. Typically, rigid flat structures have a sufficient thickness of 0.5-2 mm.

Another option related to the dynamic support element is the use of a compression spring with holes to accommodate the electrodes and restrain their lateral movement. The compression spring may be made of metallic, polymeric, or elastomeric materials and may be manufactured by molding, machining, injection molding, or other methods. At rest, the spring does not compress or is minimally compressed under the action of electrodes inserted into holes along the entire length of the spring. As the electrodes are placed, the spring is compressed in the direction of placement, while the holes ensure the sliding movement of the electrodes perpendicular to the coils of the spring. This embodiment of the invention is particularly suitable in combination with a screen or sheath used to accommodate the penetrating electrodes/needles after they have been removed from the tissue site. In these embodiments, the force imparted to the spring to move the electrodes forward can be used to position the sheath or shield over the electrodes when removing the device from the tissue.

In the implementation method illustrated in FIG. 4, a shield spring 138 may be used to partially support the electrode support 124, and in particular, the radius of the spring may be selected to match (or be slightly larger than) the radius of the wall 198 of the electrode support 124. Thus, the electrode support 124 can be inserted into the middle of the spring of the protective shield 138 during use. The electrode support 124 can then slide into the interior of the shield 134. When positioned at the center of the spring, the electrode support 124 typically maintains a position at the center of the spring, which provides a desired midpoint for supporting the electrodes, that is, approximately midway between their support point on the inner cassette cover 104 and their penetration point at the tissue interface.

Other supports for electrodes based on springs can be used. For example, a designed compression spring is located in the electrode area, which provides adaptive support for the electrode. Designed compression springs can be shaped and proportioned to precisely match the relative positions of the electrodes, thereby limiting their lateral movement. An optional bias element may be located in conjunction with the designed spring

- 38 043519 compression and can serve to displace the electrodes outward in relation to the designed compression spring. Engineered compression springs may be made of metallic, polymeric, or elastomeric materials and may be manufactured by molding, machining, injection molding, or other methods. The bias element may be made of metallic, polymeric, or elastomeric materials and may be manufactured by molding, machining, injection molding, or other methods.

Supports for electrodes designed based on other mechanisms, such as telescopic tubes, are also considered. Telescopic tubes serve to support the electrodes as they are inserted into the individual. The telescopic tubes may be sized to move freely relative to each other, or may be designed to move only when an axial force is applied to them.

Other support structures are also contemplated, for example support structures based on movable, flexible or rotating support elements. The side support members may be attached to the electrodes by optionally hinged members. The side support members may be constructed integrally with the structure in which the electrodes are located, or may be separate components attached by conventional means (by snapping, welding, gluing, fastening, etc.).

Another embodiment of the invention is the use of a compressible matrix material into which the electrodes are embedded. As the electrodes are placed, the material compresses in the direction of placement, providing lateral support along the direction of movement. Examples of compressible matrix materials are cellulose, foam plastics or rubber polymers such as micropore plastics, silicone foam or polychloroprene foam or carbon foam matrices. Since these materials are intended to contact the electrodes and/or the injection needle (if present), such materials must be selected to be compatible with indirect tissue contact.

Thus, the structures described above support the percutaneous placement of a plurality of elongated electrodes at tissue depths of up to 60 mm while maintaining the desired spatial relationship between the plurality of electrodes and, in particular embodiments of the invention, the orifice of a hypodermic needle. Such support members engage the plurality of elongate electrodes during transdermal insertion and limit deflection of the electrodes in one or more directions perpendicular to their elongate orientation. In another aspect, the present invention relates to methods and apparatus for applying biocompatible oily compounds to the surface of a plurality of electrodes to reduce the applied force required to achieve consistent transdermal placement of a plurality of elongated electrodes to a depth of up to 60 mm. In a representative embodiment of the invention, biocompatible silicone compounds, such as Dow Corning 360 Medical Fluid or Dow Corning MDX4-4159, can be conventionally sprayed or dipped onto a plurality of electrodes constituting the maritz to improve electrode insertion characteristics. The specific choice of coating and application conditions, such as coating method and thickness, depend on the number, size, composition and tip configuration of the electrodes, as well as the target tissue in which these electrodes are placed.

As shown in FIG. 4 and 7A-7B, the proximal portions 135 of the electrodes 122 may be located on the outside of the inner cassette 103 of the cassette unit 100 (FIG. 7B). Thus, if the inner cassette 103 is positioned to slide within the outer cassette 102 (FIG. 7A), the proximal portions 135 contact the respective electrode contacts 130, and in particular the internal portions 133 of the outer electrode contact cartridge 130, and these the internal portions 133 are configured to provide electrical communication with the proximal portions 135. Due to the length of the proximal portions 135 and the length of the internal portions 133 of the outer cassette, electrical communication can occur at multiple locations along their continuous interface, regardless of the longitudinal location of the inner cassette relative to the outer cassette. The electrode contacts 130 also include external contacts 131 of an external cassette configured to provide electrical communication with corresponding connections 496 on the applicator 400 (see FIG. 18C). The electrode contacts 130 are composed of conductive material sufficient to transmit an electrical signal. In some embodiments of the invention, the choice of design and material ensures that such contact will provide adequate adhesion and will produce an electrically conductive interface with the corresponding electrode within the limits of expected manufacturing variations in the position of the electrode and contact, without impeding or interfering with the direct movement of the electrodes 122 mounted on the inner cassette 103. This design must also provide consistent adhesion when exposed to expected storage conditions for the specified shelf life of the product. In a certain embodiment of the invention, the electrical contacts 130 are made of a stamped or formed metal of a suitable composition to facilitate interaction with the electrode, and may have coatings such as gold or copper to ensure the integrity of the electrical contacts and prevent corrosion. In addition to ensuring that electrical contact with the electrical

- 39 043519 trodes can be supported at multiple locations along their continuous interface, the inclusion of the electrode contacts in the outer cassette 102 in this configuration ensures that any wear due to the sliding interaction between the electrodes 122 and the electrode contacts 130 will be observed in the disposable cassette 100 , thereby providing a stable interface between the outer cassette contacts 131 and the applicator electrical contacts 496, and minimizing potential mechanical wear on the reusable applicator electrode connections 496. This configuration has the advantage of extending the shelf life of the applicator for multiple uses.

The following is a description of the remaining parts of the applicator 400. Such parts are typically parts that are independent of operation of the cassette unit 100. As shown in FIG. 13A, the applicator 400 includes an arm 402 and a multi-core cable 406 for transmitting electrical and control signals, as well as electrical signals to be applied to the fabric. Cable 406 typically terminates in a connector with a corresponding connector pin in controller 700. Applicator 400 further includes a user interface 404 in which the user can view aspects of the procedure and can direct the applicator to perform various functions, particularly depth selection. The applicator 400 further includes a treatment start button 407 that the individual presses to initiate the treatment.

Moreover, as shown in FIG. 13B, applicator 400 includes a treatment countdown timer 410 that informs the user of the remaining treatment duration and also directs the user not to remove applicator 400 until the countdown timer reaches zero. The power indicator 418 is intended to indicate a satisfactory and energized connection to the regulator 700. The procedure error indicator 414 is intended to inform the user of an error, for example, if one of the interlocks described above has not been disabled. The placement site indicator 412 is intended to inform the user that if the desired pressure has been applied to the tissue, the procedure can begin.

In fig. 14 shows a number of structural components of the applicator 400, including a connector 426 (not to scale) for connection to the regulator 700, a top housing 420, side housings 422 and 424, and an internal containment shell 432. A front cover 430 is shown along with an end cover 428. Also shown are various electromechanical subunits 450, some of which have been described above.

In fig. 15 is a more detailed view of the applicator 400, showing the cassette pressure sensor contacts 434 and sub-assemblies 452 corresponding to the cassette loading, electrode insertion and injection functions, as well as associated sensors.

In fig. 16 is a more detailed view of a group of subunits 452 including loading drive subunits 454 and a cassette loading subunits 456. The operation of subblocks was described above.

In fig. 17A is a more detailed view of the loading drive subunit 454, the general operating principle of which has been described above. In this regard, it should be noted that loading is initiated by transmitting a signal to the cassette unit 100, and that detection of the signal causes the system to be started by switch 464. The loading drive sub-unit is mounted on the applicator body using brackets 466 and 468. The action of the motor 444 is transmitted to the gear assembly 448 drive shaft 462 engine.

In fig. 17B is a more detailed view of the insertion/injection drive assembly 456. Many of these components have been described above. In this regard, it should be noted that the operating components are attached to the applicator body by means of a mounting bracket 476. After installation of the electrodes 122 and needle 105, the piston of the needle 105 is lowered by the action of the injection drive piston 484, which causes the drug to be expelled from the needle opening. The injection drive piston is driven by the injection drive motor and transmission assembly 486.

Typically, the injection drive piston driven by the injection drive assembly 486 moves forward to a point where it can no longer move forward, ie distally, indicating that it has reached the end of each step. This is typically indicated when the current used in the injection drive motor 486 increases significantly, which in turn indicates that the reservoir piston has reached the end of the reservoir and that injection is complete. However, in some cases, engine speed can be used to determine the amount of drug delivered. Such a function may be used to confirm the desired dose of medication or to notify the user in the event that the delivery volume does not match the expected total quantity, for example, when the injection reservoir 101 is expected to be full, but based on the position of the plunger rod, the reservoir is not was devastated. Such situations may arise when the user fails to press the applicator 400 against the subject's body before completing the procedure, for example, until the countdown timer reaches zero. As noted above, during an intervening period of time during the procedure, when the force applied to the individual is no longer detectable, an impedance test can be performed to determine whether the electrodes are within the individual. If this is not the case, it can be assumed that the applicator has been removed prematurely and a signal can be sent to the injection drive motor 486 to stop the injection and limit the amount of drug released from the individual.

In fig. 19 shows a controller system/assembly 700 including an electric field controller/generator 750, a lever 702, and an applicator holder 706. The regulator can be configured for both benchtop and portable use. If configured for portable use, the device desirably includes a storage bin 704. Details of the regulator in a portable configuration are shown in FIG. 20A-20D, including wheel locks to protect the regulator assembly from movement in the operating position, a tray 710 to accommodate consumables including individual preparation materials, reservoirs/vials/syringes, and the like. Applicator connection port 708 is shown as connecting applicator 400 to regulator assembly 700. The display 712 displays the status of the administration procedure, and in particular, connection to the IFU.

A cassette eject button 714 is provided to remove the cassette unit 100 from the applicator 400. Menu navigation buttons 716 allow navigation and manipulation of components as seen on the display 712. A mute button 718 is provided to mute warnings or other audible indicators if desired. The power button 722 is designed to supply power to the device, and the same button is turned on when the main power switch 726 has been turned on (Fig. 20D).

The display also includes a battery indicator 720 that provides an indication of the battery charge level where a battery backup system is provided. Such a battery backup system may be included in the regulator/generator 750 to accommodate situations in which a loss of power prevents the device from receiving power from the main power source. Such a system can also be used as a backup when the procedure was started by a power supply from the mains, but at the same time it was lost. In this case, the logic and control circuitry is implemented in such a way that it provides an almost seamless transition from mains power to battery power so that the procedure can be completed using a backup battery. It is desirable that the regulator include a battery monitoring circuit that is capable of monitoring whether the battery is sufficiently charged to complete the procedure after loss of mains power. In some embodiments of the invention, the controller also includes a display to notify the user that there is insufficient battery power to complete the procedure in the event of a power failure.

As shown in FIG. 20D, which illustrates a rear view of a regulator 750, such a regulator may include a USB port 724, an AC power switch 726, and an AC power supply 728.

As shown in FIG. 21, in one application illustrated in block diagram 800, controller/generator 750 is connected to the network and its program is automatically started (step 802). The applicator is then connected (step 804), and information or instructions for performing this action may be displayed on the user display screen 712 if the applicator has not been connected. The system may perform a self-test (step 806), wherein the self-test not only ensures reliable operation of the regulator/generator 750, but also ensures that the applicator 400 is properly connected to the regulator/generator 750.

The program may provide instructions on the user's display screen to prepare the injection site (step 808). This step can ensure the correct delivery of the drug, indication that it has not expired, any contraindications, and compliance with prescriptions/precautions.

The user then removes the reservoir cover and inserts the reservoir 101 into the cassette unit 100 (step 810). In some embodiments of the invention, an audible, tactile, or tactile signal is provided to the user (step 811) indicating correct placement of the reservoir in the cassette unit.

The user then inserts the cassette unit 100 into the applicator 400 (step 816). An error condition is then checked (step 818), for example regarding the correct placement of the reservoir, and if this error is detected, the procedure stops, an error message is displayed, and the user is instructed to correct it. If the error can be corrected, for example, if the user did not insert the reservoir correctly, or did not insert or close the back of the cassette, then the user may be instructed to remove the cassette and reinsert it correctly into the reservoir (state 820). In some cases, the cassette is ejected automatically, and in other cases, the user needs to press the cassette eject button. For other error conditions, such as when the back of the cassette has been closed, the user may be instructed to use a new cassette.

In any case, after installation of the device has been completed and an error-free state has been achieved, the user may proceed to drug administration and electroporation therapy (step 822).

- 41 043519

The primary function of the regulator is to create the electrical field necessary to achieve the desired drug delivery, to control the operation of the system during setup and use, to monitor the status of the system during setup and use, and to report the status of the system to the user during setup and use. . In some embodiments of the invention, the controller may provide recommendations and instructions for use of the device during user training, as well as during troubleshooting during normal use.

The system and method of operation of the controller can be fully implemented using any number of computing devices, including microprocessors, microcontrollers, programmable logic controllers. Typically, the instructions are located on an electronic medium, usually non-transitory, and these instructions are sufficient for a computer processor to implement the method of the invention. The electronic storage medium can be a hard disk or solid state storage medium that has instructions that are loaded into RAM when it starts up. Applications, for example, from multiple users or from any one user, can be accessed using any suitable computer input devices. For example, users may use a keypad, a conventional keyboard, a mouse, a touch screen, a joystick, a touchpad, another coordinate control device, or any other such computer device to enter data related to calculations. Data may also be entered via an on-chip memory chip, hard disk drive, interfaced flash memory, flash memory, optical media, magnetic media, or any other type of file storage media. The output may be delivered to the user via a video graphics array or embedded graphics microprocessors coupled to the user's display. Alternatively, the system may output one or more electronic document formats, or a printer may be used to output hard copies of the results. Based on this information, any number of other significant results should also be understood as implementing the disclosure of the invention. For example, the output data may be stored on a memory chip, hard disk, interface flash memory, flash memory, optical media, magnetic media, or any other type of media. It should also be noted that the disclosure can be implemented on any type of computing device, for example, personal computers, laptops, notebooks, notebook-type computers but with their own input system, PDAs, personal digital assistants, mobile phones, smartphones, tablets computers, as well as on devices specifically designed for these purposes. In one embodiment of the invention, a user of a smartphone or WiFi-connected device downloads a copy of the application to his device from a server using a wireless Internet connection. The associated authentication procedure and secure transaction process may involve payment to the provider. The application can be downloaded via a mobile connection or via WiFi or other wireless network connection. The application can then be launched by the user. Such a network system may provide suitable computer information for an implementation in which multiple users provide separate inputs to the system and method. In the applicator-controlled system below, multiple inputs allow multiple users to enter relevant data at the same time.

- 42 043519

Table of elements

# Positions Elements 100 Cassette unit 101 Storage tank 102 External cassette, also called cassette 103 Internal cassette 104 Inner cassette cover 105 Needle 106 External cassette cover 108 Leveling panel/side screen 110 External cassette cover (safety) 112 Back of the cassette 114 Cassette retaining ring 116 Reservoir detection spring 118 Detection tank cover 120 Tank lock, also known as tank insert button 122 Electrodes

- 43 043519

124 Electrode support 126 Power contact springs 128 Power contact converter 130 Electrode contacts 131 External electrode parts of the external cassette for connection to the applicator 132 Protective screen supports 133 Internal electrode parts of the outer cassette for connection with the inner cassette 134 Protective screen 135 Proximal electrode portions of the inner cassette for connection to the outer cassette 137 Distal electrode parts of the internal cassette for insertion into tissue 138 Flexible power contact circuit 139 Shoulder or bend of the electrode 140 Tank loading hole 142 Tank capacity 144 and 144’ Tank locking holes (1st or 2nd set) 146 Optical line of sight 148 Input detector, such as a PC emitter/collector sensor in the applicator 150 Internal cassette receiver 152 Needle sleeve 154 Rail 156 Output port 158 Tank cover 159 Piston stopper 160 Flexible scheme 162 First series of pads 164 Second series of pads 166 Protective screen assemblies 167 Screen openings 168 Side screen 170 Leveling panel hole for protective screen

- 44 043519

172 Initiation signal 174 Continue signala 176 External Cassette Cover Hooks 178 External surfaces of cassette cover ribs 180 Leveling panel/side screen wall hook 108 182 Protective screen mounting hooks 184 First depth support wall 186 Second depth supporting wall 188 Initial or remaining supporting wall 190 Reminder tone 192 Holes of the electrode support element 194 Hole for the needle of the electrode support element 196 Support structure with hole for electrode Electrode support structure 198 Wall of the electrode support element 400 Applicator 401 Applicator Cassette Unit Receiver Hole 402 Lever arm 403 Receiver of a cassette unit, which is surrounded by a housing 404 User interface 406 Multicore cable 407 Trigger 408 Injection depth selection indicator(s) 409 Injection depth selection button(s) (may be a switch or other forms in other embodiments) 410 Procedure countdown timer 412 Applicator placement indicator 414 Malfunction indicator during the procedure 416 Procedure completion indicator 418 Power indicator 420 Upper body 422 First side body 424 Second side building

- 45 043519

426 Electrical connector 428 End cap 430 Top cover 432 Inner containment 433 Electrical contacts for motor drive 444 434 Cassette force sensor contacts 435 Switch connectors 436 Cassette loading sensor 438 Cassette load sensor 440 Fuse or track 442 Guide rails for cassette 444 Loading drive motor 446 Engine start connector 448 Gear with gear 450 Electromechanical subunits 452 Sub-blocks for cassette loading, electrode insertion and injection 454 Drive sub-unit for loading 456 Cassette loading subunit 462 Motor drive shaft 464 System switch 466 Transmission cover bracket 468 Mounting bracket 470 Spring cap/cassette joint 471 Spring cover hole 472 Spring for insertion of electrodes/needle 474 Gear Bushing for Insertion 476 Mounting bracket 478 Insert Gear Drive Ring 479 Gear ring for insertion 480 Signal stabilizer 481 Insertion mechanism signal 482 Insertion mechanism drive motor 483 Insertion mechanism position sensor 484 Driven injection piston

- 46 043519

486 Drive motor and transmission for injection 488 Support posts (support element) 490 Channels for the first depth 491 Blocking signals 492 Channels for the second depth 494 Support wall 496 Electroporation applicator electrode contacts 700 Regulator assembly 702 Lever arm 704 Storage container 706 Applicator tray 708 Port for connecting the applicator 710 Pallet 712 Display screen 714 Cassette eject button 716 Menu navigation buttons 718 Mute button 720 Battery indicator 722 Power button 724 USB port 726 AC power switch 728 AC power port 750 Electric field generator

Examples

The following examples according to the invention are provided to further illustrate the essence of the invention. However, it should be noted that the following examples do not limit the scope of the invention, which should be determined by the attached claims.

Example 1. Obtaining the sequences of the core antigen and Pol antigen of the HBV virus and optimizing the plasmid.

T cell epitopes on the hepatitis virus core protein play an important role in clearance of hepatitis B virus infection and hepatitis B virus proteins such as polymerase and may serve to broaden the range of responses. Thus, core proteins and polymerase proteins of hepatitis B virus were selected as antigens for the development of a therapeutic vaccine against hepatitis B virus (HBV).

Derivatization of consensus sequences of core antigen and HBV polymerase antigen.

Consensus sequences of pol antigen and HBV core antigen were derived from HBV genotypes B, C and D. Various HBV sequences were obtained from different sources and aligned separately for the core and polymerase proteins. The seed sequence alignments for all subtypes (A to H) were then restricted to HBV virus genotypes, B, C, and D. Consensus sequences were determined for each protein alignment separately for each subtype and across all combined BCD sequences. At different alignment positions, the most frequently occurring amino acid was used in the consensus sequence.

Optimization of HBV core antigen.

The consensus sequence of the HBV core antigen was optimized by deletion in the native viral protein. Specifically, a deletion was made of thirty-four amino acids corresponding to the C-terminal segment with a high positive charge, which is necessary for encapsidation of pre-genomic RNA.

Optimization of HBV Pol antigen.

The HBV pol antigen consensus sequence was optimized by replacing four residues to remove reverse transcriptase and RNase H enzymatic activities. Specifically, aspartate residues (D) were replaced by aspartic residues (N) in the YXDD motif of the reverse domain.

- 47 043519 new transcriptase to eliminate any coordination function and thus bind the nucleotide/metal ion. In addition, the first aspartate residue (D) was replaced by an aspartic residue (N), and the first glutamate residue (E) was replaced by a glutamine residue (A) in the DEDD motif of the RNase H domain to eliminate Mg ²⁺ coordination. In addition, the HBV pol antigen sequence was codon optimized to scramble the internal open reading frames of envelope proteins, including the S protein and S protein variants with N-terminal extensions pre-S1 and pre-S2. As a result, the open reading frames for the envelope proteins (pre-S1, pre-S2 and protein S) and for the X protein were removed.

Optimization of HBV core antigen and pol antigen expression strategies.

To achieve maximum and equal expression of core and pol antigens from plasmid vectors, three different strategies were tested: (1) joining HBV core and pol antigens in the same reading frame to a small AGAG inserted between the coding sequences to produce a single Core-fusion protein. Pol (Fig. 25A); (2) expressing core and pol antigens from a single plasmid via a ribosomal excision site, and in particular the foot-and-mouth disease virus (FMDV) FA2 excision site, to produce a bicistronic expression vector expressing individual core and pol proteins from a single mRNA (FIG. 25B ); and (3) separating two separate plasmids encoding HBV core and pol antigens, respectively (Fig. 25C).

In vitro expression assay.

The coding sequences of the consensus core and HBV pol antigens according to each of the above three expression strategies were cloned into the commercially available expression plasmid pcDNA3.1. HEK-293T cells were transfected with the vectors, and protein expression was assessed by Western blot analysis using hbv core-specific antigen.

Optimization of post-transcriptional regulatory elements.

To increase protein expression levels by stabilizing the primary transcript, facilitating its removal from the nucleus, and/or increasing transcription-translation levels, four different post-transcriptional regulatory elements have been evaluated: (1) the woodchuck HBV post-transcriptional regulatory element (WPRE) (GenBank: J04514.1); (2) an intron/exon sequence derived from the human apolipoprotein A1 precursor (GenBank: X01038.1); (3) human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) R-U5 untranslated domain (GenBank: KM023768.1); and (4) a combination sequence consisting of the HTLV-1 LTR, a synthetic rabbit β-globin intron (GenBank: V00882.1), and a splicing enhancer (a three-component sequence). Enhancer sequences were introduced between the CMV promoter and the HBV antigen coding sequences in the plasmids. Western blot analysis did not reveal any significant differences in the expression of the core antigen when it was expressed from a plasmid in the presence and absence of the WPRE element (Fig. 25D). However, when core antigen expression in transfected HEK293T cells from plasmids having the other three post-transcriptional regulatory sequences was assessed by Western blot analysis, the triple enhancer sequence gave the highest level of core antigen expression (Fig. 25E).

Selection of signal peptide for efficient protein secretion.

Three different signal peptides introduced within the N-terminus of the HBV core antigen were evaluated: (1) Ig-gamma heavy chain signal peptide SPIgG (BAA75024.1); (2) Ig-epsilon heavy chain signal peptide SPIgE (AAB59424.1); and (3) cystatin S precursor signal peptide SPCS (NP 0018901.1). Signal peptide cleavage sites were optimized in silico for core antigen attachment using the Signal P prediction program. Secretion efficiency was determined by analyzing core protein levels in the supernatant. Western blot analysis for core antigen secretion, performed using three different signal peptides appended at the N-terminus, showed that the cystatin S signal peptide gave the most efficient protein secretion (Fig. 25F).

Optimization of the DNA vaccine vector.

Optimized expression clusters containing a three-component enhancer sequence upstream of the HBV antigen coding sequence along with the N-terminal cystatin S signal peptide sequence were cloned into the pVax-1 vaccine DNA vector (Life Technologies, Thermo Fisher Scientific, Waltham, MA). The expression cassette in pVax-1 contains the human CMV-IE promoter followed by a polyadenylation (pA) sequence derived from bovine growth hormone (BGH). Bacterial reproduction is induced by the ori pUC replicon and the kanamycin resistance gene (Kan R), under the control of a small eukaryotic promoter. The pUC ori replicon, the KanR expression cluster, and the expression cluster under the control of the CMV-IE promoter are in the same orientation within the plasmid backbone. However, a marked reduction in core antigen expression levels was observed in the pVax-1 vector compared to the expression level observed in the pcDNA3.1 vector.

- 48 043519

Several strategies have been used to increase protein expression: (1) converting the full-length pUCori-KanR cluster into a cluster in a counterclockwise orientation (pVD-core); and (2) changes in codon optimization of the KanR gene along with replacement of the Kan promoter with the Amp promoter derived from the pcDNA3.1 vector (pDK-core). Both strategies restore core antigen expression, and core antigen expression was highest in the presence of the pDK vector, which contained the codon-optimized Kan R gene, the AmpR promoter (instead of the KanR promoter), and the pUCori-KanR cluster in reverse orientation.

Four different expression clusters optimized for HBV core/pol antigen and presented in FIG. 25G was introduced into the pDK plasmid backbone to test each of the three expression strategies illustrated in FIG. 25A-25C. Plasmids were tested in vitro for core and pol antigen expression by Western blot analysis using antibodies specific for core and pol antigen. The most consistent expression profile for cellular and secreted core and pol antigens was achieved when the core and pol antigens were encoded by separate vectors, namely the separate DNA vectors pDK-core and pDK-pol (FIG. 25H). Vectors pDK-pol and pDK-core are shown schematically in FIG. 26A and 26B, respectively.

Example 2: Electroporation-Mediated Intramuscular Administration of Nucleic Acid-Based Biopharmaceuticals Using a TriGrid Systemic Delivery Device (TDS-IM).

Intracellular delivery of nucleic acid sequences to the skeletal muscles of the upper or lower extremities of an individual can be improved using an exemplary device, such as the TriGrid Systemic Delivery Device (TDS-IM) Model II described herein. In some embodiments, the TDS-IM device is used in conjunction with agents approved for research use in the TDS-IM device. In a representative embodiment of the invention, the tested agent is nucleic acids, that is, DNA or RNA. In some embodiments of the invention, the TDS-IM device is used only for the individual in need thereof. In some embodiments of the invention, the TDS-IM device is used only for an individual enrolled in an open clinical trial program through intramuscular delivery of nucleic acid by electroporation.

In order to start the system for administration, the device's main power is supplied to the stimulator and the system's battery is adequately charged for use. To do this, turn on the power switch and press the power button on the front panel. The applicator is connected to the applicator connector. Correct connection of the applicator is confirmed by the illumination of the applicator power indicator. The startup screen appears after the system has completed all self-tests. Then press the OK button to continue the insertion procedure.

To insert a syringe into a TDS cassette, remove the cap from the syringe and align the syringe flange with the syringe loading hole of the TDS cassette. The syringe should snap into place and be fully seated in the TDS cassette. After loading the syringe, press the OK button on the pulse stimulator to continue the procedure. The cassette lid must remain attached until the cassette is loaded into the applicator.

To insert a loaded syringe cassette into the applicator, the cassette is aligned with the applicator so that the syringe loading hole of the cassette is facing up. After inserting the cassette into the applicator, the cassette is automatically fed into the fully loaded position in the applicator. Successful loading of the cassette is indicated in the stimulator. After loading the cassette, the applicator is returned to its tray and the appropriate injection site for the individual is selected. In some embodiments, the injection site for intramuscular delivery of the nucleic acid is the medial deltoid muscle, located approximately three finger widths below the edge of the acromion process (humerus). In a representative embodiment of the invention, the depth of injection into the medial deltoid muscle is approximately 0.75-1.25 inches (19-30 mm). In some embodiments, the injection site for intramuscular delivery of the nucleic acid is the vastus lateralis (outer thigh) at approximately the midpoint between the hip and the knee. In a representative embodiment of the invention, the depth of injection into the vastus lateralis muscle is approximately 1.0-1.5 inches (25-38 mm). After selecting the injection site, press the applicator depth selection button, and then the injection depth selection button corresponding to the injection site/depth. In some embodiments of the invention, the injection depth selection indicator is continuously illuminated to confirm the selected injection depth. In some embodiments of the invention, the depth select button on the right side corresponds to a deeper injection depth. In one embodiment of the invention, where the originally selected injection site is to be changed, the selection button corresponding to a different injection depth is pressed, and when the injection depth selection window appears, a different injection depth is selected.

To begin administering a tested agent to an individual using the TDS-IM device,

- 49 043519 remove the cassette cover and discard it. The device is aligned and pressed firmly onto the desired injection site. When the device is firmly pressed to the desired injection site, all four bars of the applicator placement indicator light up and the procedure countdown timer begins to glow at 8 seconds, indicating the time remaining until the end of the injection procedure. To administer the agent, press the applicator start button while maintaining constant pressure. When the procedure countdown timer reaches 0, electrical stimulation is delivered. Once the injection procedure is complete, the procedure completion indicator lights up and the device can be removed from the injection site. Do not remove the device from the injection site until the procedure is completed or the malfunction indicator lights up. In some embodiments of the invention, if the device detects a malfunction during the administration procedure, the device aborts the administration procedure and the error indicator lights up. In an exemplary embodiment of the invention, if the device interrupts the insertion procedure and the device is quickly removed from the subject, then additional instructions appear on the stimulator display of the device.

To remove the cassette from the applicator after completing the administration of the agent to the individual in need of it, press the eject button located on the stimulator. The applicator automatically extends the cassette into a position where it can be manually removed from the applicator. Once the cassette stops moving, you can grab the cassette by the sides indicated by the arrows to remove the cassette from the applicator. After removing the cassette, complete completion of the injection can be verified by visually inspecting the position of the syringe plunger. To turn off the device, place the applicator in the housing and press and hold the power button on the front panel for 5 s.

Example 3: Electroporation of TDS-IM in Mice and Non-Human Primates.

Electroporation technology via TriGrid intramuscular delivery system (TDS-IM) was used to improve the delivery of DNA-based constructs into muscle tissue. The TriGrid electrode array for intramuscular (i.m.) delivery consists of four electrodes arranged in two equilateral triangles forming a diamond shape surrounding a central injection needle (FIG. 22). Combining agent delivery and electrical field propagation in a single device ensures that induction of the electroporation effect occurs at the site of agent distribution and allows the introduction of the plasmid construct and electrical pulses in a single step. Thus, i.m. delivery of plasmid DNA is achieved in an efficient and reproducible manner.

TriGrid electrode arrays for each animal model were scaled to efficiently deliver DNA plasmids, such as those prepared as described in Example 1, to the selected muscle regardless of the size and properties of the specific muscle tissue. The main electroporation device parameters that were scaled for use for each animal model were TriGrid size (i.e., distance between electrodes), applied voltage (250 V/cm, where cm is TriGrid size), electrode diameter, and electrode penetration depth. Secondary adaptations to the device include changes in syringe volume, plasmid DNA volume, and hypodermic needle gauge/length.

Mice studies were performed using a TDS-IM v1.0 device using an electrode array with an interelectrode distance of 2.5 mm. This adjustment to the size of the electrode array was made to accommodate these electrodes in a smaller mouse muscle. In fig. 23A shows a version of TriGrid TDS-IM v1.0 adapted for use in a mouse model. Studies in non-GLP (green long-tailed monkeys) monkeys were also performed on the TDS-IM v1.0 device, but with an interelectrode distance of 6 mm. In fig. 23B shows a version of the TriGrid TDS-IM v1.0 adapted for testing in non-human primates (NHPs) as models.

The device used in the GLP monkey trials is the TDS-IM v2.0 device, which is also intended for human clinical trials. In this device, the distance between the electrodes is 6 mm and the activation conditions are the same as for the TDS-IM v1.0 device. The electrode materials used in the TDS-IM v2.0 device are identical to those in the TDS-IM v1.0 device used in the NHP study described above, but the cassette support structure and the syringe used for administration are different. In addition, the insertion depth is also different for humans and non-human primates. Therefore, the TDS-IM v2.0 device was equipped with a spacer sleeve in order to adapt the TDS-IM v2.0 device for use in much smaller NHP muscles. In fig. 24 shows TriGrid TDSIM v2.0 adapted for testing on non-human primates (NHPs) as models.

In each animal test, the animals were anesthetized to immobilize them before the electroporation administration procedure was performed. The injection site was selected using regional bone anatomy as reference points, and hair was removed from the selected site using an electric hair clipper and then using an aseptic swab. The TDS-IM matrix was injected percutaneously into the selected muscle, with the major axis of the diamond-shaped configuration oriented parallel to the muscle fibers. After insertion of the electrode, an injection was carried out to distribute the DNA in the muscles. After completion of the i.m. injection, an electric field of 250 V/cm was locally applied for a total duration of 400 ms at a 10% duty cycle (ie, during these 400 ms, the voltage was only actively applied for a total of 40 ms). After completion of the electroporation procedure, the TriGrid™ matrix was removed and the animals were restored to normal condition.

Variable components for each TDS-IM variant and/or animal model are shown in Table. 1 and at 2.

Table 1

TDS-IM option and TriGrid parts

Option TDSIM Animal e-model Pa3MepTriGr id (mm) Electrode diameter (inch) Conductor length(mm) Effective penetration depth (mm) DNA injection volume (cm ³ ) Syringe volume (cm ³ ) Injection needle gauge Injection method TDS-IM vl.O mouse 2.5 0.030 3.2 3.2 0.020 3/10 cm ³ thirty Manually TDS-IM vl.O NHP 6.0 0.021 5.0 15.5 1.0 1.0 cm ³ 22 automatic eski TDS-IM v2.0 NHP 6.0 0.023 5.0 9.0* 1.0 1.0 cm ³ 22 automatic eski

* NHP body weight and muscle size required for shallower penetration depth; A 10 mm depth stop was used to reduce the penetration depth to 9 mm.

table 2

TDS-IM option and pulse stimulator details

Varian t TDS- I.M. Animal e-model Supplied electric field (V/cm) Supply Voltage Range (V) Limited by no supply current (A) Limited no submission current (A/sec) Total number of pulses Duration of active stimulation (ms) Maximum long lasting consecutive pulses (ms) TDS-IM vl.O mouse 250 59.4- 5.6 N/A* - 4 0.16 6 40.8 369.3 TDS-IM NHP 250 142.4- 157.6 0.6-4 0.16 6 40.8 369.3 vl.O TDS-IM v2.0 NHP 250 142.4-157.6 0.6-4 0.16 6 40.8 369.3

* The lower current limit is disabled to adapt to the characteristics of the mouse model. Example 4 Electroporation with TDS-IM in humans.

The TDS-IM v2.0 device was used in the clinical trial. This device is essentially the same as the device used for GLP monkeys. The depth of administration was different for humans and non-human primates. Therefore, the TDS-IM v2.0 device was equipped with a spacer sleeve in order to adapt the TDS-IM v2.0 device for use in much smaller NHP muscles. Stimulation conditions were essentially the same as for GLP monkeys, except that the penetration depth was approximately 19 mm rather than 9 mm for GLP monkeys.

Although preferred embodiments of the invention have been disclosed and described in the present invention, it will be apparent to those skilled in the art that such embodiments are presented by way of example only. It will be apparent to those skilled in the art that various changes, modifications and substitutions may be made to the present invention without departing from the scope of the invention. It should be noted that various alternatives to the embodiments described herein, or combinations of one or more of these embodiments or aspects of the invention described herein, may be used to practice the present invention. It should be borne in mind that the scope of the disclosure of the invention is determined by the following claims, which cover the methods and structures included in the scope of these claims, and their equivalents.

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Claims (15)

CLAIM 1. A device for controlled delivery of a vaccine against the hepatitis B virus (HBV) to a predetermined tissue site in an individual in need thereof, wherein said device includes: a cassette unit (100) containing an outer cassette (102); internal cassette (103); a reservoir (101) containing the HBV vaccine, wherein the reservoir receptacle (142) is located in the outer cassette (102) and is configured to be supplied to the reservoir (101); an applicator (400) comprising a cassette unit receptacle (403), a needle hub (152) and an insertion detector (148), where the insertion detector (148) detects the loading of the reservoir (101) into the reservoir receptacle (142); at least one lock (120), where such lock (120) facilitates the correct execution of the procedure for administering the HBV vaccine; at least one injection port through which the HBV vaccine is administered; a plurality of penetrating electrodes (122) located in a predetermined spatial relationship with respect to the hole; an electric field generator (750) for generating an electrical signal, operatively connected to the electrodes (122); and a controlled energy source sufficient to transport a predetermined amount of the HBV vaccine at a predetermined rate from the reservoir (101) through the opening to a predetermined site on the individual, where the HBV vaccine contains: a first nucleic acid molecule comprising a first polynucleotide encoding an HBV polymerase antigen, wherein the HBV polymerase antigen contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, and wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity ; a second nucleic acid molecule containing a second polynucleotide encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2; and a pharmaceutically acceptable carrier, wherein the first nucleic acid molecule and the second nucleic acid molecule are present in the same nucleic acid molecule or in two different nucleic acid molecules. 2. The apparatus of claim 1, wherein the electrodes (122) are a plurality of elongate electrodes and the lock (120) prevents accidental activation of the cassette function. 3. The apparatus of claim 1, wherein the at least one interlock (120) is selected from the group consisting of a mechanical interlock, a light emitter/collector, a rear cassette (112), a force interlock, a leveling panel (108), and a side shield ( 108), trigger and emergency switch blocking. 4. A device according to any one of claims 1 to 3, wherein the device comprises at least two sets of interlocks (120), wherein the first interlock is a mechanical interlock and the second interlock is selected from the group consisting of a light emitter/collector, a rear portion of the cassette (112), force lock, leveling panel (108) and side shield (108), trigger lock and safety switch, preferably where the mechanical lock is a reservoir lock, further comprising (i) at least one reservoir lock hole (144), or (ii) an indicator panel extending along one or more surfaces of the cartridge, wherein the indicator indicates proper insertion of the reservoir into the cartridge, and preferably the rear portion of the cartridge (112) provides an optical line of sight through at least one reservoir locking hole (144). 5. The device of claim 4, wherein the device further comprises a third interlock (120) which is a force interlock, wherein the force interlock detects a force applied to a predetermined area of tissue of the individual and prevents the administration of HBV vaccine to the individual if such force is insufficient, preferably where the locking force also forms an electrical lock within the applicator (400). 6. A device according to any one of claims 1 to 5, where the device also contains at least one sensor, preferably selected from the group consisting of a cassette loading sensor (436), a cassette loading sensor (438), a cassette force sensor, a mechanism position sensor insert (483), insert sensor, optical detector (148) and electrical sensor. 7. The device of claim 6, wherein the sensor detects the reservoir label and confirms the presence of HBV vaccine. 8. A device for controlled delivery of an HBV vaccine to a predetermined tissue site in an individual, wherein said device includes: a cassette unit (100) containing a housing (102); a reservoir (101) containing an HBV vaccine, wherein a reservoir receptacle (142) is located in a housing (102) and is configured to be supplied to the reservoir (101); - 52 043519 applicator (400, comprising a cassette unit receptacle (403), a needle hub (152) and an insertion detector (148), where the insertion detector (148) detects the loading of the reservoir (101) in the reservoir receptacle (142); at least one injection port through which the HBV vaccine is administered; a plurality of penetrating electrodes (122) located in a predetermined spatial relationship with respect to the hole; an electrode support structure (124) comprising an opening and a wall, where the electrode support structure prevents accidental perpendicular movement of the electrodes (122); an electric field generator (750) for generating an electrical signal, operatively connected to the electrodes (122); and a controlled energy source sufficient to transport a predetermined amount of HBV vaccine at a predetermined rate from the reservoir (101) through the opening to a predetermined site on the individual, where the HBV vaccine contains: a first nucleic acid molecule comprising a first polynucleotide encoding an HBV polymerase antigen, wherein the HBV polymerase antigen contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, and wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity ; a second nucleic acid molecule containing a second polynucleotide encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2; and a pharmaceutically acceptable carrier, and wherein the first nucleic acid molecule and the second nucleic acid molecule are present in the same nucleic acid molecule or in two different nucleic acid molecules. 9. The device of claim 8, wherein the electrode support structure (124) provides an operative connection between a conductive contact area located in a distal region (137) of the electrodes (122) and a controlled energy source if the electrodes (122) are placed in a predetermined tissue area in an individual, preferably wherein the electrode support structure (124) includes at least one opening for passage of electrodes (122) and an opening for passage of an injection needle. 10. The apparatus of claim 8 or 9, wherein the electrode support structure (124) is a planar support structure positioned perpendicular to the elongated orientation of the electrodes (122), preferably where the planar support structure includes at least one opening configured to allow passage electrode (122) through a given area of tissue. 11. The device according to any one of claims 8 to 10, wherein the electrode support structure (124) is an adaptive electrode support, preferably where the adaptive electrode support is a compression spring made of metal, polymer or elastomeric material. 12. The device according to any one of claims 8 to 11, wherein the electrode support structure (124) comprises at least one of a telescopic tube, a protective screen spring (138) and at least one side support element attached to the electrodes (122) at at least one optional hinge element. 13. The device according to any one of claims 1 to 12, wherein the HBV vaccine is capable of inducing an immune response in a mammal against at least two HBV genotypes, and preferably, the HBV vaccine is capable of inducing a T cell response in a mammal against at least HBV genotypes B, C and D, and more preferably, the HBV vaccine is capable of inducing a CD8 T cell response in humans against at least HBV genotypes A, B, C and D. 14. The device according to any one of claims 1 to 13, wherein the first non-naturally occurring nucleic acid molecule is present in the first plasmid DNA vector and the second non-naturally occurring nucleic acid molecule is present in the second plasmid DNA vector. 15. The device according to claim 14, where the vaccine against HBV includes: a) a first plasmid DNA vector comprising, from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence SEQ ID NO: 7; an enhancer sequence containing the polynucleotide sequence SEQ ID NO: 8; a sequence encoding a signal peptide and containing the polynucleotide sequence SEQ ID NO: 5; a first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 3; and a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 11; b) a second plasmid DNA vector comprising, from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence SEQ ID NO: 7; a regulatory sequence containing the polynucleotide sequence SEQ ID NO: 8; a sequence encoding a signal peptide and containing the polynucleotide sequence SEQ ID NO: 5; a second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 1; and a polyadenylation signal sequence containing a polynucleotide -