KR20200101388A - Method and device for delivery of hepatitis B virus (HBV) vaccine - Google Patents

Abstract

Methods and devices are provided for reproducibly, consistent and effective delivery of an HBV vaccine to a subject. The present invention includes a device for controlled administration of an HBV vaccine through an orifice to a subject, a plurality of through electrodes disposed in a predetermined spatial relationship with respect to the orifice, and an electrical signal generator operably connected to the electrode.

Description

Method and device for delivery of hepatitis B virus (HBV) vaccine

Cross-reference to related applications

This application claims priority to International Patent Application No. PCT/US2017/067269 filed on December 19, 2017 and US Patent Application No. 62/607,430 filed on December 19, 2017, the contents of which are disclosed herein Is incorporated by reference.

Reference to the electronically submitted sequence listing

This application contains a sequence listing in ASCII format with the file name "688097-405 Sequence Listing", created on December 10, 2018 and 46.6 KB in size, which has been submitted electronically via EFS-Web. have. Sequence listings submitted through EFS-Web are part of this specification, the entire contents of which are incorporated herein by reference.

Technical field

The present invention relates to the administration of a prophylactic and/or therapeutic hepatitis B virus (HBV) vaccine to a subject in need thereof, and more specifically, a prophylactic and/or therapeutic HBV vaccine such as a nucleic acid encoding an HBV antigen. It relates to reproducible, consistent and effective delivery, facilitated by local application of an electric field in a safe, effective and consistent manner, across a population of heterogeneous recipients with minimal user training, to a limited area of a selected tissue site of interest.

Hepatitis B virus (HBV) is a small hepatitis B virus of 3.2 kb, encoding 4 open reading frames and 7 proteins. About 2 billion people are infected with HBV, and about 220 million people have a chronic hepatitis B infection (chronic HBV) characterized by persistent viral and subviral particles in the blood for over 6 months (Cohen et al., J. Viral Hepat. (2011) 18 (6), pp. 377-383). Persistent HBV infection results in T-cell depletion in HBV-specific CD4+ and CD8+ T-cells in circulation and liver, through chronic stimulation of HBV-specific T-cell receptors with viral peptides and circulating antigens.

As a result, T-cell multifunctionality is reduced (ie, reduced levels of IL-2, tumor necrosis factor (TNF)-α, IFN-γ, lack of proliferation and loss of granules). Therapeutic vaccination has the potential to eliminate HBV from chronically infected subjects (see Michel et al. J. Hepatol. (2011) 54 (6), pages 1286-1296). Immunogenic compositions containing one or more nucleic acid molecules encoding one or more HBV antigens are used to provide therapeutic immunity to subjects in need of therapeutic immunity, such as humans with chronic HBV infection.

Naked DNA can be transferred to cells in vivo resulting in gene expression, but gene transfer efficiency is relatively low and variable. Local application of electrical signals has been shown to improve the distribution and absorption of macromolecules in living tissues. The application of such electrical signals in tissues in connection with administration of a prophylactic or therapeutic HBV vaccine may have a desirable effect on the tissue and/or the agent being delivered. Specifically, techniques such as electroporation and ion ionization have been used to significantly improve the distribution and/or uptake of nucleic acids comprising both DNA and RNA sequences. Potential clinical applications of these techniques include the delivery of nucleic acid sequences for prophylactic and therapeutic immunity, and delivery of nucleic acid sequences encoding therapeutic proteins or peptides.

The application procedure involves the administration of an agent of interest to a target tissue site in connection with the application of an electric field of sufficient size and duration to induce the desired effect on the delivery, distribution and/or efficacy of the agent. The electric field propagates through two or more electrodes in electrical communication with the tissue. Electrode configurations suitable for use in these techniques include tissue penetrating electrodes, surface contact electrodes, and air gap electrodes. Specific electrode configurations include, but are not limited to, long needle or rod electrodes, point electrodes, meander electrodes (ie, shaped wires), planar electrodes, and combinations thereof. The specific type and arrangement of electrodes is selected based on the target tissue type and the purpose of the procedure. Desirable results are best achieved when the electric field is propagated within the target tissue in the presence of the agent of interest.

A wide range of methods and devices have been described for applying an electric field to tissue in the presence of an agent of interest to enhance agent delivery in skin and muscle tissue. The device includes the use of surface and tissue penetrating electrode systems, as well as combinations thereof. Despite the prospects associated with electrically mediated drug delivery and the potential clinical applications of these techniques, there is still a need for reliable and consistent techniques to deliver nucleic acids of interest to subjects. An important source of this variability is due to differences in operator skills and skill levels. Other sources of variation not addressed by the current system include differences in physiological characteristics between subjects that may affect the application of the procedure. Other considerations for the development of suitable devices include the ease of use and the implementation of designs that reduce the frequency and importance of potential user errors.

This background description is provided to introduce a brief context for the following summary and detailed description. This background is not intended to help determine the scope of the claimed invention, and is not to be considered as limiting the claimed invention to solving some or all of the above-presented shortcomings or problems.

Cohen et al., J. Viral Hepat. (2011) 18 (6), pp. 377-383 Michel et al., J. Hepatol. (2011) 54 (6), pp. 1286-1296

Given that safe, reliable, accurate and consistent application of clinical therapy is highly desirable, the development of an improved application system is urgent. These developments should include means to minimize operator-related variability while providing a means to accommodate differences in target properties that may occur during the widespread clinical application of electrically mediated drug delivery. In other words, specific areas for refining include the ability to maintain consistent performance across heterogeneous recipient populations and the reduction in the level of training and skills required for effective use by users. In addition, the device, system or method should be designed to avoid user or device errors and minimize the impact in case of errors.

The present invention provides devices, kits and methods for reproducible, consistent and effective delivery of an HBV vaccine comprising a nucleic acid molecule encoding an HBV antigen to a subject in need thereof using electromediated therapeutic delivery (EMTAD). In the embodiments described in this application, the therapeutic agent is an HBV vaccine.

In one embodiment of the present invention, a device for delivering HBV vaccine to a predetermined location in a subject is provided, the device comprising an assembly for controlled administration of HBV vaccine to the subject, the device comprising: HBV A reservoir containing the vaccine, at least one orifice through which the medicament is administered, and a controlled energy source sufficient to deliver a predetermined amount of HBV vaccine from the reservoir through the orifice to a predetermined site in the subject at a predetermined rate. . Further, the device may include a plurality of through electrodes arranged in a predetermined spatial relationship with respect to the orifice, and an electrical signal generator operably connected to the electrodes.

In another embodiment of the present invention, a kit for delivering an HBV vaccine to a predetermined site in a subject is provided, the kit comprising an HBV vaccine and a device comprising an assembly for controlled administration of the HBV vaccine to a subject, the The device comprises a reservoir containing the HBV vaccine, at least one orifice through which the drug is administered, and a controlled energy sufficient to deliver a predetermined amount of HBV vaccine from the reservoir through the orifice to a predetermined site in the subject at a predetermined rate. Includes a circle. Further, the device may include a plurality of through electrodes arranged in a predetermined spatial relationship with respect to the orifice, and an electrical signal generator operably connected to the electrodes.

The HBV vaccine included in the device or kit of the present application,

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

A second nucleic acid molecule comprising 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

It contains a pharmaceutically acceptable carrier,

The first nucleic acid molecule and the second nucleic acid molecule exist as the same nucleic acid molecule or two different nucleic acid molecules.

Another embodiment of the present invention includes a method comprising administering an HBV vaccine in a controlled spatial and temporal relationship with electrical signal administration (ESA).

The efficacy and benefits for certain embodiments according to the invention of the present invention vary. Some embodiments allow for insertion into various types of desired tissues (eg, dermis, muscles, etc.) across heterogeneous populations of various body masses and body components, allowing selection of needle depth and electrode insertion. These examples also facilitate adaptation of methods for use in human patients with specific target populations, such as, but not limited to, chronic HBV infection and/or HBV-induced disease. Systems and methods are provided herein that include design features that make them resistant to accidental discharge or potential misuse due to, for example, falling, crushing and/or falling. In some embodiments, an apparatus configured for multiple implant depths is provided. The systems and methods according to the invention of the present invention allow numerous safety interlocks to reduce the frequency and/or impact of user errors. This includes facilitating the proper preparation and configuration of the dose to be administered, protecting the device by applying the necessary force to the subject's tissue, removing the safety cap, and so on. The systems, devices, kits and methods according to the invention of the present invention allow highly consistent therapy to be delivered regardless of the type of administer or recipient (subject). The systems, devices, kits and methods described herein can be consistently administered to recipients with a variety of skin and muscle properties, for example, such that a constant force profile is obtained before and during delivery of the therapy.

The summary description above is provided to introduce various concepts in a simplified form. The various concepts are described in detail in the Detailed Description section. Elements or steps other than those described in the summary description above are possible, and not necessarily all elements or steps are essential. This summarized description is not intended to specify key features or essential features of the claimed invention, nor is it intended to assist in determining the scope of the claimed invention. The claimed subject matter is not limited to embodiments that solve any or all drawbacks mentioned in any part of the invention.

Other embodiments, features and advantages of the present invention will become apparent from the following disclosure, including the detailed description of the present invention and preferred embodiments and the appended claims.

1 shows a potential source of spatial variability associated with conventional needle syringe injection.
2 is an overview of a system according to the invention comprising a cartridge assembly 100, an applicator 400 and a controller system 700.
3A-3B show diagrams of embodiments of the apparatus described herein. 3A shows a side view of a cartridge assembly 100 according to the present invention. 3B shows a side view of a reservoir for an HBV vaccine 101 implemented with a syringe according to the invention.
4 shows various exemplary components of a cartridge assembly 100 according to the present invention.
5A-5E show diagrams of embodiments of internal cartridges and cartridge breaches in the apparatus described herein. 5A shows a plan view of an inner cartridge 103 according to the invention. Figure 5b shows a bottom view of the inner cartridge 103 and cartridge breech according to the present invention. 5C shows a detailed side perspective view of an inner cartridge 103 according to the present invention. 5D shows a side view of a cartridge breech 112 according to the present invention. 5E shows a reservoir interlock 120 with an improved locking feature to prevent the cartridge breach 112 from accidentally advancing.
6 shows a detail of a cartridge assembly showing a rack 154, a start flag 172 and a sustain flag 174 according to the present invention.
7A-7B show diagrams of embodiments of electrodes and/or electrode contacts in the apparatus described herein. 7A shows details of an electrode contact 130 and various electrode contact portions according to the present invention. 7B shows details of the electrode 122 and various electrode contact portions according to the present invention.
8A-8B show diagrams of an embodiment of a force contact interlock system in the apparatus described herein. 8A shows the top of a force contact interlock system according to the invention. 8b shows a detail of a force contact interlock system according to the present invention.
9A-9D show diagrams of embodiments of the apparatus described herein. 9A shows a needle 105 and distal inner cartridge electrodes 137 for tissue insertion according to the present invention. 9B shows the details of a stick shield 134 according to the present invention. 9C shows the mountain alignment guide 108 and splay shield 168 according to the present invention. 9D shows a stick shield support coupled to the outer cartridge cap 106.
10A-10D show views of an embodiment of an outer cartridge cap in the apparatus described herein. 10A shows an outer cartridge cap 110 according to the present invention. 10B shows a side view of an outer cartridge cap 110 according to the present invention. 10C shows an outer cartridge cap 110 used in an alignment guide 108 and a splay shield according to the present invention. 10D shows the outer cartridge cap 110 with an extension member designed to hold the inner cartridge 103 in place during handling and loading.
11A-11C show details of a stick shield in an embodiment of the device described herein. 11A shows a stick shield retaining hook 182 of a stick shield 134 according to the present invention. 11B shows details of a stick shield 134 according to the present invention. 11C shows a stick shield support 132 holding the stick shield 134 in place according to the present invention.
12 shows details of the electrode support 124 according to the present invention.
13A-13B show views of an applicator in the device described herein. 13A shows a side view of an applicator 400 according to the present invention. 13B shows a plan view of an applicator 400 according to the present invention.
14 is an exploded view of an applicator 400 according to the present invention.
15 shows details of the side housing and electroporation electrode connection 496 of the applicator 400 according to the present invention.
16 is an exploded view of the applicator according to the present invention showing the cartridge loading sub-assembly 456.
17A-17B show views of an applicator in the device described herein. 17A is an exploded view of an applicator according to the present invention showing the loading drive sub-assembly 454. 17B shows a rack 154 in a loading drive sub-assembly 454 according to the present invention.
18A-18C show diagrams of an embodiment of an applicator in the device described herein. 18A shows the details of the cartridge loading sub-assembly 456 according to the present invention, and shows the position where the insertion/injection drive assembly of the applicator is mated with the cartridge assembly. 18B shows a cross-sectional view of the cartridge assembly according to the present invention, showing the position where the insertion/injection drive assembly of the applicator is mated with the cartridge assembly. 18C shows details of the cartridge loading, electrode insertion and injection subassembly 452 of the applicator 400 according to the present invention.
19 shows various components of the controller system according to the present invention.
20A-20D show diagrams of the apparatus described herein. 20A shows various components of the controller system according to the present invention. 20B shows details of the applicator connector port 708 and tray 710 of the controller system according to the present invention. Figure 20c shows a detail of the stimulator display screen of the controller system according to the invention. 20D shows a detail of a rear view of a controller system according to the present invention.
21 is a flow chart showing an operation method according to the present invention.
Figure 22 shows a TriGrid electrode array (cross-section) for intramuscular (IM) delivery, which consists of four electrodes arranged in two equilateral triangles to form a diamond shape surrounding a central injection needle.
23A-23B show a TDS-IM v1.0 TriGrid device suitable for use in a mouse model (FIG. 23A) and a non-human primate (NHP) model (FIG. 23B ).
24 shows a TDS-IM v2.0 TriGrid device applied for use in a non-human primate (NHP) model.
25A-25H show the design and optimization of the expression cassette and DNA plasmid encoding the HBV pol antigen and core antigen described in Example 1. 25A is a schematic diagram of an expression strategy in which the coding sequence of the HBV core and pol antigen is fused in frame. Figure 25B is a schematic diagram of an expression strategy in which the coding sequences of both core and pole antigens are expressed from a single plasmid by the ribosome FA2 slippage site. 25C is a schematic diagram of an expression strategy in which the core and pol antigens are expressed from two separate plasmids. Figure 25D is a Western blot of core antigen expression in HEK293T cells transfected with a plasmid expression core with or without a post-transcriptional regulatory element (WPRE), cell lysates (lysate, left) using α-core antibody. And expression in the supernatant (supernatant, sup; right). Figure 25e is the untranslated of human apolipoprotein A1 precursor ("AI intron"), human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) ("HTLV R"). R-U5 domain, or HTLV-1 LTR triple enhancer composite sequence (triple enhancer composite sequence), synthetic rabbit β-globin intron and splicing enhancer (splicing enhancer, "triple") derived from intron / exon sequence Western blot analysis showing a comparison of core expression in HEK293T cells transfected with the core expression plasmid; The unmarked lane is the core protein purified as a size marker; Expression was tested in both lysate (left) and supernatant (sup; right); Core antigen expression was highest in the triple enhancer complex sequence. 25F is a Western blot analysis of core antigen secretion using different signal peptides fused to the N-terminus of the HBV core antigen, and the most efficient protein secretion with cystatin S signal peptide was observed. 25G is a schematic diagram of an optimized HBV core/pol antigen expression cassette for each of the three expression strategies shown in FIGS. 25A-25C; CMVpr: human CMV-IE promoter; TRE: triple enhancer sequence; SP: cystatin S signal peptide; FA2: FMDV ribosomal slip region; pA: represents the BGH polyadenylation signal. Figure 25h is a Western blot analysis of HBV core and pol antigen expression of pDK vectors containing each of the expression cassettes shown in Figure 25G, lanes 1 and 2: pDK-core; Lanes 3 and 4: pDK-pol; Lanes 5 and 6: pDK-coreFA2Pol; Lanes 7 and 8: pDK-core-pol fusion: The most consistent expression profiles for cellular and secreted core and pole antigens were observed when antigens were encoded by separate vectors.
26A-26B show schematic diagrams of DNA plasmids according to examples of the present application; 26A shows a DNA plasmid encoding an HBV polymerase (pol) antigen according to an embodiment of the present invention; 26B shows a DNA plasmid encoding an HBV core antigen according to an embodiment of the present application; HBV core and pol antigens are expressed under the control of the CMV promoter with an N-terminal cystatin S signal peptide that is cleaved from the expressed antigen when secreted from cells; The transcriptional regulatory element of the plasmid has 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 cassette containing a kanamycin resistance gene under the control of the Ampr (bla) promoter is contained in the plasmid in reverse orientation; The origin of replication (pUC) is also included in the reverse direction.
The same reference numerals refer to the same components throughout the specification. The size of the component is not limited unless otherwise specified.

Features of the invention are specifically set forth in the appended claims. The features and advantages of the present invention will be understood in more detail with reference to the following detailed description describing exemplary embodiments in which the invention of the present invention is used and the accompanying drawings.

The following description and examples describe the embodiments of the present invention in detail. It is to be understood that the invention is not limited to the specific embodiments described herein, but may vary. Those of skill in the art will recognize that there are numerous variations and modifications of the invention that fall within the scope of the invention.

All terms are intended to be understandable to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

While various features of the invention may be described in connection with a single embodiment, the features may also be provided individually or in any suitable combination. Conversely, the present invention may be described herein in connection with a separate embodiment for clarity, but the invention can also be implemented in a single embodiment.

To assist the reader of this application, the description has been divided into various paragraphs or sections, or is set forth in relation to various embodiments of the present application. Such separation should not be regarded as separating the object of a paragraph or section or embodiment from the object of another paragraph or section or embodiment. Conversely, one of ordinary skill in the art will understand that this description applies broadly and includes all combinations of various sections, paragraphs and sentences that may be considered. The discussion of any embodiment is intended to be illustrative only and is not intended to limit the scope of the invention, including the claims, to these examples. For example, embodiments of HBV vectors (e.g., plasmid DNA or viral vectors) that can be used in the inventions described herein include specific promoter sequences, potentiators or regulatory sequences, signal peptides, HBV antigens arranged in a specific order. Special elements including (but not limited to) coding sequences, polyadenylation signal sequences, and the like, but those skilled in the art will apply the concepts disclosed herein to other elements arranged in a different order that can be usefully used in HBV vectors. You will understand that it can be applied. This application contemplates that any applicable component can be used in any combination with any sequence that can be used in HBV vectors useful in the present invention, whether or not the specific combination is explicitly described. .

The following definitions supplement the technology of the industry and relate to current applications, and should not apply until related or unrelated cases such as other patents or applications owned by the applicant. While any methods and materials similar or equivalent to those described herein can be used in practice for the testing of the present invention, preferred materials and methods are described herein. Therefore, the terms used in the present specification are intended to describe only specific embodiments and are not intended to be limited thereto.

In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that, as used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. In addition, the use of the terms "comprising" such as "comprising", "including" and "included" is not limited.

In this specification, reference to “some embodiments”, “one embodiment”, “an embodiment” or “another embodiment” indicates that a specific feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments. Meaning, but not all embodiments of the present invention are necessarily essential.

As used in this specification and claims, the words “comprising” (and any form such as “comprising”, “comprising”), “having” (and any form such as “having”) and "Containing" (and any form such as "having" and "having") or "accepting" (and any form such as "accepting") is inclusive or non-limiting-additional or additional not yet cited It does not exclude elements or method steps. Any of the embodiments discussed herein may be implemented in connection with any method or configuration of the present invention, and vice versa. In addition, the composition of the present invention can be used to achieve the method of the present invention.

The term "about" with respect to a reference value and its grammatical equivalents as used in the present invention may include the value itself and a range of plus or minus 10% from that value. For example, an amount of “about 10” includes 10 and any amount of 9 to 11. For example, the term "about" in reference to a reference value also means plus or minus 10%, 9% 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% in that value. Is in the range of.

The present invention provides an improved system for regenerable, consistent and effective delivery of HBV vaccines, such as in combination with a nucleic acid encoding an HBV antigen and an electrically modulated therapeutic agent (e.g., HBV vaccine) delivery (EMTAD), Kits, methods, and devices are provided.

In one embodiment, the present invention provides a device for delivering HBV vaccine to a predetermined site in a subject, the device comprising an external cartridge, an internal cartridge, a cartridge assembly comprising a reservoir containing the HBV vaccine, , A storage containment volume is configured to receive the storage within the external cartridge;

The device includes an applicator including a cartridge assembly receiving volume, a needle hub, and an insertion detector for detecting loading of the reservoir in the reservoir containment volume; and

At least one interlock to facilitate proper implementation of the HBV vaccine administration procedure;

At least one injection orifice through which the HBV vaccine is administered;

A plurality of through electrodes arranged in a predetermined spatial relationship with respect to the orifice;

An electric field generator for generating an electrical signal operably connected to the electrodes; And

And a controlled energy source sufficient to deliver a predetermined amount of HBV vaccine from the reservoir through the orifice to a predetermined site in the subject at a predetermined rate.

In another embodiment, the present invention provides a kit for delivering an HBV vaccine to a predetermined site in a subject, the kit comprising an HBV vaccine and a device for delivering the HBV vaccine, the device comprising: an external cartridge, A cartridge assembly comprising an inner cartridge, a reservoir containing the HBV vaccine, a reservoir containment volume configured to receive the reservoir within the outer cartridge;

The device includes an applicator including a cartridge assembly receiving volume, a needle hub, and an insertion detector for detecting loading of the reservoir in the reservoir containment volume; and

At least one interlock to facilitate proper implementation of the HBV vaccine administration procedure;

At least one injection orifice through which the HBV vaccine is administered;

A plurality of through electrodes arranged in a predetermined spatial relationship with respect to the orifice;

An electric field generator for generating an electrical signal operably connected to the electrodes; And

And a controlled energy source sufficient to deliver a predetermined amount of HBV vaccine from the reservoir through the orifice to a predetermined site in the subject at a predetermined rate.

The HBV vaccine included in the device or kit of the present application may include:

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

A second nucleic acid molecule comprising 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

Including; a pharmaceutically acceptable carrier,

The first nucleic acid molecule and the second nucleic acid molecule exist as the same nucleic acid molecule or as two different nucleic acid molecules.

In another embodiment of the invention, EMTAD is the initial, simultaneous or subsequent administration of an HBV vaccine to a biological tissue of interest, and of an electrical signal to a biological tissue to enhance migration and/or absorption of the HBV vaccine in said tissue. It can be referred to as application. The process of EMTAD consists of two components: 1) administration of a therapeutic agent (ie HBV vaccine) (TAA) and 2) application of electrical signals sufficient to induce the desired EMTAD effect (ESA). In the present invention, TAA can be achieved in a controllable manner, referred to, for example, as controlled therapeutic agent (eg, HBV vaccine) administration (CTAA). The term CTAA, as used herein, may refer to a method or device that provides spatial and/or temporal control over the administration of an HBV vaccine with respect to the induction of the EMTAD effect. Controllable dosing techniques are for conventional needle-syringes (e.g., automatic injection devices) and/or various needleless methodologies (e.g., jet injectors, transdermal/transdermal patches, oral, gel, cream or inhalation administration). You can use variations. The term ESA, as used herein, refers to the application of an electrical signal to promote or enhance the delivery of an active agent, e.g., an HBV vaccine, by improving the movement and/or absorption of the agent within the tissue, and inducing the EMTAD effect. I can. When used to promote or enhance the delivery of HBV vaccines, ESA processes such as electroporation, iontophoresis, electroosmosis, electrophoresis, electrical stimulation, electrophoresis and electroconvection all exhibit various forms of EMTAD, one or more of them. This can be included in the methods described herein.

Particular uses for 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 this application, EMTAD is initiated by injection of HBV vaccine using a conventional needle-syringe. After the formulation is administered, a device suitable for ESA is applied to the subject at the designated location. Finally, an appropriate ESA protocol is used to provide the desired promotion or enhancement in HBV vaccine delivery. However, in traditional EMTAD, the desired spatial and temporal relationship between drug administration and ESA may not be realized.

Spatial parameter

In some implementations of the systems, kits, methods and devices disclosed herein, HBV vaccine administration is performed using a conventional needle syringe. The need to deliver a specific agent with EMTAD creates additional complexity in the TAA problem. As shown in Figure 1, in any conventional needle-syringe injection, when the needle 5 is inserted into the tissue, the depth 1 and angle 2 of the insertion relative to the surface of the tissue 3 are It can be difficult to control. In addition, the point of the needle penetration 4 on the tissue surface 3 may not indicate the location of the orifice 6 in the target tissue and the area 7 of administration of the agent. As shown in the example shown, a transcutaneous intramuscular injection may not correspond to the place of insertion on the skin, as the two tissues can often move relative to each other.

While this conventional approach is generally suitable for the delivery of many different therapeutic agents that do not require EMTAD, this variability often leads to dispensing of HBV vaccines following inconsistent and/or uncertain infusions and can interfere with effective EMTAD. In certain embodiments of the present specification, the most effective use of EMTAD is to utilize a predefined relationship between HBV vaccine and ESA in a subject. As a result, in the absence of spatial control across the TAA within the target tissue, using a conventional needle syringe can reduce the effectiveness of EMTAD application compared to devices, methods or systems that provide spatial and temporal control. have. One depicted example of this concept shows the use of electroporation to facilitate delivery of HBV vaccines. When TAA and ESA are co-localized within the target area of the tissue, electroporation is typically most effective to enhance HBV vaccine delivery. In many cases, if the delivery agent and the induced electroporation effect are not co-localized within the target area of the tissue, the delivery of the agent is not optimal.

Another example of the need for adequate spatial control of TAA within EMTAD is iontophoresis. This mode of EMTAD uses an electric field to cause the movement of charged molecules. In order to achieve the desired movement of the agent, an appropriate spatial relationship between the electrode and the HBV vaccine must be realized. When the negatively charged agent is located close to the position of the positive electrode, little or no movement of the agent through the tissue is observed. In contrast, localization of the negatively charged agent near the negative electrode results in significant movement of the agent through the tissue in the direction of the positive electrode.

As explained by the previous example, it is important to control the precise positioning of the TAA relative to the application of the ESA in order to achieve the desired effect. As such, implementations of the devices and methods described herein provide precise positioning control of the TAA to the application of ESA, and are useful for achieving reproducibility, consistency and well-specified distribution of one or more HBV vaccines.

Temporal parameters

In the case of conventional needle-syringe injection, TAA results in an inconsistent distribution of the agent within the tissue, as the injection rate may vary from operator to operator. If a batch of multiple devices is required to complete the EMTAD process, additional time fluctuations occur. For example, one application of EMTAD requires the generation of an electroporation-induced electric field following administration of the plasmid DNA encoded for the therapeutic protein. Using the traditional method of EMTAD, the HBV vaccine is injected with a needle syringe followed by placement and activation of the electroporation device. As two separate device arrangements are required (the first needle syringe followed by the ESA device), this process is sensitive to subject-to-subject variations resulting from inconsistent temporal application of each device by the operator. In addition, the use of two separate device configurations creates an unavoidable time interval between the placement and activation of each device by the clinician. This is configured when multiple application sites are needed to achieve adequate delivery of the agent to a specific area within the target tissue.

This problem is particularly important for agents such as nucleic acids that can be degraded or deactivated in the extracellular environment. Degradation of nucleic acids in HBV vaccines results in decreased efficacy and consistency in the application of therapy. In addition, the rate of intersubject degradation of nucleic acids in HBV vaccines is not constant, contributing to the overall therapeutic inconsistency of conventional needle syringe injection, more specifically electroporation treatment, combined with ESA.

Due to the inherent difficulties of spatial and temporal variability due to conventional needle syringe injection used with ESA, the exact location and time of the TAA for ESA is often unknown. As a result, effective administration and dosing of HBV vaccine using EMTAD is inconsistent and cannot be reproduced. Although conventional needle syringe injection is sometimes suitable for HBV vaccine administration, the reproducible and consistent delivery of HBV vaccine is significantly improved by controlling the spatial and temporal relationship between administration of the HBV vaccine and induction of the desired EMTAD effect.

So, while traditional EMTAD processes may be suitable for certain applications, temporal and spatial control is highly desirable for clinical applications that typically require a high degree of consistency and reproducibility. In contrast to conventional EMTAD approaches, implementations of the methods, systems and devices disclosed herein facilitate CTAA and ESA to provide more beneficial methods and devices for clinical application of EMTAD. The present invention provides a reproducible, consistent, and effective HBV vaccine delivery using various aspects of CTAA in conjunction with ESA. The present invention discloses a method and apparatus for enhancing the migration and/or absorption of the vaccine in a target tissue by providing spatial and temporal control over the administration of an HBV vaccine with respect to the application of electrical signals.

In some embodiments in which the methods and devices of the present invention are provided, there is a controllable spatial relationship for the administration of HBV vaccines regarding the application of electrical signals. Prior to treatment, the optimal position of TAA relative to ESA is determined. This spatial relationship between TAA and ESA is dictated by treatment parameters, including the properties of the target tissue to which the agent is administered and the nature of the agent to be administered. In an exemplary embodiment, the electrical signal is preferentially applied distal at the site of HBV vaccine administration. In certain other embodiments, the spatial relationship is the application of an EMTAD-induced electrical signal proximal at the site of administration of the agent. In certain cases, co-localization between TAA and ESA is desirable. This is often the case where electroporation and/or iontophoresis are utilized to induce the desired EMTAD effect.

In yet another aspect of the invention, the apparatus disclosed herein provides a controllable temporal relationship for the sequence and timing of the TAA to the ESA. Prior to treatment, the optimal sequence and timing for binding of TAA and ESA are determined. As in the spatial relationship, the preferred temporal relationship between TAA and ESA is dictated by variables such as the properties of the target tissue to which the agent is administered and the nature of the agent to be administered. In certain applications, exposure to ESA-related electric fields can adversely affect HBV vaccines. In the practice of this application, the generation of this electric field follows CTAA. However, the typical temporal relationship is CTAA following ESA.

The present invention provides an improved method and apparatus for reproducible, consistent and effective delivery of HBV vaccines comprising nucleic acids based on constructs with EMTAD. This objective is achieved by controlling the spatial and temporal administration of the HBV vaccine to the application of electrical signals. In certain embodiments, EMTAD is initiated by HBV vaccine injection using a conventional needle syringe. After the formulation is administered, a device suitable for ESA is applied to the subject at the designated location. Appropriate ESA protocols are used to provide the desired ease or enhancement for HBV vaccine delivery. An exemplary ESA method that has proven to be effective for virtually all cell types is electroporation. Other exemplary methods of electrically mediated delivery include iontophoresis, electroosmosis, electropermeabilization, electrostimulation, electromigration, and electroconvection. ), but is not limited thereto. These terms are used for illustrative purposes only and are not to be construed as limitations in the present invention.

The technique of electroporation uses the application of an electric field, inducing an instantaneous increase in cell membrane permeability, and moving charged particles. By penetrating the cell membrane inside the target tissue, electroporation dramatically improves the intracellular uptake of exogenous substances administered to the target tissue. The increase in molecular migration and cell membrane permeation due to electroporation provides a method to overcome the cell membrane as an obstacle to HBV vaccine delivery. The application of electroporation as a technique to induce EMTAD is advantageous in that the physical properties of the technique allow electroporation to be applied to virtually all tissue types. Accordingly, various aspects and implementations of the present invention discuss electroporation as, but not limited to, techniques for inducing EMTAD.

HBV vaccines

The term "HBV vaccine" is used herein in its broadest sense to encompass any agent capable of providing a desirable or beneficial immune response, therapeutic and/or prophylactic effect against HBV vaccine on living tissue. do. Thus, the term encompasses both prophylactic and therapeutic HBV vaccines, as well as any other category of agents with this desirable effect. The scope of the present invention is broad enough to cover the controlled delivery of any HBV vaccine, but is classified. HBV vaccines include pharmaceuticals, vaccines, nucleic acid sequences (e.g., superhelical, loose and linear plasmid DNA, RNA, antisense constructs, artificial chromosomes or any other therapeutic-based nucleic acid) and any formulations thereof. However, it is not limited thereto. These formulations include cationic lipids, cationic and/or nonionic polymers, liposomes, saline, nuclease inhibitors, anesthetics, poloxamers. , Preservatives, sodium phosphate solutions, or other compounds capable of improving the administration, stability and/or effectiveness of the HBV vaccine, but are not limited thereto. Additional formulations include agents and additives that impart the ability to control the electrical alternating resistance and viscosity of the administered formulation.

In a preferred embodiment of the present application, the HBV vaccine used in the present invention is:

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

A second comprising a truncated HBV core antigen, preferably 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 A nucleic acid molecule, preferably a second plasmid DNA vector; And

It contains a pharmaceutically acceptable carrier,

Here, the first nucleic acid molecule and the second nucleic acid molecule are 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” refers to an immune response to an HBV core protein in a subject, eg, a humoral mediated response and/or It refers to an HBV antigen capable of inducing a cellular mediated response. Each of the terms “core,” “core polypeptide” and “core protein” refers to an HBV viral core protein. The full-length core antigen is generally 183 amino acids in length, and an assembly region (amino acids 1 to 149) and It includes a nucleic acid binding region (amino acids 150 to 183.) The 34 residue nucleic acid binding region requires pre-genomic RNA encapsidation, which also functions as a nuclear entry signal. It contains arginine residues, is highly basic, and is consistent with this function HBV core protein is a dimer in a solution in which dimers self-assemble into icosahedral capsids. Each dimer of the core protein has four α-helix bundles flanked by α-helix domains on both sides, and also a nucleic acid binding region. This lacking truncated HBV core protein is also capable of forming a capsid.

In one aspect of the present application, the HBV antigen to be used in the present invention is a truncated HBV core antigen. As used herein, “truncated HBV core antigen” refers to an HBV antigen that does not contain the full length of the HBV core protein, but is capable of inducing an immune response against the HBV core protein in a subject. For example, the HBV core antigen can be modified to delete one or more amino acids of the highly positively charged (arginine rich) C-terminal nucleic acid binding region of the core antigen, which typically contains 17 arginine (R) residues. . The truncated HBV core antigen of the present application is preferably a C-terminal truncated HBV core protein that does not contain an HBV core nuclear entry signal and/or a truncated HBV core protein in which the C-terminal HBV core nuclear entry signal is deleted. to be. In one embodiment, the truncated HBV core antigen is a deletion of the C-terminal nucleic acid binding region, such as deletion of 1 to 34 amino acid residues of the C-terminal nucleic acid binding region, e.g., 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 or 34 amino acid residues, preferably all 34 amino acid residues. In a preferred embodiment, the truncated HBV core antigen comprises a deletion of the C-terminal nucleic acid binding region, preferably of all 34 amino acid residues.

The HBV core antigen useful in the present application may be a consensus sequence derived from a plurality of HBV genotypes (eg, A, B, C, D, E, F, G and H genotypes). As used herein, "common sequence" is based on the alignment of the amino acid sequence of the homologous protein, for example, the alignment of the amino acid sequence of the homologous protein (eg, Cluster Omega). Using) means an artificial sequence of amino acids as measured by. It is based on the sequence of HBV antigens (e.g., core, pol, etc.) from at least 100 native HBV isolates, the calculation of the most frequent amino acid residues found at each position in the sequence alignment. Can be in the order of Consensus sequences can occur unnaturally and can differ from native viral sequences. Consensus sequences can be designed to align multiple HBV antigen sequences from different sources using multiple sequence alignment tools, and select the most frequent amino acids at various alignment positions. Preferably, the consensus sequence of the HBV antigen is derived from HBV genotypes B, C and D. The term “common antigen” is used to refer to an antigen having a consensus sequence.

Exemplary truncated HBV core antigens useful in the present application lack nucleic acid binding function and may be capable of inducing immune responses against at least two HBV genotypes in mammals. Preferably, the truncated HBV core antigen is capable of inducing a T-cell response to at least HBV genotypes B, C and D in mammals. More preferably, the truncated HBV core antigen is capable of inducing a CD8 T cell response to at least HBV genotypes A, B, C and D in a human subject.

Preferably, the HBV core antigen useful in the present application is a common antigen, preferably a common antigen derived from HBV genotypes B, C and D, more preferably a truncated common antigen derived from HBV genotypes B, C and D. Exemplary truncated HBV core consensus antigens according to the present application are at least 90% identical to SEQ ID NO: 4 or SEQ ID NO: 14, e.g., at least 90%, 91% and SEQ ID NO: 2 or SEQ ID NO: 14, 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%, 100% identical amino acid sequences. 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 the 34-amino acid C-terminal deletion of the highly positively charged (arginine rich) nucleic acid binding region of the natural core antigen.

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

An example of a polynucleotide sequence encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 is at least 90% identical to SEQ ID NO: 1, or, for example, at least 90%, at least 91 as SEQ ID NO: 1 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, 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%, 100% identical, preferably approximately 98 with SEQ ID NO: 1 %, approximately 99% or 100% identical polynucleotide sequences.

An example of a polynucleotide sequence encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 14 is at least 90% identical to SEQ ID NO: 15, or, for example, at least 90%, at least 91 as SEQ ID NO: 15 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, 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%, 100% identical, preferably approximately 98 with SEQ ID NO: 15 %, approximately 99% or 100% identical polynucleotide sequences.

In a specific embodiment of the present application, the HBV vaccine used in the present invention contains a non-naturally occurring nucleic acid molecule encoding a truncated HBV core antigen, and the non-naturally occurring nucleic acid molecule is SEQ ID NO: 1 and SEQ ID NO: 15 It includes the polynucleotide sequence of.

As used herein, the term "HBV polymerase antigen," "HBV Pol antigen" or "HBV pol antigen" refers to an immune response, e.g., It refers to an HBV antigen capable of inducing a humoral and/or cellular mediated response to HBV polymerase in a subject. The terms “polymerase,” “polymerase polypeptide,” “Pol” and “pol” refer to HBV viral DNA polymerase. HBV viral DNA polymerase comprises, from the N end point to the C end point, a terminal protein (TP) region that functions as a primer for the synthesis of minus strand DNA; Non-essential spacers for polymerase function; Reverse transcriptase (RT) region for transcription; And a ribonucleic acid gas decomposition enzyme H (RNase H) region.

In the embodiment of the present application, the HBV antigen includes HBV Pol antigen or any immunogenic fragment or a combination thereof. HBV Pol antigen, such as by introducing a mutation into the region of activation of the polymerase and/or ribonuclease H (RNase H) to substantially eliminate or reduce the activation of certain enzymes. Additional modifications may be included to improve the immunogenicity of the antigen.

Preferably, the HBV Pol antigen useful in the present application does not include reverse transcriptase activity and ribonuclease H (RNase H) activation, and does not contain at least two HBV genotypes in mammals. It is possible to induce an immune response against it. Preferably, HBV Pol antigen is capable of inducing T cell responses to at least HBV genotypes B, C and D in mammals. More preferably, the HBV Pol antigen is capable of inducing a CD8 T cell response to at least HBV genotypes A, B, C and D in a human subject.

Thus, in some embodiments, the HBV Pol antigen useful in the present application is an inactivated Pol antigen. In one embodiment, the inactivated HBV Pol antigen comprises one or more amino acid mutations at the site of activation of the polymerase region. In another embodiment, the inactivated HBV Pol antigen comprises one or more amino acid mutations at the site of activation of the ribonuclease H (RNase H) region. In a preferred embodiment, the inactivated HBV Pol antigen comprises one or more amino acid mutations at the site of activation of both the polymerase region and the ribonuclease H (RNase H) region. For example, the "YXDD" design (motif) in the polymerase region of HBV pol antigen, which may require nucleotide/metal ion binding, is, for example, one or more aspartate residues (D) asparagine. Replacing with a residue (N), by removing or reducing the metal coordination function, can be altered by reducing or substantially eliminating the reverse transcriptase function. "YXDD" design (motif) ) In the ribonuclease H (RNase H) region of HBV pol antigen, which requires Mg2+ coordination, alternatively or in addition to the mutation of), the "DEDD" design (motif) is, for example, one Replacing the above aspartate residue (D) with an asparagine residue (N) and/or replacing the glutamate residue (E) with glutamine (Q) results in decreased ribonuclease H (RNase H) function Or it can be mutated by being substantially removed. In certain embodiments, the HBV pol antigen is (1) a mutation of an aspartate residue (D) to an asparagine residue (N) in the "YXDD" design (motif) of the polymerase region; And (2) a first aspartate residue (D) as an asparagine residue (N) and a first glutamate residue (E) as a glutamine residue in the "DEDD" design (motif) of the ribonuclease H (RNase H) region. N) is modified by reducing or substantially eliminating both the reverse transcriptase function and RNase H function of the pol antigen. .

In a preferred embodiment of the present application, the HBV pol antigen is a common antigen, preferably a common antigen derived from HBV genotypes B, C and D, more preferably a common antigen derived from HBV genotypes B, C and D. It is an inactivated common antigen. An exemplary HBV pol consensus antigen according to the present application is at least 90% identical to SEQ ID NO: 4, e.g., at least 90%, 91%, 92%, 93%, and SEQ ID NO: 4 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%, 100% identical, preferably at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7 with SEQ ID NO: 4 %, 99.8%, 99.9%, 100% identical, at least 98% identical to SEQ ID NO: 4, including an amino acid sequence. SEQ ID NO: 4 is a pol consensus antigen derived from HBV genotypes B, C, and D containing four mutations located within the activation site of the polymerase and ribonucleic acid gas lyase H (RNase H) region. . Specifically, the four mutations are a mutation of an aspartic acid residue (D) to an asparagine residue (N) in the "YXDD" design (motif) of the polymerase region; And in the "DEDD" design (motif) of the ribonucleolytic enzyme H (RNase H) region, the first aspartate residue (D) is changed to an asparagine residue (N) and the glutamate residue (E) is converted to a glutamine residue (N). Includes mutations.

In certain embodiments of the present application, the HBV pol antigen useful in the present application includes the amino acid sequence of SEQ ID NO: 4. In another embodiment of the present application, the HBV core antigen useful in the present application consists of the amino acid sequence of SEQ ID NO: 4.

An example of a polynucleotide sequence encoding HBV pol antigen consisting of the amino acid sequence of SEQ ID NO: 4 is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16, or, for example, SEQ ID NO: 3 or at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% with SEQ ID NO: 16, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, such as 100% identical, preferably SEQ ID NO: 3 or SEQ ID NO: 16 and 98%, 99 % Or 100% identical polynucleotide sequences, including but not limited to. In a specific embodiment of the present application, the HBV vaccine useful in the present application includes a non-naturally occurring nucleic acid molecule encoding HBV pol antigen, and the non-naturally occurring nucleic acid molecule is SEQ ID NO: 3 or It contains the polynucleotide sequence of SEQ ID NO: 16.

As used herein, the term "fusion protein" or "fusion" refers to a single polypeptide chain having at least two polypeptide regions not generally present in a single, natural polypeptide. (single polypeptide chain).

In one aspect of the present application, the HBV antigen used in the present invention is preferably directed to a truncated HBV core antigen or a truncated HBV core antigen operably linked to HBV Pol antigen via a linker. It may include a fusion protein comprising an operably linked HBV Pol antigen. As used herein, the term "linker" refers to a compound or moiety that functions as a molecular bridge that operably connects two different molecules, and a linker A part of is operably connected to the first molecule, and the other part of the linker is operably connected to the second molecule. As used herein, the term “operably linked” refers to linkage or juxtaposition, wherein the components described herein are related to relationships that allow them to function in their intended manner. have. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest can direct transcription of a nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is absent. It is possible to secrete or translocate the amino acid sequence of interest over (member).

For example, in a fusion protein containing a first polypeptide and a second heterologous polypeptide, the linker serves primarily as a spacer between the first and second polypeptides. In one embodiment, the linker is made of amino acids linked together by a peptide bond, preferably 1 to 20 amino acids linked by a peptide bond, and the amino acid is selected from 20 naturally occurring amino acids. In one embodiment, amino acids from 1 to 20 are selected from glycine, alanine, proline, asparagine, glutamine and lysine. Preferably, the linker is made of major amino acids that are not sterically disturbed, such as glycine and alanine. Exemplary linkers include polyglycines, in particular (Gly)5, (Gly)8; poly(Gly-Ala) and polyalanines. One illustrative suitable linker is (AlaGly)n, where n is an integer from 2 to 5.

Preferably, the fusion proteins useful in the present application are capable of inducing an immune response against at least two HBV genotypes of HBV Pol and HBV core in mammals. Preferably, the fusion protein is capable of inducing a T-cell response to at least HBV genotypes B, C and D in mammals. More preferably, the fusion protein is capable of inducing a CD8 T-cell response to at least HBV genotypes A, B, C and D in a human subject.

In one aspect of the present application, the fusion protein useful in the present application is at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96% with SEQ ID NO: 2 or SEQ ID NO: 14 , 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%, 100% the same The same, truncated HBV core antigen having an amino acid sequence that is at least 90%, linker and at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5% with SEQ ID NO: 4 , 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%, the same as 100%, at least It may contain HBV Pol antigen having an amino acid sequence of 90%.

Preferably, the fusion protein useful in the present application is a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14, n is an integer of 2 to 5, a linker comprising (AlaGly)n, SEQ ID NO: : Contains HBV Pol antigen having an amino acid sequence of 4. More preferably, the fusion protein useful in the present application comprises the amino acid sequence of SEQ ID NO: 20.

In some embodiments of the present application, fusion proteins useful in the present application further comprise a signal sequence. Preferably, the signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19. More preferably, the fusion protein comprises the amino acid sequence of SEQ ID NO: 21.

Exemplary polynucleotide sequences encoding fusion proteins useful in the present application are,

At least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% with SEQ ID NO: 1 or SEQ ID NO: 15, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical, preferably SEQ ID NO: 1 or SEQ ID NO: 15 and 98% At least 90% of SEQ ID NO: 1 or SEQ ID NO: 15, such as 99% or 100% identical, at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5 of SEQ ID NO: 22 %, 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 Operably linked to a linker coding sequence that is at least 90% of SEQ ID NO: 22, such as 100% identical, preferably 98%, 99% or 100% identical to SEQ ID NO: 22, SEQ ID NO: 3 or sequence Numbers: 16 and 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, preferably 98%, 99% or 100% with SEQ ID NO: 3 or SEQ ID NO: 16 Includes, but is not limited to, polynucleotide sequences that are further operably linked with a polynucleotide sequence that is at least 90% of the same as SEQ ID NO: 3 or SEQ ID NO: 16. In certain embodiments of the present application, the HBV vaccine useful in the present application comprises a non-naturally occurring nucleic acid molecule encoding a fusion protein, and the non-naturally occurring nucleic acid molecule is operably linked to SEQ ID NO: 22 and It includes SEQ ID NO: 1 which is further operably linked to Number: 3.

In one aspect of the present application, in the HBV vaccine useful in the present application, the first and second nucleic acid molecules each comprise a first and a second plasmid DNA vector, and each of the first and second plasmid DNA vectors, the origin of replication , Antibiotic resistance gene, 5'to 3'end, promoter sequence, enhancer sequence, signal peptide coding sequence, first polynucleotide sequence or second polynucleotide sequence and adenylic acid polymerization reaction It includes a polyadenylation signal sequence. In a preferred embodiment of the present application, the DNA plasmid is an expression vector suitable for protein expression in a mammalian host cell. Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNA™, pcDNA3™, 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 commonly used as a plasmid in DNA vaccines and is a polya derived from bovine growth hormone (bGH) following the strong human intermediate early cytomegalovirus (CMV-IE) promoter. Denylated sequence (pA) (bovine growth hormone (bGH)-derived polyadenylation sequence (pA)) is included. pVAX-1 further contains a kanamycin resistance gene driven by a pUC origin of replication and a small prokaryotic promoter that allows bacterial plasmid proliferation. Preferably, the plasmid DNA vector comprises a codon optimized kanamycin resistance gene having a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 12, preferably 100% identical to SEQ ID NO: 12.

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

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

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

c) contains a pharmaceutically acceptable carrier,

Each of the first plasmid DNA vector and the second plasmid DNA vector comprises a kanamycin resistance gene comprising the polynucleotide sequence of SEQ ID NO: 12 and an origin of replication comprising the polynucleotide sequence of SEQ ID NO: 10,

The first plasmid DNA vector and the second plasmid DNA vector exist as the same compound or two different compounds.

In this embodiment of the present application where the HBV vaccine comprises 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. For example, the first DNA plasmid and the second DNA plasmid have a weight ratio of 10:1, 9:1, 8:1,7:1,6:1,5:1,4:1,3:1,2 Weight ratio such as :1,1:1, 1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9 or 1:10 is from 10:1 to It can exist in a ratio of 1:10. Preferably, the first and second DNA plasmids are present in a ratio of 1:1 by weight.

The combination of the compounds of the present application and immunogenicity may include additional polynucleotides or vectors encoding additional HBV antigens and/or additional HBV antigens or immunogenic fragments thereof. However, in certain embodiments, the combination of a compound of the present application and immunogenicity does not include a specific antigen. In a preferred embodiment, the HBV vaccine used in the present invention is

It encodes an HBV antigen selected from the group consisting of Hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen, and HBV L protein antigen. HBV antigen selected from the group consisting of nucleic acid molecules or hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen, and HBV L protein antigen Does not include

In addition, the HBV vaccine useful in the present application includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are non-toxic and should not interfere with the efficacy of the active ingredient. Pharmaceutically acceptable carriers may include one or more of water, glycols, sugars, oils, amino acids, alcohols, preservatives, emollients, stabilizers, coloring agents, and the like.

Other examples of HBV vaccines useful in the present application are filed on the same date as this application with attorney management number 688097-403WO1, and in an international patent application entitled "Hepatitis B virus (HBV) vaccine and its use" as the name of the invention. Disclosed, the content of which is incorporated herein by reference in its entirety.

Kits/Systems

In a general aspect, the present invention relates to a kit or system for controlling the delivery of HBV vaccine to a predetermined tissue location in an individual in need thereof, and HBV vaccine and HBV to a predetermined tissue location through electroporation. And a device for administering a vaccine. For example, the kit or system may have a packaged container such as a syringe containing a pre-measured amount of HBV vaccine, which syringe is transferred to the device described herein for subsequent administration of the HBV vaccine ( can be loaded.

How to induce 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 need thereof, and generating immunity of an HBV vaccine using the device, kit or system of the present application. And administering to the subject an effective amount. Any device, kit, or system of the present application described herein may use the method of the present application. As used herein, “infection” refers to penetration into a host by a disease-causing agent. A disease-causing agent is considered "infected" if it is capable of penetrating into the host and replicating or propagating into the host. Examples of infectious agents include certain types such as adenovirus, prions, bacteria, fungi, protozoa, and the like, and viruses such as HBV. "HBV infection" specifically refers to the penetration of a host organism such as cells and tissues of the host organism by HBV.

As used herein, "inducing an immune response" when used in conjunction with the methods described herein means a desirable immune response in an individual in need thereof against an infection, such as, for example, HBV infection, or Includes what causes the effect. Also, “inducing an immune response” includes providing therapeutic immunity for treatment against pathogenic agents, such as, for example, HBV. As used herein, the term "therapeutic immunity" or "therapeutic immune response" refers to a pathogenic agent for which the vaccinated subject has been vaccinated, for example , Means being able to control infection by immunity against HBV infection conferred by vaccination with the HBV vaccine. In one embodiment, “inducing an immune response” means producing immunity in an individual in need thereof to provide a therapeutic effect on a disease, eg, HBV infection. In certain embodiments, "inducing an immune response" means causing or improving the immunity of a cell to HBV, eg, a T cell response. In certain embodiments, “inducing an immune response” means causing or ameliorating a humoral immune response against HBV. In certain embodiments, “inducing an immune response” means causing or ameliorating a cellular immune response and a humoral immune response against HBV.

As used herein, the term "protective immunity" or "protective immune response" means that the vaccinated subject is capable of controlling infection by the vaccinated pathogenic agent. Means that. Individuals with usually developed "defensive immune response" develop only mild to severe clinical symptoms or develop no symptoms at all. Usually an individual who has a “defense immunity” or “defense immune response” to a particular agent will not die from infection with that agent.

Typically, administration of the HBV vaccine according to the embodiments of the present application will have a therapeutic purpose to develop the symptomatic characteristics of HBV infection or HIV infection, for example, to generate an immune response to the therapeutic vaccine.

As used herein, the term "immunogenically effective amount" or "immunologically effective amount" refers to an amount sufficient to induce the desired immune effect or immune response in an individual in need thereof. It refers to a compound, polynucleotide, vector or antigen. In one embodiment, an effective amount of an immunogen refers to an amount sufficient to induce an immune response in an individual in need thereof. In another embodiment, an effective amount of an immunogen means an amount sufficient to produce immunity in an individual in need thereof, for example, an amount sufficient to provide a therapeutic effect against a condition such as HBV infection. An effective amount of an immunogen can be determined by various factors, for example, the physical condition of the individual, age, weight, health, etc; Providing a specific application, for example protective immunity or therapeutic immunity; And specific diseases, such as viral infections for which immunity is required. An effective amount of an immunogen can be readily determined by a person skilled in the art in view of the present disclosure.

In certain embodiments of the present application, an immunogen effective amount refers to an amount of a compound or immunological combination (e.g., HBV vaccine) sufficient to achieve one, two, three, four or more of the following effects: (i ) Reducing or improving the intensity of HBV infection or symptoms associated therewith; (ii) reducing the duration of HBV infection or symptoms associated therewith; (iii) preventing the progression of HBV infection or symptoms related thereto; (iv) cause regression of HBV infection or symptoms associated therewith; (v) preventing the occurrence or development of HBV infection or symptoms associated therewith; (vi) preventing recurrence of HBV infection or symptoms associated therewith; (vii) reducing hospitalization of individuals infected with HBV; (viii) reducing the length of hospital stay in HBV-infected individuals; (ix) increase the survival of individuals infected with HBV; (x) removing the HBV infection of the subject; (xi) inhibiting or reducing HBV replication within the subject; And/or (xii) ameliorating or enhancing the therapeutic or prophylactic effect of another therapy.

In another specific embodiment, the immunogen effective amount is an amount sufficient to reduce HBsAg levels consistent with evolution to clinical seroconversion; Achieve sustained HBsAg clearance associated with a decrease in infected hepatocytes by the subject's immune system; Induces an HBV-antigen specific activated T-cell population; And/or achieving a continuous reduction in HBsAg within 12 months. Examples of targeting indicators include low HBsAg and/or high CD8 numbers below the threshold of 500 copies of HBsAg International Units (IU).

As a general guideline, an effective amount of immunogen when used in connection with a DNA plasmid ranges from approximately 0.1 mg/mL to 10 mg/mL of the total DNA plasmid, e.g. as 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL. mL. 0.75 mg/mL 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, or 10 mg/mL. Preferably, the immunogen effective amount of the DNA plasmid is less than 8 mg/mL, more preferably less than 6 mg/mL, and even more preferably 3-4 mg/mL. The immunogen effective amount may be derived from one vector or plasmid or a plurality of vectors or plasmids. The immunogen effective amount may be a single compound or multiple compounds, e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 compounds (e.g., tablets, capsules, or injections) may be administered, and administration of a plurality of capsules or infusions provides an effective amount of an immunogenic agent in a lump to the individual. In addition, after administering an effective amount of an immunogen to an individual, it is possible to administer different doses of an effective amount of an immunogen to the individual by a so-called prime-boost regimen. This general concept of primeboost therapy is well known to those skilled in the vaccine field. Additional booster administration can optionally be added to the therapy, if necessary.

According to the implementation of the present application, an immunological combination comprising two DNA plasmids, for example, a first DNA plasmid encoding HBV core antigen and a second DNA plasmid encoding HBV pol antigen, It can be administered to an individual by mixing both plasmids and delivering the mixture to a single anatomical location. Alternatively, two separate immunizations can be performed delivering each of a single expression plasmid. In this embodiment, both plasmids, regardless of whether they are administered in a single immunization as a mixture or in two separate immunizations, the first DNA plasmid and the second DNA plasmid are in a ratio of 10:1 to 1:10 by weight, e.g. For example, by weight 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1 It can be administered in a :3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 ratio. Preferably, the first and second DNA plasmids are administered in a ratio of 1:1 by weight.

In some embodiments, an individual treated according to the methods of the present application is an HBV-infected individual, particularly an individual having a chronic HBV infection. Acute HBV infection is subsequently characterized by the efficient activity of the innate immune system supplemented with a wide range of adaptive responses (e.g., HBV-specific T-cells, neutralizing antibodies). Which usually results in successful inhibition or elimination of replication of infected hepatocytes. In contrast, this response is impaired or reduced, due to high viral load and antigen load, e.g. HBV envelope proteins are produced in an abundance, over 1000 times greater than infectious virus. It can be released as a viral particle.

Chronic HBV infection is described as a stage characterized by viral load, liver enzyme levels (necroinflammatory activity), HBeAg or HBsAg load, or the presence of antibodies to these antigens. The cccDNA levels remain relatively constant at approximately 10 to 50 copies per cell, although viral infection can vary considerably. The persistence of cccDNA species leads to chronicity. More specifically, the stages of chronic HBV infection include: (i) a stage of immune resistance characterized by high viral load and moderate or minimally elevated liver enzymes; (ii) an immune-activated HBeAg-positive step in which a low or decreased level of viral replication with significantly elevated liver enzymes is observed; (iii) transporting inactivated HBsAg, which is a low replication state with moderate hepatic enzyme levels and low viral load in the serum capable of following HBeAg seroconversion; (iv) HBeAg-, where mutations in the pre-core promoter and/or the underlying core promoter are common, so that the fluctuations that virus replication accompanies within the liver enzyme levels occur periodically (reactivate) and HbeAg is not produced by infected cells. Voice step; includes.

As used herein, “chronic HBV infection” refers to an individual who has a detectable presence of HBV for a period of more than 6 months. Individuals with chronic HBV infection can be within any stage of chronic HBV infection. Chronic HBV infection is understood according to the general meaning of the field. Chronic HBV infection can be exemplified as being characterized by the persistence of HBsAg for a period of 6 months or longer after an acute HBV infection. For example, in chronic HBV infection referred to herein, chronic HBV infection is, for example, (i) negative for hepatitis B core antigen for IgM antibody (IgM anti-HBc), and for hepatitis B surface antigen ( HBsAg), hepatitis B e antigen (HBeAg), or positive for nucleic acid test for hepatitis B virus DNA, or (ii) positive for nucleic acid test for HBsAg or HBV DNA, or positive for HBeAg twice at least 6 months apart. It follows the definition published by the Centers for Disease Control and Prevention (CDC) according to what can be specified by laboratory standards such as those.

According to certain embodiments, an immunogenic effective amount refers to an amount of a compound or immunological combination sufficient to treat a chronic HBV infection.

According to some embodiments, an individual having a chronic HBV infection is receiving nucleoside analog (NUC) treatment and NUC is inhibited. As used herein, “NUC inhibition” refers to an individual with undetectable viral levels of HBV and stable alanine aminotransferase (ALT) levels for at least 6 months. Examples of nucleoside/nucleotide analog therapy include HBV polymerase inhibitors such as entacavir and tenofovir. Preferably, individuals with chronic HBV infection do not have advanced hepatic fibrosis or hepatic cirrhosis. These individuals may generally have a METAVIR score of less than 3 for fibrosis and a fibroscan result of less than 9 kPa. The METAVIR score is a generally used test system to evaluate the degree of inflammation and fibrosis by pathological histologic evaluation in liver biopsy of patients with hepatitis B. The test system assigns two standard numbers: one reflects the degree of inflammation and one reflects the degree of fibrosis.

It is believed that the disappearance or reduction of chronic HBV allows early disease blocking of severe liver diseases including virus-induced hepatocellular carcinoma and cirrhosis. Thus, the method of the present application is also used as a therapy for treating HBV induced diseases. Examples of HBV induced diseases include, but are not limited to, sclerosis, cancer (e.g., hepatocellular carcinoma) and fibrosis, particularly advanced fibrosis characterized by a METAVIR score higher than 3 or 3 for fibrosis. In this embodiment, the immunogen effective amount is an amount sufficient to achieve a sustained loss of HBsAg and a significant reduction in clinical disease (eg, sclerosis, hepatocellular carcinoma, etc.) for within 12 months.

The method according to the embodiment of the present application is another immunogenic agent (e.g., another HBV antigen or another antigen) or another anti-HBV agent (e.g. , A nucleoside analog or other anti-HBV agent).

When administered into an animal or human organism, the ability to induce or stimulate an anti-HBV immune response can be assessed in vitro or in vivo using a variety of assays standard in the art. For a general description of techniques by which the invention and activation of the immune response can be evaluated, see the example of Colligan et al. (1992 and 1994, current protocol in immunology; National Institutes of Health, J Wiley & Sons. Inc.). The measurement of cellular immunity is anti-specific in a sensitive subject by measurement of the activation state of immune effector cells (eg, T-cell proliferation assay by classical [3H] thymidine uptake). Those derived from CD4+ and CD8+ T-cells (e.g., IL-10 or IFN gamma-production by ELISPOT) by assays for T lymphocytes (e.g., peptide-specific lysis in cytotoxicity assays, etc.) Quantification of activated cells) can be performed by measurement of cytokine profiles secreted by activated effector cells.

The ability to stimulate cellular responses and/or humoral responses can be measured by competition in antibody binding and/or binding (see exemplarily by Harlow, 1989, Antibodies, Cold Spring Havo Newspaper). For example, the titer of an antibody produced in response to administration of a compound that provides an immunogen can be measured by enzyme-linked immunosorbent assay (ELISA). In addition, the immune response can be measured by a neutralizing antibody assay, where the neutralization of a virus is defined as loss of infectivity through reaction/inhibition/neutralization of a specific antibody and virus. In addition, the immune response can be measured by antibody-dependent cell phagocytosis (ADCP) assay.

Target tissue

Target tissues suitable for EMTAD by use of the methods, devices and systems described herein include, for example, both healthy and diseased cells located within epidermal tissue, dermal tissue, subcutaneous tissue, cuticle tissue and muscle tissue. do. The technique can also be used for application to healthy or diseased organs that must be accessed through minimally invasive methods or other surgical methods.

Such target tissues include liver, lungs, heart, blood vessels, lymph fluid, brain, kidneys, pancreas, stomach, intestines, colon, bladder and regenerable organs. In some embodiments, the desired therapeutic effect is the present invention capable of delivering a certain amount of the agent to the cell types normally located within the target tissue, as well as other cell types that may be found abnormally within the tissue (e.g., chemotherapy treatment of tumors). It can be derived by use of the method or apparatus described in the specification.

As previously described and shown in Figure 1, traditional EMTAD suffers from a lack of precision and reproducibility in the spatial and temporal relationship between administration of HBV vaccines and electrical signals. In contrast to the traditional EMTAD approach, this specification describes an apparatus and method for combining CTAA and ESA, providing more beneficial clinical applications of EMTAD. The present specification utilizes various aspects of CTAA in conjunction with ESA to provide delivery of reproducible, consistent, and effective HBV vaccines. The methods and devices provided herein improve the movement and/or absorption of the agent in the target tissue by providing spatial and temporal control over the administration of the HBV vaccine to the application of electrical signals.

Way

In one aspect, the disclosure described herein provides systems, kits, and devices for use in a method for controlling subsequent administration of an HBV vaccine by ESA to an individual in need thereof. As used herein, “individual” means any animal, preferably a mammal, most preferably a human, to be treated or to be treated by a method according to an embodiment of the present application. The term “mammal” as used herein encompasses any mammal. Examples of mammals are non-human primates such as cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys or apes ( NHPs), humans, and the like, more preferably humans, but are not limited thereto.

In yet another aspect, the disclosure described herein provides systems, kits, and devices for use in a method for controlling preferential administration of HBV vaccines by ESA. In a further aspect, the disclosure described herein provides systems and devices for use in a method for controlling the administration of an HBV vaccine with an ESA. Such methods include, but are not limited to, sequential relationships or ranges for measurement of treatment parameters, subject preparation, CTAA, ESA, and further measurements.

Measurement of treatment parameters

In some embodiments, the treatment parameters are based on the duration of administration and/or the desired dosage of the HBV vaccine. HBV vaccine administration may depend, for example, on the particular indication or therapeutic application (eg, the shape and location of the target tissue) as well as various individual parameters (eg, age or body mass). Administration of the HBV vaccine can be controlled by parameters related to the administration of the HBV vaccine and ESA. Exemplary controllable parameters related to CTAA include, but are not limited to, formulation volume, formulation viscosity, and injection rate. Exemplary controllable parameters related to the ESA include, but are not limited to, the nature of the electrical signal, the volume of tissue exposed to the electrical signal, and the electrode array type. The relative timing and location of CTAA and ESA are parameters that provide additional control over HBV vaccine administration.

Object preparation

In one implementation described herein, the methods described herein may include preparing an individual. Subject preparation, local or regional, nerve block (nerve block), spinal fluid flow obstruction (spinal block), epidural block (epidural block) or general anesthesia, including anesthesia Includes, but is not limited to, anesthetic administration and antiseptic cleansing. In the exemplary case of intramuscular (IM) ESA, protocols that minimize the effects of electrical stimulation of muscles are, for example, alternating stimulation patterns sufficient to alleviate discomfort, administration of anesthetics, thermal control (e.g. For example, it may be included in the methods described herein, including cooling the muscles). It should be understood that where an acceptable alternative exists, the subject preparation technique chosen does not adversely affect the effectiveness of the treatment. For example, in some cases, intramuscular administration of amide-based anesthetics may have undesirable effects on intramuscular delivery plasmid DNA-based therapy, due to the presumed mild myotoxicity of these agents. , It can inhibit the ability of muscle cells to express the protein encoded by the administered DNA sequence.

CTAA and ESA

In some embodiments described herein, there is a method in which CTAA and ESA are combined to allow consistent and reproducible HBV vaccine delivery. In some cases, a device comprising at least one of an automatic injection device and a jet injector is illustratively included to provide a device or kit suitable for CTAA.

The present disclosure provides methods, kits and devices that allow for transdermal placement of a plurality of elongated electrodes to a target depth in a safe and consistent manner with respect to the target location of agent secretion within receptors with heterogeneous skin thickness and composition, In order to enhance the subcutaneous, intradermal, intramuscular administration of therapeutic or prophylactic agents such as pharmaceuticals, antibodies, peptides, proteins or combinations thereof, the application of an electric field in the tissue is supported.

The system and method according to the principles of the present invention allows consistent percutaneous placement of a plurality of elongated, tissue-penetrating electrodes to a predetermined target tissue location, thereby propagating an electric field within the skin, subcutaneous tissue and/or skeletal muscle. The disclosure provided herein is that even when the process is applied to a location with varying skin characteristics, the user can bring the electrode to the target depth with minimal training, while maintaining the appropriate spatial relationship between the plurality of electrodes. Designed for consistent placement. This variability can be caused by changes in skin characteristics at different locations between heterologous receptor populations or within individual receptor populations. In other words, systems and methods in accordance with the principles of the present invention should allow for a consistent profile independent of the receptor or the administer. In certain embodiments, the placement of the electrode is accompanied by the insertion of one or more syringe needles, configured such that the HBV vaccine is administered to a target area of the tissue, and the electrode to be used for ESA is arranged in a predetermined spatial relationship. In an exemplary implementation, the electrodes are arranged such that any electrical signal from the electrode is preferentially applied away from the location of administration of the HBV vaccine by insertion of one or more syringe needles. In another implementation, the electrodes are arranged such that any electrical signal is preferentially applied to the proximal side of the location of administration of the HBV vaccine by insertion of one or more syringe needles.

One aspect of the present disclosure can be used singly or in combination to aid in the percutaneous insertion of electrodes for in vivo application of an electric field, such as intramuscular administration of nucleic acids, small molecule drugs, antigens, peptides, proteins, and combinations thereof, Intradermal and/or subcutaneous administration can be improved. In some implementations, electrode placement and subsequent electric field propagation may be performed in a manner coordinated with the distribution of the agent of interest to the target tissue location. In an exemplary embodiment, the administration of the agent and the application of one or more electric fields are performed in a controlled and monitored manner such that the probability of achieving a spatial and temporal cavity localization of the distribution of the agent at the location of the electric field application is maximized.

In general, the present disclosure provides electrodes at predetermined locations within the skin, subcutaneous tissue and/or skeletal muscle of the receptor with local application of an electric field and administration of the agent of interest to improve delivery, absorption and/or biological effects of the agent. Methods and apparatus for percutaneous placement are provided. In some implementations, the disclosure is implemented so that the setup and use of the device can be performed effectively and reliably by a user with minimal training. In yet another implementation, the disclosure also includes performing a number of engagements, sensors and feedback loops to reduce the frequency and/or potential impact of the promised potential user's error during setup and use of the device. Referring to Figure 2, the device implementation described herein includes a "cartridge assembly 100" that is detachably connected to an "applicator 400", the applicator 400 as well as diagnostic and other control routines. It is configured to be connected to the control unit 700 which functions as a source of electric energy for activating the electrode and generating an electric field subsequent thereto. The control unit 700 further includes a user interface for the applicator 400, a tray and a holster, and various other features are described. As shown in FIG. 2, the receiving portion 101 of an appropriately uniform size and general shape can be inserted into the cartridge assembly 100 when using the method.

Details of the cartridge assembly 100 present in some implementations of the devices described herein are described, for example, in FIGS. 3A-12, and in FIGS. 13A-18C, the corresponding cooperation of the applicator 400 It is described together with the parts, and subsequently described with respect to the parts of the control unit 700 in Figs. 19 to 20D. The remaining portions of the applicator 400 are subsequently described by the remaining portions of the control unit 700.

Referring to FIGS. 3A to 4, in some implementations described herein, the cartridge assembly 100 is configured to connect with the applicator 400 and is mounted on a structure forming an array body. It may include a support structure for accommodating the elongated electrode 122. In order to avoid undesired propagation of the electrical flow within the device, the design and material of the electrode mounting structure must be specified so that there is an appropriate dielectric barrier between electrodes of opposite polarity within the device. The distal region 137 of the elongated electrodes engages the mounting structure, using standardized mechanical properties and/or a bonding agent suitable for the electrode mounting structure and the material composition of the electrode.

In an exemplary implementation, the cartridge outer housing structure 102 is configured to connect to a fluid reservoir 101 containing the HBV vaccine, wherein the reservoir 101 and the cartridge housing structure 102 are the HBV vaccine within the target tissue. Is configured to be operably connected to at least one injection orifice (needle) 105 via administration. In some embodiments, such components facilitate co-localization of the distribution of the agent of interest to the location of the electric field application. do. In another implementation, this configuration facilitates the implementation of a predetermined spatial relationship between devices for ESA and CTAA. In another exemplary sphere, the syringe 101 is inserted into the cartridge 100, and when the cartridge is loaded into the applicator 400, the syringe 101 matches the needle hub 152, and the cartridge is a needle Move forward to connect to.

Certain implementations of the present disclosure may include the use of syringes, vials, ampoules, cartridges or equivalent structures to store one or more HBV vaccines. In some implementations, the reservoir may comprise at least one glass or plastic having a material selected to be compatible with the agent of interest. The coating can be applied to the reservoir used to provide the desired lubricating or protective properties. As described above, the electrodes 122 may be hollow and, in some cases, may be configured with injection orifices that may be operably connected to a fluid reservoir. Alternatively, the injection orifice may include a needleless injection port disposed against one or more hypodermic syringe needles and/or electrodes. The choice of the size and shape of the injection orifice may depend on the desired route of administration, tissue distribution and physical properties of the agent of interest. In certain implementations, the cartridge structure is designed to ensure a predetermined spatial relationship between the injection orifice and the electrode in its injection state, so that the distribution of the agent of interest occurs substantially within the tissue bounded by the guiding regions of the plurality of electrodes. . In order to minimize the need to handle sharpness by the user, in certain implementations, the cartridge 100 designed for use with a hypodermic needle is manufactured at manufacturing rather than matching the needle to the syringe in a more general rule when used. The hypodermic syringe needle is configured to match the cartridge. In certain embodiments, in certain embodiments, one aspect of the present disclosure provides features to ensure proper registration of the reservoir to the needle prior to use, as well as to ensure retention of the needle during manufacturing, dispensing, handling and use. Features may be included in the cartridge and/or needle for doing so. In some implementations, these features can minimize the risk of leakage of the agent from the reservoir or reservoir orifice interface due to breakage or improper mating.

In certain implementations, the cartridge may include a tissue contact interface located at the distal end of the subassembly. In certain implementations, the tissue contact interface comprises a substantially planar structure having one or more apertures oriented perpendicular to the elongation direction of the electrode and configured to allow the electrode to pass through the tissue contact interface. In the example of incorporating an integrated reservoir and an injection orifice, the interface also includes an injection orifice or an aperture for receiving a needleless injection. To minimize the risk of contamination of the electrode and injection needle, as well as to minimize the risk of unintended sharpness being exposed to the user or receptor, in certain implementations, the hole receiving the passage of the electrode and injection needle is provided with the electrode and injection. It is the right size to prevent accidental contact with the needle. Most commonly, the tissue contacting interface consists of one or more plastics suitable for contacting the tissue for at least a short period of time.

To avoid potential cross-contamination of biological material between the receptors, the cartridge assembly 100 may be configured for single use. In some cases, the cartridge includes one or more mechanical, electrical and/or identifying elements that limit the use of the cartridge for a single administration. One example of a mechanical element of this nature is, for example, lockouts and/or detents that secure electrode mounting structures and/or stick shields (see below) in the deployed state after use. Including, but is not limited to. Examples of electrical elements include fuses or links configured in series with one or more electrodes that are deactivated by an electrical energy source, for example at the end of initial use of the cartridge. Examples of identification elements include, for example, serialized radio frequency identification devices, barcodes or fast response codes that are configured to be read by an electrical energy source and/or applicator. The identification information for a particular cartridge can then be used by the electrical energy source and/or applicator to prevent the intended or accidental reuse of that cartridge. In some implementations, one or more redundant features are incorporated to minimize the likelihood of reusability of the cartridge.

In one implementation of autonomy described herein, as shown in FIG. 2, the applicator 400 includes a support structure configured to connect with the cartridge assembly 100, user interfaces 410 to 418; FIG. 13B, When the electrodes are disposed into the target tissue of the receptor, they include an electrically conductive electrical connection configured to provide an operable connection between the electrical energy source and the conductive contact area disposed on the distal area of the elongate electrode. In some cases, the user interface includes a handle, one or more display features designed to convey information to a user, and one or more features capable of accepting user input. In certain implementations, the display features are configured to communicate the operating status of the device during setup and during use, as well as related warning/mistake messages. Such displays may include mechanical features, lighting, letter and numeric displays, and/or electrical display screens. In some cases, a feature that can accept user input allows, at an appropriate process step, to disable stabilization features in the device to prevent accidental emissions, and allows the user to select certain parameters of the process (e.g., Injection depth), and is configured to initiate the dosing process, and may include buttons, braces, mechanical slides and/or levers.

In certain embodiments, the applicator 400 also connects with the cartridge assembly, is configured for percutaneous placement of the electrode, places an injection orifice with respect to the target tissue, and transfers the agent of interest from the reservoir through the orifice into the target tissue location. And/or an activation mechanism that transmits an electrical signal to the cartridge 100 from generating an electric field, such as the control unit 700. Applicator 400 may be configured such that energy to activate the mechanism is supplied by a user or, more preferably, the device comprises one or more non-living energy sources within the applicator operably connected with the activation mechanism. Such inanimate energy sources include, for example, electromechanical devices (solenoids, motors, lead screws), mechanical components (springs and associated devices) and compressed gases.

An exemplary implementation of the cartridge assembly 100 is shown in FIG. 3A, wherein the cartridge assembly 100 includes a reservoir loading port 140 and a reservoir containment volume 142 for containing and receiving a reservoir 101 of medicines. Include. Since the cartridge assembly 100 requires a device to generate an electromagnetic field and to be used as part of a treatment, an electric field generator such as the control unit 700 is required to be electrically connected to a device comprising an electrode configured to contact the target tissue. do. Since the control unit is configured for multiple uses and the reservoir 101 may be intended for disposable, the cartridge assembly 100 holds the electrode 122, the reservoir 101 connects with the reusable device, and is disposable. Thus, the applicator 400 may be a reusable component, and the cartridge assembly 100 may be configured for single use. The cartridge assembly 100 may also be made to be in a state that prevents subsequent use when an error or damage occurs in the insertion of the storage 101 or a defect exists.

The term reservoir 101 can be connected to a device having an orifice such as a needle 105, a needle shown in FIG. May refer to any other device that is present. The reservoir 101, given in the form of the cartridge assembly 100, generally has a normal shape and size. The various components within the cartridge assembly 100 allow margins for exact size and/or manufacturing tolerances, but generally reduce the risk of erroneous delivery of unlabeled drugs to the device for use with the cartridge 100. To do this, a normal shape and size are required. If an appropriately sized reservoir 101 is not provided by the user, one or more interlocks within the cartridge assembly 100 may not be disabled, and the system will be used until the appropriately sized reservoir 101 is inserted. Can be made not to be.

As shown in FIG. 3B, the reservoir 101 may generally be equipped with a plunger and a port 156 for discharging the drug. In addition, the removable cap 158 is configured to maintain the sterility and integrity of the formulation until the reservoir is inserted and used. The port for discharging the drug may be close to the needle hub 152 when in use, and the plunger may be opposite to the port for discharging the drug. In some implementations, such as alternately opening ports for discharging the drug, the reservoir may consist of a septum component that covers and seals the end of the container opposite the plunger. The diaphragm may be made of an elastic composition such as silicone or butyl rubber in a specific formulation and with a coating of a material selected for the stability and compatibility of the formulation contained within the reservoir. The diaphragm component is typically held in place by a crimp seal or other fastening mechanism. This diaphragm sealing configuration avoids the need for a removable cap, but requires a needle 105 to be equipped with suitable piercing members such as needles, spikes, or other features for accessing fluid contained within the reservoir. Particular implementations of this configuration include a double side needle configuration and a spike vial adapter.

The cartridge assembly 100 may not only be configured to receive the reservoir 101, but may be configured to be received by the applicator 400 within the applicator cartridge assembly receiving port 401 (see FIG. 2 ). Thus, the cartridge assembly 100 includes a device that allows the applicator 400 to be ejected, and keeps it at least partially within the inner volume. In certain implementations, the device is one or more racksons of the surface of the cartridge assembly 100 that engages the corresponding motorized pinion assembly within the applicator 400. In another implementation, the applicator 400 can connect with the cartridge assembly 100 without the need to discharge it into the inner volume. In yet another implementation, other techniques may be applied to cause the cartridge assembly 100 to engage with the applicator 400, for example, a motorized track or bracket to which the cartridge assembly 100 can connect.

The applicator 400 further includes an interface element for controlling a specific action within the cartridge assembly 100. In particular, the applicator 400 may be configured to control needle insertion, drug delivery, electrode insertion, and ]electrode activation using various sub-systems. In some cases, these steps are linked such that a single action of applicator 400 initiates such multiple steps. In some implementations, all of these steps except drug delivery and electrode activation are caused by a single action, as described in the exemplary practice below.

Upon appropriate activation, such as the use of an electrical or optical signal transmitted to a mechanical, electrical or optical element of the cartridge assembly 100, the applicator 400 can test the sub-systems within the cartridge assembly 100, manipulating it properly and However, it can be ensured that it is properly configured for drug delivery within the electric field application. For example, this sub-system ensures that the applicator 400 ensures that the cartridge assembly 100 has not been used previously, the reservoir is properly positioned within the cartridge assembly, and includes an alignment guide/splay shield; 108) to ensure the appropriate force applied against the body of the object as applied via, to ensure testing of a depth that is definitively selected by the user, and to ensure that the outer cartridge cap (11) has been removed. can do. In addition, the applicator 400 can be configured to monitor the status of cartridge functions during process execution. For example, this sub-system allows the applicator 400 to ensure that the electrodes are properly placed within the subject before starting the administration of the drug, that the plunger in the reservoir is properly activated prior to the application of the electric field, and that the user is placed on the body of the gacha during the administration process. It includes what can be tested to ensure that it maintains the proper force applied to it, etc.

In addition to the sub-system actuated by the applicator 400, the cartridge assembly 100 includes sub-systems that interact with the applicator 400, sub-systems that do not interact to achieve the goals of drug delivery and electric field applied therapy. It may include any suitable sub-system containing them. These include sub-systems to cause needle and electrode insertion, sub-systems to protect the user from sharpness following the dosage regimen, sub-systems to provide different depths of needle/electrode insertion, prior to allowing the initiation of the process, and It includes a sub-system to ensure that an appropriate force is applied to the tissue of the recipient, such as during application of the subsequent administration process. Although often described in terms of needles and electrodes that can be placed, this is not strictly required here, and systems with needles and electrodes that can be placed or fixed are also in accordance with the principles of the present invention, including the described subsystems. Note that you benefit from the system and method.

In one exemplary implementation as shown in FIG. 4, the cartridge assembly 100 includes an external cartridge 102, referred to in some cases as a housing. The outer cartridge 102 is terminated at the distal end by an outer cartridge cap 106. The outer cartridge 102 includes an inner cartridge receiving volume 150 for receiving the inner cartridge 103, which slides and is received in relation to the outer cartridge 102. The inner cartridge 103 includes a reservoir receiving volume 142 in which the reservoir 101 is placed. The inner cartridge 103 engages the inner cartridge cap 104 at its distal end. The inner cartridge cap 104 allows the electrode 122 to be fixed in place (the inner cartridge 103 itself has a seam on which the electrode 12 is placed) and provides a support surface for the rod shield 134. Includes the features of. The inner cartridge cap 104 is fixed on the inner cartridge 103.

The cartridge breech 112 is accommodated in and in a portion of the storage receiving volume 142 in the inner cartridge 103 at a portion opposite to the storage receiving volume of the inner cartridge cap 104. The reservoir detection cap 118 engages the cartridge chamber 112 through the reservoir detection spring 116. The cartridge retaining ring 114 secures the system in place containing the cartridge chamber 112 to the inner cartridge 103. The reservoir detection spring 116 also serves to push the reservoir 101 into engagement with the needle hub 152 and serves to accommodate a tolerance for the size of the reservoir 101.

The reservoir interlock 102 provides a mechanical interlock to prevent accidental or unwanted activation of cartridge functions. In particular, the reservoir interlock 102, referred to as the first reservoir insertion trigger, is disposed under the inner cartridge 103 and includes fingers extending through slots or holes defined within the inner cartridge (Fig. 5b). The finger prevents the sliding movement of the cartridge chamber 112 relative to the inner cartridge 103 before the reservoir is inserted into the reservoir receiving volume 142 and, in particular, the inner cartridge 103 towards the inner cartridge cap 104. ) To prevent movement within.

When the reservoir 101 is properly inserted into the reservoir receiving volume 142, the reservoir interlock 120 is pressed down and the fingers pressed down, so that it no longer extends into the reservoir receiving volume 142. This lower pressing or suppression of the reservoir interlock 120 may also be configured to provide an audible, tactile or haptic'click' that may inform the user of proper insertion. When restrained, then, the cartridge chamber 112, which is no longer blocked by the fingers of the reservoir interlock 120, allows movement, and in particular, allows movement in a direction towards the inner cartridge cap 104 .

The cartridge chamber 112 causes such movement by the action of the spring cap/cartridge interface 470 when the cartridge assembly 100 is inserted into the applicator 400 in the manner described below. When the cartridge chamber 112 is moved far enough forward, it is held in place, fastening the reservoir 101 within the reservoir receiving volume 142 and a complete fluid path from the reservoir 101 to the orifice of the needle 105 To ensure proper positioning relative to the needle hub 142 to ensure

In the implementation of the device in which the injection needle is integrated into the cartridge 100, the use of a standard'off the shelf' using a hypodermic syringe injection needle can be applied within the device. However, the handling and reliability properties of the device can be improved through the incorporation of custom design elements that are not present in the hypodermic syringe needle intended for traditional parenteral administration processes. Certain aspects of the needle hub 152 include material including retention properties that prevent the needle hub 152 from escaping from the inner cartridge 103 during dispensing and use, and the orientation of any warp properties within the needle relative to the hub. Includes.

Disposable injection needles, which are traditionally used once, are usually made up of injection molded polypropylene thermoplastics. However, for many applications, the impact, tensile and flexural forces of polypropylene may not be adequate to ensure the integrity of the hub when the force characteristics of needle injection and needle placement into this device are addressed. Certain malfunctioning concerns include malfunctions in the hub needle joint as well as malfunctions in the hub wall due to impact or injection forces. Adjustments to the hub's design, including the hub's geometry and wall thickness, can be utilized to address the prevention of such malfunctions, however, the hub is "International Standards Organization (ISO) ISO 80369-7:2016 Small-bore connectors for liquids. and gases in healthcare applications - Part 7: Connectors for intravascular or hypodermic application” to determine the dimensional properties required to properly fit into conical male luer slip connectors as described in the relevant standard. While maintaining, it is not always possible to realize sufficient design changes to prevent hub malfunction. In particular, when the syringe needle and hub are given the forces handled during placement, in certain implementations, materials with improved impact, tensile and flexural forces are used. One example is the use of injection molded polycarbonate plastics (e.g. ZELUX® GS, Makrolon, or Lexan) or copolyesters (e.g. Eastman Tritan® Copolyester MX731, MX711, and MX 730). to be.

When evaluated according to ISO 180:2000 Plastic-Izod impact force measurements, a notched impact force of at least 70 kJ/m2 is considered suitable for the present specification. ISO 527-1:2012 Plastics-Determination of Tensile Properties-Part 1: When evaluated according to the general principles, a tensile force of at least 30 Mpa is considered suitable for this specification. When evaluated according to ISO 178:2010 plastics-bending properties measurement, a flexural force of at least 50 Mpa is considered suitable for the present specification. In some embodiments, the particular resin selected exhibits compatibility with the intended sterilization method (eg, gamma radiation) without revealing detrimental changes in physical properties that could impair its function.

In implementations where a custom injection needle is utilized, it includes one or more mechanical features that are not normally present on a traditional syringe hub that allows the device to be inserted into the inner cartridge 103. These features may include tabs, snaps or ridges with corresponding mechanical features disposed on the inner cartridge 103. In some cases, features are implemented such that the hub mates with the inner cartridge 103 in a consistent orientation. In combination with other needle orifice features or a needle manufacturing process that can consistently orient the bevel, this can be explained by the design of the device due to the position and design of the orifice and the bias in the injection location or drug dispensing. To ensure that there is. For example, a needle with an asymmetric piercing end feature (eg, bevel cut) may exhibit a directional bias during placement into tissue due to the interaction between the tissue and the asymmetric piercing feature on the needle. If the electrodes have a symmetric through-end feature (eg, trocar tip), the electrodes cannot exhibit a corresponding deflection in their placement characteristics. Therefore, the mounting feature for the needle hub 152 on the inner cartridge 103 is at the position of the injection orifice on the needle 105 relative to the electrode 122 prior to placement to account for the expected placement characteristic of the asymmetric bevel of the needle. May include an offset of. The exact dimensions of the offset may depend on the nature of the target tissue and the expected range of penetration depth, but in certain implementations, the needles are offset by 0.5 mm to 1 mm each for every 10 mm of penetration depth. When using injection needles and electrodes with different tip profiles or where the tip profile has to be oriented consistently with the other, these features are advantageous in ensuring the co-localization of drug distribution with the application of an electric field.

The integration of the syringe measuring cap 118 mounted on the syringe measuring spring 116 allows the cartridge assembly 100 to accommodate the bearing 101 regarding the needle hub 152 beyond the expected manufacturing tolerances for the syringe 101. Ensures that they can be properly positioned. The applicator 400 causes the cartridge chamber 112 to move forward during the loading process when the spring cap/cartridge interface 470 in the applicator 400 moves distal to the cartridge assembly 100. The cartridge assembly 100 is loaded into the applicator 400, and the cartridge assembly 100 is loaded into the applicator 400, for example, by the action of a loading mechanism, for example, by a rack and pinion mechanism described below. , When pulled, this action occurs.

Movement of the cartridge chamber 112 may act as a second interlock. In particular, in one embodiment, as shown in FIGS. 5C-5D, the line of sight is, through a set of storage locking holes 144' (FIG. 5D), within the cartridge assembly receiving volume 403, by a properly configured sensor. Visible and detectable. This line of sight is seen when the cartridge assembly 100 is loaded into the applicator 400. Visualization of the gaze or its closure may act as a part of the second interlock that must deactivate the control 700 to allow activation and triggering of the device, including needle and electrode insertion, drug delivery, and electrode activation.

For example, in one embodiment, the storage lock hole 144' (FIG. 5D) must be closed for the device to operate. For example, if there is visibility of the line of sight as measured by a corresponding measuring device within the receiving volume 403 of the cartridge assembly of the IR or visible light emitter and applicator, the device may be made inoperative, and the applicator display 404 And/or it is possible to generate and display an error message on the control unit 700 including the control unit display 712, so as to not only notify the user of the state of the device, but also notify the user of the recommended step to be performed to deal with the error. have.

So, in this implementation, one error condition may be that the reservoir 101 is not loaded into the cartridge 100 or the syringe is not properly seated into the inner cartridge 103. In this case, the storage interlock 120 cannot be pressed because there is no storage 101 to perform this operation. In this case, the cartridge chamber 112 cannot move forward in the distal direction toward the inner cartridge cap 104. The structure of this component may allow the open chamber state to exceed the open line of sight 146 through the first storage lock hole 144' FIG. 5D. A secondary check is that the cartridge chamber 112 cannot be closed, which may indicate, itself, that the cartridge cannot move the required distance back or distal into the cartridge assembly receiving volume 403. Since these conditions are defined by the system as being in error conditions, this is accompanied by a message appropriate to the user on the user interface on the control 700 including, for example, the applicator display 404 and/or the control display 712, The occurrence of error messages can be identified and used. A similar error condition occurs when the storage 101 is improperly loaded or the storage interlock 120 is damaged. In general in this case, an appropriate error message may be accompanied by instructing the user to attempt to re-introduce the cartridge assembly 100 into the applicator 400 to remove the cartridge, reinstall the new reservoir.

Another error condition may be that the cartridge chamber 112 is manually moved forward by the user without the presence of the reservoir 101, and the user manually presses the reservoir interlock 120 outside the reservoir receiving volume 142. This can happen. This situation can also be defined as an error condition and can be measured since another (second) set of storage lock holes 144 (FIG. 5C) is placed on a portion of the external cartridge 102. If the reservoir is not in place, but the cartridge chamber 112 is moved forward by the action of the depressed reservoir interlock 120, the reservoir lock hole 144' FIG. 5D is aligned with the reservoir lock hole 144; FIG. 5C. In the case of listing, the eye opening and subsequent error states are recreated. This error condition also occurs if the storage interlock 120 is damaged and its fingers are no longer within the storage receiving volume 142. In certain implementations, this error condition need not be resolved by attempting to reinstall the reservoir, as the reservoir may not fit inside the reservoir receiving volume 142 with the locked cartridge chamber 112. In certain implementations, a new cartridge assembly 100 is required.

In contrast, if the reservoir 101 of the appropriate size is properly placed in place, the reservoir measuring cap 118 is pushed back against the reservoir measuring spring 116 and the movement of the reservoir detecting cap 118 is caused by the reservoir locking hole ( 144 (FIG. 5C) and the storage locking hole 144' FIG. 5D are closed. In this case, there is no error condition and allows the device to operate. The measurement of closure and closure occurs within the body of the applicator 400, after the cartridge assembly 100 is inserted, so that the user attempts to retract this interlock, regardless of whether it is caused by a defect or is intended or accidental. Does not affect things. It should be noted that this'no error' condition still occurs even if the user intentionally or accidentally closes the cartridge chamber 112 itself during handling of the cartridge 100.

Depending on which error condition occurs, the cartridge assembly 100 may remain in a usable or disabled state. If the cartridge chamber 112 is fixed in place, the cartridge assembly 100 is made unusable. However, if the cartridge chamber 112 is not locked in place, the cartridge assembly 100 can be removed from the applicator 400 and a new reservoir 101 is inserted.

The two sets of interlocks mentioned above (one using the storage interlock 120 mechanically and one using the emitter and collector and storage lockout holes 144, 144') are of particular utility in some implementations. Although it has been found to be, it will be appreciated that other types of interlocks may also be applied (FIGS. 5C and 5D ). For example, instead of generating an error condition when the storage lock hole 144 is not closed, the error condition is when the storage lock hole 144 is closed (and a clear line of sight 146 corresponds to an error-free state). It can be configured to occur (via changing the storage lock hole location and program logic). The reservoir intercor 120 may contain additional mechanical flag properties that are concave within the cartridge when the interlock is activated, but when the syringe 101 is properly inserted and the reservoir interlock 120 is pressed into the released position, a properly configured It is displayed on the sensor. In other variations, other approaches may be applied to determine whether a line of sight is present, eg, optical, acoustic, electrical, etc., as long as suitable emitters and collectors can be located within the applicator 400. Other approaches may also be applied to determine whether the reservoir 101 is properly loaded, for example, mechanical technology as can be understood by one of the skilled artisans given in this teaching. . Depending on the implementation, when an error state is measured, the applicator 400 may prevent the cartridge assembly 100 from being operated in place or the applicator 400 may prevent the cartridge assembly 100 from being accommodated in the first place. have. For example, the cartridge may be designed such that the reservoir interlock 120 may include a mechanical tab or locking feature extending from one or more cartridge surfaces until the syringe 101 is properly inserted into the cartridge 100. . Mechanical tabs are designed to interact with corresponding clasp features located within applicator 400 so that loading of cartridge 100 into applicator 400 is physically blocked as long as reservoir interlock 120 is released. . This mechanical interaction can provide feedback to a system or user that an error condition must be considered prior to proceeding with the cartridge 100 being loaded into the applicator 400. In other variations, more or less than two interlocks may be provided, but this may be correlated correspondingly with different safety profiles.

In a particular implementation, other features may also be applied in the determination or may be applied to improve the determinations. For example, when a motor is applied to push the cartridge assembly 100 into the applicator 400, the sensors may be applied as described below to detect the spatial position of the cartridge assembly 100 during the insertion process. In other words, the applicator 400 may detect where the cartridge assembly 100 is within the cartridge assembly receiving volume 403. In some cases, this may detect additional fault conditions directly or by facilitating activation of additional sensors to assess the condition of the device. For example, in a used cartridge, the cartridge chamber 112 is held in place. If the used cartridge assembly 100 is attempted to be reused, the optical detector detects the cartridge assembly 100 in a different way than the unused cartridge assembly 100. The use of an electric motor for one or more system functions also provides an opportunity to monitor operating conditions, including the voltage and current values supplied to the motor as well as the number of revolutions the motor performs during a particular operation. Measurement of these quantities during system operation can be used as a first or second method to detect potential or actual fault conditions. For example, the use of a mechanical interlock designed to block the loading process when the cartridge 100 is not properly configured can be combined with a sensor and logic circuit that monitors the motor, bringing the cartridge 100 into the applicator 400. Ensures proper loading. For example, the interaction of the mechanical features described above designed to prevent loading of the cartridge 100 without a properly inserted reservoir 102 will result in an increased load on the motor drive mechanism, resulting in higher current consumption. will be. Detection of the consumption of the elevated current by the motor will cause the loading procedure to be interrupted and a fault condition to be shown to the user, such as the fault condition shown on the stimulus display 712 and/or the applicator display 404.

There is a possibility that a drug not intended to be administered by this method may be contained within a reservoir of a similar size and configuration to the drug intended for use by this method of delivery. Therefore, additional aspects of the system include one or more methods of ensuring that the reservoir 101 inserted into the cartridge 100 by the user is specifically for use with the device. The realization of these features can reduce the risk of administering the incorrect drug to a given subject. Typically, specific information in user instructions and drug labeling includes the route and method of administration. However, in order to further reduce the likelihood of user error, it may be required to incorporate mechanical, optical and/or electrical features into the storage and device. In an implementation, the syringe may be designed to contain one or more unique mechanical features not present in other reservoirs that may be similar to those designed for use with this delivery method. For example, a reservoir may be specified to include a flange on the reservoir or a rib on a barrel or other elongate feature. In this implementation, a corresponding mating feature may be included on the reservoir interlock 120 so that the reservoir interlock can be deactivated only if a reservoir having the appropriate mating feature is properly inserted into the device. In cases where it is not possible to directly perform a feature within the design of the reservoir, an alternative implementation may include the placement of secondary mechanical components on the reservoir specific to the reservoir intended for use with the device. For example, other suitably configured features or rings designed to slide over the barrel of the reservoir may be within another cartridge 102, inner cartridge 103, reservoir interlock 120, reservoir measuring cap 118, or cartridge 100. It can be used to'enter' storage for use with the device by matching with corresponding features within other suitable features. A further implementation is a custom label of an appropriate size, color and/or electrical conductivity applied at a predetermined location on the outer surface of the reservoir intended for use with the device. Corresponding optical or electrical sensors in the applicator 400 are configured to assess the absence or presence of a label on the surface of the reservoir to verify that the drug inserted into the cartridge is to be used with the device. The measurement method may include the use of an optical or electrical signal applied to the surface to assess its absence or presence. In this way, reservoirs containing drugs that are not intended to be used with the device (and therefore no associated label) can detect or rule out the possibility of misuse.

The components of the sensor are now described as performing the cartridge loading and syringe detection measurements described above, these sensors further forming part of the cartridge loading subassembly within the applicator 400. More specifically, and further referring to FIGS. 6, 17B and 18C, an exemplary method of detecting where the cartridge assembly 100 is located is a sensor 436 for loading a cartridge and a sensor for loading the cartridge. 438, which forms part of the loading drive subassembly 454, and the subassembly 454, via a rack 154, on the base of the external cartridge 102, the cartridge assembly receiving volume It further includes a loading motor 44 and a cartridge guide rail 442 connected to the pinion gear assembly 448 which presses the cartridge assembly 100 into the 403. More specifically, when the sensor 436 for loading the cartridge detects the start flag 172 on the cartridge assembly 100 (FIG. 6), the motor may initiate loading. When the sensor 438 in which the cartridge is loaded detects the start flag, the loading may be stopped. Consecutive flags 174 may be used to require that the loading be present in sequence.

The first'teeth' of the rack 154 is configured to provide touch sensing (or audible or tactile) to the user when the user inserts the cartridge assembly 100 into the cartridge assembly receiving volume 403. I can. This configuration not only includes the shape and/or size of the rack teeth 154, but also includes the amount of curvature allowed by the placement of the rack teeth within the external cartridge. By adapting the rack tooth implementation, the required angle of tactile feedback can be achieved without providing significant force to the loading motor 444 from loading and receiving the cartridge assembly 100.

17A and as described above, the cartridge assembly 100 is inserted into the cartridge assembly receiving volume 403 inside the applicator 400. While a variety of ways can be applied to perform this insertion, one way in which particular utility has been found is to use a pinion gear assembly 448 that engages the rack 154 on the outer cartridge 102. The use of more than one rack provides additional stability, in particular torsional stability during the loading phase. See also insert/inject drive assembly 456 of FIG. The electrode/needle insertion spring 472 is further compressed through. The electrode/needle insertion spring 472 is used as the main driving force for needle and electrode insertion during drug delivery.

This hybrid motor/spring action has numerous advantages. The motor drive is advantageous because it can be controlled at a high degree and allows the cartridge 100 to be loaded into the applicator 400 in a semi-automated manner while minimizing the mechanical force required by the user. As described above, the implementation of a mechanism-based motor drive provides for monitoring the operating state of the system. For example, passing the number of turns and the number of current draws to logic and control circuits in the system provides a supplementary method for diagnosing and detecting potential fault conditions. Despite these advantages, in certain cases, electric motors are required over a sufficiently short time scale, most desirable for effective transdermal placement of an array comprising a plurality of elongated electrodes and in some selected embodiments hypodermic syringe injection needles. It may not be suitable for applying a linear force. Specifically, penetration of the skin tissue can most consistently be achieved by applying a large linear force over a short time scale. In some implementations, the most desirable insertion properties are achieved when penetrating the electrode and when there is contact of the injection needle with the skin at high speeds. This is because, at increased speed at the point of skin contact, there is less tissue deformation when a sharp cut or pierces the tissue. Therefore, in some implementations, it is required to rapidly accelerate the sharps before contacting the skin. In some implementations, multiple electrodes and injection needles are frequently utilized. In another embodiment, the speed of the electrode is at least 50 mm/s prior to contacting the skin. In another embodiment, the speed of the electrode is at least 500 mm/s prior to contacting the skin. This arrangement approach is most desirable for minimizing the discomfort perceived by the object during electrode penetration, and maintaining a consistent spatial relationship between the plurality of electrodes.

In contrast to electromechanical motors, the spring driven mechanism exhibits a more desirable discharge profile capable of delivering fast impact forces to the injection needle and electrode, which is desirable for transdermal electrode injection. In particular, the force exerted by the compression spring is at its highest point upon initial discharge. This is desirable for transdermal placement where a high speed is required at the point of skin contact, and due to the viscoelasticity of the skin tissue, the greatest force is in the penetration of the skin, especially when a plurality of electrodes and/or injection needles are in contact with the skin. Is required for In addition, spring-based mechanisms can generate these forces from simple, durable and simple forming elements that can be easy to integrate with portable device types. However, a disadvantage of spring-based mechanisms is that they typically require input of substantial mechanical force by the user in order to prepare them for operation, especially for springs with high force constants and/or large displacements. As described in this post, the use of a hybrid motor and a spring mechanism achieves desirable placement force characteristics, while simple operation by the user. The hybrid motor and spring mechanism is the preferred implementation, but depending on such implementation, one can generate a fast impact force, and the other applies an impact force mechanism, e.g., an impact force for placement of electrodes and applicable hypodermic syringe needles. In order to do this, other hybrid mechanisms incorporating two or more drive mechanisms can also be used, in which a pump can compress the gas into the chamber and then prepare a mechanism for discharging the compressed gas.

In any case, when loaded, the desired depth electrode placement and/or formulation administration is positively selected by the physician or other drug administrator, which is transferred to the applicator 400. With further reference to Figure 13B, the depth is selected by the depth select button 409 (or other equivalent interface such as a toggle switch or sliding switch), and the result is the injection depth select indicator 408 (or again another equivalent). Interface). The available injection depth is communicated to the user by the appropriately labeled applicator 400 and/or cartridge 100. In some implementations, any labeling regarding the depth of injection may be placed on the cartridge 100 and shown to the user after installation in the applicator 400. For example, the available implant depth may be labeled on the top surface of the enumeration guide/spray shield 108. In order to avoid situations where the user forgets or ignores the selection of the injection depth, it is desirable that the device does not allow the user to proceed with the dosing process until such a positive selection has been made. This can be achieved by implementing appropriate control logic in the system so that subsequent elements of device setup or use are not accessible until a valid depth selection is entered by the user. In certain implementations, when allowing the user to positively select the injection depth, the control display may convey user information regarding an appropriate method for evaluating the subject and measuring the appropriate injection depth for the selected injection location.

With further reference to FIGS. 18A-18B, upon insertion of a suitable cartridge assembly, the spring cap/cartridge interface 470 engages and also presses against the spring cover holes 471 and tabs 491. While a large spring force is pressed against the inner cartridge 103, it (inner cartridge) moves forward by engagement of a set of retaining posts 488 against the wall 494 of the outer cartridge 102. Is prevented. However, the force exerted by the spring cap/cartridge interface 470 spreads apart the tabs 491 (preventing accidental rotation of the locking ring during handling), and rotation of the cartridge locking ring 114 by the motor drive mechanism. Allow The rotation of the cartridge locking ring 114 causes rotation of the retaining post 488. The retaining post 488 can be rotated with a channel for a first depth 490 or a channel for a second depth 492. The length of the channel for the first depth 490 corresponds to one of the choices of depth, the length of the channel for the second depth 492 corresponds to the other, by the user using the button 409 One or the other depth is selected. For example, the length of channel 490 can range from 20-30 mm and the length of channel 492 can range from 12-20 mm. The user's requirement to reliably choose the depth provides another interlock. If not clearly selected, the applicator may not allow activation/needle insertion. Accordingly, rotation of the cartridge locking ring 114 is caused by a clockwise or counterclockwise rotation indicated by the applicator 400 according to a user's command. Requiring rotational movement of the set of posts into these channels to achieve deployment of the electrodes and, if present, the injection needle greatly reduces the likelihood of accidental discharge even in violent jarring or dropping.

More specifically, the rotation of the cartridge locking ring 114 is transmitted to the cartridge assembly 100 by a retaining post 488, the retaining post 488 into the slot of the insertion mechanism gear drive ring 478 into a suitable cartridge assembly ( 100) It is placed upon insertion. In Fig. 18A, the slots on the insertion mechanism gear drive ring 478 are arranged in the 3 o'clock and 9 o'clock positions. The insertion mechanism gear drive ring 478 is mounted on the partial insertion gear ring 479 driven by the insertion mechanism drive motor 482. Driving partial insertion gear ring 479 causes insertion mechanism gear drive ring 478 to rotate clockwise or counterclockwise. Flag 481 on ring 480 and accompanying insertion mechanism position. The sensor 483 is used to determine the position of the insertion mechanism gear drive ring 478, and also the insertion mechanism gear drive ring 478 at 3 o'clock and 9 o'clock when the applicator 400 needs to be reused with another cartridge. It is used to accurately return to position.

The above implementation provides various advantages. For example, the user must actively perform the step of selecting the depth before proceeding with the administration procedure. In doing so, the user should determine the appropriate injection depth for the selected injection site and prepare the site as noted in the guide or instructions for the instruction manual. As noted, the insertion mechanism, which requires rotational motion to deploy, is significantly reinforced against accidental deployment due to fall, drop, collision, etc.

Variations should be understood by a person skilled in the art. For example, two channels and two retaining posts are used for each depth, but one channel and one retaining post may be used. Various types of motors and mechanisms can be used to transmit the rotational motion required to rotate the post into the channel. Other variations, including the use of solenoids, should also be understood. Instead of using a channel, a motor-driven deployment can be used to provide variable depth, and this depth can be controlled simply by how far the motor is controlled to drive the deployment. Preferably, in this context, the described hybrid drive mechanism allows an impulse release mechanism (e.g. a spring) to be used for initial deployment through the dermis and then a motor drive mechanism to advance the electrode to the desired depth. Will be configured to be used for

One or more interlocks may be in place that must be deactivated before the insertion mechanism gear drive ring 478 rotates and rotates the retaining post 488 into the channel.

First, a force detection interlock subsystem may be in place that requires the device to be applied to the subject with a force greater than a predetermined amount before allowing the dosing procedure to be initiated. This force can be measured by a suitable mechanical or electromechanical system and the result can be fed back to the controller 700 and used as an interlock to prevent activation of the device where insufficient force is provided. In some embodiments, the controller 700 may communicate the force sensing interlock state to the user via visual, tactile, or audible signals so that the improper force state may be corrected and the user may proceed with the administration. If the user attempts to proceed with the administration under insufficient force contact (e.g., pressing the trigger 407 or other activation button), additional visual, tactile, or auditory signals are generated by the applicator 400 or controller 700. Can be provided by to inform the user that the interlock should be deactivated before proceeding with administration.

Sensing the force exerted on an object can be accomplished in several ways. 4 and 8A-8B, an alignment guide/splay shield 108 has a force contact pickup 128. Alignment guide/splay shield 108 may be mechanically biased in a distal direction (toward the object) using one or more force contact springs 126, and force contact springs 126 Four are shown in FIG. 4. The force contact pickup 128 changes position by the force applied to the alignment guide/splay shield 108. In doing so, it also changes the state of the electrical circuit formed by the force contact pickup 128, the first set of pads 162, the second set of pads 164 and the flexible circuit 160. . In particular, by testing the continuity between one or more pads 162 and one or more of each of the pads 164, how far back or proximal the alignment guide/splay shield 108 was moved by the applied force, And accordingly, it can be determined whether sufficient force is present for proper transmission. The state of the circuit is read by the applicator 400 using sensor contacts 434 (see FIG. 16). If sufficient force is displayed, the force contact interlock is deactivated, allowing the user to operate the device.

According to one implementation, at the first point of contact, the system may not register that any specific force has been applied. At the second point of contact, it can be noted that the system is at partial (but not enough) pressure. At the third point of contact, the system can record that the prescribed pressure level required to proceed with the dosing procedure has been achieved, and the interlock can be deactivated. Preferably, the state of the force contact circuit is provided to the user via the applicator display 404.

Variations should be understood by a person skilled in the art. For example, the force contact interlock may form an electrical lock that is deactivated within the controller 700 or within the applicator 400 itself. In another variation, the force contact circuit can be configured to provide information regarding the state of the device throughout the administration procedure. In particular, the system may include a feedback loop between the force contact circuit and the controller 700, wherein the reduction of the force applied by the user is the controller 700 and/or the applicator so that the state of the reduced force can be modified. Promote the generation of visual, tactile, or auditory signals by 400. In certain embodiments, a feedback loop exists between the force contact circuit and the controller 700 to detect the change in the applied force so that the system is Promote to initiate a check on whether it is properly positioned and maintained within the organization. If the check passes, the procedure proceeds normally. If the position of the electrode is no longer acceptable, the procedure can be stopped, and the user notifies the state of the device through the generation of visual, tactile, or auditory signals by the controller 700 and/or applicator 400. Can receive This feedback loop is particularly important during injection of the drug. By monitoring the position of the device and the state of the electrode, the feedback loop between the force contact circuit and the electrode resistance/impedance monitor, the system can detect if the electrode (and hence the injection needle) is no longer in the target, allowing for scanning. Allows a system halt operation of the drive mechanism 456 to stop depressing the reservoir plunger 484 and terminating the injection of the medication. This embodiment is most easily implemented with the motor-driven scan drive unit 456, but other variations may be implemented in the case of a manual-operated or spring-type drive mechanism, and the activation of the mechanical interlock is performed after detection of a fault condition. It can be used to stop the operation of the reservoir plunger. This feature may be particularly useful in preventing the HBV vaccine from inadvertently spraying into the environment when the applicator 400 is removed from the subject's tissue prior to completion of medication delivery. For drugs or therapeutics that are potentially at risk of exposure to the user or the environment, this design can avoid unintended release/exposure.

Since the dose delivered to a subject in a partial dose situation can be important in informing the clinician's decision regarding further treatment, the injection drive mechanism 456 stops a fault condition or injection stroke. It is desirable to include suitable sensors and control features to determine the position of the scan drive plunger 484 when the scan stroke is stopped due to the detection of another situation that needs to be triggered. Preferably, this is achieved through monitoring the revolution count of the motor used to drive the scan drive plunger 484, but to monitor the position of the scan drive plunger 484 comprising an optical or electrical sensor. Other methods for can be used. Based on the known dimensions of the injection driving plunger 484, the insertion depth and the reservoir 101, by using appropriate logic and control circuitry, the position of the injection driving plunger can be determined to the position of the drug remaining in the syringe at the end of the injection stroke. Can be converted to an estimate of the volume. This information may be communicated to the user through the display 712.

In addition to providing an end feature to terminate the scan stroke when a fault condition is detected, the use of motor scan drive 456 may also provide a supplemental detector for detection of fault conditions or other operational problems. have. Specifically, the incorporation of appropriate measurement and logic circuitry to monitor the circuit drawn by the motor during the injection stroke can be used to confirm that the injection has been administered within limited specifications. The expected range for the current drawn by the motor is the initial run up of the scan drive plunger 484 before contacting the plunger stopper 159, the scan drive plunger 484 and the plunger stopper 159. Including the initial interface between, the operation of the plunger stopper 159 forward in the barrel of the storage unit 101 and the termination of the scanning stroke as the plunger stopper 159 contacts the end of the barrel of the storage unit 101 It can be set for several stages of a scanning stroke. By correlating the position of the scan drive plunger 484 with the measured current drawn by the motor at the expected value during each stage of the injection of the agent, the system can identify potential fault conditions and communicate this to the user. have. For example, if the user inadvertently inserts the reservoir 101 into the cartridge 100 with the plunger stopper 159 partially actuated (thus not including the full dose of the intended drug), the system It can be detected that the expected increase in the current drawn by the scan driver 456 did not occur when the scan drive plunger 484 reached an external tolerance for setting the plunger position. In this situation, the system can terminate the dosing procedure and notify the user of the detected defect. Additional fault conditions, including, but not limited to, defective components in the applicator 400, failure of the storage 101 and/or the defective cartridge 100 can potentially be achieved through this method. Can be detected

Another'interlock' to facilitate proper execution of the dosing procedure is provided by the alignment guide/splay shield 108, which is shown in FIG. 9C. In this figure, alignment guide/splay shield 108 is shown having splay features 168 and holes 170 formed for slidable movement of stick shield 134. Stick shield 134 is shown in Fig. 9B, and electrode holes 167 are also shown. For transcutaneous insertion of the electrode and, if relevant, the injection needle, having a constant skin interface facilitates placement of the electrode into the target tissue while maintaining the desired spatial relationship between the plurality of members. In particular, proper placement is most consistently achieved when the skin is positioned perpendicular to the placement direction. In addition, misalignment of the electrode and injection needle is reduced when the skin is placed in tension in an orientation perpendicular to the placement direction. As can be seen, the splay shield 108 includes mechanical features including ribs and edges to engage the skin and position the skin in tension perpendicular to the direction of placement. A constant force applied to the skin is applied to the skin. In combination with the aforementioned force contact circuit pickup system to ensure, the alignment guide/display shield 108 ensures that the skin is oriented in a direction perpendicular to the electrode placement direction and placed under tension. These embodiments use mechanical rib features to engage the skin at the device interface, but alternative materials (e.g., rubber inserts, adhesive patches, etc.) or molded materials that have a high coefficient of friction when placed in contact with the skin. Numerous other designs and features can be used for bonding, including the use of alternative mechanical features including texture, cut outs, and toothed features that can place the skin in tension.

As can be seen, the alignment guide/splay shield 108 has a defined preferred orientation. As mentioned above, spatial and temporal co-localization of drug distribution and electric field application is desirable. In particular, the inherent structural properties of skeletal muscle result in a characteristic ellipsoid distribution pattern of intramuscular injections in which the major axis of the ellipsoid is aligned with the striation of the muscle fibers. Thus, for applications involving intramuscular injection, it is advantageous to array the electrodes to create an ellipsoid electric field profile. The use of alignment guide features is particularly useful for intramuscular administration in order to ensure that the electrode array and the resulting electric field profile are properly oriented relative to the streaks of the target skeletal muscle. The purpose of the alignment guide feature is to facilitate the placement of the device so that the orientation of the electrode array is most advantageous for muscle streaks and thus drug distribution after injection. In the case of arm injection, the alignment guide/splay shield 108 preferably wraps horizontally around the arm, such as an armband. A similar orientation is preferred for the leg so that the splay shield wraps horizontally around the leg. It has been found that configuring the alignment guide/display shield 108 in this way results in a higher than 98% (>98%) accurate drug delivery by the user even without oral instructions. The preferred orientation and resulting skin arrangement shown is a diamond-shaped electrode array (see array of distal ends of electrodes 137 in FIG. 9A which roughly matches the shape of the stick shield 134 and associated hole 167). It is particularly useful for intramuscular injection because it is properly oriented to deliver the medicament and cause electroporation of the medicament along the desired muscle streak direction. The main feature is that when properly aligned, the skin must be at the same level as the skin interface where the orifice is located, for example the needle emerges. In this way, a consistent interface to the skin is obtained.

In case of inadequate alignment to the target muscle, the arc generally causes a visually apparent gap between the alignment guide/splay shield 108 and the skin, for example 2-5 mm. The visually apparent gap can be used as a reminder to the user to reorient the applicator 400. The applicator 400 may be less stable with respect to the subject's skin. Alignment guide features where the distance between the edges of the "wings" of the alignment guide features is at least 1.3 times the vertical height of the features are desirable to facilitate proper placement. Further, in some cases, the design of the features allows the tissue interface to be placed flush with the skin in the desired orientation while exhibiting at least a 2 mm air gap when placed at an angle of 90 degrees to the desired orientation.

Variations to the design of the alignment guide should be understood by the ordinary technician. For example, instead of the arc-shaped alignment guide/splay shield 108, a “V” shape may be used. In addition, other variants allow the device to be positioned directly against the skin in the desired orientation, while a visually apparent gap of 2 mm or more between the tissue interface and the recipient's skin when the device is misaligned with respect to the streak of the target muscle. It should be understood as having the main characteristic that it exists.

As mentioned above, the orientation of the alignment guide/display shield 108 is related to the shape of the electrode array in some implementations, since the diamond symmetry of the electrode array has a specific distribution associated with it, and this distribution is It should have a certain predetermined relationship to the shape of the guide/splay shield 108. 9A-9B, there are four electrodes and more particularly distal portions 137 shown. The electrodes are arranged in a diamond-shaped array, which means that they have a quadratic symmetry, ie twofold symmetrical, so that they can be rotated and look the same in two different positions. The electrodes extend from the holes 167 formed in the stick shield 134 to this array. The use of a diamond-shaped array is particularly useful as it is generally preferred for intramuscular injection as discussed above. Repeatedly, the quadratic symmetric array leads to an applied electric field in quadratic symmetry. If the alignment guide/splay shield 108 is properly oriented, an applied electric field of this type can have a desirable orientation along the streaks of the muscle. Thus, the alignment guide/splay shield 108 and the secondary electric field orientation work together in a synergistic manner to achieve drug propagation along the muscle streak direction.

Roughly speaking, in the form of a diamond or other quadratic symmetry, there are usually a major (major axis) and a minor axis (minor axis). The axes may have a predetermined relationship with the desired axis of the alignment guide/splay shield 108. For example, if the alignment guide/splay shield 108 is thought to have a wing that has an arcuate shape of the wing surrounding the arm, the line segment connecting the center of the wing is perpendicular to the main axis of the diamond-shaped array. Can be

Variations should be understood by a person skilled in the art. For example, diamond is one possible shape for an electrode array, but another exemplary shape is rectangular, which is also quadratic symmetry. The electrodes can be placed in an appropriate position, but can be moved within a predetermined or desired tolerance, for example 5%, 10%, etc. The electrodes can be of a variety of different shapes as long as they produce a secondary rotationally symmetric electric field.

In another variation, for non-intramuscular injections, other orders of symmetry can be used. In the case of skin, for example, there is no generally preferred direction of drug propagation, so an array of circular electrodes can also be used for intradermal injection.

Other considerations for the electrode array are as follows. The simplest array configuration involves two electrodes connected to opposite poles of an electrical energy source. As disclosed in U.S. Pat. It can be used to improve the uniformity of electric field propagation within. A wide range of geometric electrode arrays and activation patterns have been developed for application of electric fields in tissues. These include electrodes arranged in a linear, rectangular, circular or triangular configuration capable of propagating an electric field in a volume of tissue approximately ellipsoid, cuboid, cylindrical or spherical. Most commonly, the electrodes are arranged parallel to each other and configured to be inserted transcutaneous in an orientation substantially perpendicular to the skin surface. In order to ensure that the target volume and the shape of the tissue are affected by the application of an electric field, the intended spatial relationship between each electrode in the array is preferably achieved after transdermal insertion. Specifically, unintended changes in the inter-electrode spacing can lead to changes in the magnitude of the electric field propagating within the target tissue, and potentially negatively affect the safety and/or efficacy of the procedure. Can be avoided.

Another interlock involves the use of an external cartridge cap 110. In particular, the alignment guide/display shield 108 is covered while being stored with the outer cartridge cap 110. The outer cartridge cap 110 is configured to function as an interlock feature itself as well as generally protect the distal end of the device until used. The protection of the distal end of the cartridge assembly 100 serves the purpose of protecting the needle and electrode from the environment, but a problem commonly encountered is that users often forget to remove these caps. One solution is to have the cap made in a bright color different from the color(s) of the other components in the cartridge 100 to inform the user of its presence and thus remind the user to remove it. Another part of this solution is to include an extension or reminder tab 190, as shown by an arcuate section adjacent to the rectangular section, which is visible even when the cartridge assembly 100 is placed against the object. have. Since the reminder tab 190 is visible, it can function as a reminder to remove the external cartridge cap 110 even when the rest of the external cartridge cap 110 is not visible.

More specifically, referring to FIGS. 10A-10B, the outer cartridge cap 110 includes an outer surface facing the object and an inner surface facing the alignment guide/display shield 108 in a distal direction. The inner surface is provided with a plurality of hooks 176 that engage a corresponding wall 180 formed in the alignment guide/splay shield 108. The hook holds the outer cartridge cap 110 in place.

However, if the outer cartridge cap 110 accidentally remains in place and the applicator 400 is pressed by force against the insertion position, the force of the alignment guide/display shield 108 against the outer cartridge cap 110 is As the cap 110 is pushed away from the hooked position by the alignment guide/splay shield 108, it may tend to cause "pop off". However, the chamfered surface 178 is provided on the hook 176, which tends to act on the stick shield 134 when pressure is applied, so that the hook 176 splays outward and the alignment guide/splay shield ( 108) to increase the holding force and prevent the outer cartridge cap 110 from protruding. In addition, the chamfered surface acts in addition to the stick shield, preventing the alignment guide/splay shield 108 from moving relative to the stick shield 134. If the alignment guide/splay shield 108 cannot move relative to the stick shield 134, the force contact pickup 128 cannot move to the third force contact point discussed above where the full pressure is sensed, so the force sensing interlock will be disabled. Can't (or there is such a problem with the second force contact). The controller can provide a report when this happens to the controller. For example, a user interface indication such as "You must remove the external cartridge cap" may be displayed, and the same may be caused by detecting a trigger pull performed in the absence of sufficient force.

Although specific interlocks have been described above, more or fewer interlocks may be provided in any given implementation. For example, different methods can be used for force sensing and can be used as a trigger signal for interlock. In addition, other types of interlocks can also be used, including trigger locking, safety switches, etc. Given this disclosure, other types should be understood by the ordinary technician.

When the insertion depth is definitively selected by the user and the outer cartridge cap 110 is removed and an appropriate force is sensed by the force interlock, activation of the trigger is performed by the cartridge locking ring 114 in the rotation direction according to the insertion depth selected by the user. Rotate clockwise or counterclockwise. The rotation causes the retaining post 488 to move into the channel of the selected depth, thereby placing the needle and electrode. In particular, the inner cartridge 103, the cartridge breech 112, the reservoir 101, the inner cartridge cap 104, the needle hub 152, the needle 105, the electrode 122 and related elements are electrode /It moves forward under the influence of the needle insertion spring 472. In this embodiment, these elements are rigidly connected to each other and move forward as a unit.

In order to further minimize the risk of sharp injury to the user, the cartridge assembly also preferably includes a mechanism for sheathe of the electrodes and any injection needles after removal from the recipient's tissue. This can be achieved through the integration of stick shield features that receive the electrode and needle (if present) prior to use and then extend across the electrode and needle (if present) after placement and removal from the subject's tissue. have. Typically, the tissue contact interface includes the distal surface of the shield feature. Although the use of manually operated shield features may be contemplated, preferably, the device automatically extends the shield features over the electrode and further locks features to keep the shield features extended if removed from the recipient's tissue. Includes a mechanism for engaging with. Examples include a shield feature that slidably engages with the cartridge outer housing and is operably connected to a source of stored energy such as a spring, which slides the shield forward when removing the electrode from the recipient's tissue. Alternatively, the mechanism may be housed within the applicator and configured to reverse the electrode placement step, retracting the electrode and any associated injection needle behind the tissue contact interface. This can be achieved using simple electromechanical mechanisms for linear motion such as motors or solenoids.

In certain embodiments, and referring back to FIGS. 4 and 11A and 11C, the stick shield 134 is configured to remain in place during drug delivery, but the inner cartridge 103 and other related components during drug delivery. The forward movement of the sticks causes compression of the stick shield spring 138. Next, the compressed stick shield spring 138 is to relax as the applicator is removed from the target, and when the applicator 400 is removed from the target, the stick shield 134 moves forward, so that the needle and Covers the electrodes and protects objects and others from uncovered sharps. In particular, these features may also prevent visualization of sharp features during pulling out, thus facilitating acceptance from subjects experiencing needle-related anxiety.

The loosened spring may otherwise be recompressed, but especially when the user moves the stick shield 134 proximally, it is mounted inside the external cartridge 102 and latched as the stick shield 134 moves forward. Doing so is prevented by the action of the stick shield support arm 132 providing a ratcheting function. In particular, referring to FIGS. 11A and 11C, the stick shield support arm 132 moves over several sequential retaining walls, and can easily do so when the stick shield 134 is moving distally. However, in the proximal direction, the stick shield 134 is prevented from moving because the stick shield support arm 132 contacts the retaining wall in this direction in a latching manner and does not allow passage.

The initial set of retaining walls 188 prevent proximal movement of the stick shield 134 prior to use of the cartridge assembly 100. The first set of depth retaining walls 184 is the stick shield after release at a selected first depth. Prevent proximal movement of 134. A second set of depth retaining walls 186 prevent proximal movement of stick shield 134 after release at a selected second depth. The stick shield 134 is prevented from being completely removed from the cartridge assembly 100 by the action of the stick shield retaining hook 182 acting on the inner cartridge 103.

Variations should be understood by ordinary technicians. For example, in one embodiment the stick shield support arm 132 may be provided by a stamped metal support arm that interfaces with the external cartridge 102. In some embodiments, the support arm features are incorporated directly into the outer cartridge cap 106 and/or alignment guide/splay shield 108 as injection molded plastic features. Certain materials must be selected to obtain sufficient rigidity to provide support to the elongated electrode while maintaining sufficient elasticity to prevent failure of the loaded component. In addition, material selection should take into account the intended sterilization method for the electrode array (eg, gamma radiation, steam sterilization, ethylene oxide or e-beam) to ensure that the features retain the appropriate material properties after sterilization. In an exemplary embodiment, as implemented, the feature should be capable of securing the stick shield 134 against proximal movement when a force of 5N or greater, more preferably 15N or greater is applied. In addition, another method of providing a ratchet function to prevent proximal movement of the stick shield, including a gear rack implemented on the stick shield 134 and a corresponding ratchet feature implemented on the outer cartridge cap 106 Can be used

Referring to FIG. 4, each elongate electrode 122 in the array includes a distal portion 137 and a proximal portion 135 in conductive communication. The distal portion is configured for tissue penetration and electric field propagation within the tissue and the proximal portion is configured to have a conductive contact zone capable of reliably achieving conductive communication with an electrical connection contained within the applicator with an appropriate source of electrical energy. The electrical energy source can cause a spatially and temporally varying electric field within the subject's body by temporal fluctuations in the power applied to the individual electrodes, which can generally be confined to the volume around the drug dispensing area, and electroporation therapy. Can be used advantageously in

The relevant properties of the electrode include shape, diameter, tip profile, length, material composition and conductivity, the details of which are selected based on the intended application. Most commonly, the electrode comprises an electrically conductive elongate rod with a curved cross section of 0.1-1.5 mm in diameter. Electrodes can be solid or hollow. Most commonly, the hollow electrode comprises an actuating connection and one or more orifices in the fluid reservoir, such that a drug or anesthetic of interest, surfactants, proteins, adjuvants or enhancers from the electrode itself It allows the administration of other related agents, including. Depending on the application, suitable metal electrode materials include, but are not limited to, titanium, gold, silver, aluminum, copper, tantalum, tungsten, molybdenum, tungsten, stainless steel, MP35N, and alloys thereof. In addition, the electrode may be composed of an electrically conductive ceramic or plastic. In order to minimize unwanted electrochemical reactions at the tissue electrode interface, it is often desirable for one or more of the electrodes to be coated with a conductive material that provides improved electrochemical stability compared to the electrode material itself. Such coating materials include, but are not limited to, platinum, iridium, palladium, osmium, gold, silver, titanium and alloys thereof.

As mentioned above, the tip of the distal portion is configured for tissue penetration. Typical tip profiles include, but are not limited to, trocars, bevels, cones, blades, lances and tapers. For transdermal electrode placement, a tip profile with one or more cutting edges is preferred. For certain applications where the penetrating tip profile may not be desirable to generate the required electric field, the tip may be composed of a non-conductive material fixed to the distal tip of the electrode or the penetrating tip may be poly(p-xylylene). Polymer, polyolefin, polyvinyl chloride, polyurethane, polyester, polyimide, silicone rubber, thermoplastic elastomer/rubber, ethylene tetrafluoroethylene, fluorinated ethylene propylene and/or perfluoroalkoxy plastics and It can be covered with an adhesive coating or tube made of the same biocompatible electrical insulating material.

There is one or more conductive zones configured for propagation of an electric field in the tissue in proximity to the penetrating tip. In general, at least a portion of the length of the electrode configured for penetration into the tissue is poly(p-xylylene) polymer, polyolefin, polychlorinated, in order to limit the propagation of the electric field to the target area of the tissue, particularly for subcutaneous and intramuscular administration. Adhesive coatings composed of biocompatible electrical insulating materials such as vinyl, polyurethane, polyester, polyimide, silicone rubber, thermoplastic elastomer/rubber, ethylene tetrafluoroethylene, fluorinated ethylene propylene and/or perfluoroalkoxy plastics, or Covered with tubing. In this way, the electrode can be configured to be activated only at a certain depth within the tissue.

Collectively, the number of electrodes and the inter-electrode distance, as well as the depth of penetration of the electrodes and the length of the conductive zone, define the volume of tissue to which the electric field is applied. These parameters are selected based on the specific purpose for the administration procedure, including the target tissue site as well as the volume, dose and viscosity of the agent to be delivered, as well as changes in skin and subcutaneous tissue thickness in the intended recipient population. Typically, for intradermal administration in human recipients, the array contains 2-16 electrodes 0.2-0.7 mm in diameter, 2-8 mm electrode-to-electrode distance, 0.5-4 mm electrode penetration depth, and 0.5-4 mm conductive length. do. Typically, for subcutaneous administration in human recipients, the array contains 2-8 electrodes with a diameter of 0.3-0.8 mm, 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, for intramuscular administration in human recipients, the array contains 2-8 electrodes with a diameter of 0.3-1.2 mm, a distance between electrodes of 4-12 mm, an electrode penetration depth of 10-60 mm and a conductive length of 2-20 mm. do.

In order to avoid undesirable visualization and exposure to electrodes during use of the device, the cartridge assembly 100 is preferably configured to recess within the device prior to insertion of the electrode 122 into the recipient's tissue. Most commonly, this is achieved by providing an outer housing structure 102 that slidably engages with the electrode mounting support, and in one embodiment includes an inner cartridge 103 on which the electrode is disposed and appears formed therein. Prior to use, the electrode is placed in a recess within the outer housing 102. During use, the sliding engagement of the electrode mounting structure, for example the inner cartridge 103 with the outer housing, causes the electrode 122 to slide forward with respect to the distal tip of the outer housing 102, so that the outer housing 102 The electrode 122 is developed from. The length of the sliding joint between the electrode mounting structure and the outer housing corresponds to the maximum desired depth of penetration of the electrode for a given application.

As can be seen in Figure 4. The electrodes 122 have a proximal portion 135 and a distal portion 137. The proximal portion is separated from the distal portion by a shoulder or bend 139. The shoulder or bend 139 is secured by and between the inner cartridge cap 104 and the inner cartridge 103. The shoulder or bend 139 serves many functions. First, the electrode at the distal end of the cartridge assembly 100 is required to form an array of a specific size and shape as described above, but the electrode 122 at the proximal end of the cartridge assembly 100 has a desired array size and shape. Placing is impractical due to the presence of the storage unit 101. In other words, the electrode 122 has to be bent "out-of-the-way" to make room for the storage 101. In addition, having the bend, especially when the bend is frictionally locked in place between the inner cartridge 103 and the inner cartridge cap 104, is the electrode 122 when the force of the needle and electrode insertion recoil in the distal direction. ) Provides a surface that the electrode 122 can abut against as it interacts with the tissue during placement. The bend can resist axial rotation, which is beneficial for the electrode to rotate away from the electrical contact pad 130 (described below) without exhibiting significant torsional motion. Also, unlike the prior art, in which a plurality of electrodes are interfaced with axially separated coaxial rings to require a different configuration for each electrode, the present system allows a common electrode type to be used for all four electrodes.

Systems and methods according to the present principles enable the propagation of an electric field in the recipient's skin, subcutaneous tissue and/or skeletal muscle, which facilitates intracellular delivery of the HBV vaccine within the recipient's tissue. In this aspect, the device includes two or more elongate electrodes 122 arranged in a predetermined spatial relationship and configured for placement into a target tissue to which the agent of interest is dispensed or to be dispensed. The desired enhancement of agonist activity depends on achieving co-localization of the agonist distribution site with the propagation of an electric field large enough to induce the desired physiological effect. Accordingly, it is desirable for the present apparatus to consistently achieve electrode placement at a target depth and at a specified interelectrode spacing. Specifically, this ensures that the electric field is propagated in the appropriate tissue site, and the magnitude of the electric field propagated in the tissue is a function of the intraelectrode spacing, thus ensuring safe and effective electric field strength. However, variations in the thickness, density and composition of skin and subcutaneous tissue between sites at a given recipient and across a population of recipients can cause significant changes in electrode placement properties. For example, recipients with increased skin thickness have increased sensitivity to electrode distortion and/or insufficient depth of penetration, which can affect co-localization with the agent dispensing site.

The system and method according to the present principles include 2-16 electrodes with a diameter of 0.2-1.3 mm with a distance between electrodes of 2-12 mm and an electrode penetration depth of 0.5-60 mm in recipients with skin of heterogeneous thickness, composition and condition. Facilitates consistent transdermal insertion of the electrode array. To allow for transdermal insertion, the tissue penetrating electrode is most commonly elongated and consists of a tissue penetrating tip and a tissue contact area in the distal portion and an electrical contact area in the proximal portion. Considering the elongate configuration, it may be vulnerable to bowing, bending, and/or buckling (collectively, torsion) when receiving the compressive force generated during transdermal insertion by the recipient. This distortion during electrode insertion is undesirable because it can lead to inadequate electrode insertion, which is characterized by insufficient penetration depth and/or excessive changes in the spacing within the electrode. Importantly, humans and animals show significant intra-species and inter-species differences in the thickness, composition, and condition of the skin, none of which can significantly affect the force experienced by the electrodes during transdermal insertion. In some embodiments, the device for transdermal insertion of elongate electrodes is designed to withstand the forces present under the most stringent electrode insertion conditions encountered in a target population of recipients. The occurrence of electrode warpage can be partially mitigated by electrode material selection as well as by reducing the length and increasing the diameter of the electrodes in the array. However, material selection may be limited due to issues of performance, cost, manufacturability and biocompatibility. In addition, reduced length electrodes may interfere with adequate coverage of target populations with heterogeneous tissue thickness, whereas larger diameter electrodes are associated with increased discomfort and tissue trauma. Accordingly, the present specification is designed to provide a method and apparatus that facilitates transdermal insertion of an electrode independent of these variables.

In the first embodiment of the present specification, the subassembly cartridge containing the electrode array physically contacts one of the electrodes during tissue insertion and restricts movement of the electrode perpendicular to the insertion direction and establishes a desired spatial relationship between the electrodes. And one or more supporting dynamic support members configured to hold. For example, referring to FIG. 12, there is shown an electrode support feature 124 provided with a hole 192 for the electrode and a hole 194 for the passage of the needle. The tendency of the elongate electrode to warp or bend under load is exacerbated by any bending, eccentricity or bending of the electrode that may have been introduced during manufacture or assembly. Thus, using a device such as electrode support feature 124 to prevent or correct any bending, eccentricity, or warpage that may appear at rest as a result of loads occurring during placement in the tissue and It is advantageous to restrain the electrode to prevent vertical motion.

In the context of the present specification, the dynamic support member provides a sliding engagement with the elongated electrode during insertion into the recipient's tissue, and the position, size during electrode insertion to maintain the desired spatial relationship of the electrode when the electrode is placed at the target tissue depth. And/or a structural element configured to undergo a change of shape. Since the electrode is subjected to the greatest loading force when it first comes into contact with the skin, the coupling of the dynamic support member with the electrode containing the array is affected before the first contact with the recipient's tissue and continues as the electrode is placed to the full penetration depth. It is desirable to provide support. In addition, the dynamic support member is preferably designed to provide support to the electrode and the injection needle while minimizing the loss of electrode penetrating force due to friction.

While the main function of the dynamic support member is to maintain the desired spatial relationship of the plurality of electrodes within the array to each other and to the injection needle, it is also desirable that the design of the dynamic support member includes features that can stabilize the entire array. Do. This is preferably achieved by providing additional structural support features that limit the lateral movement of the support member. In certain embodiments, this support feature is incorporated into a protective shield against sharps, which also serves to protect the user from accidental exposure to electrodes and/or injection needles. Hereinafter, various embodiments of the dynamic support member according to the present specification are disclosed.

Embodiments herein described as electrode support features 124 above include the use of planar support structures 124 positioned perpendicular to the elongate orientation of the electrodes. The planar support structure is configured to have one or more holes 192 corresponding to the positions of the electrodes in a specific spatial relationship. The size, shape and location of the apertures are configured to limit unwanted motion perpendicular to the electrode placement direction while allowing the support structure to slide smoothly along the long length of the electrode within the array. In general, the hole will be configured as a hole or slot 192 in a planar structure having an appropriate clearance to the electrode (at least 10% greater than the maximum cross-section of the electrode, including any coating or other adhesive material). Can However, if it is desired to be more substantial for a particular electrode, one or more of the holes may include a tubular structure 196 arranged perpendicular to the planar support structure. A hole comprising such a tubular structure increases the surface area in contact with the electrode, thereby increasing the support provided to the elongate electrode. The planar support structure can be made of any material with suitable structural properties, including metal, polymer, ceramic, or composite materials, and can be formed, processed, shaped or manufactured by other methods. In order to avoid unwanted electrical interaction with the electrode, it is preferred that the interface between the electrode and the planar support structure is not electrically conductive. In addition, the material and manufacturing method should be selected to minimize the amount of friction at the interface between the electrode and the dynamic support member. Due to a number of factors including low cost, ease of manufacture and desirable electrical properties, electrode support structures are generally made of thermoplastic materials such as polycarbonate, polystyrene, polypropylene, acrylic or polyethylene. Certain materials must be selected to achieve sufficient stiffness to support the elongated electrode while retaining sufficient elasticity to prevent failure of the loaded component. In addition, the material selection should take into account the intended sterilization method for the electrode array (eg, gamma radiation, steam sterilization, ethylene oxide or e-beam) to ensure that the dynamic support members are compatible. The specific dimensions and design of the supporting structure depend on the properties of the material chosen. However, it is desirable to minimize the dimensions of the supporting structure or interfere with other functional properties of the device so as not to unduly limit the distance over which the electrodes can be placed. Typically, a rigid, planar structure 0.5mm-2mm thick is sufficient.

Another embodiment of the dynamic support member is to use a compression spring having a hole to receive the electrode and limit lateral motion. Compression springs can be made of metal, polymer or elastomeric materials, and can be formed, processed, molded or manufactured in different ways. When seated, the spring is not compressed or is compressed to a minimum by an electrode inserted into the hole along the length of the spring. As the electrode is placed, the spring is compressed in the placement direction, and the hole accommodates the sliding motion of the electrode perpendicular to the spring coil. This embodiment is particularly useful when combined with a shield or sheath used to receive the penetrating electrode/needle after it has been removed from the tissue site. In these embodiments, the force imparted to the spring by the forward placement of the electrode may be used to place the sheath or shield over the electrode when the device is removed from the tissue.

In the embodiment of Figure 4, the stick shield spring 138 can be used to partially support the electrode support 124, in particular the radius of the spring to match the radius of the wall 198 of the electrode support 124 (or Can be configured to be slightly larger). In this way, the electrode support 124 can be inserted in the middle of the stick shield spring 138 during use. Next, the electrode support 124 may slide inside the stick shield 134. By being placed in the center of the spring, the electrode support 124 naturally maintains its position at the center of the spring, which supports the electrode approximately intermediate between the support point at the inner cartridge cap 104 and the penetration point at the tissue interface. It provides the desired "halfway point" for.

Other spring-based electrode supports are contemplated. For example, a formed compression spring is placed in the region of the electrode to provide an adaptive electrode support. The formed compression spring can be shaped and proportional to closely match the relative position of the electrode to limit lateral motion. The optional biasing element may be positioned with the formed compression spring and may serve to bias the electrode outward with respect to the formed compression spring. The formed compression spring may be made of a metal, polymer or elastomeric material, and may be formed, processed, molded or manufactured in different ways. The deflection element may be made of a metal, polymer or elastomeric material, and may be formed, processed, shaped or manufactured in other ways.

Electrode supports based on other mechanisms are also contemplated, for example telescoping tubes. The telescopic tube serves to support the electrode while being inserted into the object. The telescopic tubes can be sized to move freely with respect to each other, or they can be sized to move only when an axial force is applied.

Other support structures are contemplated, for example support structures based on movable, flexible or pivoting support members. Lateral support members can be attached to the electrodes at optional hinge features. The side support member may be integrally formed with the structure receiving the electrode or may be a separate component attached by conventional methods (snap, welding, adhesive, fastener, etc.).

Another embodiment is to use a compressible matrix material with an electrode embedded therein. As the electrode is placed, the material is compressed in the placement direction to provide lateral support along the direction of movement. Examples of compressible matrix materials include microcellular plastics, foamed silicones or cellulose such as foamed polychloroprene or carbon foamed matrices, foamed plastics or rubber polymers. Since the material is designed to contact the electrode and/or injection needle (if applicable), the material must be selected to be compatible with indirect tissue contact.

Thus, the structures described above support the transdermal placement of the plurality of elongated electrodes at a tissue depth of up to 60 mm while maintaining the desired spatial relationship between the plurality of electrodes and the orifice of the hypodermic needle in certain embodiments. This support member engages the plurality of elongate electrodes during transdermal insertion and limits the deflection of the electrode in one or more directions perpendicular to the elongate orientation. Another aspect of the present specification is a method that utilizes the application of a biocompatible lubricating compound to the surface of a plurality of electrodes to reduce the applied force required to achieve a constant transdermal placement of the plurality of elongate electrodes to a depth of up to 60 mm, and Provide the device. In an exemplary embodiment, a biocompatible silicone compound such as Dow Corning 360 Medical Fluid or Dow Corning MDX4-4159 comprises an array by conventional spray or dip coating to improve the insertion properties of the electrode. Can be applied to multiple electrodes. The specific choice of coating and application conditions, such as coating method and thickness, depends on the number, size, composition and tip configuration of the electrodes as well as the target tissue in which the electrodes are placed.

4 and 7A-7B, the proximal portion 135 of the electrode 122 may be located outside the inner cartridge 103 of the cartridge assembly 100 (FIG. 7B). In this way, when the inner cartridge 103 is slidably disposed in the outer cartridge 102 (Fig. 7A), the proximal portion 135 is the corresponding electrode contact 130, in particular the outer cartridge of the electrode contact 130 In contact with the inner portion 133, this inner portion 133 is configured to communicate in power with the proximal portion 135. Due to the length of the proximal portion 135 and the length of the outer cartridge inner portion 133, the electrical communication can take place at multiple locations along a continuous interface, regardless of the longitudinal position of the inner cartridge with respect to the outer cartridge. The electrode contact 130 further includes an external cartridge outer contact 131 configured to power communication with a corresponding connector 496 on the applicator 400 (see FIG. 18C). The electrode contact 130 is made of a conductive material sufficient to transmit an electrical signal. In certain embodiments, design and material selection is made with the corresponding electrode over a range of expected manufacturing variations in the electrode and contact location without inhibiting or impeding forward movement of the electrode 122 mounted on the inner cartridge 103. Ensure that the contacts provide proper mating to ensure an electrically conductive interface. In addition, it should be designed to allow this combination to persist when exposed to the expected storage conditions over the indicated shelf life of the product. In certain embodiments, the electrical contact 130 is made of a stamped or formed metal with suitable properties to facilitate engagement with the electrode, and with gold or copper to ensure the integrity of the electrical contact and avoid corrosion. The same coating may be included. In addition to ensuring that electrical contact can be maintained with the electrode at multiple locations along a continuous interface, incorporation of the electrode contact into the external cartridge 102 in this configuration allows the contact between the electrode 122 and the electrode contact 130 It ensures that wear due to the sliding interaction occurs within the cartridge 100 designed for disposable, thereby enabling a static interface between the external cartridge contact 131 and the applicator electrical contact 496, Minimizes potential mechanical wear on the electrode connection 496 of the applicator designed for multi-use. This configuration has the advantage of extending the useful functional life of the versatile applicator.

The remaining portions of applicator 400 are now described. These parts are generally parts independent of the operation of the cartridge assembly 100. Referring first to Fig. 13A, the applicator 400 includes a handle 402 and a multi-conductor cable 406 designed to carry power and control signals as well as electrical signals to be applied to the tissue. Cable 406 generally ends at a connector having a corresponding connector interface in controller 700. The applicator 400 further includes a user interface 404, in which the user can see aspects of the procedure, and the user can instruct the applicator to perform various functions, especially depth selection. The applicator 400 further includes a procedure activation trigger 407 used by the subject to initiate the procedure.

13B, the applicator 400 includes a procedure countdown timer 410, and the procedure countdown timer informs the user of the remaining duration of the procedure, and the countdown trigger counts down to zero. Implicitly indicates to the user that applicator 400 should not be removed from the subject. A power indicator 418 is provided to indicate a satisfactory and powered connection with the controller 700. In the event of a failure, for example, if one of the aforementioned interlocks has not been deactivated, a procedural failure indicator 414 is provided to indicate to the user. An application placement indicator 412 is provided to inform the user that appropriate pressure has been obtained against the subject's tissue to allow the procedure to begin.

14 is an applicator comprising a controller 700, an upper housing 420, a connector 426 (not sized) for connection to the side housings 422, 424 and the inner protective shell 432 Represents a plurality of structural elements of 400. A front cap 430 is provided with an end cap 428. In addition, various electromechanical subassemblies 450 are provided, some of which have been described above.

FIG. 15 shows a more detailed view of the applicator 400, showing the cartridge pressure sensor contacts 434 and subassemblies 452 corresponding to the cartridge loading, electrode insertion and injection functions, and associated sensors.

16 shows a more detailed view of a group of subassemblies 452, including a loading drive subassembly 454 and a cartridge loading subassembly 456. The operation of the subassemblies has been described above.

17A shows a more detailed view of the loading drive subassembly 454, and general details of the operation of the loading drive subassembly have been described above. Here, loading is triggered by a flag on the cartridge assembly 100 and detection of the flag causes the system to be triggered at the switch 644. The loading drive subassembly is mounted to the applicator housing using brackets 466 and 468. The operation of motor 444 is transmitted to pinion gear assembly 448 by motor drive shaft 462.

17B shows a more detailed view of the insertion/scan drive assembly 456. Many of these components have been described above. Here, it is noted that the actuating components are mounted to the applicator housing by means of a mounting bracket 476. After insertion of the electrode 122 and the needle 105, the plunger of the needle 105 is driven by the injection drive plunger 484. It is pressed, and the drug is released from the orifice of the needle by its action. The scan drive plunger is driven by a scan drive motor and gear assembly 486.

In general, the scan drive plunger driven by the scan drive assembly 486 moves forward to a point where it can no longer move distally, indicating that the end of each stroke has been reached. This indication is generally provided as the current used in the scan drive motor 486 increases substantially, indicating that the reservoir plunger has reached the end of the reservoir and the scan is complete. However, in some cases, the number of revolutions of the motor may be used to determine how much drug has been delivered. This feature is to support the administration of the metered drug or when the delivery volume is not within the expected total, for example, the entire volume of the storage unit 101 was expected to be injected but not emptied based on the position of the plunger rod. It can be used to inform the user if not. This situation may occur when the user fails to hold the applicator 400 against the subject's body until the procedure is complete, for example, the countdown timer counts down to zero. As mentioned above, in an intermediate-procedure time frame in which the force on the object is no longer detected, an impedance check can be performed to determine if the electrode is still within the object. If not, it can be assumed that the applicator has been removed prematurely, and a signal is transmitted to the scan drive motor 486 to stop the injection, thereby limiting the amount of the drug released to the outside of the subject.

Referring to FIG. 19, the controller system/assembly 700 can be seen to include an electric field controller/generator 750 and a handle 702 and an applicator cradle 706. The controller can be configured for table top and cart mounting applications. In a cart mounted configuration, it is advantageous to include a storage bin 704. Details of the controller in a cart-mounted configuration include a wheel lock to secure the controller assembly against movement in an operational setup, a tray 710 for placing supplies including a subject preparation supply. , Storage/vial/syringe, etc. shown in FIGS. 20A-20D. Applicator connector port 708 is shown to connect applicator 400 to controller assembly 700. Display 712 indicates the status of the dosing procedure, particularly with respect to the attached IFU.

A cartridge eject button 714 is provided to eject the cartridge assembly 100 from the applicator 400. The menu navigation button 716 allows navigation and manipulation of components as shown in the display 712. A mute button 718 is provided to mute an alert or other audible indicator if desired. A power button 722 is provided to supply power to the unit, which is operable when the main power switch 726 (FIG. 20D) is turned on.

In addition, the display includes a battery indicator 720 that provides an indication of the battery level for which the battery backup system is provided. Such a battery backup system can be included in the controller/generator 750 to accommodate situations in which power loss prevents the mains from supplying power to the unit. Also, this can be used as a backup in case the procedure was initiated with the main power, but main power loss occurred. In this case, logic and control circuitry are implemented to provide an essentially seamless transition from main power to battery power so that the procedure can be completed using battery backup. The controller preferably includes a battery monitoring circuit capable of monitoring whether the battery has a sufficient amount of charge to complete the procedure after main power loss. In some embodiments, the controller also includes a display to notify the user if the current state of charge of the battery is not sufficient to complete the procedure in case of main power loss.

Referring to FIG. 20D, which is a rear view of the controller 750, it can be seen that a USB port 724, a main power switch 726, and a main power input unit 728 are included.

Referring to Figure 21, in one method of use, as shown by flow chart 800, controller/generator 750 is powered up and the program automatically starts (step 802). If the applicator is connected (step 804) and the applicator is not yet connected, an indication or instruction to the user to perform this operation may be displayed on the display screen 712. The system can perform a self test (step 806), and the self test not only ensures proper operation of the controller/generator 750, but also the controller/generator 750 of the applicator 400. Ensure the correct connection.

The program may cause the display screen to provide instructions to the user regarding preparation of the administration site (step 808). These steps may include ensuring that correct medication is being delivered, that medication has not expired, contraindications have been reviewed, and warnings/precautions have been followed.

Next, the user removes the storage cap and inserts the storage unit 101 into the cartridge assembly 100 (step 810). In some embodiments, the user experiences an audible, tactile, or tactile click (step 811) indicating proper placement of the reservoir within the cartridge assembly.

Next, the user inserts the cartridge assembly 100 into the applicator 400 (step 816). Next, an error condition is tested (step 818), for example, tested for proper storage placement, and if one is detected, the procedure is stopped, an error message is displayed, and a remedial action is taken to the user. Instructed to take. If the error condition can be corrected, for example, if the user has inserted the reservoir improperly but has not engaged with the cartridge breach or has not closed the cartridge breach, the user may be instructed to remove the cartridge and reinsert the reservoir correctly. (Step 820). In some cases the cartridge will be ejected automatically, in other cases the user may have to press the "Eject cartridge" button to get the same result. In other error conditions, for example the cartridge breach closed, the user is given a new cartridge. Can be instructed to use.

In any case, if the device setup is complete and the "no error" status is achieved, the user can proceed with medication administration and electroporation therapy (step 822).

The main functions of the controller are to generate the electric field required to achieve the desired drug delivery, control the system operation during setup and use, monitor the system status during setup and use, and communicate the system status to the user during installation and use. will be. In some embodiments, the controller may provide recommendations and instructions for use of the device during resolution of fault conditions during normal use, as well as in terms of user training.

The controller system and the controller operating method can be fully implemented using a variety of computing devices, including microprocessors, microcontrollers, and programmable logic controllers. In general, instructions are placed on a computer-readable medium, and are generally non-transitory, and such instructions are sufficient to enable a processor to implement the methods herein on a computing device. The computer-readable medium may be a hard drive or solid state storage with instructions loaded into random access memory when executed. For example, input from multiple users or from any one user to an application may be by any number of suitable computer input devices. For example, a user may use a keypad, keyboard, mouse, touch screen, joystick, trackpad, other pointing device, or other computer input device to input data related to calculation. In addition, data may be input by an inserted memory chip, hard drive, flash drive, flash memory, optical medium, magnetic medium or any other type of file-storage medium. The output may be delivered to the user by means of a video graphics card or an integrated graphics chipset coupled to a user viewable display. Alternatively, the system can output one or more electronic document formats or a printer can be used to output a hard copy of the result. Given this teaching, it is understood that any number of other substantial outputs will also be contemplated by this specification. For example, the output may be stored on a memory chip, hard drive, flash drive, flash memory, optical medium, magnetic medium, or any other type of output device. In addition, the present specification may be any number such as a personal computer, a laptop computer, a notebook computer, a netbook computer, a handheld computer, a personal digital assistant, a mobile phone, a smart phone, a tablet computer. It should be noted that it can be implemented in various types of computing devices, and in devices specifically designed for this purpose. In one implementation, a user of a smart phone or wi-Fi connected device downloads a copy of the application from a server using a wireless Internet connection to the device. Appropriate authentication procedures and secure transaction processes can be provided for payments made to the seller. The application can be downloaded via a mobile connection or WiFi or other wireless network connection. Then the application can be executed by the user. Such a network system can provide a computing environment suitable for implementations in which multiple users provide separate inputs to the present system and method. In the system below where the control of the applicator is considered, multiple inputs are multiple users. Can be made to enter the relevant data at the same time.

Examples

The following examples of the present invention are intended to further illustrate the essence of the present invention. It is to be understood that the examples below do not limit the present invention and that the scope of the present invention should be determined by the appended claims.

Example 1. Generation of HBV core and Pol antigen sequences and plasmid optimization

T-cell epitopes on the hepatitis core protein are believed to be important in the elimination of hepatitis B infection, and hepatitis B virus proteins such as polymerase may play a role in enhancing the breadth of the response. Therefore, the hepatitis B core and polymerase proteins were selected as antigens for the design of a therapeutic hepatitis B virus (HBV) vaccine against the antigen.

Derivation of HBV core and polymerase antigen consensus sequence

HBV pol and core antigen consensus sequences were generated based on HBV genotypes B, C and D. Different HBV sequences were obtained from different sources and were aligned separately for the core and polymerase proteins. Subsequently, the original sequence alignments for all subtypes (A-H) were restricted to HBV genotypes B, C and D. Consensus sequences were defined for each protein alignment individually in each subtype and in all joint BCD sequences. At variable alignment positions, the most frequent amino acids were used in the common sequence.

Optimization of HBV core antigen

The HBV core antigen consensus sequence was optimized by removal of the native viral protein. In particular, the removal of 34 amino acids was achieved in response to a largely positively charged segment of the C-terminus, which is required for pre-genomic RNA encapsulation.

Optimization of HBV Pol antigen

The HBV pol antigen consensus sequence was optimized by changing four residues to eliminate reverse transcriptase and RNAseH enzyme activities. In particular, aspartate (aspirate) residue (D) is changed from "YXDD" motif (motif) of the reverse transcriptase domain to asparagines residue (N) to any coordination function (coordination function) and thus nucleotide / metal ion The binding was removed. In addition, the first aspartate residue (D) is changed to an asparagine residue (N), and the first glutamate residue (E) is changed from the "DEDD" motif of the RNAseH domain to a glutamine residue (A). Changed to remove the Mg2+ coordination. In addition, the sequence of the HBV pol antigen scrambled the inner open reading frame for the envelope protein, including versions of the S protein and the S protein with N-terminal extension pre-S1 and pre-S2. It has been optimized to scrambe codons. As a result, open reading frames for envelope proteins (pre-S1, pre-S2 and S proteins) and X protein were removed.

Optimization of HBV core and Pol antigen expression strategy

Three different strategies were tested to obtain maximal and identical expression of both the core and pol antigens from the plasmid vector: (1) fusion of the HBV core and pol antigens in a small AGAG and frame inserted between the coding sequences. Resulting in a single core-Pol fusion protein (FIG. 25A); (2) individual from a single mRNA by expression of both core and pol antigens from one plasmid by the slippage site of the ribosome, in particular the FA2 slip site from foot-and-mouth disease (FMDV) Generating a biscistronic expression vector expressing the core and pol proteins (FIG. 25B); And (3) two separate plasmid encodings for the HBV core and pol antigen (FIG. 25C ).

In vitro expression analysis

The coding sequences of the common HBV core and pol antigen according to each of the above three expression strategies were cloned with a commercially available expression plasmid pcDNA3.1. HEK-293T cells were transfected with a vector and protein expression was converted to HBV core. -Evaluated by Western blot using a core-specific antigen.

Optimization of regulatory elements after transcription

Four different post-transcriptional regulatory elements are evaluated for enhancement of protein expression by stabilizing the primary transcript, facilitating nuclear export, and/or improving transcriptional-translational coupling. It became: (1) Woodchuck HBV post-transcriptional regulatory element (WPRE) (GenBank: J04514.1); (2) an intron/exon sequence derived from a human apolipoprotein A1 precursor (GenBank: X01038.1); (3) untranslated R-U5 domain of human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) (GenBank: KM023768.1); And (4) a composite sequence of HTLV-1 LTR, a synthetic rabbit globin intron (GenBank: V00882.1) and a splicing enhancer (triple composite sequence). The enhancer sequence was introduced between the CMV promoter and the HBV antigen coding sequence in the plasmid. No significant difference was observed by Western blot in the expression of the core antigen when expressed from the plasmid in the presence and absence of the WPRE element (Fig. 25D). However, when the expression of the core antigen in HEK293T cells infected cells from the plasmid with the other three post-transcriptional regulatory sequences was evaluated by Western blot, the triplet enhancer sequence resulted in the strongest core antigen expression (Fig. 25E).

Selection of signal peptides for efficient protein secretion

Three different signal peptides introduced into the framework at the N-terminus of the HBV core antigen were evaluated: (1) Ig heavy chain gamma signal peptide SPIgG (BAA75024.1); (2) Ig heavy chain epsilon signal peptide SPIgE (AAB59424.1); And (3) cystatin S precursor signal peptide SPCS (NP_0018901.1). The signal peptide cleavage site was optimized in silico for core fusion using the signal P prediction program. The secretion efficiency was determined by analyzing the core protein level of the supernatant. Western blot analysis of the secretion of the core antigen using three different signal peptides fused at the N-terminus resulted in the cystatin S signal peptide being the most efficient protein secretion. Proved to be (FIG. 25F).

DNA vaccine vector optimization

The optimized expression cassette comprising the triple complex enhancer sequence upstream of the HBV antigen coding sequence with the N-terminal cystatin S signal peptide sequence is the DNA vaccine vector pVax-1 (Life Technologies, Thermo Fisher Scientific, Waltham, Massachusetts)) The expression cassette of pVax-1 contains the human CMV-IE promoter followed by a bovine growth hormone (BGH) induced polyadenylation sequence (pA). Bacterial propagation is driven by pUC duck replicon and kanamycin resistance gene (Kan R) driven by small eukaryotic promoter. driven by pUC duck replication, KanR expression cassette and CMV-IE promoter All of the resulting expression cassettes are in the same orientation within the plasmid backbone. However, a significant reduction in core antigen expression was observed in the pVax-1 vector compared to the expression level observed in the pcDNA3.1 vector.

Several strategies have been used to improve protein expression: (1) reversal of the entire pUCori-KanR cassette in a counterclockwise orientation (pVD-core); And (2) altering the codon use of the KanR gene with the replacement of the Kan promoter from the pcDNA3.1 vector with the Amp promoter (pDK-core). Both strategies restore core antigen expression, but core antigen expression was highest with the pDK vector, which included the codon-regulated Kan R gene, the AmpR promoter (instead of the KanR promoter) and reverse orientation of the pUCori-KanR cassette.

As shown in Figure 25g, four different HBV core/pol antigen-optimized expression cassettes were introduced into the pDK plasmid backbone to test each of the three expression strategies illustrated in Figures 25A-25C. Plasmids were tested in vitro for core and pol antigen expression by Western blot analysis using core and pol specific antibodies. The most consistent expression profiles of cells and for secreted core and pol antigens were achieved when the core and pol antigens were encoded by separate vectors, i.e. the individual DNA vectors pDK-core and pDK-pol (Figure 25h). pDK- Schematic diagrams of the pol and pDK-core vectors are shown separately in Figures 26A and 26B.

Example 2. Electroporation-mediated intramuscular administration of nucleic acid-based biopharmaceuticals by TriGrid delivery system (TDS-IM) device

Intracellular delivery of nucleic acid sequences in the skeletal muscle of the upper or lower extremities of a subject can be enhanced using an exemplary device, for example the TriGrid Delivery System (TDS-IM) Model II as provided herein. In some embodiments, the TDS-IM device is used with a formulation approved for irradiation use with the TDS-IM device. In an exemplary embodiment, the approved agent is a nucleic acid, i.e. DNA or RNA. In some embodiments, the use of a TDS-IM device is limited to those who need it. In some embodiments, the use of the TDS-IM device is limited to subjects enrolled in open clinical trials of electroporation mediated intramuscular nucleic acid delivery.

To start the system for administration, the main power of the device is connected to a stimulator and the system battery is sufficiently charged for use. The main power switch is turned on and the front panel power button is pressed. The applicator is connected to the applicator connector. Correct connection of the applicator is verified by the illumination of the applicator power indicator. When the system has completed all self-checks, a welcome screen appears. The OK button is pressed to proceed with the dosing procedure.

To insert the syringe into the TDS cartridge, the syringe cap is removed from the syringe and the syringe flange is aligned with the TDS cartridge syringe loading port. The syringe should snap into place and fully seat on the TDS cartridge. When the syringe is loaded, the OK button is pressed on the pulse stimulator to continue. The cartridge cap should remain attached to the cartridge until the cartridge is loaded into the applicator.

To insert the syringe loaded cartridge into the applicator, the cartridge is aligned with the applicator so that the cartridge syringe loading port is facing up. When the cartridge is inserted into the applicator, the cartridge is automatically pulled out of the applicator to the fully loaded position. When the cartridge is successfully loaded, it is indicated on the stimulator. When the cartridge is loaded, the applicator returns to the cradle, and an appropriate injection site for the subject is selected. In some embodiments, the injection site for intramuscular nucleic acid delivery is the medial deltoid muscle approximately 3 fingers wide below the edge of the acromion process (shoulder bone). In an exemplary embodiment, the depth of injection in the medial deltoid muscle is about 0.75"-1.25" (19-30 mm). In some embodiments, the injection site for intramuscular nucleic acid delivery is the vastus lateralis muscle (lateral thigh) at approximately midpoint between the hip and knee. In an exemplary embodiment, the injection depth in the lateral vastus is about 1.0"-1.5" (25-38mm). When the injection site is selected, the applicator depth selection button is pressed following the injection depth selection button corresponding to the injection site/depth. In some embodiments, the scanning depth selection indicator turns into solid illumination to confirm the selected scanning depth. In some embodiments, the depth select button on the right corresponds to a deeper scan depth. In one embodiment, the initially selected injection site has to be changed, a select button corresponding to a different scan depth is pressed, and a different scan depth is selected when the select scan depth screen returns.

To begin administering the approved formulation to the subject via the TDS-IM device, the cartridge cap is removed and discarded. The device is aligned with the target injection site and pressed tightly against the site. When the device is pressed firmly against the target injection site, all four bars of the applicator placement indicator illuminate and the procedure countdown timer illuminates "8" seconds, indicating the time remaining in the dosing procedure. The applicator trigger is depressed so that the formulation is administered while the pressure is maintained consistently. When the procedure countdown timer reaches "0", electrical stimulation is delivered. When the dosing procedure is complete, the procedure completion indicator lights up and the device can be removed from the injection site. The device cannot be withdrawn from the injection site until the procedure is complete or the procedure fault indicator is lit. In some embodiments, the device detects a problem during the dosing procedure, and the device stops the dosing procedure and illuminates a procedure failure indicator. In an exemplary embodiment, the device stops the dosing procedure and the device is immediately removed from the subject, and the device's stimulator display provides additional instructions.

The eject button located on the stimulator is pressed to eject the cartridge from the applicator after completing the drug administration to the subject in need. The applicator automatically advances the cartridge to a position that can be manually removed from the applicator. When the cartridge stops moving, it can be pulled out of the applicator by holding the side of the cartridge indicated by the arrow. After the cartridge is removed, the completion of the entire injection can be confirmed by inspecting the syringe plunger position. To turn off the device, the applicator is placed in a holster, and the front panel power button is pressed for 5 seconds.

Example 3. TDS-IM electroporation for rat and non-human primates

TriGrid Delivery System-Intramuscular (TDS-IM) electroporation technology is used to enhance the delivery of DNA-based structures in muscle tissue. The TriGrid electrode array for intramuscular (IM) delivery consists of four electrodes arranged in two equilateral triangles to form a diamond shape surrounding a central injection needle (Fig. 22). The integration of agent delivery and electric field propagation into a single device ensures that the induction of the electroporation effect occurs at the site of agent dispensing and allows injection of the plasmid structure and application of electrical pulses in a single step. In this way, IM delivery of plasmid DNA is achieved in an effective and reproducible manner.

The TriGrid electrode array for each animal model is scaled to efficiently deliver DNA plasmids such as those prepared in Example 1 to selected muscles, regardless of size and overlying tissue characteristics. The scaled primary electroporation device parameters for use in each animal model are TriGrid size (i.e. electrode spacing), applied voltage (250V/cm, where cm is the TriGrid size), electrode diameter, and electrode penetration depth. Secondary adaptations to the device include changes in syringe volume, plasmid DNA volume and hypodermic syringe needle gauge/length.

Rat studies were performed with a TDS-IM v1.0 device using an electrode array with 2.5 mm spacing between the electrodes. This adjustment of electrode array size is intended to accommodate smaller sized rat muscles. 23A depicts a version of TDS-IM v1.0 TriGrid suitable for use in a rat model. In addition, non-GLP monkey studies were performed with a TDS-IM v1.0 device, but with 6 mm electrode array spacing. 23B shows a TDS-IM v1.0 TriGrid version suitable for use in a non-human primate (NHP) model.

The device used in the GLP monkey study was a TDS-IM v2.0 device also aimed at human clinical studies. These devices have 6mm electrode spacing and the same activation conditions as the TDS-IM v1.0 device. The electrode material of the TDS-IM v2.0 device is the same as the TDS-IM v1.0 device used in the NHP study described above, but The cartridge support structures and syringes used for administration are different. Also, the depth of insertion for humans and non-human primates is different. Thus, a spacer was mounted on the TDS-IM v2.0 device to adjust the TDS-IM v2.0 device for use in much smaller NHP muscles. 24 shows a TDS-IM v2.0 TriGrid version suitable for use in a non-human primate (NHP) model.

In each animal study, animals were anesthetized to immobilize the animals prior to the electroporation administration procedure. The site of administration was positioned using the local bone anatomical landmark as a reference point, the hair was removed over the selected area with an electric hair clipper and followed by a sterile swab. A TDS-IM array with a diamond-shaped major axis oriented parallel to the muscle fibers. It was inserted percutaneously into selected muscles. After the electrode was inserted, the injection was started and the DNA was distributed to the muscle. After the IM injection was completed, a 250V/cm electric field was applied locally for a total of 400 ms time at a 10% duty cycle (i.e., a total of 40 ms out of 400 ms time). While voltage is actively applied). Upon completion of the electroporation procedure, the TriGridTM array was removed and the animals recovered. The variable elements for each TDS-IM version and/or animal model are as in Tables 1 and 2 below.

Table 1: TDS-IM version and TriGrid details TDS-IM version Animal model TriGrid size (mm) Electrode diameter (in) Conductive length (mm) Effective penetration depth (mm) DNA injection volume (cc) Syringe volume (cc) Injection needle gauge Injection method TDS-IM v1.0 rat 2.5 0.030 3.2 3.2 0.020 3/10 cc 30 manual TDS-IM v1.0 NHP 6.0 0.021 5.0 15.5 1.0 1.0 cc 22 Automatic TDS-IM v2.0 NHP 6.0 0.023 5.0 9.0* 1.0 1.0 cc 22 Automatic * NHP weight and muscle size required shorter penetration depth; A 10mm depth limiter was used to reduce the penetration depth to 9mm.

Table 2: TDS-IM version and pulse stimulator details TDS-IM version Animal model Applied electric field (V/cm) Applied voltage range (V) Applied current limit (A) Applied current limit (A/sec) Total number of pulses Active stimulation time (ms) Maximum sequence time (ms) TDS-IM v1.0 rat 250 59.4-65.6 N/A*-4 0.16 6 40.8 369.3 TDS-IM v1.0 NHP 250 142.4-157.6 0.6-4 0.16 6 40.8 369.3 TDS-IM v2.0 NHP 250 142.4-157.6 0.6-4 0.16 6 40.8 369.3 *Low current limit cannot be used to accommodate rat model characteristics.

Example 4. TDS-IM electroporation for humans

The TDS-IM v2.0 device is used in clinical studies. The device is essentially the same device used in the GLP monkey. The depth of insertion for humans and non-human primates is different. Thus, a spacer was mounted on the TDS-IM v2.0 device to tune the TDS-IM v2.0 device for use in much smaller NHP muscles. Stimulation conditions were essentially the same as those used for GLP monkeys, except that the penetration depth was about 19 mm instead of 9 mm as in GLP monkeys.

While the preferred embodiments herein have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, changes and substitutions will occur to the skilled artisan without departing from this specification. It should be understood that various alternatives to the embodiments described herein, or combinations of one or more of these embodiments or aspects described herein, may be used in practicing the invention. The following claims define the scope of the invention, and methods and structures within the scope of these claims, and equivalents thereto, are intended to be covered by the scope of the invention.

Table of elements
Number of parts
100 cartridge assembly
101 storage
102 external cartridge (or housing)
103 internal cartridge
104 inner cartridge cap
105 needles
106 outer cartridge cap
108 Alignment Guide/Display Features
110 outer (safe) cartridge cap
112 cartridge breech
114 cartridge locking ring
116 Reservoir detection spring
118 reservoir sensing cap
120 Storage interlock (or trigger to insert storage)
122 electrodes
124 electrode support
126 force contact spring
128 force contact pickup
130 electrode contacts
131 outer cartridge electrode portion on the outside for bonding to the applicator
132 Stick Shield Support
133 inner outer cartridge electrode portion for mating to inner cartridge
134 stick shield
135 Proximal inner cartridge electrode portion for bonding to an outer cartridge
137 Distal inner cartridge electrode portion for tissue insertion
138 Force Contact Flexible Circuit
139 Electrode shoulder or bend
140 Storage loading port
142 Storage volume
144, 144' storage lockout hole (first or second set)
146 optical gaze
148 Insertion detector (e.g. emitter/collector IR sensor in applicator)
150 internal cartridge receiving volume
152 needle hub
154 rack
156 exit port
158 reservoir cap
159 plunger stopper
160 flexible circuit
162 first pad set
164 second pad set
166 Stick Shield Nerve
167 stick shield hole
168 Splay Features
170 Alignment guide hole for stick shield
172 start flag
174 consecutive flags
176 outer cartridge cap hook
178 outer cartridge cap chamfer surface
180 Alignment guide/hook-engagement wall of display shield (108)
182 stick shield retaining hook
184 First Depth Maintenance Wall
186 Second Depth Maintenance Wall
188 Initial or Rest Retaining Wall
190 reminder tab
192 electrode support feature electrode hole
194 electrode support feature needle hole
196 Electrode support feature Electrode hole support structure
198 electrode support feature wall
400 applicator
401 applicator cartridge assembly receiving port
402 handle
Cartridge assembly receiving volume defined by 403 housing
404 user interface
406 multi-conductor cable
407 trigger
408 scan depth selection indicator(s)
409 Scan Depth Select Button(s) (may be a toggle or other form in other implementations)
410 procedure countdown timer
412 Apply placement indicator
414 procedural fault indicator
416 procedure completion indicator
418 power indicator
420 upper housing
422 first side housing
424 second side housing
426 electrical connector
428 end cap
430 front cap
432 inner protective shell
Electrical contact for 433 motor drive 444
434 cartridge force sensor contacts
Connector for 435 switch
436 cartridge loading sensor
438 cartridge loaded sensor
440 guide or track
442 cartridge guide rail
444 loading drive motor
446 motor trigger connector
448 pinion gear assembly
450 Electromechanical subassembly
452 cartridge loading, electrode insertion and injection subassembly
454 loading drive subassembly
456 cartridge loading subassembly
462 motor drive shaft
464 system trigger switch
466 gear cover bracket
468 mounting bracket
470 spring cover/cartridge interface
471 spring cover hole
472 electrode/needle insertion spring
474 insert gear bushing
476 mounting bracket
478 Insertion mechanism gear drive ring
479 insert gear ring
480 flag holder
481 Insertion mechanism flag
482 Insertion Mechanism Drive Motor
483 Insertion Mechanism Position Sensor
484 injection driven plunger
486 scan drive motor and gearing
488 retention posts (retention features)
490 Channel for the first depth
491 locking tab
492 Channel for second depth
494 contact wall
496 Applicator Electroporated Electrode Contact
700 controller assembly
702 handle
704 storage bin
706 applicator cradle
708 applicator connector port
710 tray
712 display screen
714 cartridge eject button
716 menu navigation buttons
718 mute button
720 battery indicator
722 power button
724 USB port
726 main power switch
728 main power port
750 electric field generator

SEQUENCE LISTING <110> JANSSEN SCIENCES IRELAND UNLIMITED COMPANY ICHOR MEDICAL SYSTEMS, INC. <120> METHODS AND APPARATUS FOR THE DELIVERY OF HEPATITIS B VIRUS (HBV) VACCINES <130> 688097-405WO1 <150> PCT/US2017/067269 <151> 2017-12-19 <150> US 62/607,430 <151> 2017-12-19 <160> 24 <170> PatentIn version 3.5 <210> 1 <211> 444 <212> DNA <213> Artificial Sequence <220> <223> HBV truncated core antigen gene <400> 1 gacatcgacc cttacaagga gttcggcgcc agcgtggaac tgctgtcttt tctgcccagt 60 gatttctttc cttccattcg agacctgctg gataccgcct ctgctctgta tcgggaagcc 120 ctggagagcc cagaacactg ctccccacac cataccgctc tgcgacaggc aatcctgtgc 180 tggggggagc tgatgaacct ggccacatgg gtgggatcga atctggagga ccccgcttca 240 cgggaactgg tggtcagcta cgtgaacgtc aatatgggcc tgaaaatccg ccagctgctg 300 tggttccata ttagctgcct gacttttgga cgagagaccg tgctggaata cctggtgtcc 360 ttcggcgtct ggattcgcac tccccctgct tatcgaccac ccaacgcacc aattctgtcc 420 accctgcccg agaccacagt ggtc 444 <210> 2 <211> 148 <212> PRT <213> Artificial Sequence <220> <223> HBV truncated core antigen <400> 2 Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu Ser 1 5 10 15 Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp Thr 20 25 30 Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser 35 40 45 Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu 50 55 60 Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser 65 70 75 80 Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile 85 90 95 Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu 100 105 110 Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 115 120 125 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu 130 135 140 Thr Thr Val Val 145 <210> 3 <211> 2529 <212> DNA <213> Artificial Sequence <220> <223> HBV pol antigen gene <400> 3 atgcccctgt cttaccagca ctttagaaag cttctgctgc tggacgatga agccgggcct 60 ctggaggaag agctgccaag gctggcagac gaggggctga accggagagt ggccgaagat 120 ctgaatctgg gaaacctgaa cgtgagcatc ccttggactc ataaagtcgg caacttcacc 180 gggctgtaca gctccacagt gcctgtcttc aatccagagt ggcagacacc atcctttccc 240 aacattcacc tgcaggagga catcattaat agatgcgaac agttcgtggg acctctgaca 300 gtcaacgaaa agaggcgcct gaaactgatc atgcctgcca ggttttaccc aaatgtgact 360 aagtatctgc cactggataa gggcatcaag ccttactatc cagagcacct ggtgaaccat 420 tacttccaga ctagacacta tctgcatacc ctgtggaagg ccggaatcct gtacaaacga 480 gaaactaccc ggagtgcttc attttgtggc tccccatatt cttgggaaca ggagctgcag 540 catggcaggc tggtgttcca gaccagcaca cgccacgggg atgagtcctt ttgccagcag 600 tctagtggca tcctgagcag atcccccgtg gggccttgtc tgcagtctca gctgcggaag 660 agtagactgg gactgcagcc acagcaggga cacctggcac gacggcagca gggaaggtct 720 ggcagtatcc gggctagagt gcatcccaca actagaaggc ctttcggcgt cgagccatca 780 ggaagcggcc acaccacaaa caccgcatca agctcctcta gttgcctgca tcagtcagcc 840 gtgagaaagg ccgcttacag ccacctgtcc acatctaaaa ggcactcaag ctccgggcat 900 gctgtggagc tgcacaacat ccctccaaat tctgcacgca gtcagtcaga aggacccgtg 960 ttcagctgct ggtggctgca gtttcggaac tcaaagcctt gcagcgacta ttgtctgagc 1020 catattgtga atctgctgga ggattggggc ccttgtaccg agcacgggga acaccatatc 1080 aggattccac gaacaccagc acgagtgact ggaggggtgt tcctggtgga caagaacccc 1140 cacaatacta ccgagagccg gctggtggtc gatttcagtc agttttcaag aggcaacaca 1200 agggtgtcat ggcccaaatt cgccgtccct aatctgcaga gtctgactaa cctgctgtct 1260 agtaatctga gctggctgtc cctggacgtg tccgcagcct tttaccacct gcctctgcat 1320 ccagctgcaa tgccccatct gctggtgggg tcaagcggac tgagtcgcta cgtcgcccga 1380 ctgtcctcta actcacgcat cattaatcac cagcatggca ccatgcagaa cctgcacgat 1440 agctgttccc ggaatctgta cgtgtctctg ctgctgctgt ataagacatt cggcagaaaa 1500 ctgcacctgt acagccatcc tatcattctg gggtttagga agatcccaat gggagtggga 1560 ctgagcccct tcctgctggc acagtttacc tccgccattt gctctgtggt ccgccgagcc 1620 ttcccacact gtctggcttt ttcctatatg aacaatgtgg tcctgggcgc caaatccgtg 1680 cagcatctgg agtctctgtt cacagctgtc actaactttc tgctgagcct ggggatccac 1740 ctgaacccaa ataagactaa acgctggggg tacagcctga atttcatggg atatgtgatt 1800 ggatcctggg ggaccctgcc acaggagcac atcgtgcaga agatcaagga atgctttcgg 1860 aagctgcccg tcaacagacc tatcgactgg aaagtgtgcc agcggattgt cggactgctg 1920 ggcttcgccg ctccctttac ccagtgcggg tacccagcac tgatgcccct gtatgcctgt 1980 atccagtcta agcaggcttt cacctttagt cctacataca aggcattcct gtgcaaacag 2040 tacctgaacc tgtatccagt ggcaaggcag cgacctggac tgtgccaggt ctttgcaaat 2100 gccactccta ccggctgggg gctggctatc ggacatcagc gaatgcgggg cacattcgtg 2160 gcccccctgc ctattcacac tgctcagctg ctggcagcct gctttgctag atctaggagt 2220 ggagcaaagc tgatcggcac cgacaatagt gtggtcctgt caagaaaata cacatccttc 2280 ccatggctgc tgggatgtgc tgcaaactgg attctgaggg gcaccagctt cgtgtacgtc 2340 ccctcagccc tgaatcctgc tgacgatcca tcccgcgggc gactgggact gtaccgacct 2400 ctgctgagac tgcccttcag gcctacaact ggccggacat ctctgtatgc cgattcacca 2460 agcgtgccct cacacctgcc tgacagagtc cactttgctt cacccctgca cgtcgcttgg 2520 cggcctcca 2529 <210> 4 <211> 843 <212> PRT <213> Artificial Sequence <220> <223> HBV pol antigen <400> 4 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp Asp 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pro 65 70 75 80 Asn Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Glu Gln Phe Val 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pro 100 105 110 Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Leu Val Asn His Tyr Phe Gln Thr 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu 165 170 175 Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Thr Arg His 180 185 190 Gly Asp Glu Ser Phe Cys Gln Gln Ser Ser Gly Ile Leu Ser Arg Ser 195 200 205 Pro Val Gly Pro Cys Leu Gln Ser Gln Leu Arg Lys Ser Arg Leu Gly 210 215 220 Leu Gln Pro Gln Gln Gly His Leu Ala Arg Arg Gln Gln Gly Arg Ser 225 230 235 240 Gly Ser Ile Arg Ala Arg Val His Pro Thr Thr Arg Arg Pro Phe Gly 245 250 255 Val Glu Pro Ser Gly Ser Gly His Thr Thr Asn Thr Ala Ser Ser Ser 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Ala Ala Tyr Ser His 275 280 285 Leu Ser Thr Ser Lys Arg His Ser Ser Ser Gly His Ala Val Glu Leu 290 295 300 His Asn Ile Pro Pro Asn Ser Ala Arg Ser Gln Ser Glu Gly Pro Val 305 310 315 320 Phe Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Asp 325 330 335 Tyr Cys Leu Ser His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cys 340 345 350 Thr Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Thr 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Asn Thr 385 390 395 400 Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Ala 420 425 430 Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Met Pro His Leu Leu 435 440 445 Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn 450 455 460 Ser Arg Ile Ile Asn His Gln His Gly Thr Met Gln Asn Leu His Asp 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Thr 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Phe 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gln 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cys 530 535 540 Leu Ala Phe Ser Tyr Met Asn Asn Val Val Leu Gly Ala Lys Ser Val 545 550 555 560 Gln His Leu Glu Ser Leu Phe Thr Ala Val Thr Asn Phe Leu Leu Ser 565 570 575 Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Ser 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gln 595 600 605 Glu His Ile Val Gln Lys Ile Lys Glu Cys Phe Arg Lys Leu Pro Val 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Leu 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pro 645 650 655 Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Thr 660 665 670 Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn Leu Tyr Pro Val Ala 675 680 685 Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asn Ala Thr Pro Thr 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg Gly Thr Phe Val 705 710 715 720 Ala Pro Leu Pro Ile His Thr Ala Gln Leu Leu Ala Ala Cys Phe Ala 725 730 735 Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Val 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Leu 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pro 785 790 795 800 Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gly Arg Thr Ser Leu Tyr 805 810 815 Ala Asp Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Phe 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 <210> 5 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Cystatin S signal peptide coding sequence <400> 5 atggctcgac ctctgtgtac cctgctactc ctgatggcta ccctggctgg agctctggcc 60 agc 63 <210> 6 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Cystatin S signal peptide sequence <400> 6 Met Ala Arg Pro Leu Cys Thr Leu Leu Leu Leu Met Ala Thr Leu Ala 1 5 10 15 Gly Ala Leu Ala Ser 20 <210> 7 <211> 586 <212> DNA <213> Artificial Sequence <220> <223> hCMV promoter sequence <400> 7 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 60 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 120 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 180 actttccatt gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat 240 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 300 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 360 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 420 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 480 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 540 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctc 586 <210> 8 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> triple enhancer regulatory sequence <400> 8 ggctcgcatc tctccttcac gcgcccgccg ccctacctga ggccgccatc cacgccggtt 60 gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt 120 aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga gcctacctag 180 actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctagt tctctcgtta 240 acttaatgag acagatagaa actggtcttg tagaaacaga gtagtcgcct gcttttctgc 300 caggtgctga cttctctccc ctgggctttt ttctttttct caggttgaaa agaagaagac 360 gaagaagacg aagaagac 378 <210> 9 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Bla promoter sequence <400> 9 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 60 ccctgataaa tgcttcaata atattgaaaa aggaagagt 99 <210> 10 <211> 671 <212> DNA <213> Artificial Sequence <220> <223> pUC ori sequence <400> 10 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 60 ttgcaaacaa aaaaaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 120 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 180 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 240 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 300 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 360 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 420 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 480 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 540 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 600 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 660 tttgctcaca t 671 <210> 11 <211> 225 <212> DNA <213> Artificial Sequence <220> <223> bGH polyadenylation signal sequence <400> 11 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225 <210> 12 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> Kan R gene <400> 12 atgattgagc aagatggtct tcacgctggc tcgccagctg cgtgggtgga acgcctgttt 60 ggttatgatt gggcgcagca gactattgga tgttccgacg cggctgtatt tcggctgtct 120 gctcagggtc gccccgtgct gtttgtgaag acggatttgt ctggcgcatt aaatgagtta 180 caggacgagg cggctcgtct gagttggttg gccaccaccg gcgtgccctg cgccgcagtg 240 ctggatgtcg tgacagaagc aggccgcgat tggctccttc tcggcgaagt gccgggccag 300 gacctgctca gcagccactt ggcaccggca gaaaaagttt ctatcatggc cgacgccatg 360 cgtcgtcttc acactctcga tccggccacg tgcccctttg accaccaggc caagcatcgt 420 attgaacgtg cgcgtactcg gatggaagca ggtttagtag accaggacga tttggatgag 480 gaacatcaag gcctggcccc ggctgaactg tttgcgcgct taaaagcgtc gatgccagat 540 ggcgaagatt tggtagtcac ccatggagat gcgtgtttgc caaacatcat ggttgaaaat 600 ggccgcttct caggctttat tgactgtggg cgcctgggtg ttgccgaccg ctatcaagat 660 attgcgctcg caactcgtga catcgctgaa gagctgggcg gagaatgggc tgaccgtttc 720 ctggtactgt atggcattgc agcgcccgat tcccaacgca tcgcatttta tcgtctgctg 780 gatgagtttt tctaa 795 <210> 13 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> Kan R protein <400> 13 Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val 1 5 10 15 Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser 20 25 30 Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe 35 40 45 Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala 50 55 60 Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val 65 70 75 80 Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu 85 90 95 Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys 100 105 110 Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro 115 120 125 Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala 130 135 140 Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu 145 150 155 160 Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala 165 170 175 Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys 180 185 190 Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp 195 200 205 Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala 210 215 220 Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe 225 230 235 240 Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe 245 250 255 Tyr Arg Leu Leu Asp Glu Phe Phe 260 <210> 14 <211> 149 <212> PRT <213> Artificial Sequence <220> <223> HBV truncated core antigen <400> 14 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val 145 <210> 15 <211> 447 <212> DNA <213> Artificial Sequence <220> <223> HBV truncated core antigen gene <400> 15 atggacatcg acccttacaa ggagttcggc gccagcgtgg aactgctgtc ttttctgccc 60 agtgatttct ttccttccat tcgagacctg ctggataccg cctctgctct gtatcgggaa 120 gccctggaga gcccagaaca ctgctcccca caccataccg ctctgcgaca ggcaatcctg 180 tgctgggggg agctgatgaa cctggccaca tgggtgggat ccaatctgga ggaccccgct 240 tcacgggaac tggtggtcag ctacgtgaac gtcaatatgg gcctgaaaat ccgccagctg 300 ctgtggttcc atattagctg cctgactttt ggacgagaga ccgtgctgga atacctggtg 360 tccttcggcg tctggatccg cactccccct gcttatcgac cacccaacgc accaattctg 420 tccaccctgc ccgagaccac agtggtc 447 <210> 16 <211> 2529 <212> DNA <213> Artificial Sequence <220> <223> HBV pol antigen gene <400> 16 atgcccctgt cttaccagca ctttagaaag ctgctgctgc tggacgatga agccgggcct 60 ctggaggaag agctgccaag gctggcagac gaggggctga accggagagt ggccgaagat 120 ctgaatctgg gaaacctgaa cgtgagcatc ccttggactc ataaagtcgg caacttcacc 180 gggctgtaca gctccacagt gcctgtcttc aatccagagt ggcagacacc atcctttccc 240 aacattcacc tgcaggagga catcattaat agatgcgaac agttcgtggg acctctgaca 300 gtcaacgaaa agaggcgcct gaaactgatc atgcctgcca ggttttaccc aaatgtgact 360 aagtatctgc cactggataa gggcatcaag ccttactatc cagagcacct ggtgaaccat 420 tacttccaga ctagacacta tctgcatacc ctgtggaagg ccggaatcct gtacaaacga 480 gaaactaccc ggagtgcttc attttgtggc tccccatatt cttgggaaca ggagctgcag 540 catggcaggc tggtgttcca gaccagcaca cgccacgggg atgagtcctt ttgccagcag 600 tctagtggca tcctgagcag atcccccgtg gggccttgtc tgcagtctca gctgcggaag 660 agtagactgg gactgcagcc acagcaggga cacctggcac gacggcagca gggaaggtct 720 ggcagtatcc gggctagagt gcatcccaca actagaaggc ctttcggcgt cgagccatca 780 ggaagcggcc acaccacaaa caccgcatca agctcctcta gttgcctgca tcagtcagcc 840 gtgagaaagg ccgcttacag ccacctgtcc acatctaaaa ggcactcaag ctccgggcat 900 gctgtggagc tgcacaacat ccctccaaat tctgcacgca gtcagtcaga aggacccgtg 960 ttcagctgct ggtggctgca gtttcggaac tcaaagcctt gcagcgacta ttgtctgagc 1020 catattgtga atctgctgga ggattggggc ccttgtaccg agcacgggga acaccatatc 1080 aggattccac gaacaccagc acgagtgact ggaggggtgt tcctggtgga caagaacccc 1140 cacaatacta ccgagagccg gctggtggtc gatttcagtc agttttcaag aggcaacaca 1200 agggtgtcat ggcccaaatt cgccgtccct aatctgcaga gtctgactaa cctgctgtct 1260 agtaatctga gctggctgtc cctggacgtg tccgcagcct tttaccacct gcctctgcat 1320 ccagctgcaa tgccccatct gctggtgggg tcaagcggac tgagtcgcta cgtcgcccga 1380 ctgtcctcta actcacgcat cattaatcac cagcatggca ccatgcagaa cctgcacgat 1440 agctgttccc ggaatctgta cgtgtctctg ctgctgctgt ataagacatt cggcagaaaa 1500 ctgcacctgt acagccatcc tatcattctg gggtttagga agatcccaat gggagtggga 1560 ctgagcccct tcctgctggc acagtttacc tccgccattt gctctgtggt ccgccgagcc 1620 ttcccacact gtctggcttt ttcctatatg aacaatgtgg tcctgggcgc caaatccgtg 1680 cagcatctgg agtctctgtt cacagctgtc actaactttc tgctgagcct ggggatccac 1740 ctgaacccaa ataagactaa acgctggggg tacagcctga atttcatggg atatgtgatt 1800 ggatcctggg ggaccctgcc acaggagcac atcgtgcaga agatcaagga atgctttcgg 1860 aagctgcccg tcaacagacc tatcgactgg aaagtgtgcc agcggattgt cggactgctg 1920 ggcttcgccg ctccctttac ccagtgcggg tacccagcac tgatgcccct gtatgcctgt 1980 atccagtcta agcaggcttt cacctttagt cctacataca aggcattcct gtgcaaacag 2040 tacctgaacc tgtatccagt ggcaaggcag cgacctggac tgtgccaggt ctttgcaaat 2100 gccactccta ccggctgggg gctggctatc ggacatcagc gaatgcgggg cacattcgtg 2160 gcccccctgc ctattcacac tgctcagctg ctggcagcct gctttgctag atctaggagt 2220 ggagcaaagc tgatcggcac cgacaatagt gtggtcctgt caagaaaata cacatccttc 2280 ccatggctgc tgggatgtgc tgcaaactgg attctgaggg gcaccagctt cgtgtacgtc 2340 ccctcagccc tgaatcctgc tgacgatcca tcccgcgggc gactgggact gtaccgacct 2400 ctgctgagac tgcccttcag gcctacaact ggccggacat ctctgtatgc cgattcacca 2460 agcgtgccct cacacctgcc tgacagagtc cactttgctt cacccctgca cgtcgcttgg 2520 cggcctcca 2529 <210> 17 <211> 684 <212> DNA <213> Artificial Sequence <220> <223> hCMV promoter sequence <400> 17 accgccatgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt 60 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 120 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 180 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 240 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 300 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 360 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 420 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 480 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 540 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt 600 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca 660 ccgggaccga tccagcctcc gcgg 684 <210> 18 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> immunoglobulin secretion signal coding sequence <400> 18 atggagttcg gcctgtcttg ggtctttctg gtggcaatcc tgaagggcgt gcagtgtgaa 60 gtgcagctgc tggagtctgg a 81 <210> 19 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> immunoglobulin secretion signal sequence <400> 19 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly 20 25 <210> 20 <211> 996 <212> PRT <213> Artificial Sequence <220> <223> HBV core-pol fusion antigen sequence <400> 20 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Ala Gly Ala Gly Met Pro Leu Ser Tyr Gln His 145 150 155 160 Phe Arg Lys Leu Leu Leu Leu Asp Asp Glu Ala Gly Pro Leu Glu Glu 165 170 175 Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg Val Ala Glu 180 185 190 Asp Leu Asn Leu Gly Asn Leu Asn Val Ser Ile Pro Trp Thr His Lys 195 200 205 Val Gly Asn Phe Thr Gly Leu Tyr Ser Ser Thr Val Pro Val Phe Asn 210 215 220 Pro Glu Trp Gln Thr Pro Ser Phe Pro Asn Ile His Leu Gln Glu Asp 225 230 235 240 Ile Ile Asn Arg Cys Glu Gln Phe Val Gly Pro Leu Thr Val Asn Glu 245 250 255 Lys Arg Arg Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr Pro Asn Val 260 265 270 Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu 275 280 285 His Leu Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu His Thr Leu 290 295 300 Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser 305 310 315 320 Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu Gln His Gly Arg 325 330 335 Leu Val Phe Gln Thr Ser Thr Arg His Gly Asp Glu Ser Phe Cys Gln 340 345 350 Gln Ser Ser Gly Ile Leu Ser Arg Ser Pro Val Gly Pro Cys Leu Gln 355 360 365 Ser Gln Leu Arg Lys Ser Arg Leu Gly Leu Gln Pro Gln Gln Gly His 370 375 380 Leu Ala Arg Arg Gln Gln Gly Arg Ser Gly Ser Ile Arg Ala Arg Val 385 390 395 400 His Pro Thr Thr Arg Arg Pro Phe Gly Val Glu Pro Ser Gly Ser Gly 405 410 415 His Thr Thr Asn Thr Ala Ser Ser Ser Ser Ser Cys Leu His Gln Ser 420 425 430 Ala Val Arg Lys Ala Ala Tyr Ser His Leu Ser Thr Ser Lys Arg His 435 440 445 Ser Ser Ser Gly His Ala Val Glu Leu His Asn Ile Pro Pro Asn Ser 450 455 460 Ala Arg Ser Gln Ser Glu Gly Pro Val Phe Ser Cys Trp Trp Leu Gln 465 470 475 480 Phe Arg Asn Ser Lys Pro Cys Ser Asp Tyr Cys Leu Ser His Ile Val 485 490 495 Asn Leu Leu Glu Asp Trp Gly Pro Cys Thr Glu His Gly Glu His His 500 505 510 Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu 515 520 525 Val Asp Lys Asn Pro His Asn Thr Thr Glu Ser Arg Leu Val Val Asp 530 535 540 Phe Ser Gln Phe Ser Arg Gly Asn Thr Arg Val Ser Trp Pro Lys Phe 545 550 555 560 Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu 565 570 575 Ser Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His Leu Pro Leu 580 585 590 His Pro Ala Ala Met Pro His Leu Leu Val Gly Ser Ser Gly Leu Ser 595 600 605 Arg Tyr Val Ala Arg Leu Ser Ser Asn Ser Arg Ile Ile Asn His Gln 610 615 620 His Gly Thr Met Gln Asn Leu His Asp Ser Cys Ser Arg Asn Leu Tyr 625 630 635 640 Val Ser Leu Leu Leu Leu Tyr Lys Thr Phe Gly Arg Lys Leu His Leu 645 650 655 Tyr Ser His Pro Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val 660 665 670 Gly Leu Ser Pro Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser 675 680 685 Val Val Arg Arg Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asn 690 695 700 Asn Val Val Leu Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe 705 710 715 720 Thr Ala Val Thr Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro 725 730 735 Asn Lys Thr Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val 740 745 750 Ile Gly Ser Trp Gly Thr Leu Pro Gln Glu His Ile Val Gln Lys Ile 755 760 765 Lys Glu Cys Phe Arg Lys Leu Pro Val Asn Arg Pro Ile Asp Trp Lys 770 775 780 Val Cys Gln Arg Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr 785 790 795 800 Gln Cys Gly Tyr Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser 805 810 815 Lys Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys Lys 820 825 830 Gln Tyr Leu Asn Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys 835 840 845 Gln Val Phe Ala Asn Ala Thr Pro Thr Gly Trp Gly Leu Ala Ile Gly 850 855 860 His Gln Arg Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr 865 870 875 880 Ala Gln Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys 885 890 895 Leu Ile Gly Thr Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser 900 905 910 Phe Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr 915 920 925 Ser Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser 930 935 940 Arg Gly Arg Leu Gly Leu Tyr Arg Pro Leu Leu Arg Leu Pro Phe Arg 945 950 955 960 Pro Thr Thr Gly Arg Thr Ser Leu Tyr Ala Asp Ser Pro Ser Val Pro 965 970 975 Ser His Leu Pro Asp Arg Val His Phe Ala Ser Pro Leu His Val Ala 980 985 990 Trp Arg Pro Pro 995 <210> 21 <211> 1023 <212> PRT <213> Artificial Sequence <220> <223> HBV core-pol fusion antigen sequence with Ig signal sequence <400> 21 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Met Asp Ile Asp Pro 20 25 30 Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu Ser Phe Leu Pro Ser 35 40 45 Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu 50 55 60 Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr 65 70 75 80 Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Asn Leu Ala 85 90 95 Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu Leu Val 100 105 110 Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Leu Leu 115 120 125 Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu 130 135 140 Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg 145 150 155 160 Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val 165 170 175 Ala Gly Ala Gly Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu 180 185 190 Leu Leu Asp Asp Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu 195 200 205 Ala Asp Glu Gly Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly 210 215 220 Asn Leu Asn Val Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr 225 230 235 240 Gly Leu Tyr Ser Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr 245 250 255 Pro Ser Phe Pro Asn Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys 260 265 270 Glu Gln Phe Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys 275 280 285 Leu Ile Met Pro Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro 290 295 300 Leu Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Leu Val Asn His 305 310 315 320 Tyr Phe Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile 325 330 335 Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro 340 345 350 Tyr Ser Trp Glu Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr 355 360 365 Ser Thr Arg His Gly Asp Glu Ser Phe Cys Gln Gln Ser Ser Gly Ile 370 375 380 Leu Ser Arg Ser Pro Val Gly Pro Cys Leu Gln Ser Gln Leu Arg Lys 385 390 395 400 Ser Arg Leu Gly Leu Gln Pro Gln Gln Gly His Leu Ala Arg Arg Gln 405 410 415 Gln Gly Arg Ser Gly Ser Ile Arg Ala Arg Val His Pro Thr Thr Arg 420 425 430 Arg Pro Phe Gly Val Glu Pro Ser Gly Ser Gly His Thr Thr Asn Thr 435 440 445 Ala Ser Ser Ser Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Ala 450 455 460 Ala Tyr Ser His Leu Ser Thr Ser Lys Arg His Ser Ser Ser Gly His 465 470 475 480 Ala Val Glu Leu His Asn Ile Pro Pro Asn Ser Ala Arg Ser Gln Ser 485 490 495 Glu Gly Pro Val Phe Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys 500 505 510 Pro Cys Ser Asp Tyr Cys Leu Ser His Ile Val Asn Leu Leu Glu Asp 515 520 525 Trp Gly Pro Cys Thr Glu His Gly Glu His His Ile Arg Ile Pro Arg 530 535 540 Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro 545 550 555 560 His Asn Thr Thr Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser 565 570 575 Arg Gly Asn Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu 580 585 590 Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu 595 600 605 Asp Val Ser Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Met 610 615 620 Pro His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg 625 630 635 640 Leu Ser Ser Asn Ser Arg Ile Ile Asn His Gln His Gly Thr Met Gln 645 650 655 Asn Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu 660 665 670 Leu Tyr Lys Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile 675 680 685 Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe 690 695 700 Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala 705 710 715 720 Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asn Asn Val Val Leu Gly 725 730 735 Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val Thr Asn 740 745 750 Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg 755 760 765 Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly 770 775 780 Thr Leu Pro Gln Glu His Ile Val Gln Lys Ile Lys Glu Cys Phe Arg 785 790 795 800 Lys Leu Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile 805 810 815 Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro 820 825 830 Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr 835 840 845 Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn Leu 850 855 860 Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asn 865 870 875 880 Ala Thr Pro Thr Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg 885 890 895 Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr Ala Gln Leu Leu Ala 900 905 910 Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp 915 920 925 Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu 930 935 940 Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val 945 950 955 960 Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly 965 970 975 Leu Tyr Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gly Arg 980 985 990 Thr Ser Leu Tyr Ala Asp Ser Pro Ser Val Pro Ser His Leu Pro Asp 995 1000 1005 Arg Val His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 1010 1015 1020 <210> 22 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> linker coding sequence <400> 22 gccggagctg gc 12 <210> 23 <211> 248 <212> DNA <213> Artificial Sequence <220> <223> ApoAI gene fragment <400> 23 ttggccgtgc tcttcctgac gggtaggtgt cccctaacct agggagccaa ccatcggggg 60 gccttctccc taaatccccg tggcccaccc tcctgggcag aggcagcagg tttctcactg 120 gccccctctc ccccacctcc aagcttggcc tttcggctca gatctcagcc cacagctggc 180 ctgatctggg tctcccctcc caccctcagg gagccaggct cggcatttcg tcgacaagct 240 tagccacc 248 <210> 24 <211> 130 <212> DNA <213> Artificial Sequence <220> <223> SV40 polyadenylation signal sequence <400> 24 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120 tatcatgtct 130

Claims (92)

A device for controlled delivery of an HBV vaccine to a predetermined tissue site in a subject in need thereof, the device comprising:
A cartridge assembly comprising an outer cartridge, an inner cartridge, and a reservoir containing the HBV vaccine, wherein a reservoir containment volume is accommodated within the outer cartridge and is configured to receive the reservoir; and
An applicator comprising a cartridge assembly receiving volume, a needle hub and an insertion detector, the insertion detector being adapted to detect loading of the reservoir in the storage container containment volume;
At least one interlock to facilitate proper implementation of the HBV vaccine administration procedure;
At least one injection orifice through which the HBV vaccine is administered;
A plurality of through electrodes arranged in a predetermined spatial relationship with respect to the orifice;
An electric field generator for generating an electrical signal operably connected to the electrodes; And
A controlled energy source sufficient to deliver a predetermined amount of HBV vaccine from the reservoir through the orifice to a predetermined site in the subject at a predetermined rate; and
The HBV vaccine,
A first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is 98% or more identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity. molecule;
A second nucleic acid molecule comprising 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
Including; a pharmaceutically acceptable carrier,
An apparatus for controlled delivery of an HBV vaccine, wherein the first nucleic acid molecule and the second nucleic acid molecule exist as the same nucleic acid molecule or two different nucleic acid molecules.
A kit for controlled delivery of an HBV vaccine to a predetermined tissue site in a subject in need thereof, the kit comprising an HBV vaccine and a device for delivering the HBV vaccine,
The HBV vaccine,
A first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is 98% or more identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity. molecule;
A second nucleic acid molecule comprising 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
Including; a pharmaceutically acceptable carrier,
The first nucleic acid molecule and the second nucleic acid molecule exist as the same nucleic acid molecule or two different nucleic acid molecules,
The device,
A cartridge assembly comprising an outer cartridge, an inner cartridge, and a reservoir containing the HBV vaccine, wherein a reservoir containment volume is accommodated within the outer cartridge and is configured to receive the reservoir; and
An applicator comprising a cartridge assembly receiving volume, a needle hub and an insertion detector, the insertion detector being adapted to detect loading of the reservoir in the storage container containment volume;
At least one interlock to facilitate proper implementation of the HBV vaccine administration procedure;
At least one injection orifice through which the HBV vaccine is administered;
A plurality of through electrodes arranged in a predetermined spatial relationship with respect to the orifice;
An electric field generator for generating an electrical signal operably connected to the electrodes; And
A kit for controlled delivery of an HBV vaccine comprising a controlled energy source sufficient to deliver the HBV vaccine at a predetermined rate from a reservoir to a predetermined site in a subject from a reservoir of a predetermined amount of HBV vaccine.
The device according to claim 1 or the kit according to claim 2, wherein the electrode consists of a plurality of elongated electrodes.
The device according to claim 1 or the kit according to claim 2, wherein the interlock is adapted to prevent accidental activation of the cartridge function.
The one or more interlocks are selected from the group consisting of mechanical interlocks, emitters/receivers, cartridge breaches, force interlocks, alignment guides and splay shields, trigger locks and safety switches. The device according to any one of claims 4 or a kit according to any of claims 2 to 4.
The device comprises a set of two or more interlocks, the first interlock being a mechanical interlock, the second interlock being a light emitter/receiver, a cartridge breach, a force interlock, an alignment guide and a splay shield, a trigger safety device and a safety switch. The device according to any one of claims 1, 3 to 5, or the kit according to any one of claims 2 to 5, selected from.
The device or kit according to claim 5 or 6, wherein the mechanical interlock is a reservoir interlock.
The device or kit according to claim 7, wherein the reservoir interlock further comprises one or more reservoir locking holes.
The device or kit according to any of claims 5 to 8, wherein the cartridge breach provides an optical line of sight through one or more reservoir locking holes.
The device or kit according to any one of claims 7 to 9, wherein the reservoir interlock further comprises a tab extending from at least one cartridge surface, the tab ensuring that the reservoir is properly inserted into the cartridge.
The device or kit according to claim 6, wherein the device further comprises a third interlock.
The device or kit according to claim 11, wherein the third interlock is a force interlock.
The device or kit according to claim 12, wherein the force interlock senses a force applied to a predetermined tissue site of the subject and prevents administration of the HBV vaccine to the subject when insufficient force is provided.
The device or kit according to claim 12 or 13, wherein the force interlock forms an electrical lock within the applicator.
The device further comprises a key to the reservoir, the key adapted to slide over the barrel of the reservoir to ensure proper engagement within the cartridge assembly. A device according to claim 1 or a kit according to claim 2.
The splay shield comprises one or more ribs and one or more edges for positioning the device with a tension that engages with a predetermined tissue site of the subject and acts at right angles to the needle placement direction for administration of the HBV vaccine. Device or kit according to claim 6.
The device according to claim 1 or the kit according to claim 2, wherein the cartridge assembly further comprises a stick shield.
The device or kit according to any one of claims 5, 6 and 16, wherein the splay shield comprises at least one hole for sliding movement of the stick shield.
When loading the cartridge assembly into the applicator, the reservoir moves forward to engage the needle hub and contacts the cartridge to the injection needle upon administration of the HBV vaccine. The device according to any one of the preceding claims or the kit according to any one of claims 2 to 18.
The device according to any one of claims 1, 3 to 19 or the kit according to any one of claims 2 to 19, wherein the inner cartridge is adapted to move in a sliding manner in relation to the outer cartridge. .
The inner cartridge is coupled with the inner cartridge cap at the distal end, the inner cartridge cap holding the electrode in place and providing a bearing surface for the stick shield, any one of claims 1, 3 to 20 A device according to or a kit according to any one of claims 2 to 20.
The device according to claim 1 or the kit according to claim 2, wherein the device comprises one or more sensors.
The sensor according to claim 22, wherein the sensor is selected from the group consisting of a cartridge loading sensor, a cartridge loaded sensor, a cartridge force sensor, an insertion mechanism position sensor, an insertion detector, an optical detector and an electrical sensor. According to the device or kit.
The apparatus or kit according to claim 23, wherein the cartridge loading sensor and the cartridge loaded sensor form part of a loading drive subassembly.
The device or kit according to claim 24, wherein the loading drive subassembly further comprises at least one cartridge guide rail and a loading motor.
The loading drive subassembly according to claim 24 or 25 having a connection to a pinion gear assembly that pulls the cartridge assembly through at least one rack on the base of the external cartridge into the cartridge assembly receiving volume. Device or kit.
The device or kit according to claim 26, wherein the pinion gear assembly engages a rack of the external cartridge.
An apparatus or kit according to claim 26 or 27, wherein the rack comprises one or more rack teeth.
The device or kit according to any one of claims 26 to 28, wherein the first rack teeth provide a tactile sensation when the cartridge assembly is inserted into the cartridge assembly receiving volume.
An apparatus or kit according to any of claims 26 to 29, wherein the rack teeth provide torsional stability.
The device or kit according to any one of claims 23 to 26, wherein the cartridge loading sensor senses a start flag in the cartridge assembly to start loading.
The device or kit according to any one of claims 23 to 26, wherein the cartridge loading sensor senses a start flag in the cartridge assembly to stop loading.
33. The device according to any one of claims 1, 3 to 32 or the kit according to any one of claims 2 to 32, wherein the device further comprises a continuation flag for continuing loading the cartridge.
The device or kit according to claim 23, wherein the insertion detector is a light emitter/receiver IR sensor.
The device or kit according to any one of claims 22 to 34, wherein the sensor detects a reservoir label and identifies an HBV vaccine.
A device for controlled delivery of an HBV vaccine to a predetermined tissue site in a subject in need thereof, the device comprising:
A cartridge assembly comprising a housing, a reservoir containing the HBV vaccine, wherein a reservoir containment volume is received within the housing and is configured to receive the reservoir; and
An applicator comprising a cartridge assembly receiving volume, a needle hub and an insertion detector, the insertion detector being adapted to detect loading of the reservoir in the storage container containment volume;
At least one injection orifice through which the HBV vaccine is administered;
A plurality of through electrodes arranged in a predetermined spatial relationship with respect to the orifice;
An electrode support structure that prevents accidental vertical movement of the electrode and includes a hole and a wall;
An electric field generator for generating an electrical signal operably connected to the electrodes; And
A controlled energy source sufficient to deliver a predetermined amount of HBV vaccine from the reservoir through the orifice to a predetermined site in the subject at a predetermined rate; and
The HBV vaccine,
A first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is 98% or more identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity. molecule;
A second nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2; And
Including; a pharmaceutically acceptable carrier,
An apparatus for controlled delivery of an HBV vaccine, wherein the first nucleic acid molecule and the second nucleic acid molecule exist as the same nucleic acid molecule or two different nucleic acid molecules.
As a kit comprising an HBV vaccine and a device,
(i) the HBV vaccine,
A first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is 98% or more identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity. molecule;
A second nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2; And
Including; a pharmaceutically acceptable carrier,
The first nucleic acid molecule and the second nucleic acid molecule exist as the same nucleic acid molecule or two different nucleic acid molecules,
(ii) the device,
A cartridge assembly comprising a housing, a reservoir containing the HBV vaccine, wherein a reservoir containment volume is received within the housing and is configured to receive the reservoir; and
An applicator comprising a cartridge assembly receiving volume, a needle hub and an insertion detector, the insertion detector being adapted to detect loading of the reservoir in the storage container containment volume;
At least one injection orifice through which the HBV vaccine is administered;
A plurality of through electrodes arranged in a predetermined spatial relationship with respect to the orifice;
An electrode support structure that prevents accidental vertical movement of the electrode and includes a hole and a wall;
An electric field generator for generating an electrical signal operably connected to the electrodes; And
A controlled energy source sufficient to deliver a predetermined amount of HBV vaccine from said reservoir to a predetermined site in a subject through said orifice at a predetermined rate.
The device or device according to claim 36, wherein the electrode support structure provides an operative connection between the controlled energy source and a conductive contact area located in a distal area of the electrode when the electrode is disposed at a predetermined tissue site within the object. Kit according to item 37.
The device according to claim 36 or 38 or the kit according to claim 37 or 38, wherein the electrode support structure comprises at least one hole for passing the electrode and a hole for passing the injection needle.
The device according to any one of claims 36, 38 to 39, or any one of claims 37 to 39, wherein the electrode support structure is a planar support structure positioned perpendicular to the long orientation of the electrode. Kit according to.
The device or kit according to claim 40, wherein the planar support structure comprises at least one opening configured to allow passage of the electrode through a predetermined tissue site.
The device or kit according to claim 40 or 41, wherein the opening comprises at least one tubular structure arranged perpendicular to the planar support structure.
The device or kit according to any one of claims 40 to 42, wherein the planar support structure is oriented perpendicular to the electrode.
The device according to any one of claims 36, 38 to 43 or the kit according to any one of claims 37 to 43, wherein the electrode support structure is an adaptive electrode support.
The device or kit according to claim 44, wherein the compliant electrode support is a compression spring.
The device or kit according to claim 45, wherein the compression spring is made of a metal, polymer or elastomeric material.
The device according to any one of claims 36, 38 to 46 or the kit according to any one of claims 37 to 46, wherein the electrode support structure comprises one or more telescopic tubes.
The device according to any one of claims 36, 38 to 47 or the kit according to any one of claims 37 to 47, wherein the electrode support structure further comprises a stick shield spring.
The device according to claim 36 or the kit according to claim 37, wherein the electrode support structure further comprises at least one side support member attached to the electrode together with at least one optional hinge structure.
The device according to any one of claims 36, 38 to 49 or any one of claims 37 to 49, wherein the electrode support structure is made of a metal, polymer, ceramic, composite or compressible matrix material. Kit according to.
The device or kit according to claim 50, wherein the compressible matrix material is selected from the group consisting of cellulose, foamed plastics, rubber polymers, microporous plastics, foamed silicones, foamed polychloroprene and carbon foamed matrices.
The device according to any one of claims 36, 38 to 51 or the kit according to any of claims 37 to 51, wherein the electrode support structure is made of a non-conductive material.
The device according to any one of claims 36, 38 to 52, or the kit according to any of claims 37 to 52, wherein the electrode support structure is made of a thermoplastic material.
The device or kit according to claim 53, wherein the thermoplastic material is selected from the group consisting of polycarbonate, polystyrene, polypropylene, acrylic and polyethylene.
The device according to any one of claims 36, 38 to 54, or any of claims 37 to 54, wherein the electrode support structure supports transdermal placement of the electrode and maintains a tissue depth of up to 60 mm. A kit according to one of the preceding claims.
Any one of claims 36, 38-55, wherein the electrode proximal portion is an electrode contact portion and is located outside of the inner cartridge of the cartridge assembly, wherein the electrode contact portion is configured to be in electrical power communication with a corresponding connection on the applicator. A device according to or a kit according to any one of claims 37 to 55.
The device or kit according to claim 56, wherein the electrode contact further comprises an external cartridge external contact.
The device or kit according to claim 56 or 57, wherein the electrode contacts provide an electrically conductive interface with the corresponding electrode without interfering with the forward movement of the electrode mounted on the inner cartridge.
The applicator further comprises an injection drive assembly, wherein the injection drive assembly engages the cartridge assembly, the device according to any one of claims 1, 3 to 36, 38 to 58, or The kit according to any one of claims 2 to 35 and 37 to 58.
The device according to any one of claims 1, 3 to 36, 38 to 58, or the device according to any one of claims 2 to 35, 37 to 37, further comprising a depth selector button. The kit according to claim 58.
The device or kit according to claim 60, wherein the depth selection button is selected from the group consisting of a toggle, a switch and a sliding switch.
The device according to any one of claims 1, 3 to 36, 38 to 58 or 2 to 35, wherein the device further comprises a plurality of channels and a plurality of retaining posts. The kit according to any one of claims 37 to 58.
The device according to any one of claims 1, 3 to 36, 38 to 58 or the device according to any of claims 2 to 35, 37, wherein the device further comprises an insertion mechanism gear drive ring. The kit according to any one of claims to 58.
The apparatus or kit according to claim 63, wherein rotation of the insertion mechanism gear ring rotates the retaining post into the channel.
The cartridge assembly is disposable, the device according to any one of claims 1, 3 to 36, 38 to 64 or any of claims 2 to 35, 37 to 64 The kit according to any one of the preceding claims.
The applicator can be used repeatedly, the device according to any one of claims 1, 3 to 36, 38 to 64, or any of claims 2 to 35, 37 to 64 The kit according to any one of the preceding claims.
The device according to any one of claims 1, 3 to 36, 38 to 66, wherein the applicator further comprises an upper housing, a side housing, an inner protective shell, a front cap and an end cap. Or the kit according to any one of claims 2 to 35 and 37 to 66.
The applicator further comprises a user interface, a process activation trigger, a process countdown timer, a process fault indicator, or an application placement indicator, any one of claims 1, 3 to 36, 38 to 67. A device according to claim 2 or a kit according to any one of claims 2 to 35, 37 to 67.
The device according to any one of claims 1, 3 to 36, 38 to 68 or the device according to any of claims 2 to 35, 37 to 68, further comprising a controller. The kit according to any one of claims.
The device according to any one of claims 1, 3 to 36, 38 to 69, or the device according to any of claims 2 to 35, wherein the applicator further comprises a connector for connection to the controller. The kit according to any one of claims 37 to 69.
The device or kit according to claim 69 or 70, wherein the controller further comprises an electric field controller.
The device according to any one of claims 1, 3 to 36, 38 to 71, wherein the through electrode and/or injection orifice is in contact with a predetermined tissue site at a rate of 50 mm/sec or more, or The kit according to any one of claims 2 to 35 and 37 to 71.
The device according to any one of claims 1, 3 to 36, 38 to 71, or wherein the through electrode and/or injection orifice is in contact with a predetermined tissue site at a rate of at least 500 mm/sec. The kit according to any one of claims 2 to 35 and 37 to 71.
The HBV vaccine is capable of inducing an immune response against two or more HBV genotypes in mammals, preferably the HBV vaccine is T for at least HBV genotypes B, C and D, more preferably for HBV in mammals. Claims 1, 3-36, 38, wherein the HBV vaccine is capable of inducing a cellular response, and is capable of inducing a CD8 T cell response in a human subject to at least HBV genotypes A, B, C and D. The device according to any of claims 2 to 73 or the kit according to any of claims 2 to 35 and 37 to 73.
The device or kit according to claim 74, wherein the first non-natural nucleic acid molecule is in the first plasmid DNA vector and the second non-natural nucleic acid molecule is in the second plasmid DNA vector.
Each of the first and second plasmid DNA vectors comprises an origin of replication, an antibiotic resistance gene, and a 5'to 3'end, a promoter sequence, an enhancer sequence, a signal peptide coding sequence, a first polynucleotide sequence or a second poly. A device or kit according to claim 75 comprising a nucleotide sequence and a polyadenylation signal sequence.
The device or kit according to claim 76, wherein the antibiotic resistance gene is a kanamycin resistance gene having a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 12, preferably 100% identical to SEQ ID NO: 12.
The HBV vaccine,
a) a first plasmid DNA vector comprising a 3'-end to a 5'-end, a promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 7, an enhancer sequence comprising the polynucleotide sequence of SEQ ID NO: 8, and A signal peptide coding sequence comprising a polynucleotide sequence, 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 a 3'-end to a 5'-end, a promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 7, a regulatory sequence comprising the polynucleotide sequence of SEQ ID NO: 8, and A signal peptide coding sequence comprising a polynucleotide sequence, a second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 1, and a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 11; And
c) a pharmaceutically acceptable carrier; includes,
Each of the first plasmid DNA vector and the second plasmid DNA vector further comprises a kanamycin resistance gene having the polynucleotide sequence of SEQ ID NO: 12, and a replication original having the polynucleotide sequence of SEQ ID NO: 10, and the first plasmid The device or kit according to claim 77, wherein the DNA vector and the second plasmid DNA vector are present in the same composition or in two different compositions.
The HBV vaccine includes a nucleic acid molecule encoding an HBV antigen selected from the group consisting of a hepatitis B surface antigen (HBsAg), an HBV envelope (Env) antigen, and an HBV L protein antigen; And an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen, and HBV L protein antigen; the device or kit according to claim 78 which does not contain.
A device according to any one of claims 1, 3 to 36, 38 to 79, or a kit according to any one of claims 2 to 35, 37 to 79. A method for inducing an immune response against HBV infection in a subject in need of the vaccine, comprising the step of delivering the aHBV vaccine to a predetermined tissue site in the subject,
The method of inducing an immune response against HBV infection, wherein the subject is preferably a human suffering from asynchronous HBV infection.
A device according to any one of claims 1, 3 to 36, 38 to 79, or a kit according to any one of claims 2 to 35, 37 to 79. A method of treatment for HBV-induced disease in a subject in need of a vaccine, comprising delivering the HBV vaccine to a predetermined tissue site in the subject using
Preferably the subject is a human subject, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).
82. The method of claim 80 or 81, wherein the predetermined tissue site is located in the skeletal muscle of the subject, a method of inducing an immune response or a method of treating an HBV-induced disease.
83. The method of claim 82, wherein the subject's skeletal muscle is a medial deltoid muscle.
The method of claim 83, wherein the depth of injection of the medial deltoid muscle is about 3-30 mm.
The method of claim 82, wherein the skeletal muscle of the subject is a vatus lateralis muscle, a method for inducing an immune response or a method for treating an HBV-induced disease.
88. The method of claim 85, wherein the depth of injection in the lateral vastus muscle is about 3-38 mm. The method of inducing an immune response or treatment for an HBV-induced disease.
The immune response according to any one of claims 80 to 86, wherein the electrical signal operably connected to the electrode comprises an electric field strength of 100 V/cm to 400 V/cm, preferably 250 V/cm. Method of inducing or treatment for HBV-induced disease.
89. The method of claim 87, wherein the voltage of the electrical signal operably connected to the electrode is 50 to 200 V, preferably about 150 V. The method of inducing an immune response or treatment for an HBV-induced disease.
The method of claim 88, wherein the electrical signal operably connected to the electrode has a current of 0.5 to 5 A/sec, preferably 0.6 to 4 A/sec, more preferably 0.16 A/sec. Method or treatment method for HBV-induced disease.
89. The method of claim 89, wherein the electrical signal operably connected to the electrode has about 1 to 10 electrical pulses, preferably 6 pulses, of inducing an immune response or treatment for an HBV-induced disease.
91. The method of claim 90, wherein the electrical signal operably connected to the electrode has an activation duration of 30 to 50 milliseconds, preferably 40.8 milliseconds, and an activation duration of 200 to 500 milliseconds, preferably about 370 milliseconds. A method of inducing an immune response or a method of treatment for an HBV-induced disease, applied as a total duration.
In order to induce an immune response against at least two HBV genotypes, in particular for use in the treatment of HBV-induced diseases, the device is applied to the electroporation of the HBV vaccine to a subject in need of an HBV vaccine, preferably to a human The device according to any one of claims 1, 3 to 36, 38 to 79 or according to any one of claims 2 to 35, 37 to 79 Kit.

Images