Systems and devices for administration of RNA vaccines and methods of use thereof

Abstract

Methods of delivering pharmaceutical compositions containing one or more nucleic acids via electroporation are disclosed. The methods include (i) delivering a pharmaceutical composition containing one or more nucleic acids to a target site of the subject; and (ii) electroporating the target site of the subject where the pharmaceutical composition was delivered. Electroporating the target site facilitates the uptake of the nucleic acids by and expression of an immunogenic protein in cells of the subject. Typically the nucleic acid is RNA such as non-replicating mRNA or circular RNA. The methods can deliver two or more nucleic acids, wherein at least one of the nucleic acids is an mRNA or a circular RNA. The disclosed methods are suitable treating or preventing the development of one or more symptoms of a disease or disorder such as an infection or cancer in a subject.

Description

[0001] SYSTEMS AND DEVICES FOR ADMINISTRATION OF RNA VACCINES AND

[0002] METHODS OF USE THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 712,227, filed October 25, 2024, the contents of which is specifically incorporated by reference herein in its entirety.

[0005] REFERENCE TO THE SEQUENCE LISTING

[0006] The Sequence Listing submitted as an XML file named “PIEZO 103_PCT_ST26. xml” created on October 27, 2025, and having a size of 18,611 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0007] FIELD OF THE INVENTION

[0008] The disclosed invention is generally in the field of devices for nucleic acid expression and more specifically in the area of the mRNA and circular RNA vaccines.

[0009] BACKGROUND OF THE INVENTION

[0010] For mRNA vaccines, and RNA therapeutics more broadly, to be effective, they must bypass the cell membrane and reach the cytoplasm, where translation and protein expression occur. Several delivery methods can achieve this, including LNPs, electroporators, and gene guns. While LNPs have demonstrated good efficacy and safety, they still pose several challenges. First, their production is expensive and complex, with LNPs accounting for more than 50% of the cost of goods sold due to the over 20-step process required to formulate mRNA. Second, research and development timelines are extended because there is no universal LNP formulation, necessitating extensive screening to find suitable LNPs for each application. Third, LNPs raise concerns about reactogenicity, including off-target effects, nonlocalized biodistribution, and heightened inflammatory responses. Finally, LNPs have poor thermostability, requiring ultracold storage and complicated logistics. As a result, the use of LNPs makes mRNA vaccines costly, time-consuming, and complex to develop and manufacture at scale, while also contributing to unfavorable tolerability in patients. Addressing these challenges through an alternative delivery platform can significantly improve the safety, affordability, speed, and scalability of mRNA vaccines, enabling their broader and continued application.

[0011] Delivering DNA vaccines (due to their lower cost and greater stability) to circumvent problems associated with mRNA delivery has been used as an option. However, DNA vaccines elicit suboptimal immune responses in higher species unless administered through electroporation. Additionally, DNA vaccines require a dose 20 to 100 times higher than that of mRNA, to achieve a comparable immune response, which poses challenges for their translation to clinical settings. These challenges include lower transcription efficiency. DNA must first enter the nucleus of the cell, where the DNA can be transcribed into mRNA. This additional step, compared to mRNA vaccines that go directly to the cytoplasm for translation into protein, is less efficient. Not all DNA makes it to the nucleus, which reduces the overall efficiency of the vaccine and necessitates a higher dose to ensure enough mRNA is produced to stimulate an immune response. Also, DNA is less immunogenic compared to mRNA, which is more readily recognized by the immune system. Therefore, higher doses of DNA are often needed to generate a comparable immune response to that induced by smaller doses of mRNA.

[0012] Electroporation (EP) is a nonchemical method that uses electric pulses to temporarily permeabilize cell membranes and facilitate the uptake of molecules like RNA and DNA EP has emerged an alternative approach for nucleic acid delivery. The delivery of mRNA vaccines via electroporation has been less explored and primarily limited to self-replicating mRNA due to the instability of 'naked' mRNA and the suboptimal electroporation parameters and delivery systems available for RNA. Thus, effective electroporation-based delivery of 'naked' RNA vaccines an electroporator / electroporation technique which ensures delivered mRNA remains intact following delivery, to induce adequate innate immune stimulation.

[0013] Furthermore, the use of EP has been significantly constrained by various factors, including the high cost of equipment (in the thousands of dollars), lack of portability (equipment is typically placed on rolling carts or benchtop boxes), challenges in scalability and manufacturing, and the need for a reliable electricity source. Although some EP devices have been adapted into handheld formats, they remain prohibitively expensive, require continuous access to power, are often painful for patients, and frequently depend on docking stations for recharging or reuse, which adds to the complexity of their use. Thus, more effective electroporation-based methods capable of delivering “naked” RNA vaccines are needed.

[0014] It is an object of the invention to provide more effective methods of delivering RNA compositions via electroporation to a subject.

[0015] It is also an object of the present invention to provide more effective methods of treating a disease via electroporation in a subject.

[0016] It is still an object of the invention to provide improved systems for the delivery of RNA compositions to a subject. SUMMARY OF THE INVENTION

[0017] Disclosed are methods and systems for delivering nucleic acids to a site in a subject in need thereof.

[0018] The disclosed methods are based on the Applicant's discovery of an electroporation system (device and method) for delivering naked and non-replicating nucleic acids, such as mRNAs and circular RNAs, which ensure delivery of effective amounts of the nucleic acid to elicit an immune response in the subject, at levels that are comparable to an immune response obtained when the nucleic is encapsulated prior to delivery.

[0019] Generally, the method includes the following steps:

[0020] (i) administering to a target site of a subject, a composition containing an effective amount of one or more nucleic acid (s), wherein the one or more nucleic acid (s) encodes one or more antigen (s); and

[0021] (ii) electroporating the target site of the subject, wherein electroporating increases delivery of the one or more nucleic acid (s) to cells in the target site.

[0022] The method can further include a step (iii) of monitoring the expression of one or more protein (s) encoded by the one or more nucleic acids in the target site over a predetermined period.

[0023] The composition can be administered via intradermal or subcutaneous injection to the target site on a subject. Generally, the subject is a mammal, preferably a human.

[0024] Generally, the target site is mammalian skin, e.g., mammalian skin cells. Exemplary skin cells include but are not limited to keratinocytes, Langerhans cells, melanocytes, fibroblasts, and epidermal dendritic cells.

[0025] The one of or more nucleic acids can be of the same type or they can be different. In some forms, the composition can include two different types of nucleic acids, for example an RNA and a DNA.

[0026] In some forms, the isolated nucleic acid encodes more than one protein or peptides, such that more than one proteins or peptides are expressed as a fusion protein / peptide. The one or more nucleic acid (s) can optionally be / or include an RNA or DNA encoding or expressing an adjuvant. Exemplary adjuvants include but arc not limited to interleukins, interferons, Granulocyte-Macrophage Colony-Stimulating Factor (GMCSF), Retinoic Acid-Inducible Gene I, chemokines, pattern recognition receptors, flagellin, bacterial toxins, tumor necrosis factors, and heat shock proteins. In some forms, the isolated nucleic acid is an RNA such as messenger RNA (mRNA) and / or circular RNA. In some forms, the RNA is not a self-replicating RNA. In some forms, the RNA is “naked” RNA, in that it is not encapsulated in a carrier such as nanoparticles, liposomes, microspheres, or microcapsules, etc.

[0027] In some forms, the nucleic acid has a molecular weight from about 10 base pairs to about 100 kilobases.

[0028] The one or more nucleic acid(s) can optionally include one or more modified nucleotide(s), for example, 5-methylcytidine (m5C), 2-thiouridine (s2U), 5 -methyluridine (m5U), pseudouridine (\|t), 5 -methoxyuridine (5moU) and N1 -methylpseudouridine (mly).

[0029] The disclosed systems and methods can be used in treating or preventing the development of one or more symptoms of a disease or disorder in a subject.

[0030] In some forms, the disease or disorder can be an infection caused by a virus, bacterium, fungus, parasite, or protozoa. In some forms, the infection is caused by a virus such as orthomyxovirus, rhinovirus, paramyxovirus, coronavirus, adenovirus, human metapneumovirus (hMPV), enterovirus, bocavirus, flavivirus, retrovirus, or para retrovirus. In other forms, the disease or disorder is cancer for example, skin cancer or liver cancer.

[0031] Generally, the Steps (i) and (ii) of the disclosed methods are performed using a device. Steps (i) and (ii) of the disclosed methods can be performed by:

[0032] (a) positioning the device adjacent to a target site on the subject, wherein the device comprises: a piezoelectric pulse generator; an array of biocompatible and conductive microeleclrodes electrically coupled to the piezoelectric pulse generator; a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses;

[0033] (b) contacting the array of biocompatible and conductive microelectrodes comprising the at least one needle embedded therein into the biological tissue on the subject;

[0034] (c) activating the switch to generate and deliver one or more electrical pulses through the array of biocompatible and conductive microelectrodes into the target site to electroporate the cells; and

[0035] (d) delivering the composition into the target site of the subject. In some forms, step (c) and step (d) are performed concurrently. In other forms, step (d) is performed following step (c). In yet other forms, step (c) is performed following step (d). Generally, step (c) is repeated at least once. In one exemplary form, the device contains a syringe having a container holding the composition; wherein the array of biocompatible and conductive microelectrodes further includes a syringe and at least one needle for delivery of the composition embedded therein; and wherein the syringe is coupled to at least one needle for administering the composition into the target site by actuation of the syringe.

[0036] In a second exemplary form, the device contains a detachable syringe having a container for holding the composition; wherein the detachable syringe can be removed from the main body of the device after the composition is delivered.

[0037] In a third exemplary form, the microelectrodes of the array of biocompatible and conductive microelectrodes are microneedle electrodes, wherein the microneedle electrodes are coated with the composition and / or coated with one or more therapeutic, prophylactic, or diagnostic agents.

[0038] In some forms, the one or more electrical pulses have a peak voltage absolute value between about 100 V and 1000 V, a peak current absolute value between about 0.001 A and 50 A, a peak static voltage absolute value between about 1 ,000 V and 35,000 V, an initial pulse length of between about 1 ps and 100 ps, or a combination thereof.

[0039] The disclosed systems includes:

[0040] (i) a pharmaceutical composition containing an effective amount of one or more nucleic acid (s), wherein the one or more nucleic acid (s) encodes one or more antigen (s), and

[0041] (ii) device containing: a piezoelectric pulse generator; an array of biocompatible and conductive microeleclrodes electrically coupled to the piezoelectric pulse generator; and a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIGs. 1A-1C are photos of an exemplary device for the delivery of non-replicating mRNA and circular RNA.

[0044] FIG. 2A shows a non- limiting illustrative view of a device 100 having a needle component 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, an array of biocompatible and conductive microelectrodes 200, a piezoelectric pulse generator 300, and a mechanical switch 310 for activating the piezoelectric pulse generator. FIG. 2B shows a nonlimiting illustrative head-on view of a device 100 having a needle component 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, and an array of biocompatible and conductive microelectrodes 200. FIG. 2C shows a non- limiting illustrative side view of a device 100 having a needle component 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, an array of biocompatible and conductive microelectrodes 200, a piezoelectric pulse generator 300, and a switch 310 for activating the piezoelectric pulse generator.

[0045] FIG. 3A shows a non-limiting illustration of a microneedle electrode array (MEA) 200 having an array of biocompatible and conductive microelectrodes 210 (see Figure 3B), an opening for at least one needle component therein 220, and a base 230. FIG. 3B shows a nonlimiting illustration of an array of biocompatible and conductive microelectrodes 210. FIG. 3C shows a non- limiting illustration of a needle component 110 having a needle end tip.

[0046] FIG. 4A shows a non- limiting illustration of a needle component 110 placed into base 230 through the opening / hole such that the needle tip of the needle component 110 is exposed adjacent to the microelectrodes of the array 210. FIG. 4B shows a non-limiting illustration of a needle component 110 placed into base 230 through the opening / hole such that the needle tip of the needle component 110 is exposed adjacent to the microelectrodes of the array 210.

[0047] FIG. 5A shows a non- limiting illustration of a piezoelectric pulse generator 300 and a switch 310, which is coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate electrical pulse(s). FIG. 5B shows a non-limiting illustration of a syringe 120 having a container 126, which can hold one or more agents, a plunger 125, which can be used to administer the agents when actuated, and a delivery end 127 which optionally includes an attachment or lock, which can be used for connecting and attaching a needle component. FIG. 5C shows a non-limiting illustration of a syringe 120 coupled to a needle component 110 and an MEA 200.

[0048] FIGs. 6A and 6B are non- limiting illustrations of an exemplary electroporator having a microneedle array.

[0049] FIGs. 7A and 7B are line graphs showing that Piezopen delivery achieves results compared with top-of-the-line mRNA-LNPs, such as Modema’s and Pfizer’s LNPs when delivering mRNA-Luc in vivo intramuscularly and intradermally.

[0050] FIGs. 8A and 8B are line graphs showing that Piezopen delivery achieves results compared with top-of-the-line mRNA-LNPs, such as Moderna’s and Pfizer’s LNPs when delivering mRNA-Luc in vivo intradermally (ID only). FIGs. 9A and 9B are dose ranging studies indicating variability in expression magnitude and kinetics with Piezopen vs SMI 02 LNP Delivery of mRNA-Luc in vivo.

[0051] FIG. 10 is a line graph showing exemplary proof-of-concept on saRNA delivery at lose dose validates Piezopen’ s cross platform potential.

[0052] FIG. 11 is a line graph showing that circRNA delivery at low doses validates Piezopen’s cross platform potential and enhanced stability of circRNA.

[0053] FIG. 12A is a schematic of an experimental timeline. FIGs. 12B-12D are graphs showing that Piezopen administration induced robust immune responses (namely, IgG and Nabs) to naked mRNA, comparable to LNP responses at a higher dose. Day 35 serum was used to determine IgG and NAbs. FIG. 12B is a line graph of Absorbance vs dilution curve for IgG, FIG. 12C is a bar graph of Log transformed anti-SARS-CoV-2 antibody IgG, FIG. 12D is a line graph of an inhibition rate vs dilution curve for NAbs and showing log transformed neutralization titer for 30% inhibition rate. N=3 for each group.

[0054] FIGs. 13A-13D present data from Corbet el al. (2020), used to conduct a comparative analysis with Moderna’s results, as both studies utilized the same vaccine.

[0055] FIG. 14 is a bar graph showing that intradermal injection of OVA mRNA paired with an adjuvant followed by electroporation. Intradermal injections and electroporation was done using OVA mRNA (SC2346 GenScript); OVA mRNA + IFN-g mRNA (SC2325-IVT; GenScript): OVA mRNA + IL-6 mRNA (SC2325-IVT; GenScript); OVA RNase Inhibitor (N2615; Promega) per animal was added for experimental groups. Each treatment used a total of 5 pg of mRNA. For the animals that received adjuvants, a weight ratio of 1 pg adjuvant to 4 pg of mRNA OVA was maintained, for a total of 5 pg mRNA. Immunization was done twice, once on Day 0 and once on Day 7.

[0056] FIG. 15A is a line graph showing quantification of corresponding luciferase gene expression using IVIS. Total flux vs. time plot. FIGs. 15B-15D are bar graphs showing quantification of binding (FIG. 15B) , neutralizing antibodies (FIG. 15C), and IFN-g spots (FIG. 15D) for a SARS-CoV-2 mRNA vaccine using ELISA IgG, NAbs sVNT, and IFN-g ELISpot kits.

[0057] FIGs. 16A-16D are graphs showing that an exemplary Piezopen drives intracellular delivery of naked RNA (namely, mRNA (FIGs. 16C and 16D), saRNA (FIG. 16A), and circRNA (FIG. 16B)) across species (namely, mice and rats) in vivo and in live human skin ex vivo. (Shown are quantification of corresponding gene expression using IVIS via Total flux vs time plots. n>3 / group, mean ± SEM, 2-way or 1-way ANOVA, and Tukey’s post hoc. *p < 0.05, **p < 0.01, ****p < 0.0001; color-coordinated asterisks above plots indicate a pairwise comparison.

[0058] FIGs. 17A and 17B are graphs showing neutralizing antibody titers and T-cell responses following CHIMERA vaccination. FIG. 17A shows neutralizing antibody titers against SARS- CoV-2 measured at Days 49, 90, and 180 post-prime across vaccine groups including CHIMERA formulations, mRNA-1273 equivalent, and reference standards. FIG. 17B shows spike-specific IFNy-secreting T-cell responses measured by ELISpot at Day 180 (6 months) in nonhuman primates receiving CHIMERA or comparator vaccines. Data are shown as mean ± SD.

[0059] FIGs. 18A and 18B are line graphs showing Piezopen delivery of CHIMERA-Luc achieves sustained antigen expression exceeding mRNA-Luc. FIG. 18A shows luciferase expression kinetics following Piezopen delivery of DNA-Luc, mRNA-Luc, or CHIMERA-Luc in vivo. FIG. 18B shows comparison of Piezopen delivery of DNA and mRNA (5 pg each) Data are presented as mean ± SD (n = 3).

[0060] FIG. 19 is a bar graph showing neutralizing antibody responses following CHIMERA vaccination. Neutralizing antibody titers (ID30) measured at Day 49 post-prime in animals receiving CHIMERA formulations (vl or v2, prime or prime-boost) compared with WHO convalescent sera and Modema’s Spikevax (prime + boost).

[0061] DETAILED DESCRIPTION OF THE INVENTION

[0062] I. DEFINITIONS

[0063] The term “conductive" refers to materials that allow for the flow of electric current with low or minimal resistance. Such materials can be made of or contain one or more metals or can be made of other materials, such as conductive polymers / plastics.

[0064] The term “about,” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0065] As used herein, the term “heterologous” refers to elements occurring where they are not normally found. For example, a promoter may be linked to a heterologous nucleic acid sequence, c.g., a sequence that is not normally found operably linked to the promoter. When used herein to describe a promoter element, heterologous means a promoter element that differs from that normally found in the native promoter, either in sequence, species, or number. For example, a heterologous control element in a promoter sequence may be a control / regulatory element of a different promoter added to enhance promoter control, or an additional control element of the same promoter. The term “heterologous” thus can also encompass “exogenous” and “non-native” elements. The terms “heterologous nucleotide sequence” and “heterologous nucleic acid” are used interchangeably herein and refer to a sequence that is not naturally occurring in the virus. Generally, the heterologous nucleic acid comprises an open reading frame that encodes a polypeptide or non-translated RNA of interest (e.g., for delivery to a cell or subject).

[0066] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0067] A “polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA or DNA- RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotide) and can be either single or double stranded DNA sequences.

[0068] As used herein, the term “percent (%) sequence identity” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0069] As used herein, the term “binds” in reference to the interaction of a binding protein and an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, a binding protein recognizes and binds to a specific antigen structure rather than to antigens generally. If a binding protein binds to epitope "A", the presence of a molecule containing epitope “A” (or free, unlabeled “A”), in a reaction containing labeled “A” and the binding protein, will reduce the amount of labeled “A” bound to the binding protein.

[0070] As used herein, the term “carrier” or “excipient” refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient in a formulation, with which one or more active ingredients arc combined. The carrier or excipient would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0071] As used herein, the term “individual,” “subject,” and “patient” are used interchangeably to refer to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term does not denote a particular age or sex.

[0072] As used herein, the terms “treat” or “treating” refer to the medical management of a disease, pathological condition, or disorder in a subject or patient with the intent to cure, ameliorate, stabilize, or prevent the disease, pathological condition, or disorder. These terms include active treatment, that is, treatment directed specifically toward the improvement of the disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, these terms include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. These terms may also refer to the reduction of one or more symptoms of the disease, pathological condition, or disorder in the subject or patient.

[0073] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0074] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0075] II. METHODS FOR DELIVERING RNA VACCINES

[0076] Disclosed arc methods of delivering compositions containing one or more nucleic acid (s) via electroporation, such as intradermal or subcutaneous electroporation, to a site in a subject in need thereof. The disclosed methods are suitable for treating a subject having a disease such as an infectious disease or cancer. Electroporation facilitates uptake of the one or more nucleic acid (s) by and systemic expression of one or more immunogenic protein (s) in cells of the subject.

[0077] An exemplary method of treating a disease or condition in a subject includes the following steps: (i) delivering or administering the composition to a target site of the subject: and (ii) electroporating the target site of the subject where the composition was delivered or administered. Generally, electroporating the target site increases the delivery of one or more nucleic acid (s) to cells in the target site. For example, electroporating cells in the target site facilitates uptake of the one or more nucleic acid (s) by cells in the target site.

[0078] In some embodiments, the methods can be used treat or prevent the development of one or more symptoms of an infectious disease such as a viral infection by delivering to a target site, e.g., a region of the skin, of the subject, a pharmaceutical composition containing an effective amount of one or more nucleic acid (s), and electroporating the target site. In another example, the methods treat one or more symptoms of skin cancer by delivering to a target site, e.g., a region of the skin, of the subject, a pharmaceutical composition containing an effective amount of one or more nucleic acid (s), and electroporating the target site. In a third example, the methods treat one or more symptoms of a distal tumor such as liver cancer by administering to a target site, e.g., a region of the skin, of the subject, a pharmaceutical composition containing an effective amount of one or more nucleic acid (s), and electroporating the target site of the subject.

[0079] Also disclosed are methods of delivering an immunogenic composition to cells at a target site e.g., a region of the skin, of a subject by: (i) administering a composition containing an effective amount of one or more nucleic acid (s) to the target site of the subject; and (ii) applying electroporation to the target site. Generally, the electroporation of the target site is effective in increasing delivery of the one or more nucleic acid (s) to the target site.

[0080] The composition can be delivered via subcutaneous injection, intratumoral injection, or intradermal injection. Typically, electroporation is intradermal electroporation or by subcutaneous electroporation. Typically, electroporation is not administered via intramuscular electroporation. This may be due in part because mRNA vaccines may not work as well as intradermal electroporation since the skin is rich in immune cells.

[0081] In some forms, the methods further include step (iii) monitoring the expression of one or more protein (s) and / or peptide (s) encoded by the one or more nucleic acid (s) in a biological sample from the subject over a predetermined period. For example, monitoring the expression of the one or more protein (s) and / or peptide (s) can include measuring the amount of the one or more protein (s) and / or peptide (s) in a biological sample from the subject, comparing the level of the one or more protein (s) and / or peptide (s) in the biological sample from the subject to a control, and determining whether the biological sample from the subject has increased levels of the one or more protein (s) compared to the control.

[0082] In some forms, the measuring can be accomplished by performing an enzyme-linked immunosorbent assay (ELISA). In some forms, the measuring can be measuring the level of mRNAs that encode the one or more protein (s) and / or peptide (s) in the sample. In some forms, the measuring can be accomplished by performing a hybridization assay or RT, PCR, or qPCR.

[0083] In some forms, the subject can be monitored for expression of the one or more protein (s) and / or peptide (s) by testing the subject for the presence of the one or more protein (s) and / or peptide (s) two or more times. In some forms, at least two of the two or more times at which the subject is tested for the expression of the one or more protein (s) and / or peptide (s) can be separated by an interval of time. In some forms, the interval of time can be between one week and twelve months, inclusive. In some forms, the interval of time can be between one week, two weeks, three weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months and two weeks, three weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, inclusive. In some forms, the interval of time can be one week, two weeks, three weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Different intervals can be the same length, different lengths, or a combination thereof.

[0084] In some forms, the control can be a standard. In some fomrs, the control is a subject not being delivered or administered the pharmaceutical composition. In some forms, the control is the subject being delivered or administered the pharmaceutical composition prior to delivery or administration of the pharmaceutical composition. The biological sample can be serum, plasma, blood, saliva, or tissue sample.

[0085] A. Compositions of Nucleic Acid (s)

[0086] The disclosed compositions contain an effective amount of one or more isolated nucleic acid (s). Generally, the one or more isolated nucleic acid (s) encode one or more protein (s) and / or peptide (s) such as one or more antigen (s). Typically, the one or more protein (s) and / or peptide (s) are effective for increasing an immune response in a subject.

[0087] In some forms, the composition is a pharmaceutical composition and it further includes as pharmaceutically acceptable excipients, and additional agents such , such as agent for specific delivery, for increasing half-life, or other therapeutic agents.

[0088] In some fomis, the composition contains one nucleic acid. In these forms, the nucleic acid is ribonucleic acid (RNA). In some fomrs, the composition contains two or more nucleic acids. For example, the composition can contain two or more nucleic acids. In some forms, when the composition contains two nucleic acids, one of the two nucleic acids is an RNA and the other is DNA. Alternatively, both nucleic acids can be RNA. For example, one nucleic in the composition can be an mRNA molecule and the other can be a circRNA molecule. Alternatively, both nucleic acids in the composition can be mRNA molecules. Alternatively, both nucleic acids in the composition can be circRNA molecules. In some forms, the composition can contain three nucleic acid molecules, four nucleic acid molecules, five nucleic acid molecules, six nucleic acid molecules, up to forty nucleic acid molecules.

[0089] In some forms the one or more nucleic acids is a modified nucleic acid. In some forms, the isolated nucleic acid encodes more than one protein or peptides, such that more than one proteins or peptides are expressed as a fusion protein / peptide.

[0090] 1. Nucleic Acids

[0091] The nucleic acid can have any suitable molecular weight as long as it encodes a protein or peptide that can elicit an immune response in the subject when delivered to the subject. For example, the nucleic acid can have a molecular weight from about 10 base pairs (bp) to about 100 kilobases (kb). In some forms, the nucleic acid can have a molecular weight ranging from about 10 bp pairs to about 90 kb, from about 10 bp pairs to about 80 kb, from about 10 bp pairs to about 60 kb, from about 10 bp pairs to about 50 kb, from about 10 bp pairs to about 40 kb, from about 10 bp pairs to about 30 kb, from about 10 bp pairs to about 20 kb, from about 10 bp pairs to about 10 kb, from about 10 bp pairs to about 5 kb, from about 10 bp pairs to about 3 kb, from about 10 bp pairs to about 1 kb, from about 10 bp pairs to about 900 bp, from about 10 bp pairs to about 800 bp, from about 10 bp pairs to about 700 bp, from about 10 bp pairs to about 600 bp, from about 10 bp pairs to about 500 bp, from about 10 bp pairs to about 400 bp, from about 10 bp pairs to about 300 bp, from about 10 bp pairs to about 200 bp, from about 10 bp pairs to about 100 bp, from about 10 bp pairs to about 90 bp, from about 10 bp pairs to about 80 bp, from about 10 bp pairs to about 70 bp, from about 10 bp pairs to about 60 bp, from about 10 bp pairs to about 50 bp, from about 10 bp pairs to about 40 bp, from about 10 bp pairs to about 30 bp, or from about 10 bp pairs to about 20 bp. All sizes between the range of 10 bp and 100 kb not mentioned are also contemplated.

[0092] The RNA can be messenger RNA (mRNA) or circular RNA. In some forms, the composition contains one RNA molecule. In some forms, the composition contains two RNA molecules. For example, the composition can contain two mRNA molecules. In another example, the composition can contain two circular RNA molecules. In another example, the composition can contain one mRNA molecule and a circular RNA molecule. In some forms, when the composition contains a DNA molecule and an RNA molecule, the RNA molecule is either a mRNA molecule or a circular RNA molecule. i. messenger RNA

[0093] Messenger RNA (mRNA) is a single-stranded molecule that carries genetic information from DNA to the ribosome, where it guides the synthesis of proteins. It serves as a template that determines the sequence of amino acids in a protein, based on the genetic code. mRNA can be engineered to instruct cells to produce specific proteins or peptides that can elicit an immune response.

[0094] In a vaccine context, mRNA vaccines can be of three types: self-amplifying mRNA vaccines, non-replicating mRNA vaccines, and in vitro dendritic cell non-replicating mRNA vaccines. mRNA vaccine constructs have common features, including a 5' cap sequence, 5' and 3' translated regions (UTRs), an open reading frame (ORF) carrying coding sequence (CDS), and a 3' poly (A) tail. Non- replicating mRNA vaccines include an mRNA sequence coding for a target antigen, which is flanked by 3’ and 5’ untranslated regions (UTRs). The mRNA sequence is small in size and easy to construct as it does not contain any additional protein-coding sequences to support the self-replication of the mRNA. In self-replicating mRNA vaccines, a viral genome is used, in which the viral gene sequence responsible for coding structural proteins is replaced by the antigen sequence of interest. The viral RNA sequence generated in this way can still replicate and be transcribed using viral RNA polymerase. Dendritic cells are antigen- presenting cells that express antigens on the cell surface so that specialized immune cells, such as T cells, can recognize the antigen and initiate cellular immune responses. For in vitro dendritic cell non-replicating mRNA vaccines, dendritic cells are isolated from the patient’s blood, transfected with the mRNA sequence of interest, and administered back to the patient to induce desired immune responses.

[0095] Typically, the mRNA included in the composition is a non-replicating mRNA. In some forms, the mRNA included in the composition is linear non-replicating mRNA. Alternatively, the mRNA included in the composition can be linear replicating mRNA. In some forms, the mRNA included in the composition is not linear replicating mRNA. Typically, the mRNA included in the composition is not a dendritic cell non-replicating mRNA as dendritic cell vaccines are electroporated in vitro and then the cells are injected into the subject e.g., a human patient. ii. Circular RNA

[0096] The RNA in the composition can be an isolated and / or modified circular RNA (herein referred to as a synthetic circRNA). Circular RNA (circRNA) is a type of noncoding RNA (ncRNA) characterized by its covalently closed circular structure, lacking the typical 5 ’—3’ ends or a polyadenylated tail found in linear RNA molecules. circRNAs are stable, evolutionarily conserved, and exhibit diverse expression patterns that are often specific to certain tissues or developmental stages. circRNAs can originate from various regions of the genome, leading to a wide range of lengths. Most circRNAs are derived from coding exons, though some arise from the 5’ or 3’ untranslated regions (5’UTRs or 3’UTRs), while others come from noncoding RNA regions. Some circRNAs are formed from introns, excised during pre-mRNA processing; these are referred to as circular intronic RNAs (ciRNAs). The formation of ciRNAs depends on specific sequence motifs, including a GU-rich element near the 5’ splice site and a C-rich element near the branch point. Additionally, circRNAs can include both exons and retained introns, forming what is known as exon-intron circRNAs (EIciRNAs).

[0097] Like long non-coding RNAs, circRNAs can be categorized based on their location relative to neighboring genes into five broad groups: (1) sense or exonic, originating from one or more exons on the same strand as the linear transcript; (2) intronic, when entirely derived from an intron; (3) antisense, overlapping exons on the opposite strand: (4) bidirectional or intragenic, transcribed from the same gene locus but not classified as sense or intronic; and (5) intergenic, situated between two genes. circRNAs are known to function as microRNA (miRNA) sponges, influencing splicing or transcription regulation, and they can also interact with RNA binding proteins (RBPs). They have been implicated in physiological processes such as aging, insulin secretion, and tissue development. Furthermore, circRNAs play significant roles in various pathological conditions, including atherosclerotic vascular disease, neurological disorders, cardiac hypertrophy, and cancer. More detailed information regarding the structure and function of natural circRNAs can be found at least in Liu, et al., Journal of Control Release, 348:84-94 (2022), all of which are incorporated by reference herein in their entireties.

[0098] In some forms, the synthetic circRNA included in the composition is believed to provide improved stability against degradation such as from RNAses and temperature. Methods of designing and generating synthetic circRNAs are known (see Liu, et al., Journal of Control Release, 348:84-94 (2022); Obi and Chen, The design and synthesis of circular RNAs, Methods, 196:85-103 (2021)). In some embodiments, the synthetic circRNA included in the composition is designed to improve circularization and increase protein or peptide translation efficiency. Methods for improving circRNA circularization, such as modifying pre-mRNA splicing mechanisms and utilizing specific intronic sequences or RNA-binding proteins (RBPs), are known in the art. These methods may involve strategies to enhance pre-mRNA back splicing or the use of constructs such as permuted intron-exon (PIE) systems, which promote self-back splicing. Additionally, incorporating self-cleaving ribozyme sequences can further increase circularization efficiency.

[0099] The covalently closed structure of circRNAs confers improved stability compared to linear RNAs, providing resistance to degradation by exonucleases, and thus improving the potential therapeutic efficacy of circRNAs as vaccines or other treatments. In some forms, the synthetic circRNA included in the composition can include one or more altered translation initiation sequences to increase the translation efficiency of the synthetic circRNAs, such as modified internal ribosome entry sites (IRES), m6A modifications, or Kozak consensus sequences. Such improvements may facilitate the production of proteins or peptides with increased expression levels and tissue specificity (Liu, et al., Journal of Control Release, 348:84-94 (2022)).

[0100] A number of chemical and intracellular approaches have been developed for circRNA synthesis. In general, circRNA is synthesized via the synthesis of one or multiple precursor linear RNA, followed by RNA circularization mediated by chemical or enzymatical ligation (Petkovic and Muller, Methods Mol. Biol. 1724: 167-180 (2018)). The precursor linear RNAs used for circRNA synthesis can be synthesized via chemical or enzymatic methods (Usman and Cedergren, Trends Biochem. Sci. 17 (9) (1992) 334-339; Rong et al., Proc. Natl. Acad. Sci. U. S. A. 95 (2) (1998) 515-519). Chemical synthesis is based on phosphoroamidite chemistry, which involves natural nucleoside triphosphate derivatives and protecting groups to favor the formation of 3'-5' phosphodiester linkages. However, chemical methods typically generate RNA shorter than 70-80 nucleotides. Alternatively, in vitro transcription (IVT) is commonly used to synthesize longer RNAs (up to kilobases in length) using DNA templates and RNA polymerases such as T7 RNA polymerases (Petkovic and Muller, Methods Mol. Biol. VITA'. 167-180 (2018); Rong M, et al., Proc. Natl. Acad. Sci. U. S. A. 95 (2) (1998) 515-519).

[0101] For circRNA synthesis, precursor linear RNA can be circularized either chemically or enzymatically, with the latter involving protein enzymes or ribozymes. In chemical RNA circularization, agents like cyanogen bromide (BrCN) or l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are commonly used to induce the bonding of the 5'-end phosphate with the 3'-end hydroxyl group of linear RNA (Dolinnaya NG, et al., Nucleic Acids Res. (11) (1991) 3067-3072). However, chemical ligation of RNA oligomers is usually less efficient than that of DNA due to side reactions like the formation of unwanted 2'-5' phosphodiester bonds. To improve this process, 2'-deoxynucleotides can be substituted at the 3'-end of linear RNA (Wang and Kool, Nucleic Acids Res. 22 (12) (1994) 2326-2333). Additionally, reactive chemical groups can be attached to both ends of the RNA, enabling more efficient circularization through non- natural linkages (Edupuganti et al., J. Organomet. Chem. 68 (22) (2003) 8708-8710). RNA circularization is often limited by competition between intramolecular and intermolecular reactions. Using low concentrations of linear RNA can promote intramolecular circularization over intermolecular oligomerization, which improves circRNA yield (Petkovic and Muller, Methods Mol. Biol. 1724: 167-180 (2018)). Moreover, complementary oligonucleotide splints can be introduced to hybridize with the two ends of linear RNA, bringing them close together and facilitating circularization through either chemical or enzymatic ligation (Dolinnaya, et al., Nucleic Acids Res. 21 (23) (1993) 5403-5407; Kershaw and O’Keefe, Methods Mol. Biol. 941 (2012) 257-269).

[0102] Protein enzymatic RNA circularization is widely used due to its high efficiency. Enzymes like DNA and RNA ligases from T4 bacteriophage are commonly employed to ligate linear RNAs with terminal monophosphate groups. T4 DNA ligase is highly selective and only ligates RNAs that are fully complementary to DNA splints, making it useful for creating precise circRNAs from precursor RNAs synthesized by in vitro transcription (IVT), which often produces heterogeneous RNA termini (Moore MJ, Query CC, Methods Enzymol. 317 (2000) 109-123; Gholamalipour et al., Nucleic Acids Res. 46 (18) (2018) 9253-9263). T4 RNA ligase catalyzes the ligation of a 5 '-monophosphate on a donor RNA to a 3'-hydroxyl on an acceptor RNA, with T4 RNA ligase 1 being specific to single-stranded RNAs and favoring cytidine and adenosine at the ligation site (Romaniuk E, et al., Eur. J. Biochem. 125(3) (1982) 639-643). To improve circularization efficiency, partially complementary nucleic acid splints can be used to bring reactive ends into proximity. T4 RNA ligase 2, however, is more efficient at joining nicks in double-stranded RNA substrates. A truncated version of T4 RNA ligase 2 was developed to ligate the pre- adenylated 5'-end of DNA or RNA to the 3'-end of RNA without the need for ATP (Ho CK, et al., Structure 12 (2) (2004) 327-339). Since these enzymes require monophosphates, IVT-generated RNAs with 5 '-triphosphates must be dephosphorylated and rephosphorylated. Alternatively, RNA 5' pyrophosphohydrolase (RppH) can be used to directly remove the f> and y phosphates (Deana A, et al., Nature 451 (7176) (2008) 355-358). circRNA can also be synthesized using ribozymes, which are RNA enzymes that catalyze the ligation of precursor linear RNAs. One example is the permuted group I intron system, where intron sequences arc fused with exons in organisms like bacteria (Obi and Chen, Methods 196 (2021) 85-103). The Anabaena pre-tRNALeu gene was used to create the permuted intron exon (PIE) system, which allows the self-cleavage of group I introns and ligation of exons to form circRNAs (Puttaraju and Been, Nucleic Acids Res. 20 (20) (1992) 5357-5364). The PIE system has been adapted for various RNAs, and recently, an engineered version demonstrated the ability to circularize RNAs up to 5 kilobases in vitro (Wesselhoeft, et al., Nat. Commun. 9 (1) (2018) 2629). This system uses complementary "homology arms" to bring splice sites close together, enhancing efficiency. However, the use of native exons in the PIE system may affect protein expression of inserted genes. In contrast, group II introns from yeast can produce circRNAs without native exons (Mikheeva, et al., Nucleic Acids Res. 25 (24) (1997) 5085-5094). PIE- generated circRNAs may induce immune responses due to extraneous double-stranded RNA fragments, which activate protein kinase K. In comparison, circRNAs produced by T4 RNA ligase have minimal immune response activation because they lack these extraneous RNA fragments, allowing them to serve as inhibitors of PKR. Intracellular synthesis of exogenous circRNA involves creating DNA templates, such as plasmids, that encode artificial exons with the desired RNA sequences flanked by introns. After transcription, the precursor linear RNA is circularized. To facilitate this, complementary intronic sequences, similar to those found in natural circRNAs, are inserted into pre-mRNA to bring donor and acceptor splice sites close together for efficient ligation (Dubin et al., Gene 167 (1-2) (1995) 245-248). For example, a green fluorescent protein (GFP) exon has been expressed using Drosophila Laccase 2 introns. RNA-binding protein (RBP) sites in flanking introns can also bring RNA regions together by binding the same RBP, promoting circularization (Ashwal-Fluss, et al., Mol. Cell 56 (1) (2014) 55-66). Another method uses the permuted intron-exon (PIE) system in living cells, where selfsplicing ribozymes generate circRNAs in organisms like E. coli and yeast (Ford and Ares, Proc. Natl. Acad. Sei. U. S. A. 91 (8) (1994) 3117-3121). A modified tRNA-based system in metazoan cells has also been used to improve circRNA yield by inserting customized RNA into intronic or exonic cleavage sites (Lu Z, et al., RNA 21 (9) (2015) 1554-1565; Litke and Jaffrey, Nat. Biotechnol. 37 (6) (2019) 667-675). Additionally, a ribozyme-based approach using "Twister" self-cleaving ribozymes generates precursor linear RNA, followed by RNA circularization with the RtcB ligase. These circRNAs have been studied for real-time monitoring of intracellular bioanalytes (Litke and Jaffrey, Nat. Biotechnol. 37 (6) (2019) 667-675; Dey et al., Nat. Chem. Biol. 18 (2021) 180-190). iii. Nucleic Acid Modifications

[0103] Originally, unmodified RNA was considered advantageous for vaccines due to its innate immunostimulatory properties. However, excessive immune stimulation can disrupt protein production and impair subsequent immune responses, potentially due to the overproduction of Type I interferons. While unmodified RNA can still be effective, its heightened reactogenicity — especially when combined with the reactogenicity already associated with lipid nanoparticles (LNPs) — poses significant challenges. Existing research indicates that modified RNA increases protein production compared to unmodified RNA. For example, pseudouridine-modified RNA increases protein production compared to unmodified RNA (Kariko, et al., Molecular Therapy, 16(11):1833-1840 (2008); Kariko, et al., Immunity, 23(2): 165-75 (2005)). This modification reduces the excessive immune activation seen with unmodified RNA, allowing for more controlled and efficient vaccine responses. One of the challenges in mRNA vaccine design is balancing reactogenicity and immunogenicity. The vaccine needs enough reactogenicity to stimulate an effective immune response but must avoid overstimulation, which can impair the adaptive immune response. Modifications such as pseudouridine help achieve this balance by increasing protein production while mitigating excessive immune reactions, making them critical for improving mRNA vaccine efficacy.

[0104] Thus, the one or more nucleic acid (s) in the composition can optionally contain one or more modifications. In some forms, the one or more nucleic acid (s) can contain one or more modified nucleotide (s). Exemplary modified nucleotides include but are not limited to inosine, 5-methylcytidine (m5C). 2-thiouridine (s2U), 5 -methyluridine (m5U), pseudouridine (y), 5- methoxyuridine (5moU), N1 -methyladenosine (mlA), N4-Acetylcytidine (ac4C), 7- Methylguanosine (m7G), 1 -methylguanosine (mlG), and 2-methylguanosine (m2G), N6- Methyladenosine (m6A), A6,25-O-Dimethyladenosine (m6am), 3 -Methylcytidine (m3c), 2'-O- Methylation (refers to 2'-O-methyl nucleosides; Nm), 5-Hydroxymethylcytidine (hm5c), and N1 -methylpseudouridine (ml\| / ). For example, the modified nucleotide can be Nl- methylpseudouridine (ml\|i). N1 -methylpseudouridine (ml\| / ) is the methylated derivative of pseudouridine and increases immune evasion and protein production. The chemical structure, molecular formula, site of action, and function of exemplary modified nucleotides are detailed in Table 1. More detailed description of nucleic acid modifications and their effects on translation is reviewed, for example, in Roy (2021) “Chapter 20: The Effects of mRNA modifications on Translation: An Overview”, In Mary McMahon (Ed.), RNA Modifications: Methods and Protocols, Methods in Molecular Biology, 2298: pp. 327-355, Springer; Boo and Kim, Nature: Experimental and Molecular Medicine, 52: 400-408 (2020); Flamand, et al., Annual Review of Biochemistry, 92:145-173 (2023); Gilbert and Nachtergaele, Annual Review of Biochemistry, 92:175-198 (2023); and Mei and Wang, Clinical and Experimental Medicine, 23: 1917-1931 (2023), the contents of which arc incorporated by reference herein in their entireties. iv. Nucleic Acid Encoding Adjuvants

[0105] The one or more nucleic acid (s) in the composition can optionally include one or more nucleic acid (s) encoding or expressing an adjuvant. Generally, adjuvants are additional immunostimulant compounds besides antigens (i.e., the immunizing agent) in vaccines, which activate innate immunity and provide the “help” needed to increase the magnitude and quality of the adaptive responses against pathogens (Coffman et al., Immunity 33, 492-503 (2010)). The use of adjuvants during vaccination helps to increase the vaccine immunogenicity, improve immunological memory, and decrease the vaccine dose and administration frequency (Zhi et al., Bioprep. Profit. Diagn. Lech., 1 (53), 15 - 20 (2015)).

[0106] In some forms, the one or more nucleic acid (s) encoding or expressing an adjuvant can be an RNA encoding or expressing an adjuvant. Exemplary RNA-encoded adjuvants include but are not limited to interleukins, interferons, Granulocyte-Macrophage Colony-Stimulating Factor (GMCSF), Retinoic Acid-Inducible Gene I, chemokines, pattern recognition receptors, flagellin, bacterial toxins, tumor necrosis factors, and heat shock proteins.

[0107] In other forms, the one or more nucleic acid (s) encoding or expressing an adjuvant can be a DNA encoding or expressing an adjuvant. Exemplary DNA-encoded adjuvants include but are not limited to interleukins, interferons, Granulocyte-Macrophage Colony-Stimulating Factor (GMCSF), Retinoic Acid-Inducible Gene I, chemokines, pattern recognition receptors, flagellin, bacterial toxins, tumor necrosis factors, and heat shock proteins.

[0108] In some forms, the RNA- or DNA-encoded adjuvant is a cytokine. Cytokines are regulators of immune-response development at all its stages for any antigenic stimulus and can be considered as potential immunoadjuvants for vaccination. Cytokines are involved in all steps of the immune response including differentiation of immunocompetent precursor cells, AG presentation, cell activity and proliferation, and adhesion molecule expression. Cytokine synthesis is induced, and their receptors are expressed upon activation of myeloid, lymphoid, and reticuloendothelial cells. Cytokines affect cells locally or systemically at very low concentrations. Cytokines have been introduced as recombinant proteins or cytokine encoding plasmids to modify the performance of memory cells, or as a traditional adjuvant to strengthen the immune response induced by vaccination. Cytokines are subdivided into several classes e.g., interleukins (IL), interferons, cell growth factors, and non-lymphoid cell growth factors. More detailed description of cytokines as adjuvants are reviewed in Alphatova et al.. Pharmaceutical Chemistry Journal, 53(11): 3-8 (2020); Rahman et al., Cytokine, 169: 156268 (2023), the contents of which arc incorporated herein by reference in their entireties.

[0109] In some forms, the RNA- or DNA-encoded adjuvant can be an interferon. Exemplary interferons that can be used as the RNA- or DNA-encoded adjuvant include but are not limited to IFN-a, IFN-P, IFN-y, IFN-s, IFN-K, IFN-M, IFN-X2, IFN-X3, and IFN-co. For example, Audsley and colleagues demonstrated that inclusion of 1FN-P as an adjuvant in a vaccine containing anti-PD-Ll checkpoint blockade therapy, significantly boosted T cell expansion and delayed tumor progression (Audsley, et al., Frontiers in Immunology, 12: Article 735133 (2021).

[0110] In some forms, the RNA-or DNA-encoded adjuvant can be an interleukin. Exemplary interleukins that can be used as the RNA- or DNA-encoded adjuvant include but are not limited to IL-1, IL-1 IL-2, IL-4, IL-6, IL-7, IL-10, IL-11, IL-12, IL-L5, IL-18, IL-21, and IL-33.

[0111] In some forms, the RNA- or DNA-encoded adjuvant is a colony stimulating factor. Exemplary colony stimulating factors that can be used as the RNA- or DNA-encoded adjuvant include but are not limited to Granulocyte colony-stimulating factor (G-CSF), Granulocytemacrophage colony-stimulating factor (GM-CSF), and Macrophage colony-stimulating factor (M-CSF).

[0112] In some forms, the RNA- or DNA-encoded adjuvant is a tumor necrosis factor. These cytokines are characterized by a P-sandwich structure and generally regulate inflammation, cell proliferation, and apoptosis. Exemplary tumor necrosis factors include but are not limited to TNF-a (Tumor Necrosis Factor-alpha), TNF-P (Lymphotoxin-alpha), CD40 Ligand (CD40L), 0X40 Ligand (OX40L), 4-1BB Ligand (4-1BBL), BAFF (B-cell Activating Factor), APRIL (A Proliferation-Inducing Ligand), and RANK Ligand (Receptor Activator of Nuclear Factor KB Ligand).

[0113] In some forms, the RNA- or DNA-encoded adjuvant is a chemokine. Example chemokines that can be used as the RNA- or DNA-encoded adjuvant include but are not limited to CXCL-1, CXCL-10, CXCL12, CCL2, CCL3, CCL4, and CCL5. Other Exemplary chemokines that can be used as an adjuvant are reviewed in Bobanga et al., Vaccines, 1(4):444- 462 (2013). v. Fusion Peptides

[0114] Nucleic acids included in the disclosed compositions can encode a fusion protein / peptide. Methods of making nucleic acids encoding proteins with different biological activities are known in the art.

[0115] To recombinantly produce a fusion protein of interest, a nucleic acid containing a nucleotide sequence encoding the polypeptide can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (c.g., an insect, yeast, or mammalian cell). In general, nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding a fusion proteins of interest. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product, but instead affect the expression of the nucleic acid sequences to which they are operably linked. The nucleotide sequences encoding the fusion protein are usually inserted into a recombinant vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. The vector is preferably an expression vector in which the DNA sequence encoding the fusion protein is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, “operably linked” indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the fusion protein. Expression vectors for use in expressing the fusion protein will comprise a promoter capable of directing the transcription of a cloned gene or cDNA. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Expression vectors for use in expressing the fusion protein will comprise a promoter capable of directing the transcription of a cloned gene or cDNA. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA in mammalian cells are the SV40 promoter (Subramani el al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), the CMV promoter (Boshart et al., Cell 41 :521-530, 1985) or the adenovims 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982).

[0116] Exemplary Combinations of Nucleic Acids

[0117] In some forms, each nucleic acid molecule in the composition can have one or more nucleotide modifications. In one exemplary form, the nucleic acid can be an mRNA encoding a spike protein of a virus without any modification. In a second exemplary form, the nucleic acid can be an mRNA encoding a spike protein of a vims having a 5moU base modification. In a third exemplary form, the nucleic acid can be an mRNA encoding a spike protein of a vims having a mly modification. In a fourth exemplary form, the composition can contain two nucleic acids, wherein one nucleic acid is a circRNA encoding a spike protein and the other nucleic acid is mRNA having a ml\| / base modification. In a fifth exemplary form, the composition can contain two nucleic acids, wherein one nucleic acid is ml\| / modified mRNA encoding a spike protein and the other nucleic acid is a DNA encoding IFN-y. In a sixth exemplary form, the composition can contain two nucleic acids, wherein one nucleic acid is circRNA encoding a spike protein and the other nucleic acid is a DNA encoding IFN-y.

[0118] In some forms, the composition can contain one or more nucleic acid molecule (s) encoding the same or different antigens. Employing different combinations of nucleic acids can improve antigen delivery and immune activation. These various combinations offer significant flexibility in designing vaccines that elicit potent and tailored immune responses, for example, in the context of rapidly evolving viruses such as SARS-CoV-2 and influenza.

[0119] In one exemplary form, the composition can include a N1 -methylpseudouridine (ml\| / ) modified mRNA encoding the Spike protein (Nl\| / mRNA-Spike) and a circRNA encoding the Spike protein (circRNA-Spike). This combination leverages the enhanced protein production of ml\| / -modified mRNA and the stability of circRNA to elicit a robust immune response against the same antigen. In one exemplary form, the ml -modified Spike mRNA can be a synthetic construct corresponding to the SARS-CoV-2 Spike sequence used in the Modema (mRNA- 1273) vaccine (GenScript, RP-A00065). The mRNA includes a Capl structure with high capping efficiency and 100% substitution of uridine with Nl-methyl-pseudouridine (ml\| / ), which enhances expression while reducing innate immunogenicity. This exemplary transcript includes a 100-adenine poly(A) tail to mimic mature mRNA and is linearized with a restriction site for translational performance. In some forms, the composition can include such ml\| / - modified Spike mRNA as the mRNA component of a dual-modality vaccine formulation (e.g., CHIMERA-type constructs), combined with a DNA or circRNA encoding the same antigen to enhance both humoral and cellular immune responses. An exemplary nucleic acid sequence for the is provided in Section II(A)(vi)(a) below as SEQ ID NO:1.

[0120] In another exemplary form, the composition can include a circRNA encoding Spike protein (circRNA-Spike) and a DNA encoding Spike protein (DNA-Spike). This combination utilizes the different mechanisms of RNA and DNA vaccines to diversify antigen presentation, potentially boosting the breadth and duration of the immune response. In a third exemplary form, the composition can include a Nlxp mRNA encoding Spike protein (Nl\| / mRNA-Spike), a Nl\| / mRNA encoding Hemagglutinin (Nl\p mRNA-HA), a DNA encoding Spike protein (DNA- Spike), and a DNA encoding Hemagglutinin (DNA-HA). This form allows for simultaneous immunization against both the Spike protein, commonly associated with coronaviruses, and Hemagglutinin (HA), a key antigen in influenza vaccines. Such a strategy provides broader immune protection, enabling the vaccine to target multiple pathogens or viral variants. In another exemplary form, the composition can contain two nucleic acids. In some forms, both nucleic acids are mRNAs, wherein each mRNA encodes different antigens. For example, one mRNA can encode Hemagglutinin (mRNA-HA) and the other mRNA can encode IFN-y (mRNA-IFN-y). Optionally, one or both of these mRNAs can include a nucleotide modification. For example, mRNA-HA can include a Mly modification and the mRNA-IFN-y can include a 5MoU modification.

[0121] In other forms, one nucleic acid can be a circRNA encoding a first antigen and the second nucleic acid can be an mRNA encoding a second antigen. For example, the circRNA can encode the spike protein of SARS-CoV-2 (circRNA-Spike), while the mRNA can encode an adjuvant, such as IL-2 (mRNA-IL2). Optionally, one or both of these nucleic acids can incorporate modified nucleotides. For instance, the circRNA-Spike may remain unmodified, while the mRNA-IL2 could include an Ml\| / modification. This combination could enhance the effectiveness of a COVID vaccine by leveraging the durability of circRNA and boosting T-cell response through the inclusion of IL-2.

[0122] In some forms, the composition can contain more than two nucleic acids. In one exemplary form, the composition can contain two mRNA molecules and one DNA molecule. For example, the first nucleic acid can be a MIT-modified mRNA encoding the SARS-CoV-2 spike protein (MIT-mRNA-Spike), the second nucleic acid can be DNA encoding the spike protein (DNA-Spike), and the third nucleic acid can be a M IT-modified mRNA encoding IL-6 (M 1 T-mRNA-IL-6). The mRNA and DNA forms of the spike protein provide both immediate and prolonged antigen expression, broadening the antibody response. IL-6 serves as an adjuvant, improving T-cell activation and promoting stronger, longer-lasting immunity. Overall, this combination can improve both the breadth and durability of immune protection.

[0123] In another example, the first nucleic acid can be a MIT-modified mRNA encoding the RSV F glycoprotein (M 1 T-mRNA-RSV-F), the second nucleic acid can be DNA encoding the RSV F glycoprotein (DNA-RSV-F), and the third nucleic acid can be a MIT- modified mRNA encoding IL-6 (MlT-mRNA-IL-6). This combination provides both mRNA and DNA forms of the RSV F glycoprotein, which can provide immediate and prolonged antigen expression, thereby broadening the antibody response against RSV. The MIT-modified mRNA encoding IL-6 acts as an adjuvant to boost T-cell activation, improving the overall immune response. Together, this composition can provide a more robust and durable immune protection against RSV.

[0124] In a second exemplary form, the composition can include three mRNA molecules and two DNA molecules. For example, the first nucleic acid can be a MIT-modified mRNA encoding the SARS-CoV-2 spike protein (M 1 T-mRNA-Spike), the second nucleic acid can be DNA encoding the spike protein (DNA-Spike), the third nucleic acid can be a MIT-modified mRNA encoding the hemagglutinin protein (M IT-mRNA-I lemagglutinin), the fourth nucleic acid can be DNA encoding the hemagglutinin protein (DNA-Hemagglutinin), and the fifth nucleic acid can be a MIT-modified mRNA encoding IL-6 (M 1 T-mRNA-IL-6). This combination provides both mRNA and DNA forms of the spike and hemagglutinin proteins, ensuring immediate and prolonged expression of antigens from both SARS-CoV-2 and influenza, thereby broadening the antibody response against both viruses. IL-6 acts as an adjuvant, improving T-cell activation and supporting stronger, more durable immunity. Overall, this combination can create a combination COVID / Flu vaccine with broader and longer-lasting immune protection.

[0125] In a third exemplary form, the composition can contain multiple nucleic acids targeting different strains of influenza. For example, the composition can include several MIT-modified mRNA molecules and DNA molecules. Specifically, the first nucleic acid can be a MIT- modified mRNA encoding hemagglutinin from Influenza A (H1N1) (MlT-mRNA- Hemagglutinin H1N1), the second can be a MIT-modified mRNA encoding hemagglutinin from Influenza A (II3N2) (MIT-mRNA-I lemagglutinin II3N2), the third can be a MIT-modified mRNA encoding hemagglutinin from Influenza B (Victoria lineage) (MlT-mRNA- Hemagglutinin Victoria), and the fourth can be a MIT- modified mRNA encoding hemagglutinin from Influenza B (Yamagata lineage) (MIT-mRNA-Hemagglutinin Yamagata). Additionally, the composition can include DNA encoding conserved epitopes of hemagglutinin (DNA- Hemagglutinin) and DNA encoding conserved epitopes of neuraminidase (DNA- Neuraminidase). Finally, the composition can include MIT-modified mRNA encoding IL-6 (MlT-mRNA-IL-6) and MIT-modified mRNA encoding IL-12 (MlT-mRNA-IL-12). This combination offers a broad-spectrum, multivalent flu vaccine, providing antigenic coverage across multiple lineages of influenza A and B. The inclusion of DNA encoding conserved epitopes of hemagglutinin and neuraminidase helps induce a more durable immune response by targeting highly conserved regions of the virus. The MlT-mRNA-IL-6 and MlT-mRNA-IL-12 act as adjuvants, enhancing both B cell and T cell responses, resulting in a more robust and long- lasting immunity. Overall, this composition aims to improve both the breadth and durability of protection compared to conventional flu vaccines by targeting multiple lineages and enhancing immune activation.

[0126] In a third exemplary form, the composition can contain a large panel of nucleic acids encoding tumor-associated antigens (TAAs) and immune modulators. In some forms, the composition can include multiple MIT-modified mRNA molecules and DNA molecules. For example, the first set of nucleic acids can be MIT-modified mRNAs encoding a range of patient-specific tumor-associated antigens (MIT-mRNA-TAAl, MlT-mRNA-TAA2, up to MlT-mRNA-TAA40). Additionally, the composition can include DNA encoding the same panel of tumor-associated antigens (DNA-TAA1 to DNA-TAA40), and a MIT-modified mRNA encoding IL- 12 (MlT-mRNA-IL-12) to serve as an immune adjuvant.

[0127] For example, the chimeric constructs can also be applied to encode consensus or personalized tumor antigens, facilitating individualized immunotherapy approaches that target unique neoantigen landscapes. Also, chimeric formulations can encode B-cell epitopes of selfantigens such as PCSK9, ANGPTL3, or lipoprotein(a) (Lpa) to induce tolerogenic or neutralizing immune responses for preventing or treating cardiovascular disease. Similarly, chimeric constructs can encode antigens associated with infectious diseases or autoimmune disorders, providing a flexible platform for vaccine and immunomodulatory applications across multiple therapeutic areas.

[0128] This neoantigen cancer vaccine can be designed to encode a broad panel of patientspecific TAAs using both mRNA and DNA backbones, thereby improving antigen presentation and eliciting diversified immune responses. The dual delivery system ensures both immediate and sustained expression of TAAs, helping to stimulate both B cell and T cell responses. The MlT-mRNA-IL- 12 further acts as an adjuvant, promoting localized immune activation to boost the overall anti-tumor response. This combination aims to induce a robust and durable immune attack against a wide array of tumor antigens, tailored to the specific tumor profile of the patient. vi. Antigens

[0129] The one or more isolated nucleic acid (s) contained in the composition encodes one or more protein (s) and / or peptide (s), which function as antigen (s). The one or more protein (s) and / or peptide (s) are generally effective to induce an immune response in the subject.

[0130] In some forms, the one or more nucleic acid (s) encoding the one or more antigen (s) is isolated from a pathogenic microorganism. For example, a nucleic acid encoding an antigen can be isolated from a virus, bacterium, protozoan, parasite, or a fungus. Exemplary antigens are described below.

[0131] In other forms, the one or more nucleic acid (s) encoding the one or more antigen (s) is a cancer antigen. For example, a nucleic acid encoding a cancer antigen can be isolated directly from tumor tissue such as directly from cancer cells or tumor biopsy samples, where the DNA or RNA encoding tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs) is present. These antigens may arise from mutated proteins, overexpressed proteins, or cancer-specific splice variants. In another example, cancer antigens can be isolated from circulating tumor DNA in blood samples as part of liquid biopsies. ctDNA is released from dying cancer cells into the bloodstream and can contain mutations or other alterations specific to cancer antigens. The nucleic acid encoding the antigen can also be isolated from cancer cell lines and cancer vaccine libraries. Exemplary cancer antigens are provided below. a. Exemplary Pathogenic Antigens

[0132] The antigen encoded by the nucleic acid can be a viral antigen, bacterial antigen, parasitic antigen, protozoan antigen, or a fungal antigen.

[0133] In preferred forms, the antigen is a viral antigen. Exemplary viral families from which the nucleic acid encoding the antigen can be isolated include but are not limited to Adenoviridae, Arenaviridae , Astroviridae, Bornaviridae , Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae. Rhabdoviridae, and Togaviridae. In some forms, the nucleic acid encoding the antigen is derived from a vims of the Coronaviridae family. For example, the nucleic acid encoding the antigen can be isolated from a coronavirus. Exemplary coronaviruses include but are not limited to SARS-CoV (Severe Acute Respiratory Syndrome coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV-2 (the vims responsible for COVID-19), HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKUl.

[0134] In some forms, the nucleic acid encoding the antigen is derived from a vims of the Flaviviridae family. For example, the nucleic acid encoding the antigen can be isolated from a flavivims. Exemplary flavivimses include but are not limited to Dengue vims, Zika vims, West Nile vims, Yellow Fever vims, Japanese Encephalitis vims, and Tick-home Encephalitis vims.

[0135] In some forms, the nucleic acid encoding the antigen is derived from a vims of the Orthomyxoviridae family. For example, the nucleic acid encoding the antigen can be isolated from a orthomyxovirus. Exemplary orthomyxoviruses include but are not limited to Influenza A vims, Influenza B vims, and Influenza C vims.

[0136] In some forms, the nucleic acid encoding the antigen is derived from a vims of the Paramyxoviridae family. For example, the nucleic acid encoding the antigen can be isolated from a paramyxovims. Exemplary paramyxoviruses include but are not limited to Respiratory syncytial vims (RSV) and parainfluenza vims.

[0137] In some forms, the nucleic acid encoding the antigen is derived from a vims of the Retroviridae family. For example, the nucleic acid encoding the antigen can be isolated from a retrovirus. Exemplary retroviruses include but are not limited to human immunodeficiency vims, and human t-lymphotrophic vims type 1 (HTLV-1).

[0138] In some forms, the nucleic acid encoding the antigen is derived from a vims of the Adenoviridae family. For example, the nucleic acid encoding the antigen can be isolated from an adenovims. Exemplary adenovimses include but are not limited to Human adenovims A (HAdV-A), Human adenovims B (HAdV-B), Human adenovims C (HAdV-C), Human adenovims D (HAdV-D), Human adenovims E (HAdV-E), Human adenovims F (HAdV-F), and Human adenovims G (HAdV-G).

[0139] In some forms, the nucleic acid encoding the antigen is derived from a vims of the Arenaviridae family. For example, the nucleic acid encoding the antigen can be isolated from an arenavirus. Exemplary arenaviruses include but are not limited to Lassa vims, Junin vims, Machupo vims, and Guanarito vims.

[0140] In some forms, the nucleic acid encoding the antigen is derived from a vims of the Filoviridae family. For example, the nucleic acid encoding the antigen can be isolated from a filovims. Exemplary filovimses include but are not limited to ebola vims and Marburg vims.

[0141] In some forms, the nucleic acid encoding the antigen is derived from a vims of the Herpesviridae family. For example, the nucleic acid encoding the antigen can be isolated from a herpes vims. Exemplary herpes vimses include but are not limited to Herpes simplex vims 1 (HSV-1), Herpes simplex vims 2 (HSV-2), Varicella- zoster vims (VZV), Epstein-Barr vims (EBV), Cytomegalovims (CMV), Human herpesvirus 6 (HHV-6), Human herpesvirus 7 (HHV- 7), and Kaposi's sarcoma-associated herpesvirus (KSHV / HHV-8).

[0142] Other exemplary vimses from which the nucleic acid encoding the antigen can be derived include but are not limited to Human parvovirus B 19, Human picobirnavims, poliovims, rhinovimses, enterovims such as enterovims D68, hepatitis A vims, coxsackievirus, Human respiratory syncytial vims (HRSV), Human metapneumovims (HMPV), BK vims (BKV), JC vims (JCV), Merkel cell polyomavims (MCV), Variola vims, Vaccinia vims. Monkeypox vims, rotavims, Colorado tick fever vims, rabies vims, rubella, and chikungunya vims.

[0143] RNA sequences, including mRNA sequences and circRNA sequences that can be used in a vaccine can be commercially obtained. For example, the GenScript Spike mRNA containing Nl-Mcthylpscudouridinc / mlvP is represented by SEQ ID NO:1. This Spike mRNA sequence is a synthetic constmct HCV1145 Modema (mRNA-1273) vaccine sequence. This mRNA is capped with Capl stmeture with high capping efficiency. It has 100% substituted with Nl-methyl- pseudo-Uridine for enhanced expression and reduced immunogenicity. The mRNA has a 100A tail in its sequence, mimicking a mature mRNA. mRNA ORF Sequence for SARS-COV2 Spike Protein (4111 nt)

[0144] ATGTTCGTGTTCCTGGTCTCTGCTCTCCCCTGGTCTAGCAGCCAGTGCGTGAACCTGACC

[0145] ACCCGGACCCAGCTGCCACCAGCCTACACCAACAGCTTCACCCGGGGCGTCTACTA

[0146] CCCCGACAAGGTGTTCCGGAGCAGCGTCCTGCACAGCACCCAGGACCTGTTCCTGCC

[0147] CTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAACGGCAC

[0148] CAAGCGGTTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGCAC

[0149] CGAGAAGAGCAACATCATCCGGGGCTGGATCTTCGGCACCACCCTGGACAGCAAGA

[0150] CCCAGAGCCTGCTGATCGTGAATAACGCCACCAACGTGGTGATCAAGGTGTGCGAG

[0151] TTCCAGTTCTGCAACGACCCCTTCCTGGGCGTGTACTACCACAAGAACAACAAGAGC

[0152] TGGATGGAGAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTA

[0153] CGTGAGCCAGCCCTTCCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACC

[0154] TGCGGGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCAC

[0155] ACCCCAATCAACCTGGTGCGGGATCTGCCCCAGGGCTTCTCAGCCCTGGAGCCCCTG

[0156] GTGGACCTGCCCATCGGCATCAACATCACCCGGTTCCAGACCCTGCTGGCCCTGCAC

[0157] CGGAGCTACCTGACCCCAGGCGACAGCAGCAGCGGGTGGACAGCAGGCGCGGCTG

[0158] CTTACTACGTGGGCTACCTGCAGCCCCGGACCTTCCTGCTGAAGTACAACGAGAACG

[0159] GCACCATCACCGACGCCGTGGACTGCGCCCTGGACCCTCTGAGCGAGACCAAGTGC

[0160] ACCCTGAAGAGCTTCACCGTGGAGAAGGGCATCTACCAGACCAGCAACTTCCGGGT

[0161] GCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGG

[0162] CGAGGTGTTCAACGCCACCCGGTTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGA

[0163] TCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACC

[0164] TTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTG

[0165] TACGCCGACAGCTTCGTGATCCGTGGCGACGAGGTGCGGCAGATCGCACCCGGCCA

[0166] GACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCG

[0167] TGATCGCCTGGAACAGCAACAACCTCGACAGCAAGGTGGGCGGCAACTACAACTAC

[0168] CTGTACCGGCTGTTCCGGAAGAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCAC

[0169] CGAGATCTACCAAGCCGGCTCCACCCCTTGCAACGGCGTGGAGGGCTTCAACTGCT

[0170] ACTTCCCTCTGCAGAGCTACGGCTTCCAGCCCACCAACGGCGTGGGCTACCAGCCCT

[0171] ACCGGGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCAGCCACCGTGTGTGGCC

[0172] CCAAGAAGAGCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGC

[0173] CTTACCGGCACCGGCGTGCTGACCGAGAGCAACAAGAAATTCCTGCCCTTTCAGCA

[0174] GTTCGGCCGGGACATCGCCGACACCACCGACGCTGTGCGGGATCCCCAGACCCTGG

[0175] AGATCCTGGACATCACCCCTTGCAGCTTCGGCGGCGTGAGCGTGATCACCCCAGGC

[0176] ACCAACACCAGCAACCAGGTGGCCGTGCTGTACCAGGACGTGAACTGCACCGAGGT GCCCGTGGCCATCCACGCCGACCAGCTGACACCCACCTGGCGGGTCTACAGCACCG

[0177] GCAGCAACGTGTTCCAGACCCGGGCCGGTTGCCTGATCGGCGCCGAGCACGTGAAC AACAGCTACGAGTGCGACATCCCCATCGGCGCCGGCATCTGTGCCAGCTACCAGAC

[0178] CCAGACCAATTCACCCCGGAGGGCAAGGAGCGTGGCCAGCCAGAGCATCATCGCCT ACACCATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACAGCAACAACAGCATCGCC ATCCCCACCAACTTCACCATCAGC:GTGACCACCGAGATTCTGCCCGTGAGCATGACC AAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGACAGCACCGAGTGCAGCAA CCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAACCGGGCCCTGACCGGCAT CGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTCGCCCAGGTGAAGCAGATCT ACAAGACCCCTCCCATCAAGGACTTCGGCGGCTTCAACTTCAGCCAGATCCTGCCC:G ACCCCAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAGGTG ACCCTAGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGCCTCGGCGACATAGC CGCCCGGGACCTGATCTGCGCCCAGAAGTTCAACGGCCTGACCGTGCTGCCTCCCCT

[0179] GCTGACCGACGAGATGATCGCCCAGTACACCAGCGCCCTGTTAGCCGGAACCATCA

[0180] CCAGCGGCTGGACTTTCGGCGCTGGAGCCGCTCTGCAGATCCCCTTCGCCATGCAGA

[0181] TGGCCTACCGGTTCAACGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAACCAG AAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAG CAGCACCGCTAGCGCCCTGGGCAAGCTGCAGGACGTGGTGAACCAGAACGCCCAGG CCCTGAACACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCATCAGCAGC:GTG CTGAACGACATCCTGAGCCGGCTGGACCCTCCCGAGGCCGAGGTGCAGATCGACCG GCTGATCACTGGCCGGCTGCAGAGCCTGCAGACCTACGTGACCCAGCAGCTGATCC GGGCCGCCGAGATTCGGGCCAGCGCCAACCTGGCCGCCACCAAGATGAGCGAGTGC GTGCTGGGCCAGAGCAAGCGGGTGGACTTCTGCGGCAAGGGCTACCACCTGATGAG

[0182] CTTTCCCCAGAGCGCACCCCACGGAGTGGTGTTCCTGCACGTGACCTACGTGCCCGC

[0183] CCAGGAGAAGAACTTCACCACCGCCCCAGCCATCTGCCACGACGGCAAGGCCCACT TTCCCCGGGAGGGCGTGTTCGTGAGCAACGGCACCCACTGGTTCGTGACCCAGCGG AACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGAGCGGCAACTG CGACGTGGTGATCGGCATCGTGAACAACACCGTGTACGATCCCCTGCAGCCCGAGC TGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAATCACACCAGCCCCGAC

[0184] GTGGACCTGGGCGACATCAGCGGCATCAACGCCAGCGTGGTGAACATCCAGAAGGA GATCGATCGGCTGAACGAGGTGGCCAAGAACCTGAACGAGAGCCTGATCGACCTGC AGGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGC TTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGCATGACC AGCTGCTGCAGCTGCCTGAAGGGCTGTTGCAGCTGCGGCAGCTGCTGCAAGTTCGA CGAGGACGACAGCGAGCCCG TGC TGAAGGGCG TGAA GC TGCACTACACC IGA TAA (SEQ ID NO:1).

[0185] Another chimeric construct includes combining the N-terminal domain (NTD) of SARS- CoV-2 with the receptor-binding domain (RBD) and S2 region of SARS-CoV, including a ml'P- modification, thereby creating a hybrid spike protein capable of eliciting cross-neutralizing antibodies against both pandemic and epidemic SARS-related coronaviruses. SEQ ID NO:2 represents an exemplary amino acid sequence encoded by this chimeric construct.

[0186] MFVFLVLLPLVSSQCGIISRKPQPKMAQVSSSRRGVYYNDDIFRSDVLIILTQDYFLPFDS NETQYFSENVDSDRYTYFDNPIEDFGDGVYFAATEKSNVIRGWIFGSSFDNTTQSAVIVN NSTHIIIRVCNFNLCKEPMYTVSRGTQQNAWVYQSAFNCTYDRVEKSFQLDTTPKTGNF KDEREYVFKNRDGFESVYQTYTAVNEPRGEPTGFSVLKPIEKEPFGINITSYRVVMAMFS QTTSNFEPESAAYYVGNEKYSTFMERFNENGTITDAVDCSQNPEAEEKCTIKNFTVEKGI YQTSNFRVQPTESIVRFPNITNECPFGEVFNATKFPSVYAWERKKISNCVADYSVEYNST FFSTFKCYGVSATKENDECFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKEPDDFM GCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCY WPENDYGFYTTTGIGYQPYRVVVESFEELNAPATVCGPKKSTNEVKNKCVNFNFNGET GTGVETESNKKFEPFQQFGRDIADTTDAVRDPQTEEIEDITPCSFGGVSVITPGTNTSNQV AVEYQDVNCTEVPVAIHADQETPTWRVYSTGSNVFQTRAGCEIGAEHVNNSYECDIPIG AGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPV SMTKTSVDCTMYICGDSTECSNLEEQYGSFCTQENRAETGIAVEQDKNTQEVFAQVKQI YKTPPIKDFGGFNFSQIEPDPSKPSKRSFIEDEEFNKVTEADAGFIKQYGDCEGDIAARDEI CAQKFNGETVLPPEETDEMIAQYTSAELAGTITSGWTFGAGAAEQIPFAMQMAYRFNGI GVTQNVEYENQKLIANQFNSAIGKIQDSESSTASAEGKEQDVVNQNAQALNTEVKQESS NFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK

[0187] MSECVEGQSKRVDFCGKGYHLMSFPQSAPHGVVFEHVTYVPAQEKNFTTAPAICHDGK AHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPEQPEED SFKEEEDKYFKNHTSPDVDEGDISGINASVVNIQKEIDRENEVAKNENESEIDEQEEGKY EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL KGVKLHYT (SEQ ID NO: 2)

[0188] Another chimeric construct includes combining the NTD and S2 region of SARS-CoV-2 with the RBD from SARS-CoV, including a ml'P-modification, thereby influencing immune recognition and breadth of protection across Sarbecoviruses. SEQ ID NOG represents an exemplary amino acid sequence encoded by this chimeric construct. MFIFLLFLTLTSGSDLDRCTrFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFL

[0189] PFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNST NVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKH LREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQ DIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQ TSNFRVVPSGDVVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFP LQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKLSTDLIKNQCVNFNFNGLTGTG VLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVL YQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGIC ASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVD CNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYKTPTLK YFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKF NGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQ NVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGA ISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECV LGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPR EGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELD KYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP WYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHY T (SEQ ID N0:3)

[0190] Another chimeric construct includes combining the NTD and S2 region of SARS-CoV combined with the RBD from bat SARS-like CoV RsSHC014, a pre-emergent bat coronavims with zoonotic potential, including a ml'P-modification. SEQ ID NO:4 represents an exemplary amino acid sequence encoded by this chimeric construct.

[0191] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD LEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATKFPSVYAWERKKISN CVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIA DYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDLSNVPFSPDG KPCTPPALNCYWPENDYGFYTTTG1GYQPYRVVVESFEELNAPATVCGPKKSTNLVKN KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH VNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPT NFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQN AQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRA AEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEK NFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIV NNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNL NESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSC CKFDED DSEPVLKGVKLHYT (SEQ ID N0:4).

[0192] Another chimeric construct includes combining the NTD and S2 region of bat S ARS-like CoV HKU3-1 with the RBD of SARS-CoV-2, including a ml'P-modification. SEQ ID NO:5 represents an exemplary amino acid sequence encoded by this chimeric construct.

[0193] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD LEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFPSVYAWERKRISN CVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIA DYNYKLPDDFLGCVLAWNTNSKDSSTSGNYNYLYRWVRRSKLNPYERDLSNDIYSPGG QSCSAVGPNCYNPLRPYGFFTTAGVGHQPYRVVVLSFELLNAPATVCGPKKSTNLVKN KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH VNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPT NFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP

[0194] FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQN AQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRA AEIRASANEAATKMSECVEGQSKRVDFCGKGYHEMSFPQSAPHGVVFEHVTYVPAQEK NFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIV NNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNL NESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSC CKFDED DSEPVLKGVKLHYT (SEQ ID N0:5). b. Exemplary Cancer Antigens

[0195] In some forms, the antigen is a cancer antigen or a nucleic acid thereof encoding a cancer antigen. A cancer antigen is an antigen that is typically expressed preferentially by cancer cells (i.e., it is expressed at higher levels in cancer cells than on non-cancer cells; cancer-associated antigen) and in some instances it is expressed solely by cancer cells (cancer-specific antigen). Cancer antigen may be expressed within a cancer cell or on the surface of the cancer cell. Exemplary cancer antigens include tumor-associated antigens (TAAs), tumor specific antigens (TSAs), tissue-specific antigens, viral tumor antigens, cellular oncogene proteins, and / or tumor- associated differentiation antigens. These antigens can serve as targets for the host immune system and elicit responses which result in tumor destruction. (1990) . / . Biol. Response Mod. 9:499 511.

[0196] In some forms, the cancer antigen is a tumor-associated antigen (TAA). Tumor associated antigens proteins are overexpressed in cancer cells but are also present in normal cells, although typically at lower levels. Exemplary tumor associated antigens include but are not limited to carcinoembryonic antigen (CEA) commonly found in colorectal, pancreatic, gastric, and lung cancers; Alpha-fetoprotein (AFP) which is elevated in liver cancer and germ cell tumors; prostate-specific antigen (PSA), which is overexpressed in prostate cancer; HER2 / neu (ERBB2) which is overexpressed in breast and gastric cancers; Mucin- 1 (MUC1) which is found in breast, ovarian, and lung cancers; Wilms tumor protein (WT1), which is overexpressed in several cancers, including leukemia and ovarian cancer; Survivin, which is found in many cancers, including lung, breast, and colorectal cancers; Melanoma-associated antigens (MAGE-A3), commonly found in melanoma, as well as other cancers like lung and bladder; and Cancer-testis antigens (e.g., NY-ESO-1), expressed in various tumors, including melanoma, ovarian, and lung cancer.

[0197] In some forms, the cancer antigen is a tumor specific antigen. Tumor specific antigens arc unique to cancer cells and result from mutations, making them specific to individual tumors. Exemplary tumor specific antigens include but are not limited to mutant KRAS, a common mutation in pancreatic, colorectal, and lung cancers; BCR-ABL fusion protein, found in chronic myeloid leukemia (CML); EGFRvIII, a mutant form of the EGFR protein, found in glioblastoma; mutant p53, often mutated in a wide range of cancers; BRAI V600E, a mutation found in melanoma and other cancers; ALK fusion proteins, which is present in lung cancer and neuroblastoma; and mutant CDKN2A (pl 6), found in various cancers, including melanoma and pancreatic cancer.

[0198] One exemplary chimeric tumor antigen is CHIM-TA-Poly-A, which combines epitopes from NY-ESO-1, WT1, Survivin, and mutant TP53 (R175H). This construct encodes amino acids 157-165 of NY-ESO-1 (sequence: SLLMWITQC) (SEQ ID NO:7), 126-134 of WT1 (sequence: RMFPNAPYL), (SEQ ID NO;8) 95-104 of Survivin (sequence: ELTLGEFLKL) (SEQ ID NO:9), and 168-176 of TP53 (R175H variant) (sequence: HMTEVVRHC) (SEQ ID NOTO). The epitopes can be joined by AAY linkers to facilitate proteasomal processing and MHC class I presentation, producing the amino acid sequence: SLLMWITQCAAYRMFPNAPYLAAYELTLGEFLKLAAYHMTEVVRHC (SEQ ID NO:6). vii. Effective Amounts of Nucleic Acids

[0199] When used for vaccinating a subject against an infectious agent(s) or cancer agent(s), the quantity of nucleic acid(s) included in a pharmaceutical dosage unit or otherwise administered to the subject can be an amount sufficient to effectively reduce infection, viability, proliferation, or any combination thereof, of the infectious agent(s) or cancer agent(s) when used together with the disclosed electroporator. The phrase "dosage unit form" refers to a physically discrete unit of conjugate appropriate for the patient to be treated. It will be understood, however, that the total single administration of the compositions will be decided by the attending physician within the scope of sound medical judgment. The therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rats, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information should then be useful to determine useful doses and routes for administration in humans.

[0200] Exemplary formulations of the one or more nucleic acid (s) include liquids and dry powders. In some forms, the one or more nucleic acid (s) are present in the pharmaceutical composition in an amount from about 1% to about 90%, inclusive, from about 1% to about 80%, from about 1% to about 50%, preferably from about 1% to about 40% by weight, more preferably from about 1% to about 20% by weight, most preferably from about 1% to about 10% by weight. The ranges above are inclusive of all values from 1% to 90%.

[0201] The one or more nucleic acid (s) is present in the pharmaceutical composition at a ratio of total nucleic acid to solution volume ranging from about 0.001X to about 20X. In some forms, the ratio of the one or more nucleic acid (s) to solution volume ranges from about 0.001X to about 0.01X, from about 0.001X to about 0.1X, from about 0.001X to about l.OX, from about 0.001X to about 1.5X, from about 0.001X to about 2.0X, from about 0.001X to about 2.5X, from about 0.001X to about 3. OX, from about 0.001X to about 3.5X, from about 0.001X to about 4.0X, from about 0.001X to about 4.5X, from about 0.001X to about 5. OX, from about 0.001X to about 5.5X, from about 0.001X to about 6.0X, from about 0.001X to about 6.5X, from about 0.001X to about 7. OX, from about 0.001X to about 7.5X, from about 0.001X to about 8.0X, from about 0.001X to about 8.5X, from about 0.001X to about 9. OX, from about 0.001X to about 9.5X, from about 0.00 IX to about 9.6X, from about 0.00 IX to about 9.7X, from about 0.00 IX to about 9.8X, from about 0.001X to about 9.9X, from about 0.001X to about 18X, from about 0.001X to about 16X, from about 0.001X to about 14X, from about 0.001X to about 12X, or from about 0.00 IX to about 1 IX.

[0202] The amount of the one or more nucleic acid (s) in the pharmaceutical composition is effective when delivered to a subject, to , to facilitate expression of the heterologous polypeptide of interest for from about one day to about eight months. In some forms, the amount of the one or more nucleic acid (s) in the pharmaceutical composition is effective when delivered to a subject, to facilitate expression of the heterologous polypeptide of interest for from about one day to about seven months, for from about one day to about six months, for from about one day to about five months, for from about one day to about four months, for from about one day to about three months, for from about one day to about eight weeks, for from about one day to about seven weeks, for from about one day to about six weeks, for from about one day to about five weeks, for from about one day to about four weeks, for from about one day to about three weeks, from about one day to about two weeks, or from about one day to about seven days. For example, the amount of the one or more nucleic acid (s) in the pharmaceutical composition is effective when delivered to a subject, to facilitate expression of the heterologous polypeptide of interest for at least about one day, at least one about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about five weeks, at least about six weeks, at least about seven weeks, at least about eight weeks, at least about nine weeks, at least about ten weeks, at least about eleven weeks, at least about twelve weeks, at least about three months, at least about four months, at least about five months, or at least about six months.

[0203] The amount of the one or more nucleic acid (s) in the pharmaceutical composition is effective, when delivered to a subject, to facilitate an increase in the expression of the one or more antigen (s) by about 5% to about 100% compared to a control. In some forms, the amount of the one or more nucleic acid (s) in the pharmaceutical composition is effective, when delivered to a subject, to facilitate an increase in the expression of the one or more antigen (s) by about 5% to about 99%, by about 5% to about 98%, by about 5% to about 96%, by about 5% to about 95%, by about 5% to about 90%, by about 5% to about 85%, by about 5% to about 80%, by about 5% to about 75%, by about 5% to about 70%, by about 5% to about 65%, by about 5% to about 60%, by about 5% to about 55% , by about 5% to about 50%, by about 5% to about 45%, by about 5% to about 40%, by about 5% to about 35%, by about 5% to about 30%, by about 5% to about 25%, by about 5% to about 20%, or by about 5% to about 15% compared to a control. In some forms, the control is a subject not being delivered or administered the pharmaceutical composition. In some forms, the control is the subject being delivered or administered the pharmaceutical composition prior to administration of the pharmaceutical composition. viii. Pharmaceutically Acceptable Carriers

[0204] In some forms, the pharmaceutical composition includes a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable’’ refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. A “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, involved in carrying or transporting the subject composition from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient.

[0205] Exemplary materials which can serve as pharmaceutically-acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as peptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (24) C2-C12 alcohols, such as ethanol; and (25) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants can also be present in the formulation. In one form, the one or more nucleic acid (s) are formulated in phosphate buffered saline (PBS). In another form, the one or more nucleic acid (s) are formulated in nuclease free water. In a third form, the one or more nucleic acid (s) are formulated in Ringer’s solution.

[0206] Typically, the one or more nucleic acid (s) are not encapsulated in nanoparticles, microspheres, microcapsules, or liposomes. For example, the one or more nucleic acid (s) is not encapsulated in a liposome, nanoemulsion, PLG micro- and nanoparticles, lipoplexes, chitosan micro- and nanopaiticles and other polyplexes. In some forms, the pharmaceutical carrier and / or delivery system does not include liposomes, nanoemulsions, PLG micro- and nanoparticles, lipoplexes, chitosan micro- and nanoparticles and other polyplexes. ix. Treatment Regimens

[0207] A treatment regimen can include one or multiple administrations of a pharmaceutical composition including the described nucleic acids, optionally together with one or more additional active agents, for achieving a desired physiological change in a subject, such as a human. In some forms, the desired physiological change is the reduction in the amount of the pathogen e.g., virus or bacteria, in the body (i.e., in a bodily location or site, such as the lungs, gastrointestinal tract, blood and / or other tissues and organs of the subject). In some forms, the desired physiological change is the reduction in number of cancer cells in the body of the subject. In other forms, the desired physiological change is the prevention of the onset one or more symptoms associated with a disease or disorder such as an infection or cancer.

[0208] An effective amount of a pharmaceutical composition containing the one or more nucleic acid (s) may be administered or delivered repeatedly to a subject (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more). In some forms, the dosage is daily, every other day, every three days, every four days, every five days, or every six days. In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The dosing regimen (including the agents used) can vary over time.

[0209] In some forms, for an adult subject of normal weight, doses ranging from about 0.1 to 1000 mg / kg may be administered or delivered. In some forms, the dose is between about 0.001 mg to about 100 mg. In some forms, the dose for a human is between about 0.1 mg / kg to about 10 mg / kg. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular subject and that subject's medical history, as well as the properties of the agent (such as the half-life of the agent).

[0210] In some forms, pharmaceutical compositions such as vaccines are administered to a subject as a one -part, two-part, or three-part, or more than three-part vaccination schedule. It will also be apparent to one of ordinary skill in the art that the preferred course of treatment can be ascertained using conventional course of treatment determination tests. In some forms, the methods administer a priming dose of a vaccine. The priming dose may be followed by a booster dose. The booster may be for the purpose of revaccination. In some fomrs, the vaccine is administered at least once, twice, three times or more to the same subject. In some forms, the subject is a naive recipient, i.e., a subject being seronegative for particular target strain(s) of the pathogen (e.g., virus) or cancer types.

[0211] Typically, a vaccination schedule includes a first (e.g., “priming”) dose and a second (e.g., “boosting”) dose. For example, in some forms, it will be desirable to have several or multiple administrations of a vaccine including the described nucleic acids. For example, in some forms, vaccines including the nucleic acids are administered to the same subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some forms, the administrations are from about one to about twelve-week intervals, and in certain forms from about one to about four- week intervals. In some forms, periodic re- administration is desirable in the case of recurrent exposure to a particular pathogen targeted by the nucleic acids.

[0212] 2. Devices for Delivering Agents and Components Thereof

[0213] The pharmaceutical compositions containing the one or more nucleic acid (s) are delivered using a device. Exemplary devices that can be used to deliver the pharmaceutical compositions are described in International Application No. PCT / US 2021 / 034959, the contents of which are incorporated herein in their entirety.

[0214] An exemplary device which can deliver a pharmaceutical composition containing the one or more nucleic acid (s) (also referred to as “the pharmaceutical composition”), and which incorporate electroporation includes: a piezoelectric pulse generator; an array of biocompatiblc and conductive microclcctrodcs electrically coupled to the piezoelectric pulse generator; and a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses; wherein the array of biocompatible and conductive microelectrodes comprises an opening for insertion of at least one needle therein for delivery of the pharmaceutical composition containing the self- replicating DNA vectors; wherein insertion of the array of biocompatible and conductive microelectrodes into the biological tissue and activation of the piezoelectric pulse generator generates the one or more electrical pulses through the array of biocompatible and conductive microelectrodes to electroporate cells in the biological tissue to permit administration of the pharmaceutical composition into the electroporated cells.

[0215] In some forms, the device includes a syringe having a container for holding the pharmaceutical composition, which can be coupled to the at least one needle and the pharmaceutical composition can be administered or delivered into a biological tissue by actuation of the syringe. In some instances, the syringe (loaded with a pharmaceutical composition containing the one or more nucleic acid (s)) is coupled to the device and the device is used to administer the pharmaceutical composition intradermally, but without applying electric pulses or toggling the piezoelectric pulse generator. For example, the device can be configured for integrated vertical intradermal injection, featuring an embedded syringe coupled with a depth-limited needle and a microelectrode array (MEA) for electroporation. In an exemplary embodiment, the device includes both a syringe and a luer lock for drug delivery, embedded within the microneedle array. In this configuration, the syringe can be used by trained healthcare personnel. In this configuration, the device integrates injection and electroporation in two steps but delivers a liquid pharmaceutical composition via a single device.

[0216] In some forms, the device includes a detachable syringe, allowing for the delivery of the pharmaceutical composition separately from the application of electrical pulses. This configuration allows the syringe to be removed from the main body of the device after the injection is administered. Once the pharmaceutical composition is delivered, the electrical pulses, typically used for electroporation, can then be applied independently. An exemplary embodiment of such a device is shown in Figure 1C.

[0217] In some forms, the device includes an array of microneedle electrodes electrically coupled to the piezoelectric pulse generator, wherein the device, following insertion of the micronccdlc electrodes into the tissue c.g., skin, is configured to generate and deliver one or more electrical pulses, produced by the piezoelectric pulse generator, through the microneedle electrodes effective to electroporate cells in the biological tissue and allow delivery of the pharmaceutical composition into the electroporated cells. In some forms, the device is configured for self-administration, having microelectrodes which are microneedle electrodes that are coated with an active pharmaceutical ingredient (API). The API may be the one or more nucleic acid (s) described herein and may also include one or more therapeutic, prophylactic, or diagnostic agents, as well as pharmaceutical compositions of such agent(s). The pharmaceutical composition is delivered via the microneedle electrodes, followed by the application of electrical pulses. This embodiment integrates both injection and electroporation, making it suitable for self-administration by the user. In this configuration, the device does not include a syringe or luer lock needle, as the pharmaceutical composition is, for example, a dried formulation that is coated directly onto the microneedle electrodes.

[0218] In other forms, instead of coated microneedles, the device is used in conjunction with a dissolvable patch containing the API, as described above. In this form, the user applies the patch to a targeted area of biological tissue, such as a region of skin, and then applies electric pulses by toggling the piezoelectric pulse generator which facilitates cellular uptake of the pharmaceutical composition to the tissue.

[0219] Figure 2 A shows a non- limiting illustrative view of a device 100 having a needle 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, an array of biocompatible and conductive microelectrodes 200 (also referenced as a microelectrode array “MEA”), a piezoelectric pulse generator 300, and a mechanical switch 310 for activating the piezoelectric pulse generator. Figure 2B shows a non-limiting illustrative head-on view of a device 100 having a needle 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, and an array of biocompatible and conductive microelectrodes 200. Figure 1C shows a non-limiting illustrative side view of a device 100 having a needle 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, an array of biocompatible and conductive microelectrodes 200 (also referenced as a microelectrode array, “MEA”), a piezoelectric pulse generator 300, and a switch 310 for activating the piezoelectric pulse generator.

[0220] In some instances, the array of biocompatiblc and conductive microclcctrodcs extends from a base and the housing contains the piezoelectric pulse generator and the switch. In some other instances, the array of biocompatible and conductive microelectrodes extends from a base and the housing contains the base, the piezoelectric pulse generator, and the switch. In some instances, the base and the array of biocompatible and conductive microelectrodes may form part of a cartridge configured to be releasably coupled to the housing that contains the piezoelectric pulse generator. The base and array can form an independent and self-contained MEA unit which can be coupled to the housing of the device by any suitable means, such as with pins or screws. In some instances, the device includes an optional screw that can be used to hold a syringe which is placed into the device, as shown, in Figures 2A-2C. In some cases, the screw can be replaced by other holding components, such as pins, clips, and the like to hold the syringe in place.

[0221] In certain instances, the device is designed, sized, and shaped to be handheld and manually operable, such as with a single operator hand. For instance, the device may include an optional finger rest 140, as shown in Figure 2A-2C.

[0222] The device can be applied to any suitable biological tissue(s) of a subject. That is, the electrical pulse and one or more agents may be delivered to the tissue, and cells thereof, that are at or near a physically accessible part of the body of the subject. The delivery of electrical pulses and at least one agent may be to cells in the skin, cells in epithelial layers of the body, or cells in the body’s interior that are accessible for example laparoscopically or due to a surgical intervention. In some instances, the biological tissue comprises mammalian skin. In some instances, the target tissue site on which the device is used includes the dermis or epidermis. In some other instances, the biological tissue includes a mucosal membrane. A mucosal membrane, also known as a mucous membrane, is a type of epithelial tissue that lines various cavities and structures within the body that are exposed to the external environment. In some cases, the target tissue site may be in the mouth, nose, eye, gastrointestinal tract, or vagina.

[0223] In some instances, the device may weigh under 300 g, under 150 g, under 100 grams, or under 50 grams and may have a total volume of less than about 1000 cm3, less than 750 cm3, less than 500 cm3, less than 250 cm3, less than 100 cm3, less than 50 cm3, or less than 20 cm3, and requires no battery or power sources beyond the piezoelectric crystal of the generator, which is triggered by the mechanical input of a user.

[0224] In some instances, the array of biocompatible and conductive microelectrodes is replaceable and disposable, and / or the piezoelectric pulse generator and the switch are each independently intended for single-use or are intended to be reusable. In certain instances, the array of biocompatiblc and conductive microclcctrodcs and syringe may each independently be reusable.

[0225] In some instances, the device may further include a patch and / or a coating, which are each optionally dissolvable. Without limitation, the microelectrodes can be microneedle electrodes coated with an agent (such as a drug, i.e., therapeutic, prophylactic, or diagnostic agent(s)), such that when they are inserted into tissue (such as skin), the coating releases the agent(s) which diffuse into the tissue. This can be achieved, for example, where a coating is formed by dipping the microelectrodes into a solution containing active agent(s) and drying it to form a coating thereon. Various different coating methods are known. For example, see J Pharmacol Exp Ther. 2019 Sep; 370(3): 555-569. In some instances, a dissolvable coating is provided on a separate dissolvable micro-needle patch containing an agent(s) (such as a drug(s)), that can be housed with (such as below) the microelectrode array and is used when the electroporation device is inserted, such that it first causes insertion of the dissolvable microneedle patch which delivers the agent(s) / drug(s), and then insertion of the microelectrode array with triggering is used to administer electrical pulses. In still other instances, the MEA may itself be a dissolvable micro-needle patch, which otherwise meets the requirements of the MEA described.

[0226] The therapeutic, prophylactic, or diagnostic agent(s) referenced herein may be coated onto the microneedle electrodes of the device or onto a patch, as discussed. Non-limiting examples of such therapeutic, prophylactic, or diagnostic agents can include, without limitation, corticosteroids, methylxanthines, phosphodiesterase-4 inhibitors, anti-angiogenesis agents, antibiotics, antioxidants, anti-viral agents, anti-fungal agents, anesthetic agents, antiinflammatory agents, immunosuppressant agents, anti-allergic agents, and combinations thereof. Such agents, and pharmaceutical compositions thereof, are known in the art. i. Microelectrode Array (MEA)

[0227] In one non- limiting instance, a microelectrode array (MEA) includes: an array of biocompalible and conductive microelectrodes comprising an opening for at least one needle therein; wherein a syringe comprising a container for holding the pharmaceutical composition containing the one or more nucleic acid (s) can be coupled to the at least one needle for delivery of the pharmaceutical composition.

[0228] In some forms, the MEA array is around the at least one needle that fits through the opening, such that the opening is present through the base of the MEA. In some instances, there is also a possibility for the at least one needle that fits through the opening to be adjacent to the array, such as for injecting through a side port needle.

[0229] In some instances, the microelectrodes of the array are tissue-penetrating microelectrodes such as, microneedle electrodes. In some instances, the MEA does not include flat microelectrodes. In a second non-limiting instance, a microelectrode array (MEA) includes an array of biocompatible and conductive microelectrodes and a separate opening that allows for the insertion of a syringe, to which a needle can be attached. This configuration allows for the use of both the microneedle array for electrical stimulation and the syringe for delivery of a pharmaceutical composition via injection.

[0230] In some instances, the MEA includes a base from which the array of biocompatible and conductive microelectrodes extends. For instance, Figure 3A shows a non-limiting illustration of an MEA 200 having an array of biocompatible and conductive microelectrodes 210, an opening for at least one needle therein 220, and a base 230.

[0231] Figure 3B shows a non- limiting illustration of an array of biocompatible and conductive microelectrodes 210. In some instances, the biocompatible and conductive electrodes are formed of a metal; or wherein the biocompatible and conductive electrodes are formed of a mixture of epoxy and graphite; or a mixture of glass and platinum; or wherein the biocompatible and conductive electrodes are formed of a conductive ceramic, such as those known in the art. In some instances, the metal is stainless steel. Other suitable metals can include, without limitation, nickel, iron, titanium, copper, silver, gold, magnesium, cobalt, chromium, or other metals or alloys thereof. In some cases, the electrodes can be made of a cobalt-chromium alloy.

[0232] In some instances, the biocompatible and conductive microelectrodes of the array extend from one or more metal plates. The one or more plates can form part of a base and are typically a separate component from the base. In some instances, the one or more plates can be perpendicular to the microelectrode array central axis. In some other instances, the one or more metal plates can be parallel to the microelectrode array central axis. It is understood that on each plate, some of the microelectrodes may be positive and some may be negative. In some instances, a linear array of the biocompatible and conductive microelectrodes extends from one edge of each of the one or more metal plates. In some instances, the one or more plates are parallel to each other and spaced apart from one another. In some other instances, the biocompatible and conductive microelectrodes of the array extend from a single metal plate, and the array forms a two-dimensional array. It is understood that when the microelectrodes of the array extend from a single metal plate there would be at a minimum two metal plates / sheets where one functions as an anode and another as a cathode. In some instances, the biocompatiblc and conductive microelectrodes of the array extend from one or more metal plates and the array forms a two-dimensional array. All of the microelectrodes on a plate may not be connected to each other. The connections may be selective among the microelectrodes, and there can be multiple connections / electrode groupings. In some instances, the plates are fixed in the base, which acts as an insulating holder, made, for example, of a non-conducting polymeric material. For example, the insulating holder may be a cartridge body having a series of slots into which the plates forming part of the array may be fixed and where the microelectrodes extend from a surface of the cartridge body (i.e., base).

[0233] In still other instances, the biocompatible and conductive microelectrodes of the array extend from at least one non-electrically conductive plate, and electrical connections are provided between the biocompatible and conductive microelectrodes, where the electrical connections can be located on a surface of the at least one non-electrically conductive plate. In such instances, for example, the non-electrically conductive plate and microelectrodes can be part of the same piece, where a single piece of metal can be chem-etched, which forms a base plate from which the microneedle electrodes extend and are associated, joined, or inserted with the non-electrically conductive plate. In some instances, the electrical connections cross from a first side of the at least one non-electrically conductive plate to an opposed second side of the plate through holes in the at least one non-electrically conductive plate.

[0234] In some instances, two or more microelectrodes of the array with the same polarity (i.e., cathodes or anodes) can form part of the same piece of electrically conducting materials (e.g., metal), for example, formed from a single piece of material and cut, etched or otherwise processes to achieve the microelectrode array geometry. The material could be a sheet and the microelectrodes could be in the same plane as the sheet, or they could be at a non-zero angle, such as about 90 degrees, from the plane of the sheet.

[0235] In some instances, the microelectrodes of the array may be formed of a non-electrically conductive material and the array further includes a conductive material (e.g., metal) deposited and / or patterned onto the surface of the array in such a way that all of the anodes are electrically connected and all of the cathodes are electrically connected, but the anodes and cathodes and electrically isolated from each other on the microelectrode array. In still other instances, the microelectrodes of the array are made of a conductive material and are connected to a substrate that is not conductive, such that the microelectrodes are electrically connected to each other by patterning conductive material on one or more of the surfaces of the substrate and / or by electrical connections, which may involve wires electrically connected to the microelectrodes via the array surface and / or through holes in the substrate.

[0236] In certain instances, the base forms a cartridge that contains the array of biocompatible and conductive microelectrodes and further includes a first receptacle for mating engagement with a first electrode and a second receptacle for mating engagement with a second electrode, the first and second receptacles being in electrical communication with the microelectrodes of the array.

[0237] In some instances, the biocompatible and conductive microelectrodes of the array define and have an area of between about 1 mm2and 50 cm2or about 1 mm2and 25 cm2, as well as sub-ranges or individual values contained within. The area defined by the biocompatible and conductive microelectrodes of the array refers to the maximum area on which the microelectrodes can form contact with a tissue (such as skin); i.e. , skin contact area. In some instances the array of biocompatible and conductive microelectrodes can include from: (1) about 2 to 3000 microelectrodes, about 2 to 2000 microelectrodes, or about 2 to 1000 microelectrodes, as well as sub-ranges or individual values contained within the aforementioned ranges; or (2) at least: about 25 microelectrodes, about 50 microelectrodes, about 100 microelectrodes, about 150 microelectrodes, about 200 microelectrodes, about 250 microelectrodes, about 300 microelectrodes, about 350 microelectrodes, about 400 microelectrodes, about 450 microelectrodes, about 500 microelectrodes, about 550 microelectrodes, about 600 microelectrodes, about 650 microelectrodes, about 700 microelectrodes, about 750 microelectrodes, about 800 microelectrodes, about 850 microelectrodes, about 900 microelectrodes, about 950 microelectrodes, about 1000 microelectrodes, about 1500 microelectrodes, about 2000 microelectrodes, about 2500 microelectrodes, or about 3000 microelectrodes.

[0238] In some instances, the biocompatible and conductive microelectrodes of the array can each independently have a length from 10 pm to 8 mm, as well as sub-ranges or individual values contained within; or the biocompatible and conductive microelectrodes of the array each independently have a length of at least about 500 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm, 1050 pm, 1100 pm, 1150 pm, 1200 pm, 1250 pm, 1300 pm, 1350 pm, 1400 pm, 1450 pm, or 1500 pm. The length of the microelectrodes typically refers to the longest dimension of the microelectrode(s) measured from the tip, which penetrates a tissue.

[0239] The microelectrodes of the array can be spaced in the array in a manner effective to penetrate into the target tissues (e.g., through the stratum corneum) and to achieve electroporation of cells with the electrical pulses generated by the piezoelectric pulse generator. The microclcctrodcs arc mechanically robust to prevent shearing off, or otherwise damaging, the microelectrodes during insertion or withdrawal in a tissue. The microelectrodes may include a straight or tapered body, which may be cylindrical or square-shaped, and may include a tapered tip portion. The microelectrodes may be manufactured using any suitable method known in the art, for example, lithographic etching technology, photochemical etching, electroplating, wet or dry etching, 3D printing, micro-molding, and laser cutting. The microelectrodes may be solid, have a hollow bore extending from a base to the tip or may have one or more grooves in the sides of the microelectrodes. Method of making the microelectrodes, as well as other components of the MEA, such as the base, and hole for receiving at least one needle, are known to those skilled in the art. For example, molding, 3-D printing, and other suitable techniques can be used to form the base having a hole separately from the microelectrode array, where these parts can be combined to form the MEA, as shown in Figure 3A.

[0240] The microelectrodes are typically closely spaced to achieve a high field strength in conjunction with microsecond pulses for electroporation of cells in biological tissues. Because the piezoelectric pulses are of microseconds duration, effective electroporation benefits from a field strength >500 V / cm. To achieve such a high field strength, the microelectrodes of the array should have a close spacing, for example, of less than 1 mm apart, i.e., where spacing refers to the distance between a microelectrode (which acts as a cathode) and its nearest neighboring microelectrode (which acts as an anode), and vice versa. This close spacing allows piezoelectric pulses of hundreds of volts to achieve the required field strengths. In some cases, the microelectrodes of the array of the device have a spacing in the array between about 0.1 mm and 10 mm, between 0.2 mm and 5 mm, and between 0.3 mm and 2 mm. In some cases, the spacing is in the range of about 0.5 mm to 1.5 mm. In still other instances, the biocompatible and conductive microelectrodes of the array have: a spacing in the array between about 0.1 mm and 3 mm, between about 0.2 mm and 2.5 mm, between about 0.3 mm and 2 mm, between about 0.5 mm and 1.5 mm, between about 0.5 mm and 1.0 mm, between about 0.5 mm and 0.9 mm, between about 0.5 mm and 0.8 mm, between about 0.5 mm and 0.7 mm, or between about 0.5 mm and 0.6 mm, as well as sub-ranges or individual values contained within; and / or the microelectrodes of the array are arranged in a rectangular, square, triangular, or circular orientation or pattern. Other orientations and patterns are possible. In some instances, the microelectrodes may be in alternating rows, or may be in a checkerboard pattern, or may be in a concentric circle pattern, or may be arranged in other suitable patterns. Rows or columns of microelectrodes can function as positive and negative electrodes.

[0241] In some instances, electroporation may require electric fields on the order of 103V / cm, with shorter pulses requiring larger field strengths. This means that more closely spaced microelectrodes can electroporate with lower voltages. Close electrode spacing can also decrease the electric field penetration depth into the tissue, which can facilitate epidermal targeting and reduce nerve stimulation when used on skin. The pulse length (or decay constant) can be in the order of microseconds (e.g., 1 - 1000 microseconds), and possibly nanoseconds (e.g., 1 - 1000 nanoseconds), depending on the piezoelectric crystal utilized. The pulse length can be manipulated, for example, through choosing different dopants utilized in the piezoelectric crystal's manufacturing process, choosing different dopants, different piezoelectric materials, and other methods known in the art. The pulse length may be in the range of about 1 ns to 1 ms or in the range of about 1 to 100 ps.

[0242] An exemplary embodiment of a device with a microneedle array is shown in Figures 6A and 6B. For example, in use, the MEA was pressed against the skin so that the microneedles penetrated across the skin’s stratum comeum barrier to enter the viable epidermis and superficial dermis, after which the thumb toggle was pressed to administer the pulses (Figure 6A). Figure 6A shows electroporator 80 being held in a position by a hand 82 before activation. The electroporator 80 comprises a housing 86 having a toggle switch 84, which may be activated, for example, by a user’ s thumb. The electroporator 80 further comprises an MEA cartridge 88. Figure 7B is an exploded view of electroporator 90 comprising a piezoelectric pulse generator and a metal MEA, known as an ePatch. The piezoelectric pulse generator comprises a crystal case 96 housing the piezoelectric crystal 94. MEA cartridge 1 20 comprising microneedles 1000 is connected to the piezoelectric pulse generator via copper wires 98. The electroporator 90 further comprises a hand toggle 92. a. Optional Needle Opening and Needle Component

[0243] As shown in Figure 3A, the MEA can include at least one opening / hole which can receive at least a needle component 110, such as shown in Figure 3C. The needle component has a needle tip. In some instances, the needle component is a hypodermic needle, a hollow microneedle, an insulin syringe needle, or a pen needle. Other needles known in the art may be used. In some instances, the microelectrodes are microneedle electrodes which can be coated and / or dissolvable, as detailed above.

[0244] As shown in Figures 4A and 4B, the needle component 110 is placed into the base 230 through the opening / hole such that the needle tip is exposed adjacent to the microelectrodes of the array 210. In some instances, the MEA may include two, three, four, or more needle components. ii. Piezoelectric Generator and Switch

[0245] The device includes a piezoelectric pulse generator 300, as shown in Figure 6, having a switch 310. The piezoelectric generator includes a piezoelectric crystal which can be selected, without limitation, from lead zirconate titanate (PZT), silicon nitride, barium titanate, quartz, zinc oxide, sodium tungstate, sodium potassium tartrate, tourmaline, lithium niobate, gallium arsenide, aluminum nitride, and combinations thereof. The piezoelectric crystal is a material which exhibits a piezoelectric effect of large magnitude, that is the production of an electric field and thereby a large voltage output with a short time constant in the form of a pulse. This may occur through a mechanical force, pressure, or compression exerted against the crystal’s surface, creating a temporary deformation resulting in the formation of electric charges which are then released as an electrical pulse. Piezoelectric pulse generators are known to those skilled in the art. Suitable piezoelectric pulse generator can be selected for use in the devices which provide the desired performance properties, as detailed below. These generators may be obtained from commercial sources. In addition, methods for their manufacture are also known to those skilled in the art.

[0246] The piezoelectric pulse generator is coupled to a switch, as shown in Figure 5A, for activating the piezoelectric pulse generator and causing the generation of one or more electrical pulses. The term “switch” refers to a non-electrical switch, which is a mechanical device or mechanism used to control and activate the generation of electrical currents, and pulses thereof, by the piezoelectric pulse generator. A non-electrical switch is preferred for the device as it does not require the use of electricity or electronic components therein.

[0247] In some instances, the switch triggers a spring-latch hammer mechanism configured to strike a surface of the piezoelectric crystal of the piezoelectric pulse generator. In some instances, there is a pin (such as a metal pin) disposed between the spring-latch hammer mechanism and the piezoelectric crystal.

[0248] In some instances, the switch is a toggle switch with a latch configured to release a hammer, configured to strike a surface of the piezoelectric crystal of the piezoelectric pulse generator effectively, such as driven by decompression of a spring. In some instances, a pin (such as a metal pin) disposed between the latch and the piezoelectric crystal. In some instances, the latch can be a wedge controlling latch, where the wedge acts to unlock the hammer (pushes it out of a lock), which results in a spring decompressing the hammer and launching it at the piezoelectric crystal to produce an electric force.

[0249] In some instances, the piezoelectric pulse generator includes a casing for the piezoelectric crystal and electrical connections inclusive of a first electrode and a second electrode which extend from the casing, where the first and second electrodes provide electrical contact points where the MEA can be connected or mated to in order to receive the electrical pulsc(s) generated by the piezoelectric pulse generator.

[0250] The array of biocompatible and conductive microelectrodes is electrically coupled to the piezoelectric pulse generator. The piezoelectric pulse generator may include any mechanism effective to generate a suitable electrical pulse from a piezoelectric crystal. In some instances, the piezoelectric pulse generator includes (i) a piezoelectric crystal, (ii) a spring-latch hammer mechanism configured to strike a surface of the piezoelectric crystal effective to generate the electrical pulse, and (iii) electrical connections for conducting the electrical pulse to the microelectrodes of the array.

[0251] In certain instances, the piezoelectric pulse generator, when activated by the switch, generates one or more electrical pulses having a peak voltage absolute value of between about 10 V and 35,000 V, 10 V and 30,000 V, 10 V and 20,000 V, 10 V and 10,000 V, 20,000 V and 30,000 V, between about 50 V and 5,000 V, between about 100 V and 1,000 V, or between about 200 V and 500 V when measured in air, or sub-ranges or individual values contained within the aforementioned ranges. In some instances, the piezoelectric pulse generator, when activated by the switch, generates one or more electrical pulses having a peak voltage absolute value of between about 20,000 V and 30,000 V or 23,000 V and 28,000 V, when measured in air, or sub-ranges or individual values contained within the aforementioned ranges. In some instances, the one or more electrical pulses have a peak voltage absolute value of between about 100 V and 1000 V, about 100 V and 900 V, about 100 V and 800 V, about 100 V and 700 V, about 100 V and 600 V, about 100 V and 500 V, about 100 V and 400 V, about 100 V and 300 V, about 100 V and 200 V, about 200 V and 400 V, between about 200 V and 350 V, between about 200 V and 300 V, or between about 200 V and 250 V when measured in a tissue, or subranges or individual values contained within the aforementioned ranges.

[0252] In some instances, the one or more electrical pulses have a ratio of absolute value of peak voltage to absolute value of peak-to-peak voltage between about 0.1 and 10, between about 0.3 and 5, or between about 0.5 and 2, or sub-ranges or individual values contained within the aforementioned ranges. In certain instances, the one or more electrical pulses have a peak current absolute value between about 0.001 A and 1,000 A, between about 0.01 A and 500 A, between about 0.1 A and 100 A, or between about 1 A and 50 A when measured in a tissue, or sub-ranges or individual values contained within the aforementioned ranges. In some instances, the one or more electrical pulses have a peak static voltage absolute value of between about 100 V and 35,000 V, between about 1,000 V and 30,000 V, or between about 15,000 V and 35,000 V when measured in air, or sub-ranges or individual values contained within the aforementioned ranges. In still other instances, the one or more electrical pulses can produce an electric field strength of between about 100 V / cm and 30,000 V / cm, between about 200 V / cm and 10,000 V / cm, between about 300 V / cm and 5,000 V / cm, or between about 500 V / cm and 3,500 V / cm, or sub-ranges or individual values contained within the aforementioned ranges. In yet other instances, the one or more electrical pulses generated can have an initial pulse length of between about I ps and 10,000 ps, between about 1 ps and 1,000 ps, between about 1 ps and 100 ps, between about 3 ps and 100 ps , between about 5 ps and 50 ps , or between about 10 ps and 30 |_is, or sub-ranges or individual values contained within the aforementioned ranges. In some instances, the one or more electrical pulses generated can have a ratio of initial pulse length to total pulse length between about 1.5 and 100, between about 2 and 50, or between about 3 and 20. Total pulse length should be greater than initial pulse length.

[0253] The device may include a casing surrounding the piezoelectric crystal, which includes electrical connections for relaying the pulse to the microelectrodes of the array. The casing may include a lower electrode and a side electrode, which extends from the casing.

[0254] In some instances, the device may operate as follows: After the microelectrodes of the array are inserted into a tissue, such as skin, mucosa, or other biological tissue, a user exerts a force against the switch, such as a thumb-toggle switch, which compresses a lower spring, and pushes a wedge towards the hammer locked in a latch. When the user pushes all the way, the wedge forces the hammer out of the latch which subsequently strikes a pin sitting against the piezoelectric crystal to concentrate the force. The voltage output generated by the piezoelectric pulse generator may then be directed to the microelectrodes of the array of the device. An upper spring may then be decompressed to reset the hammer and latch into the original locked state. The user may repeat the operation to generate additional electrical pulses, as needed to provide an effective electroporation of the tissue(s) for delivery of the pharmaceutical composition containing the one or more nucleic acid (s) into the tissue and cells thereof.

[0255] The electroporation portion of the devices described herein provides an electrical pulse capable of increasing delivery of the pharmaceutical composition containing the one or more nucleic acid (s), into the tissue and cells therein. The use of piezoelectric crystals as the source of the electric pulses induces membrane permeabilization through a high-voltage, short timeconstant pulse (e.g., microseconds), which is believed to induce a temporary change in the cell membranes of cells in the electric field produced by the microelectrodes of the array during an electric pulse(s), which includes cells in contact with, or in the vicinity of the microelectrodes, allowing the pharmaceutical composition containing the one or more nucleic acid (s) to enter the tissue cells, to produce an intended effect.

[0256] In some instances, the piezoelectric pulse generator produces bipolar, oscillatory pulses, which may electroporate cells more effectively compared to conventional monopolar or exponential-decay or square-wave pulses.

[0257] The foregoing pulse values refer to pulses in biological tissues, such as the skin, unless they are identified as static voltages (i.e., peak static voltage, peak-to-peak static voltage). It is noted that similar, conventional piezoelectric devices are designed to make sparks in air. In contrast, the piezoelectric pulse generators described herein are coupled with microelectrodes of an array which are used to pass current through a conductive medium (i.e., no sparks). In certain instances, a bipolar oscillating pulse is used for electroporation instead of a monopolar pulse, often in the form of an exponential-decay or square wave pulse, which is more conventionally used for electroporation. This bipolar oscillating pulse may be a natural result of the compression and extension of piezoelectric crystals induced by a spring shock in the case containing the piezoelectric crystal. Compared to conventional monopolar pulses, bipolar oscillating pulses are not only able to produce a dielectric breakdown of the cell membrane but can also produce a sonicating motion in the cell membrane, inducing more effective cell poration. Further, oscillating pulses may provide better cell viability by avoiding polarizing the cell membrane beyond the critical potential for an extensive period, therefore, preventing irreversible rupture of the cell membrane. While for non-oscillatory pulses, the initial and total pulse lengths are the same, the pulses of the devices described herein may be oscillatory, such that the initial and total pulse lengths may not be the same. Moreover, while conventional electroporators generate substantially monopolar pulses, the devices and methods described herein may use bipolar pulses, wherein each pulse alternates between positive and negative. iii. Optional Syringe Component

[0258] In some instances, the devices described herein may include a syringe component. As shown in Figure 5B, the syringe may include a container 126, which can hold a pharmaceutical composition containing the one or more nucleic acid (s) , a plunger 125 which can be used to administer the pharmaceutical composition containing the one or more nucleic acid (s) when actuated, and a delivery end which optionally includes an attachment or lock, such as a Luer lock, used for connecting and attaching a needle component. For example, Figure 5C shows a non-limiting illustration of a syringe coupled to a needle component 110 and an MEA 200, where such an assembled unit may be placed directly into device casing 130. In some instances, the assembled unit can optionally be held in the device casing by pressure (clicks into the device casing) and / or may be held by screws or pins.

[0259] In some other instances, the syringe component need not rely on a plunger, which is manually pressed to cause delivery of a pharmaceutical composition containing the one or more nucleic acid (s) contained within the syringe but can rely instead on, for example, air pressure to cause delivery of the pharmaceutical composition from the syringe container. Such pressurebased systems (i.e., jet injectors) are known to those skilled in the art and can be used in the device in lieu of the common plunger-style syringe illustrated. iv. Method of using the device for delivering nucleic acid

[0260] In one non-limiting instance, the device may be used in a method of delivering the pharmaceutical composition containing the one or more nucleic acid (s) into or across a biological tissue, the method including the steps of:

[0261] (a) positioning a device adjacent to a biological tissue site, the device including: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses;

[0262] (b) contacting the array of biocompatible and conductive microelectrodes;

[0263] (c) activating the switch to generate and deliver one or more electrical pulses through the array of biocompatible and conductive microelectrodes into the target biological tissue site to electroporate cells at the target biological tissue site; and

[0264] (d) administering the pharmaceutical composition containing the one or more nucleic acid (s) into the target biological tissue site.

[0265] In some instances of the method, step (c) and step (d) are performed concurrently. In some instances, step (d) is performed following step (c). In other instances, step (c) is performed following step (d). In still other instances, step (c) can be repeated at least once or two or more times.

[0266] In some forms, the array of biocompatible and conductive microelectrodes further include al least one needle for delivery of the pharmaceutical composition embedded therein. In some forms, the device further includes a syringe comprising a container holding the pharmaceutical composition containing the one or more nucleic acid (s). In some forms, the syringe is coupled to the at least one needle for administering the pharmaceutical composition into a biological tissue by actuation of the syringe. In some forms, step (b) further includes contacting the at least one needle embedded therein into the target biological tissue site.

[0267] In some forms, step (d) includes administering the pharmaceutical composition containing the one or more nucleic acid (s) into the target biological tissue site through the at least one needle by actuation of the syringe. In some other forms, step (d) is performed by contacting microneedle electrodes coated with the pharmaceutical composition and / or coated with one or more therapeutic, prophylactic, or diagnostic agents with the target biological tissue. In some forms, step (d) is performed by contacting a dissolvable patch containing the pharmaceutical composition with the target biological tissue. In some instances, contacting during step (b) refers to insertion and / or penetration of the microelectrodes of the array into the target biological tissue.

[0268] Typically, the methods include manually applying a force to the device effective to penetrate a biological tissue site surface with the array of biocompatible and conductive microelectrodes, and then manually pressing the switch on the device that triggers a mechanical force onto a piezoelectric pulse generator to produce one or more electric pulses effective to induce electroporation (i.e., pore formation) in the cells of the biological tissue site. In some forms, depressing the plunger of the syringe can actuate release of the pharmaceutical composition, from the syringe container. In some forms, dispensing the pharmaceutical composition coated onto the microneedle electrodes onto the target biological tissue can facilitate release of the pharmaceutical composition, from the microneedles.

[0269] The methods described herein are used to administer a pharmaceutical composition containing the one or more nucleic acid (s), to a subject's biological tissue site with the aid of electroporation.

[0270] In some instances of the method, the target biological tissue site includes mammalian skin. In some instances, the target biological tissue site is mammalian skin dermis. In still other instances, the target biological tissue site is mammalian skin epidermis. In certain instances, the target biological tissue site is or includes a mucosal membrane. A mucosal membrane, also known as a mucous membrane, is a type of epithelial tissue that lines various cavities and structures within the body that are exposed to the external environment.

[0271] In some instances of the method, the one or more electrical pulses have a peak voltage absolute value between about 100 V and 1000 V, a peak current absolute value between about 0.001 A and 50 A, a peak static voltage absolute value between about 15,000 V and 35,000 V, an initial pulse length of between about I s and 100 ps, or a combination thereof.

[0272] The methods described can be used to deliver the one or more nucleic acid (s) for the delivery of large molecules into body tissues of a subject using electroporation without the use of batteries, capacitors, or other conventional electric power storage devices, and without the use of electricity from an external source (e.g., not plugged into an electrical outlet).

[0273] In some instances, the electric field is localized to the target biological tissue site, such as the skin and especially to the epidermis, where localization to the epidermis means that the majority of cells experiencing electroporation conditions are located in the epidermis and not in the dermis. This localization is facilitated by the combination of limiting penetration of the electric field to deeper tissues and by the fact that cell density in viable epidermis is much greater than in dermis. Targeting the electric field and resulting electroporation to the skin, for example, and especially the epidermis improves delivery of the one or more nucleic acid (s) and allows for reduced side effects. Unlike dermis, which is largely acellular, the epidermis is densely populated with cells, including keratinocytes as well as melanocytes. Targeting certain agents, such as an immunogenic protein, to these epidermal cells facilitate increased and longer- term expression of the immunogenic proteins such as a viral antigen e.g., Covid- 19 spike protein, compared to intramuscular injection. Nevertheless, diffusion of immunogenic proteins produced in the upper dermis can also be beneficial, e.g., fibroblasts.

[0274] Moreover, localizing the electric field to the epidermis can reduce nerve stimulation, thereby making electroporation more tolerable. Of particular concern is stimulation of motor nerves and muscle cells below the skin, which can cause violent twitching reported for skin or muscle electroporation in other contexts. Such contractions are not expected when electroporating with the MEAs described herein which can localize the electric field superficially, far away from muscles.

[0275] In most instances, the present microelectrodes of the array can be used for controlled and selective penetration into the epidermis, and possibly a portion of the dermis, as needed. In most instances, the microelectrodes would not fully cross the dermis and would not contact tissues below the dermis. Because the electric field produced when pulsing these microelectrodes is strongest in the biological tissue between oppositely charged electrodes, tissue deeper than the penetration depth of the microelectrodes receives weaker electric fields that is less likely to cause electroporation.

[0276] III. IMPROVED SYSTEMS FOR DELIVERING RNA VACCINES

[0277] Also disclosed are systems for delivering pharmaceutical compositions containing one or more nucleic acid (s) via electroporation, such as intradermal or subcutaneous electroporation.. Generally, the system includes:

[0278] (i) a pharmaceutical composition containing an effective amount of one or more nucleic acid (s), wherein the one or more nucleic acid (s) encodes one or more antigen (s), and

[0279] (ii) device containing: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; and a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses. The disclosed systems are suitable for treating a subject having a disease such as an infectious disease or cancer. All of the variations of the pharmaceutical composition and the device in the disclosed system are described in the Section II above.

[0280] The disclosed system can include, for example, a dosage supply of the pharmaceutical composition containing the nucleic acids as an antigen or vaccine, and optionally one or more additional active agents, or a combination thereof in separately or together in the same admixture. The active agents can be supplied alone (e.g., lyophilized), or in a pharmaceutical composition. The active agents can be in a unit dosage, or in a stock that should be diluted prior to administration. In some forms, the system includes a supply of pharmaceutically acceptable carriers. The system can also include additional devices for administration of the active agents or compositions, for example, additional syringes.

[0281] The system can also include printed instructions for administering the compound in a use as described above. The instructional material can include a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the kit.

[0282] The disclosed compositions, devices, and kits can further be understood through the following numbered paragraphs:

[0283] Paragraph 1. A method of treating or preventing the development of one or more symptoms of a disease or disorder in a subject, the method comprising:

[0284] (i) administering to a target site of a subject, a pharmaceutical composition comprising an effective amount of one or more nucleic acid (s), wherein the one or more nucleic acid (s) encodes one or more antigen (s); and

[0285] (ii) electroporating the target site of the subject, wherein electroporating increases delivery of the one or more nucleic acid (s) to cells in the target site.

[0286] Paragraph 2. A method for delivering one or more nucleic acid (s) to cells at a target site of a subject, comprising:

[0287] (i) administering a pharmaceutical composition comprising an effective amount of one or more nucleic acid (s) to the target site of the subject, wherein the one or more nucleic acid (s) encodes one or more antigen (s); and

[0288] (ii) applying electroporation to the target site, wherein the electroporation is effective to increase delivery of the one or more nucleic acid (s) to cells in the target site.

[0289] Paragraph 3. The method of paragraph 1 or paragraph 2, wherein the pharmaceutical composition is administered via intradermal injection. Paragraph 4. The method of any of paragraphs 1-3, wherein the cells are skin cells selected from the group consisting of keratinocytes, Langerhans cells, melanocytes, fibroblasts, epidermal dendritic cells and combinations thereof.

[0290] Paragraph 5. The method of any of paragraphs 1-4, wherein Steps (i) and (ii) are performed using a device.

[0291] Paragraph 6. The method of paragraph 5, wherein Steps (i) and (ii) are performed by:

[0292] (a) positioning the device adjacent to a target site on the subject, wherein the device comprises: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses;

[0293] (b) contacting the array of biocompatible and conductive microelectrodes comprising the at least one needle embedded therein into the biological tissue on the subject;

[0294] (c) activating the switch to generate and deliver one or more electrical pulses through the array of biocompatible and conductive microelectrodes into the target site to electroporate the cells; and

[0295] (d) delivering the pharmaceutical composition into the target site of the subject. Paragraph 7. The method of paragraph 6, wherein the device comprises a syringe comprising a container holding the pharmaceutical composition of paragraph 1 or paragraph 2; wherein the array of biocompalible and conductive microelectrodes further comprises a syringe and at least one needle for delivery of the pharmaceutical composition embedded therein; and wherein the syringe is coupled to at least one needle for administering the pharmaceutical composition of paragraph 1 or paragraph 2 into the target site by actuation of the syringe. Paragraph 8. The method of paragraph 6, wherein the device comprises a detachable syringe comprising a container for holding the pharmaceutical composition of paragraph 1 or paragraph 2; wherein the detachable syringe can be removed from the main body of the device after the pharmaceutical composition of paragraph 1 or paragraph 2 is delivered. Paragraph 9. The method of paragraph 6, wherein the microelectrodes of the array of biocompatible and conductive microelectrodes are microneedle electrodes, wherein the microneedle electrodes are coated with the pharmaceutical composition of paragraph 1 or paragraph 2 and / or coated with one or more therapeutic, prophylactic, or diagnostic agents.

[0296] Paragraph 10. The method of paragraph 6, wherein step (c) and step (d) are performed concurrently.

[0297] Paragraph 11. The method of paragraph 10, wherein step (d) is performed following step (c). Paragraph 12. The method of paragraph 11, wherein step (c) is performed following step (d). Paragraph 13. The method of any one of paragraphs 1-12, wherein step (c) is repeated at least once.

[0298] Paragraph 14. The method of any one of paragraphs 1-13, wherein the target site is mammalian skin.

[0299] Paragraph 15. The method of any one of paragraphs 1-14, wherein the target site is mammalian skin dermis and / or epidermis.

[0300] Paragraph 16. The method of any one of paragraphs 1-15, wherein the one or more electrical pulses have a peak voltage absolute value between about 100 V and 1000 V, a peak current absolute value between about 0.001 A and 50 A, a peak static voltage absolute value between about 15,000 V and 35,000 V, an initial pulse length of between about 1 ps and 100 ps, or a combination thereof.

[0301] Paragraph 17. The method of any one of paragraphs 1-16, further comprising: (iii) monitoring the expression of one or more protein (s) encoded by the one or more nucleic acids in the target site over a predetermined period.

[0302] Paragraph 18. The method of any one of paragraphs 1-17, wherein the disease or disorder is an infection caused by a virus, bacterium, fungus, parasite, or protozoa.

[0303] Paragraph 19. The method of any one of paragraphs 1-18, wherein the subject has an infection or is at risk of an infection.

[0304] Paragraph 20. The method of any of paragraphs 1-19, wherein the infection is caused by a virus selected from the group consisting of orthomyxovirus, rhinovirus, paramyxovirus, coronavirus, adenovirus, human metapneumovirus (hMPV), enterovirus, bocavirus, flavivirus, retrovirus, or para retrovirus.

[0305] Paragraph 21. The method of paragraph 20, wherein the orthomyxovirus is an influenza virus, optionally wherein the influenza virus is selected from the group consisting of Influenza

[0306] A, Influenza B, and Influenza C.

[0307] Paragraph 22. The method of paragraph 20, wherein the paramyxovirus is selected from the group consisting of Respiratory syncytial vims (RSV) and parainfluenza vims. he method of paragraph 20, wherein the coronavirus is selected from the group consisting of SARs-CoV, MERS-CoV, and SARS-CoV-2 (CO VID 19).

[0308] Paragraph 23. The method of paragraph 20, wherein the retrovirus is human immunodeficiency virus, and human t-lymphotrophic virus type 1 (HTLV-1).

[0309] Paragraph 24. The method of paragraph 20, wherein the para retrovirus is hepatitis B virus.

[0310] Paragraph 25. The method of paragraph 20, wherein the flavivirus is dengue virus, yellow fever virus, West Nile virus, Zika virus, Japanese encephalitis virus, tick-borne encephalitis virus, and hepatitis C virus.

[0311] Paragraph 26. The method of any one of paragraphs 1-25, wherein the disease or disorder is cancer.

[0312] Paragraph 27. The method of any one of paragraphs 1-26, wherein the pharmaceutical composition comprises an isolated nucleic acid encoding a protein or peptide, wherein the protein or peptide is effective to induce an immune response in the subject.

[0313] Paragraph 28. The method of paragraph 27, wherein the nucleic acid comprises RNA, and wherein the RNA is not encapsulated in a nanoparticle, liposomes, microspheres, or microcapsules.

[0314] Paragraph 29. The method of paragraph 28, wherein the RNA comprises messenger RNA (mRNA) and / or circular RNA.

[0315] Paragraph 30. The method of paragraph 29, wherein the messenger RNA is not self-replicating RNA.

[0316] Paragraph 31. The method of any one of paragraphs 1-30, wherein the pharmaceutical composition comprises one or more nucleic acid(s), and wherein the one or more nucleic acid(s) optionally comprise one or more modified nucleotide(s).

[0317] Paragraph 32. The method of paragraph 31, wherein the one or more modified nucleotide (s) is selected from the group consisting of 5-methylcytidine (m5C), 2-thiouridine (s2U), 5- methyluridine (m5U), pseudouridine (\| / ), 5-methoxyuridine (5moU) and Nl- methylpseudouridine (inly).

[0318] Paragraph 33. The method of any one of paragraph 31 or 32, wherein the one or more nucleic acid (s) comprise an RNA encoding or expressing an adjuvant.

[0319] Paragraph 34. The method of any one of paragraphs 31-33, wherein the one or more nucleic acid (s) comprise DNA encoding or expressing an adjuvant.

[0320] Paragraph 35. The method of paragraph 33, wherein one or more RNA-encoded adjuvant (s) are selected from the group consisting of interleukins, interferons, Granulocyte-Macrophage Colony-Stimulating Factor (GMCSF), Retinoic Acid-lnducible Gene I, chemokines, pattern recognition receptors, flagellin, bacterial toxins, tumor necrosis factors, and heat shock proteins. Paragraph 36. The method of paragraph 34, wherein one or more DNA-encoded adjuvant (s) are selected from the group consisting of interleukins, interferons, Granulocyte-Macrophage Colony-Stimulating Factor (GMCSF), Retinoic Acid-Inducible Gene I, chemokines, pattern recognition receptors, flagellin, bacterial toxins, tumor necrosis factors, and heat shock proteins.

[0321] Examples

[0322] Example 1: Exemplary Piezopen Electroporation Increases Humoral Immune Responses to Naked mRNA Vaccines

[0323] Objective

[0324] The goals of the present were to: (1) demonstrate that Piezopen electroporation (EP) can lead to detectable levels of immune responses in a dose-dependent manner; (2) demonstrate that Piezopen EP leads to higher humoral immune responses compared to ID injection alone; (3) demonstrate at least comparable immunogenicity of Piezopen compared to delivery of antigens encapsulated in lipid nanoparticles (LNP).

[0325] Methods

[0326] Experimental Design

[0327] A baseline blood collection was performed for serum processing a day prior to prime dose injections (pre-bleed. Day -1). To evaluate the impact of Piezopen EP , we set up the following groups: ID injection of Ca2+-free PBS (Naive; 14190-136, ThermoFisher Scientific), ID injection of naked Spike mRNA (SC2325; GenScript) at a dose of 0.5 pg, 2 pg, and 5 pg, ID injection of naked Spike mRNA at a dose of 0.5 pg, 2 pg, and 5 pg followed by Piezopen EP , and IM injection of Spike LNP (SM102) (SC2337; GenScript) at a dose of 0.5 pg, 2 pg, and 5 pg in mice in vivo (Day 0). A second blood collection was performed for serum processing on Day 14 followed by a booster dose on Day 21. On Day 35, a final blood collection and necropsy were performed, collecting blood for plasma and serum processing and harvesting the spleen for future testing. Animal weight was monitored throughout the study.

[0328] Microneedle Injections

[0329] The Spike mRNA was delivered to female BALB / c mice (6-8 weeks old) using ID injections with or without Piezopen electroporation and intramuscular injection of LNP encapsulated with Spike mRNA (SM102) (Day 0). Injections were performed on the left flank (for ID) or left quadricep (for IM) of each mouse. For ID injections, 0.5 mg, 2 mg, or 5 mg of Spike mRNA was added to 20 mL Ca2+-free PBS (i.e., 10 mL FLuc mRNA + 40 mL Ca2+-free PBS). For IM injections, 0.5 mg, 2 mg, or 5 mg of Spike-mRNA was added to 50 mL Ca2+-free PBS. mRNA was stored in a -20 °C cryo-box after preparation and during transport to the animal facility to minimize any degradation risk.

[0330] Experimental Groups

[0331] The experimental group contains 30 animals and were divided into: (1) Naive (Ca2+-free PBS), (2) ID injection of naked Spike mRNA (0.5 pg), (3) ID injection of naked Spike mRNA (2 pg), (4) ID injection of naked Spike mRNA (5 pg), (5) IM LNP SM102 (0.5 pg), (6) IM LNP SM102 (2 pg), (7) IM LNP SM102 (5 pg), (8) ID injection of naked Spike mRNA (0.5 pg) + Group C-3 (5 pulses), (9) ID injection of naked Spike mRNA (2 pg) + Group C-3 (5 pulses), and (10) ID injection of naked Spike mRNA (5 pg) + Group C-3 (5 pulses).

[0332] IgG Antibody

[0333] Spike protein-specific IgG levels in Day 35 serum were measured using the Mouse Anti- SaRS-CoV-2 Antibody IgG kit (RAS-T023, AGRO Biosystems) according to the manufacturer's protocol. 15 dilutions: 1 / 200, 1 / 400, 1 / 800, 1 / 1600, 1 / 3200, 1 / 6400, 1 / 12800, 1 / 25600, 1 / 51200, 1 / 102400, 1 / 204800, 1 / 409600, 1 / 819200, 1 / 1638400, and 1 / 32762800 were tested.

[0334] Neutralization Antibody

[0335] Levels of neutralization antibody in Day 35 serum were measured using the SARS-CoV- 2 (2019-nCoV) Neutralization Antibody Kit (BSKV0004, Bioss Antibodies). The manufacturer's protocol was followed with some modifications. Specifically, plates were incubated at 30°C for 40 minutes after HRP and substrate addition instead of following incubation at room temperature. Seven different dilutions were tested: 1 / 10, 1 / 50, 1 / 100, 1 / 200, 1 / 500, 1 / 1000, and 1 / 2000.

[0336] Results

[0337] It was observed that Piezopen delivery led to higher humoral immune responses compared to naked ID injection. Piezopen delivery of naked Spike mRNA led to at least comparable immunogenicity to the current state-of-the-art delivery platform, namely LNPs.

[0338] A graph of absorbance vs. dilution for IgG antibodies was plotted to show how the amount of IgG antibody and binding affinity changed with different dilutions between the treatments (Figure 11B). Piezopen delivery of naked Spike mRNA induced robust immune responses greater than ID injection alone regardless of dose. Piezopen delivery induced clear dose-dependent responses, detectable responses even at a low 0.5 pg dose. In addition, Piezopen delivery of 5.0 pg mRNA was comparable to 0.5 pg SM102 LNPs (mRNA-1273) in terms of binding antibody responses. Notably, IM SM102 (5 pg and 2 pg) exhibited the most robust IgG antibody responses, followed by Piezopen delivery of naked mRNA (5 pg) and IM SM102 (0.5 pg). Moreover, Piezopen delivery of naked mRNA (5 pg) appeared to induce a comparable antibody response to LNPs at - (0.5 pg).

[0339] The endpoint IgG titer curve was plotted to understand the binding interactions between IgG and antigen (Figure 11C). The titer was defined as the reciprocal of the last serum dilution that produced an optical density greater than 4 times the value of the background (i.e., the average optical density of saline-administered mice). Unlike Piezopen or LNP delivery, naked mRNA alone produced significant variability with some detectable responses. However, Piezopen and LNPs induced robust immune responses at all tested doses. Piezopen delivery of 5.0 pg mRNA was comparable to 0.5 pg SM102 LNPs (mRNA-1273). Also, Piezopen delivery of 0.5 pg mRNA is comparable to 5.0 pg ID mRNA, demonstrating the adjuvant effect of Piezopen EP despite negligible improvements in gene expression at the same dose. IM SMI 02 (5 pg and 2 pg) exhibited the highest antibody titer, followed by IM SM102 (0.5 pg) and Piezopen (5 pg).

[0340] Next, neutralizing antibodies were measured and plotted corresponding inhibition rates against different antibody concentrations and log -transformed neutralization titers to visualize the performance of the different vaccines (Figure 1 ID). A 30% inhibition rate was used to determine the neutralization antibody cutoff (as per the kit’s instructions). In general, the higher the inhibition rate, the better the vaccines perform in neutralizing the virus. The endpoint titers were: 1 / 1000 for IM SM102 (5 pg), 1 / 500 for IM SM102 (2 pg), 1 / 100 for Piezopen (5 pg), and 1 / 50 for IM SM102 (0.5 pg). Low neutralizing antibodies were detected for Piezopen at 0.5 pg and 2 pg.

[0341] Figures 13A-13D present data from Corbett et al. (2020), used to conduct a comparative analysis with Moderna’s results, as both studies utilized the same vaccine. The findings indicate that the binding antibody responses observed in Corbett et al. are within the same order of magnitude as those reported by Moderna at similar doses. This suggests that the positive controls are accurate and that Piezopen’s performance relative to lipid nanoparticles (LNPs) can be appropriately contextualized. In summary, Piezopen delivers naked mRNA encoding the Spike protein (without adjuvants) and achieves robust immune responses comparable to those elicited by Moderna’s LNPs, albeit at a higher dose.

[0342] Figure 14 is a bar graph showing that intradermal injection of OVA mRNA paired with an adjuvant followed by electroporation. Intradermal injections and electroporation were done using OVA mRNA (SC2346 GenScript); OVA mRNA + IFN-g mRNA (SC2325-IVT; GenScript); OVA mRNA + IL-6 mRNA (SC2325-IVT; GenScript); OVA RNase Inhibitor (N2615; Promega) per animal was added for experimental groups. Each treatment used a total of 5 pg of mRNA. For the animals that received adjuvants, a weight ratio of 1 pg adjuvant to 4 pg of mRNA OVA was maintained, for a total of 5 pg mRNA. Immunization was done twice, once on Day 0 and once on Day 7. A terminal blood collection was performed on day 14 to collect serum for antibodies. The ELISA kit used was the mouse anti-ova IgG antibody assay kit (Catalog # 3011, Chondrex, Inc.)

[0343] Conclusions

[0344] Piezopen induces robust immune responses to a naked mRNA vaccine, achieving comparability to SM102 LNPs at a higher dose. Piezopen induced robust humoral responses exceeding ID injection alone at all tested doses, with comparable binding antibody response between 0.5 pg Piezopen and 5.0 pg ID mRNA (~10-fold dose sparing). Piezopen delivery of 5.0 pg mRNA is comparable to 0.5 pg mRNA-1273, in terms of both binding and neutralizing antibody responses. This study is the first to demonstrate naked mRNA delivery has achieved humoral responses with a similar order of magnitude to LNPs.

[0345] Example 2: An Exemplary Electroporator For Increased “Naked” RNA Vaccine Delivery Materials and Methods

[0346] Animal Studies

[0347] Female 6-8-week-old Balb / c mice or ~8-week-old Wistar rats were used for the in vivo studies. For immunogenicity studies, blood collection was performed via the lateral tail vein at various time points and processed for serum. During terminal procedures, blood was collected via a cardiac puncture or inferior vena cava and spleens were harvested.

[0348] All procedures were approved by the Georgia State Institutional Animal Care and Use Committee (IACUC) Review Board. Studies were carried out at the Division of Animal Resources (DAR), Georgia State University, Atlanta, GA.

[0349] Ex vivo Human Skin Studies

[0350] Live human skin samples were purchased from Genoskin and maintained using the manufacturer’s protocol. Genoskin utilizes donated surgical human skin biopsies and stabilizes them in a proprietary matrix to preserve tissue viability and live skin responses for 7 days.

[0351] Piezopen Administration

[0352] Piczopcn includes a piezoelectric electroporator coupled with micronccdlc electrodes that localize electric fields to the epidermis, as described previously4,5Briefly, a 20 pL intradermal (ID) injection was administered via the Mantoux method (insulin syringe) followed by Piezopen application of 5 or 10 pulses. Bleb formation served as visual confirmation for a successful ID injection. For naked RNA injections, payloads were prepared by adding an RNase inhibitor to Ca2+-free PBS as described previously27

[0353] Reporter Expression Kinetics Studies

[0354] Animals (mice and rats) and human skin samples were injected with 5 pg FLuc-mRNA (SC2325, GenScript). For ID injections, a volume of 20 pL was used while for IM injections a volume of 50 pL. FDA-approved LNP formulations, namely SM102, ALC0315, and MC3 were used (LNP-formulated mRNA and saRNA were purchased from GenScript).6 LNPs were administered ID for reporter studies. Mice were injected with 1 pg FLuc-saRNA (SC2346, GenScript) and 1 pg FLuc-circRNA (SC2339, GenScript).

[0355] In Vivo Imaging System (IVIS)

[0356] IVIS was used 24 hrs after FLuc-RNA delivery. For animal studies, luciferin was injected intraperitoneally while for human skin samples, luciferin was injected via ID administration at the injection site. Samples were incubated for 10-15 minutes before imaging. IVIS was performed using an IVIS® Spectrum Imaging System. For animal reporter kinetics studies, animals were monitored for 14 days post-administration. For human skin reporter kinetics studies, human skin samples were imaged 1 day post-administration.

[0357] Vaccination Studies

[0358] Spike mRNA (SC2346, Genscript) was delivered to mice using ID injections with or without Piezopen electroporation and IM injection of LNPs encapsulated with Spike mRNA (SM102, ALC0315, and MC3) (Day 0). Injections were performed on the left flank (for ID) or left quadricep (for IM) of each animal at different doses (0.5 pg, 1 pg, 3 pg, 5 pg) depending on the group. A booster dose was administered on Day 21.

[0359] Blood and Tissue Collection

[0360] A baseline blood collection was performed for semm processing a day prior to prime dose injections (pre -bleed, Day -1). A second blood collection was performed for semm processing on Day 14. On Day 35, a final blood collection and necropsy were performed, collecting blood for semm processing and harvesting the spleen for testing. Animal weight was monitored throughout the study.

[0361] ELISA IgG

[0362] Spike protein-specific IgG levels in Day 35 semm were measured using the Mouse Anti- SARS-CoV-2 Antibody IgG kit (RAS-T023, AGRO Biosystems) according to the manufacturer's protocol. Endpoint titers were calculated as the highest dilution emitting an optical density greater than 4x background. Neutralizing Antibodies

[0363] Levels of neutralization antibodies in Day 35 serum were measured using the SARS- CoV-2 Surrogate Virus Neutralization Test (sVNT) Kit (L00847-A, GenScript) according to the manufacturer’s protocol. Of note, not all replicates in SM102 (1 pg) and ALC0315 (1 pg) achieved endpoint titers due to limited assay availability to run additional dilutions. Endpoint titers were calculated as the highest dilution emitting an inhibition rate exceeding 30% based on the manufacturer’s protocol.

[0364] ELISpot

[0365] IFN-y ELISPOT assays were performed on Day 35 using a commercial kit Mouse IFN-y Single-Color ELISPOT (96-well, precoated, strip, CTL) following the manufacturer’s protocol. In brief, 500,000 cells (from spleens) were stimulated overnight (17-18 hrs.) with 2 pg / mL Spike peptide (5823-45-01, InvivoGen). SARS-CoV-2 Spike Glycoprotein-crude (RP30020, GenScript) was used as a positive control at a concentration of 2 pg / mL.

[0366] Reactogenicity Array

[0367] A mouse cytokine array was performed 1-day post-administration with serum according to the manufacturer’s instructions (Mouse Cytokine Array G3 kit, AAM-CYT-G3-4, RayBiotech). The array was run and the analysis was performed by RayBiotech.

[0368] Statistical Analysis

[0369] For animal reporter studies, a two-way ANOVA with Tukey’s multiple comparisons test was performed. For human skin reporter studies, ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed. For vaccination studies, a two-way ANOVA with Tukey’s multiple comparisons test was performed. Each data point corresponds to either an independent subject (animal or human skin sample) or the average of technical replicates as stated in the figure caption. Error bars indicate the corresponding standard error of the mean. Data were analyzed using GraphPad Prism 7 (GraphPad Software).

[0370] Reported p values are multiplicity adjusted to account for multiple comparisons. For all cases, significance was defined as p < 0.05 (*) orp < 0.01 (**), orp < 0.001 (***).

[0371] Results

[0372] Piezopen Significantly Augments Gene Expression and Induces Immune Responses to Naked mRNA with Comparability to LNPs In Vivo

[0373] To evaluate whether Piezopen (Figure 1A) facilitates successful intracellular delivery of naked mRNA in vivo superior to intradermal (ID) injection alone and comparable to lipid nanoparticles (LNPs), reporter expression kinetics studies were conducted using luciferase. Benchmarking against two accepted LNP formulations, SMI 02 and ALC03156, it was demonstrated that Piezopen delivery of mRNA-Luc achieved comparable expression magnitude and improved durability and kinetics relative to LNPs in a dose-dependent manner (Figure 15 A). Gene expression following Piezopen administration persisted at levels 10- to 100-fold higher on Day 14 compared to LNPs, indicating enhanced antigen availability and potential for improved vaccine durability. Collectively, these data show that Piezopen (1) facilitates successful intracellular delivery of naked mRNA and (2) mitigates mRNA degradation concerns.

[0374] Following confirmation that Piezopen increases intracellular delivery and gene expression of naked mRNA to levels comparable with LNPs, the platform was evaluated for its ability to induce immune responses to naked mRNA vaccination. Comparative studies using a SARS-CoV-2 mRNA vaccine demonstrated that Piezopen delivery elicited humoral (antigenspecific IgG and neutralizing antibody) and cellular (spike-specific IFN-y) responses comparable to those induced by LNP formulations. Dose ranges between 0.5 pg and 1 pg for LNP groups aligned with preclinical studies of approved COVID-19 vaccines, serving as benchmarks for evaluating the higher-dose naked mRNA used as an initial proof of concept.

[0375] Piezopen delivery of naked mRNA using Piezopen A (the configuration associated with the highest mRNA-Luc expression) induced robust immune responses significantly greater than those observed under control conditions, including both naive and intradermal (ID) injection alone (Figures L5B, 15C, 15D). Manipulation of the electroporation (EP) field strength (Piezopen B) further enhanced immune responses despite a 40% reduction in the administered mRNA dose, indicating potential for optimization to achieve reduced dosing while maintaining or improving immune activation (Piezopen A: 5 pg vs. Piezopen B: 3 pg). Delivery using both Piezopen A (5 pg) and Piezopen B (3 pg) produced humoral and cellular immune responses comparable to those elicited by MC3 (1 pg) and slightly lower than SM102 (1 pg) and ALC03L5 (1 pg). Notably, neutralizing antibody titers induced by both Piezopen configurations were similar to those reported for mRNA-1273 and BNT162b2 vaccines analyzed using the same assay, supporting clinical translatability.78

[0376] Quantification of a panel of relevant cytokines demonstrated a favorable systemic reactogenicity profile across all Piezopen and LNP treatment groups (Figure 15E). Given the typically inflammatory nature of LNPs, this observation was unexpected.

[0377] Taken together, these findings demonstrate that Piezopen delivery of naked mRNA (1) achieves gene expression kinetics comparable to benchmark LNPs, (2) induces robust humoral and cellular immune responses comparable to existing LNP formulations, and (3) produces minimal reactogenicity. Piezopen Significantly Augments Gene Expression to Diverse Arrays of Naked RNA Payloads with Comparability to LNPs Across Species In Vivo and in Live Human Skin Ex Vivo

[0378] Leveraging Piezopen across different RNA subtypes permits a broad range of applications. In this study, Piezopen delivery was evaluated using self-amplifying RNA (saRNA) and circular RNA (circRNA), both of which have been reported to exhibit increased antigen expression at lower doses (saRNA)9 13or improved stability and prolonged antigen yield with durable immune responses (circRNA)14 15relative to canonical linear mRNA. Validation of saRNA delivery using luciferase reporters showed comparable expression magnitude and kinetics to state-of-the-art LNP formulations (SM102) at an equivalent 1 pg dose (Figure 16A). Piezopen delivery of naked circRNA also resulted in higher expression than intradermal injection alone at the same 1 pg dose (Figure 16B). Collectively, these results indicate that Piezopen allows efficient delivery of naked mRNA, saRNA, and circRNA with expression performance comparable to LNP formulations, supporting its utility as a payload-agnostic delivery platform.

[0379] To demonstrate that the results are translatable across species, mRNA delivery was evaluated in rats and preserved human skin explants, the latter providing a physiologically relevant model for human application. Piezopen delivery significantly enhanced gene expression compared to injection alone in rats (Figure 16C) and achieved expression levels comparable to SM102 LNP delivery in human skin ex vivo (Figure 16D), even at a low subtherapeutic dose (5 pg). These findings indicate efficient intracellular delivery with minimal mRNA degradation. Because the same Piezopen device was used in both in vivo and ex vivo experiments, the results confirm cross-species translatability and support clinical feasibility for human use. Furthermore, comparable expression to LNPs at low doses in human skin suggests that Piezopen allow dose reduction in clinical mRNA vaccination by overcoming the greater tissue thickness and broader immune-cell repertoire of human skin relative to mice.

[0380] Discussion

[0381] The findings of the present study demonstrate that naked RNA vaccination is feasible without the use of lipid nanoparticles (LNPs) or other formulation approaches traditionally considered necessary for RNA protection and intracellular delivery.6-8The increased performance observed with Piezopen is likely attributable to its electroporation-based mechanism, which rapidly delivers RNA into cells within seconds.16Piezopen achieved immunogenicity and expression kinetics comparable to LNPs even at low doses, representing a significant advancement given that improved expression durability has been strongly associated with enhanced vaccine efficacy.17 18

[0382] Importantly, Piezopen exhibited no measurable reactogenicity despite administration of higher doses compared to LNP formulations. This outcome underscores the favorable tolerability of the electroporation-based delivery platform and suggests potential for broader therapeutic use where formulation-free RNA administration is desirable. Collectively, these results highlight Piezopen as a versatile, clinically translatable delivery technology capable of inducing robust and durable immune responses to naked RNA while mitigating formulation-related complexity and inflammatory risk.

[0383] Piezopen delivery offers advantages over lipid nanoparticles (LNPs) by permitting multivalent or combination vaccine formulations through increased tolerability and dosing thresholds. The system accommodates a broad range of pay load sizes, from approximately 1,000 base pairs of mRNA to ~10 kilobase pairs of self- amplifying RNA (saRNA) or DNA, demonstrating the capacity to deliver large, complex, or multiple antigens. Notably, Piezopen induced strong immune responses to unmodified linear mRNA, which represents one of the least stable and least immunogenic RNA platforms,19underscoring its potential utility for circRNA and saRNA delivery. These findings highlight the advantages of Piezopen in increasing mRNA vaccine durability via improved antigen persistence and intrinsic adjuvant effects, addressing an important limitation of current mRNA vaccine technologies.

[0384] The development of effective mRNA medicines remains challenged by an incomplete understanding of the specific cell types that must be targeted to achieve effective immune protection. Studies have suggested that antigen-presenting cell (APC) transfection efficiency is closely associated with antigen-specific immune responses,20whereas non-APCs can also play a substantial role in initiating broad and durable immune activation through cross-presentation mechanisms observed with other physical delivery systems.21-24These observations imply that antigen cross-presentation by non-APCs to dendritic cells, rather than direct transfection of dendritic cells, may be a major driver of the humoral and cellular immune responses induced by physical delivery approaches.

[0385] LNP -based delivery systems typically transfect immune cell populations such as T cells, natural killer (NK) cells, macrophages, and dendritic cells at the injection site as well as within draining lymph nodes, spleen, and liver. In contrast, Piezopen directs electrical pulses and subsequent antigen expression to the epidermis, targeting resident antigen-presenting cells, keratinocytes, and Langerhans cells to enhance localized immunogenicity while minimizing systemic biodistribution. The observed differences in immune responses between Piezopen and LNPs are likely attributable to these distinct cellular targets and to variations in the number and type of APCs transfected by each delivery mechanism.

[0386] The observed effects from Piezopen electroporation potentially arise from a combination of efficient intracellular delivery, prolonged antigen expression, localized immune cell transfection, and adjuvant activity associated with cell death,26which collectively enhance both innate and adaptive immunity. The present study serves as a proof of concept for successful naked RNA intracellular delivery and vaccination, revealing additional advantages of Piezopen in improving RNA vaccine performance. These benefits include (1) enhanced durability through increased antigen persistence and intrinsic adjuvant effects, and (2) improved tolerability through localized administration rather than systemic distribution, addressing important limitations of LNP-based RNA vaccines. Overall, the findings highlight Piezopen as a potent, cost-effective, and versatile delivery platform capable of advancing the acceptability and global translation of mRNA vaccines.

[0387] References

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[0389] 2. Pardi, N„ et al., Nat Rev Drug Discov 17, 261-279 (2018).

[0390] 3. Ndeupen, S., et al., iScience 24, 103479 (2021).

[0391] 4. Xia, D„ et al., Proc Natl Acad Sci U S A 118, e2110817118 (2021).

[0392] 5. Lu, C. Y., et al., Bioeng Transl Med 9, el0662 (2024).

[0393] 6. Wang, L„ et al., JAMA 327, 678-680 (2022).

[0394] 7. Tangsathapornpong, A., et al., Vaccines (Basel) 11, 553 (2023).

[0395] 8. Liu, Y„ et al., medRxiv 2023.06.02.23290871 (2023).

[0396] 9. Bloom, K., et al., Gene Therapy 2020 28, 117-129 (2020).

[0397] 10. Ballesteros-Briones, M. C., et al., Curr Opin Virol 44, 145-153 (2020).

[0398] 11. Ci, Y., et al., Vaccines (Basel) 6, 367-383 (2018).

[0399] 12. Vogel, A. B., et al., Molecular Therapy 26, 446-455 (2018).

[0400] 13. Saraf, A., et al., Nature Medicine 2024 30:5, 1363-1372 (2024).

[0401] 14. Chen, R., et al., Nature Biotechnology 202241:2, 262-272 (2022).

[0402] 15. Liu, X., et al., J Control Release 348, 147-154 (2024).

[0403] 16. Knudsen, et al., Methods Mol Biol 1121, 147-154 (2014).

[0404] 17. Joyce, J. C., et al., J Control Release 304, 135 (2019).

[0405] 18. Palin, A. C., et al., Nat Immunol 23, 1665 (2022).

[0406] 19. Mokuda, S„ et al., BioRxiv 2022.03.22.435933 (2022).

[0407] 20. Chen, J., et al., Proc Natl Acad Sci U S A 120, e2309472120 (2023). 21. Laittebach, B., et al., The Journal of Immunology 176, 4600-4607 (2006).

[0408] 22. Cho, J. H., et al., The Journal of Immunology 167, 5549-5557 (2001).

[0409] 23. Vij, R„ et al., PLoS One 13, e0197962 (2018).

[0410] 24. Kim, B. S., et al., J Invest Dermatol 129, 2805-2817 (2009).

[0411] 25. Kim, E. H., et al., Eur J Immunol 2451008 (2024).

[0412] 26. Batisha Topnjak, et al., Bioelectrochemistry 141, 107871 (2021).

[0413] 27. Huysmans, H., et al., Mol Ther Nucleic Acids 17, 388-395 (2019).

[0414] Example 3: An Exemplary CHIMERA Dual-Modality Vaccination Induces Durable Humoral and Cellular Immunity in Nonhuman Primates

[0415] Materials and Methods

[0416] Vaccine Constructs and Formulation

[0417] The CHIMERA vaccine platform was designed to combine DNA and mRNA encoding identical SARS-CoV-2 Spike antigens to enhance the breadth and durability of the immune response. Two candidate formulations were evaluated, designated CHIMERA 1 and CHIMERA 2, which differed in construct design and expression characteristics. Specifically, CHIMERA 1 included co-administration of DNA and mRNA in the same injection site, whereas CHIMERA 2 utilized two adjacent injection sites to deliver the DNA and mRNA components separately, allowing assessment of localized versus spatially distinct expression effects. Each formulation was evaluated under a prime-only and a prime-boost regimen. A comparator arm included an mRNA- 1273 equivalent formulation, along with WHO and NIH standards. All constructs encoded full-length, prefusion-stabilized SARS-CoV-2 Spike glycoprotein. Unlike mRNA-1273, which typically requires both a priming and a boosting dose to achieve high antibody titers, CHIMERA was designed to generate robust immune responses following a single administration at lower, well-tolerated doses.

[0418] Animal Study Design

[0419] Mice were randomized into treatment groups receiving either CHIMERA 1 , CHIMERA 2, or mRNA-1273 equivalent formulations via intramuscular injection. For prime-boost regimens, animals received a booster dose 28 days after the initial priming dose. Serum samples were collected on Days 49, 90, and 180 post-prime for humoral immune assessments. Peripheral blood mononuclear cells (PBMCs) were collected at Day 180 for cellular immune assays.

[0420] Neutralizing Antibody Assay

[0421] SARS-CoV-2 neutralizing antibody titers were quantified using a pseudovirus neutralization assay. Titers were expressed as the reciprocal serum dilution resulting in 30% inhibition of infection (ID30). Titers were log -transformed for analysis and plotted as mean ± standard deviation (SD) for each time point.

[0422] IFNy ELI Spot Assay

[0423] Spike-specific T cell responses were evaluated at 6 months (Day 180) by IFNy ELISpot. PBMCs were stimulated with overlapping peptide pools spanning the Spike protein. Results were reported as IFNy spot-forming units (SFUs) per IxlO5cells after background subtraction. Results

[0424] Humoral Immune Response

[0425] As shown in Figure 17A, CHIMERA vaccination elicited measurable neutralizing antibody responses against SARS-CoV-2 that were sustained through 6 months post-prime. The prime-only CHIMERA 1 group generated modest neutralization titers comparable to baseline standards (ID30 ~ 2.2). By contrast, CHIMERA 1 and CHIMERA 2 prime-boost regimens induced approximately 1.3-1.5-fold higher titers (ID30 ~ 3.0-3.2) at Day 49, approaching levels observed with the mRNA-1273 equivalent comparator. Although titers declined slightly by Day 180, both CHIMERA prime-boost groups maintained detectable neutralizing activity comparable to standard benchmarks, indicating durable humoral immunity. The mRNA-1273 comparator achieved saturating antibody titers after a prime-boost, confirming its strong humoral potency; however, CHIMERA demonstrated comparable antibody levels with a single dose, suggesting reduced dependence on boosting and lower reactogenicity.

[0426] Cellular Immune Response

[0427] At 6 months, Spike-specific IFNy-secreting T cells were detected in all vaccine groups (Figure 17B). The CHIMERA 2 prime-boost group produced the highest frequency of IFNy+spots (—120 SFU / lxlO5cells), exceeding responses observed with the mRNA-1273 equivalent (~80 SFU / lxlO5cells). Both CHIMERA prime-boost regimens induced stronger cellular responses than the prime-only arm, indicating that boosting enhanced T cell memory persistence. CHIMERA elicited stronger T-cell responses at Day 180 than the mRNA-1273 comparator, which showed high antibody titers but relatively weaker cellular responses. The enhanced T-cell activity observed with CHIMERA is attributed to inclusion of the DNA-encoded antigen, which supports sustained cellular immune activation. The broad range of responses and large error bars suggest inter- animal variability typical of NHP studies but confirm the induction of a durable Th 1- skewed immune profile. Piezopen Delivery of CHIMERA-Luc Achieves Sustained Expression Kinetics

[0428] Exceeding mRNA-Luc

[0429] To assess antigen expression kinetics following delivery of different nucleic acid vaccine modalities, luciferase reporter constructs were administered using the Piezopen electroporation (EP) system. Bioluminescent imaging was performed at defined time points to quantify total photon flux as a measure of antigen expression over time.

[0430] As shown in Figures 18A and 18B, delivery of the CIIIMERA-Luc construct (encoding both DNA and mRNA components of luciferase) achieved sustained and durable antigen expression compared to mRNA-Luc alone. mRNA-Luc induced rapid and robust expression at early time points but declined sharply within 10-15 days post-administration, approaching background levels by Day 30. In contrast, CHIMERA-Luc maintained elevated expression for the full 30-day observation period, with expression kinetics closely resembling those of DNA- Luc, which exhibited the most prolonged signal persistence.

[0431] Quantitatively, Piezopen-mediated delivery of DNA-Luc alone resulted in continuous luciferase expression exceeding 107p / s at early time points and remaining above 106p / s for over 25 days. CIIIMERA-Luc delivery sustained similar levels of expression, exceeding 105- 106p / s at Day 30, while mRNA-Luc expression declined by nearly two orders of magnitude within the same period. The Naive control group showed negligible background signal.

[0432] These results demonstrate that combining DNA and mRNA components in a single CHIMERA formulation enhances and prolongs antigen expression kinetics beyond those achieved by mRNA alone. The findings suggest that Piezopen electroporation facilitates efficient intracellular delivery of both nucleic acid forms, supporting continuous antigen production and improved expression durability, features advantageous for next-generation nucleic acid vaccine platforms.

[0433] CHIMERA Vaccination Induces Potent Neutralizing Antibody Responses Comparable to Convalescent Sera and Commercial mRNA Vaccines

[0434] To evaluate the magnitude of humoral responses induced by CHIMERA vaccination, neutralizing antibody titers were assessed 7 weeks post-prime (Day 49) using a validated pscudovirus neutralization assay. As shown in Figure 1 , a single CHIMERA dose elicited neutralizing antibody titers comparable to those measured in human convalescent sera, approximating the levels associated with 80-90% predicted protection against symptomatic SARS-CoV-2 infection. Both CHIMERA vl and CHIMERA v2 formulations generated neutralization titers similar to those achieved with the prime-boost regimen of Modema’s Spikevax (mRNA-1273). Analysis of systemic cytokine responses indicated that CHIMERA vaccination produced cytokine profiles consistent with a balanced immune response, with no detectable elevations in inflammatory cytokines within the first 4-6 hours post-administration (data not shown). These findings suggest that CHIMERA vaccination effectively induces potent neutralizing antibody responses with favorable tolerability, supporting its potential as a singledose or prime-boost nucleic acid vaccine platform.

[0435] Conclusion

[0436] The CHIMERA platform, which integrates DNA and mRNA vectors encoding identical antigens, effectively elicited both neutralizing antibody and IFNy-producing T cell responses in nonhuman primates. Prime-boost regimens demonstrated superior immunogenicity and durability relative to prime-only administration, achieving responses comparable to or exceeding those induced by an mRNA-1273-equivalent benchmark. The results of the present study confirm that dual-modality vaccination can generate balanced humoral and cellular immunity. Overall, CHIMERA demonstrated the combined advantages of both vector systems, robust antibody and T-cell immunity, favorable tolerability, and the potential for single-dose efficacy, representing a significant step toward broad and durable immunogenicity.

[0437] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

77CLAIMSWe claim:

1. A method of treating or preventing the development of one or more symptoms of a disease or disorder in a subject, the method comprising:(i) administering to a target site of a subject, a pharmaceutical composition comprising an effective amount of one or more nucleic acid (s), wherein the one or more nucleic acid (s) encodes one or more antigen (s); and(ii) electroporating the target site of the subject, wherein electroporating increases delivery of the one or more nucleic acid (s) to cells in the target site.

2. A method for delivering one or more nucleic acid (s) to cells at a target site of a subject, comprising:(i) administering a pharmaceutical composition comprising an effective amount of one or more nucleic acid (s) to the target site of the subject, wherein the one or more nucleic acid (s) encodes one or more antigen (s); and(ii) applying electroporation to the target site, wherein the electroporation is effective to increase delivery of the one or more nucleic acid (s) to cells in the target site.

3. The method of claim 1 or claim 2, wherein the pharmaceutical composition is administered via intradermal injection.

4. The method of any of claims 1-3, wherein the cells are skin cells selected from the group consisting of keratinocytes, Langerhans cells, melanocytes, fibroblasts, epidermal dendritic cells and combinations thereof.

5. The method of any of claims 1-4, wherein Steps (i) and (ii) are performed using a device.

6. The method of claim 5, wherein Steps (i) and (ii) are performed by:(a) positioning the device adjacent to a target site on the subject, wherein the device comprises: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses;78(b) contacting the array of biocompatible and conductive microelectrodes comprising the at least one needle embedded therein into the biological tissue on the subject;(c) activating the switch to generate and deliver one or more electrical pulses through the array of biocompatible and conductive microelectrodes into the target site to electroporate the cells; and(d) delivering the pharmaceutical composition into the target site of the subject.

7. The method of claim 6, wherein the device comprises a syringe comprising a container holding the pharmaceutical composition of claim 1 or claim 2; wherein the array of biocompatible and conductive microelectrodes further comprises a syringe and at least one needle for delivery of the pharmaceutical composition embedded therein; and wherein the syringe is coupled to at least one needle for administering the pharmaceutical composition of claim 1 or 2 into the target site by actuation of the syringe.

8. The method of claim 6, wherein the device comprises a detachable syringe comprising a container for holding the pharmaceutical composition of claim 1 or claim 2; wherein the detachable syringe can be removed from the main body of the device after the pharmaceutical composition of claim 1 or 2 is delivered.

9. The method of claim 6, wherein the microelectrodes of the array of biocompatible and conductive microelectrodes are microneedle electrodes, wherein the microneedle electrodes are coated with the pharmaceutical composition of claim 1 or 2 and / or coated with one or more therapeutic, prophylactic, or diagnostic agents.

10. The method of claim 6, wherein step (c) and step (d) are performed concurrently.

11. The method of claim 10, wherein step (d) is performed following step (c).

12. The method of claim 11, wherein step (c) is performed following step (d).

13. The method of any one of claims 1-12, wherein step (c) is repeated at least once.

14. The method of any one of claims 1-13, wherein the target site is mammalian skin.

15. The method of any one of claims 1-14, wherein the target site is mammalian skin dermis and / or epidermis.

16. The method of any one of claims 1-15, wherein the one or more electrical pulses have a peak voltage absolute value between about 100 V and 1000 V, a peak current absolute value between about 0.001 A and 50 A, a peak static voltage absolute value between about 15,000 V and 35,000 V, an initial pulse length of between about I ps and 100 ps, or a combination thereof.7917. The method of any one of claims 1-16, further comprising: (iii) monitoring the expression of one or more protein (s) encoded by the one or more nucleic acids in the target site over a predetermined period.

18. The method of any one of claims 1-17, wherein the disease or disorder is an infection caused by a virus, bacterium, fungus, parasite, or protozoa.

19. The method of any one of claims 1-18, wherein the subject has an infection or is at risk of an infection.

20. The method of any of claims 1-19, wherein the infection is caused by a virus selected from the group consisting of orthomyxovirus, rhinovirus, paramyxovirus, coronavirus, adenovirus, human metapneumo virus (hMPV), enterovirus, bocavirus, flavivirus, retrovirus, or para retrovirus.

21. The method of claim 20, wherein the orthomyxovirus is an influenza virus, optionally wherein the influenza virus is selected from the group consisting of InfluenzaA, Influenza B, and Influenza C.

22. The method of claim 20, wherein the paramyxovirus is selected from the group consisting of Respiratory syncytial virus (RSV) and parainfluenza virus. he method of claim 20, wherein the coronavirus is selected from the group consisting of SARs- CoV, MERS-CoV, and SARS-CoV-2 (CO VID 19).

23. The method of claim 20, wherein the retrovirus is human immunodeficiency virus, and human t-lymphotrophic virus type 1 (HTLV-1).

24. The method of claim 20, wherein the para retrovirus is hepatitis B virus.

25. The method of claim 20, wherein the flavivirus is dengue virus, yellow fever virus, West Nile virus, Zika virus, Japanese encephalitis virus, tick-bome encephalitis virus, and hepatitis C vims.

26. The method of any one of claims 1-25, wherein the disease or disorder is cancer.

27. The method of any one of claims 1-26, wherein the pharmaceutical composition comprises an isolated nucleic acid encoding a protein or peptide, wherein the protein or peptide is effective to induce an immune response in the subject.

28. The method of claim 27, wherein the nucleic acid comprises RNA, and wherein the RNA is not encapsulated in a nanoparticlc, liposomes, microsphcrcs, or microcapsules.

29. The method of claim 28, wherein the RNA comprises messenger RNA (mRNA) and / or circular RNA.

30. The method of claim 29, wherein the messenger RNA is not self-replicating RNA.8031. The method of any one of claims 1 -30, wherein the pharmaceutical composition comprises one or more nucleic acid(s), and wherein the one or more nucleic acid(s) optionally comprise one or more modified nucleotide(s).

32. The method of claim 31, wherein the one or more modified nucleotide (s) is selected from the group consisting of 5-methylcytidine (m5C), 2-thiouridine (s2U), 5-methyluridine (m5U), pseudouridine (\| / ), 5 -methoxyuridine (5moU) and N1 -methylpseudouridine (mly).

33. The method of any one of claim 31 or 32, wherein the one or more nucleic acid (s) comprise an RNA encoding or expressing an adjuvant.

34. The method of any one of claims 31-33, wherein the one or more nucleic acid (s) comprise DNA encoding or expressing an adjuvant.

35. The method of claim 33, wherein one or more RNA-encoded adjuvant (s) are selected from the group consisting of interleukins, interferons, Granulocyte-Macrophage Colony- Stimulating Factor (GMCSF), Retinoic Acid-Inducible Gene I, chemokines, pattern recognition receptors, flagellin, bacterial toxins, tumor necrosis factors, and heat shock proteins.

36. The method of claim 34, wherein one or more DNA-encoded adjuvant (s) are selected from the group consisting of interleukins, interferons, Granulocyte -Macrophage Colony- Stimulating Factor (GMCSF), Retinoic Acid-Inducible Gene I, chemokines, pattern recognition receptors, flagellin, bacterial toxins, tumor necrosis factors, and heat shock proteins.

37. The method of any one of claims 1-36, wherein the nucleic acid has a molecular weight from about 10 base pairs to 100 kilobases.

38. The method of any one of claims 1-37, wherein the pharmaceutical composition comprises two or more nucleic acids.

39. The method of claim 38, wherein the pharmaceutical composition comprises two nucleic acids, wherein one of the two nucleic acids is RNA and the other is DNA.

40. The method of claim 39, wherein the pharmaceutical composition comprises two nucleic acids, wherein the two nucleic acids are RNA.

41. The method of any one of claims 1-40, wherein the nucleic acid encoding the antigen is isolated from a pathogenic microorganism.

42. The method of claim 41, wherein the pathogenic microorganism is a virus, bacteria, protozoa, parasite, or fungus.

43. The method of any one of claims 1-17 and 27-40, wherein the antigen is a cancer antigen.

44. The method of any one of claims 1-43, further comprising a step of administering a second active agent prior to, during, or after steps (i) and / or (ii).8145. A system for use in the method of any one of claims 1-44, comprising:(i) a pharmaceutical composition comprising an effective amount of one or more nucleic acid (s), wherein the one or more nucleic acid (s) encodes one or more antigen (s), and(ii) device comprising: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; and a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses.

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