Methods and systems for transdermal immunotherapy

Abstract

Disclosed herein are patches, methods, devices, and systems for delivering RNA encapsulated lipid nanoparticles into a subject. In some aspect, the patch includes a backing, a reservoir comprising lipid nanoparticles and one or more RNA molecules disposed within the reservoir, and a release liner. In other aspects, the method includes opening at least one channel in the subject's skin, applying the patch described herein, thereby delivering lipid nanoparticles to an immune cell.

Description

METHODS AND SYSTEMS FOR TRANSDERMAL IMMUNOTHERAPYField

[0001] This disclosure relates to patches, devices, systems and methods for transdermal drug delivery, and in particular, lipid nanoparticles composition encapsulating RNA molecules and methods for administering the compositions to the subjects by a transdermal microporation device.BACKGROUND

[0002] Emerging technologies associated with immunotherapy hold tremendous promise. However, the delivery of a therapeutic compound to a subject is important for its therapeutic effects, and usually, it can be impeded by the limited ability of the compound to reach targeted cells and tissues. RNA-based immunotherapies are a potential new class of RNA medicines. RNA vaccines can be developed more rapidly than traditional vaccines in response to infectious disease outbreaks. RNA immunotherapies can be generated using either messenger RNA (mRNA) or self-amplifying RNA. Both mRNA and self-amplifying RNA immunotherapies provide genetic instructions to the body's cells to unleash a potent immune response against infectious organisms. However, one of the significant obstacles in immunotherapy development is delivering DNA or RNA to target tissues.SUMMARY OF THE DISCLOSURE

[0003] One aspect of the disclosure relates to a patch for delivering RNA into a biological membrane (e.g.. skin) of a subject. In some embodiments, the patch includes a backing, a reservoir comprising lipid nanoparticles and one or more RNA molecules, and a release liner, wherein the release liner is configured to be removed before application of the patch to the subject’s skin. In some embodiments, the lipid nanoparticles further comprise one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, an anionic lipid, a neutral lipid, an aminolipid, a sphingolipid, a helper lipid, a lipid vesicular core, a phospholipid, and a conjugated lipid, and combinations thereof. In some embodiments, the cationic lipid is selected from the group consisting of ALC-0315, SM-102, (15Z, 18Z)-N,N-dimethyl-6-(9Z,12Z)- octadeca-9,12-dien-l-yl) tetracosa-15.18-dien-l-amine, (15Z, 18Z)-N,N-dimethyl-6-((9Z,12Z)- octadeca-9, 12-dien- 1 -y 1) tetracosa-4, 15.18-trien-l-amine. ( 15Z.18Z)-N,N-dimethyl-6-((9Z, 12Z)- octadeca-9,12-dien-l-yl) tetracosa-5,15,18-trien-l-amine, DSDMA, DLin-K-DMA, , DLin-K- C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA, DLin- M-C3-DMA. DLin-MP-DMA, l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1 ,2-dimyristoy 1-3 -trimethylammoniumpropane (DMTAP), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 3P-[N- (N,N-dimethylamino-ethane)carbamoyl] cholesterol (DC-Chol), N,N-dimethyl-N,N-di-9-cis- octadecenylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB). N-(l-(2.3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA). N.N- dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-Dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y-DLenDMA), 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP), l,2-Dilinoleyloxy-3- (dimethylamino)acetoxypropane (Dlin-DAC), l,2-Dilinoleyloxy-3-morpholinopropane (Dlin- MA), l,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3- dimethylaminopropane (Dlin-S-DMA), l-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (Dlin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (Dlin-TMA.Cl), 1,2- Dilinoleoyl-3-trimethylaminopropane chloride salt (Dlin-TAP.Cl), l,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (Dlin-MPZ), 3-(N,N-Dilinoleylamino)-l,2-propanediol (DlinAP), 3- (N,N-Di oleylamino)-!, 2-propanediol (DOAP), l,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (Dlin-EG-DMA). 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane (Dlin-K-DMA) or analogs thereof. (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), l,l’-(2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (Dlin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (Dlin-K- DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-l- amine (MC3 Ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N- dimethylbutan-1 -amine (MC4 Ether), N-(l-(2,3-dioleyloxy)propyl)-N-2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), Dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (XTC), and combinations thereof. In some embodiments, the anionic lipid is selected from the group consisting of phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N- dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG). and combinations thereof. In some embodiments,the helper lipid is selected from the group consisting of 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-di-(9Z-octadecenoyl)-sn- glycero-3-phosphoethanolamine (DOPE), cholesterol, l-palmitoyl-2-oleoyl-sn- glycero-3phosphocholin (POPC), 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), and combinations thereof. In some embodiments, the phospholipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N- maleimidomethy l)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethyl -phosphatidyl ethanol amine, dimethylphosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), ly sophos phatidylcholine, dilinoleoylphosphatidylcholine. In some embodiments, the one or more RNA molecules are encapsulated in the lipid nanoparticles. In some embodiments, the one or more RNA molecules comprise mRNA encoding a biologically active protein, or a nucleotide sequence homologous to an mRNA in a target cell. In some embodiments, the lipid nanoparticles are in an amount in the range from about 0.01 mg / cm2to about 200 mg / cm2in the reservoir, for example, from 0. 1 mg / cm2to 20 mg / cm2, from 0.1 mg / cm2to 10 mg / cm2, or from 0. 1 mg / cm2to 5 mg / cm2. In further embodiments, the RNA (e.g., mRNA) encapsulated lipid nanoparticles are in an amount in the range from about 0.01 mg / cm2to about 200 mg / cm2in the reservoir, for example, from 0. 1 mg / cm2to 20 mg / cm2, from 0. 1 mg / cm2to 10 mg / cm2, from 0.1 mg / cm2to 5 mg / cm2, from 0.2 mg / cm2to 5 mg / cm2, from 0.5 mg / cm2to 5 mg / cm2, or from 1 mg / cm2to 5 mg / cm2. In some embodiments, the reservoir further comprises at least one sugar. In some embodiments, the at least one sugar is selected from a non-reducing sugar, a reducing sugar, or a combination thereof. In some embodiments, the non-reducing sugar is sucrose, trehalose, mannitol, or sorbitol, or a combination thereof. In some embodiments, the reducing sugar is lactose, maltose, or a combination thereof. In some embodiments, the weight ratio of the at least one sugar to lipid nanoparticles is greater than 0.02. In some embodiments, the weight ratio of the at least one sugar to lipid nanoparticles is from about 0.02 to about 0.4. In some embodiments, the reservoir further comprises a drug delivery7modifier. In some embodiments, the drug delivery7modifier is an organic acid, a salt thereof, or a combinationthereof. In some embodiments, the drug delivery modifier is citric acid or its salt form, or a combination thereof. In some embodiments, the reservoir further comprises a preservative. In some embodiments, the preservative is an anti-microbial agent. In some embodiments, the antimicrobial agent is selected from the group consisting of methyl paraben, propylparaben, benzalkonium chloride, and sodium benzoate, and combinations thereof. In some embodiments, the reservoir further comprises at least one of sucrose, lactose, disodium citrate sesquihydrate, methylparaben, propylparaben, and benzalkonium chloride. In some embodiments, the reservoir further comprises at least one sugar, a drug delivery modifier, and a preservative as described herein. In some embodiments, the reservoir comprises at least one fiber or a laminated material of film, or a combination thereof. In some embodiments, the at least one fiber is a non-woven fiber having a thickness of less than 300 pm. In some embodiments, the at least one fiber has a weight of less than 100 g / m2. In some embodiments, the reservoir has a water-holding capacity of less than 20 mg / cm2, for example, from 0.1 mg / cm2to 20 mg / cm2, from 1 mg / cm2to 15 mg / cm2, or from 1 mg / cm2to 10 mg / cm2.

[0004] In some aspect, the disclosure provides a device for delivering RNA into a biological membrane (e.g., a skin) of a subject through a plurality of micropores. In some embodiments, the device includes a porator comprising an array of conductive filaments, an applicator electrically connected to the conductive filaments and configured to supply a predetermined electrical energy to the array of conductive filaments to create the plurality of micropores in an micropore area of the skin by heating the filaments, and a patch as described herein. In some embodiments, the device is configured to generate thermal energy' based on a current flowing through the array of conductive filaments, and provide the thermal energy to a biological membrane positioned adjacent to the device. In some embodiments, the applicator is configured to supply a predetermined electrical energy' to the array of conductive filaments for creating the plurality of micropores. In some embodiments, the device creates between about 25 to about 500 micropathways / cm2In some embodiments, the device creates about 400 micropathways / cm2. In some embodiments, the device has a filament density from about 300 to about 500 filament / cm2In some embodiments, the device has a filament density from about 400 filament / cm2In some embodiments, the device has a poration energy' from about 2 to about 10 mJ / filament. In some embodiments, the device has a poration energy of about 2 mJ / filament, 4 mJ / filament, or 8 mJ / filament. In some embodiments, the porator is configured to open at least one channel in the subject’s skin and has an area from about 0.01 cm2to about 4 cm2. In some embodiments, the porator is configured to open at least one channel in the subject’s skin and has an area of about 0. 1 cm2, about 0.25 cm2, about 0.65 cm2, or about 1.0 cm2. In some embodiments, the porator is configured to open at least one channel in the subject’s skin and has an area lessthan about 1.0 cm2, about 0.5 cm2, or about 0.25 cm2. In some embodiments, the at least one channel is one or more micropores, wherein one micropore is about 0.5% to about 12.5% of the total microporation area. In some embodiments, the porator is a microneedle, laser, or radio frequency porator. In some embodiments, the porator is configured to produce at least 50 pores in a subject’s skin.

[0005] In some aspect, the disclosure provides a system for transdermal RNA delivery into a biological membrane (e.g., a skin) of a subject through a plurality of micropores. In some embodiments, the system includes a substrate having an upper substrate surface and defining a poration area, the substrate comprising a filament array having a plurality of filaments that are disposed in the poration area, wherein each filament is capable of conductively delivering thermal energy via direct contact to the tissue membrane to form a plurality of micropores in a micropore area of the skin, an applicator electrically connected to the filament array and configured to supply a predetermined electrical energy to the filaments in order to create the plurality of micropores in the micropore area of the skin by heating the filaments, a power supply circuit configured to provide electric current to the applicator; and a patch as described herein configured for application on the micropore area. In some embodiments, the applicator is configured to generate thermal energy based on the electric current flowing through the array of conductive filaments. In some embodiments, the transdermal microporation device creates between about 25 to about 500 micropathways / cm2In some embodiments, the device creates about 400 micropathways / cm2In some embodiments, the device has a filament density from about 300 to about 500 filament / cm2In some embodiments, the device has a filament density from about 400 filament / cm2In some embodiments, the transdermal microporation device has a poration energy’ from about 2 to about 10 mJ / filament. In some embodiments, the applicator has a poration energy of about 2 mJ / filament, about 4 mJ / filament or about 8 mJ / filament. In some embodiments, opening at least one channel in the subject’s skin has an area from about 0.01 cm2to about 4 cm2. In some embodiments, the substrate is configured to open at least one channel in the subject’s skin and has an area of about 0.1 cm2, about 0.25 cm2, about 0.65 cm2, or about 1.0 cm2. In some embodiments, the porator is configured to open at least one channel in the subject’s skin and has an area less than about 1.0 cm2, about 0.5 cm2, or about 0.25 cm2. In some embodiments, the one or more micropores is about 0.5 to about 12.5% of the total poration area. In some embodiments, the transdermal microporation device produces at least 50 pores in a subject’s skin.

[0006] Some aspect relates to a method of providing RNA to a subject. In some embodiments, the method includes opening at least one channel in the subject’s skin, and applying a patch as described herein to the subject's skin. Some aspect relates to a method for triggering an immune response in a subject in need thereof. In some embodiments, the method includesopening at least one channel in the subject’s skin, and applying a patch as described herein to the subject’s skin. Some aspect relates to a method for treating, reducing, or ameliorating a pathogenic antigen in a subject. In some embodiments, the method includes opening at least one channel in the subject’s skin, and applying a patch as described herein to the subject’s skin. Some aspect relates to a method for treating, reducing, preventing or ameliorating cancer (e.g., tumor) in a subject. In some embodiments, the method includes opening at least one channel in the subject’s skin, and applying a patch as described herein to the subject’s skin. In some embodiments, opening at least one channel in the subject’s skin comprises applying a trans dermal microporation device as described herein to the subject’s skin. In some embodiments, the RNA is mRNA comprising a sequence encoding an antigenic peptide or protein, or a fragment, variant or derivative thereof. In some embodiments, the sequence encoding the antigenic peptide or protein, or the fragment, variant or derivative thereof is selected from the group consisting of pathogenic antigens, tumor antigens, allergenic antigens and autoimmune self-antigens.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below and, together with the description, serve to explain the principles of the disclosure. Like numbers represent the same elements throughout the figures.

[0008] FIG. 1 is a cross-section diagram illustrating patch structures applied after microporation treatment for the mRNA transdermal immunotherapy.

[0009] FIG. 2 is a cross-section diagram illustrating a ly ophilized and tablet dry patch structure applied after microporation treatment for the mRNA transdermal immunotherapy.

[0010] FIG. 3 is a graph illustrating transdermal microporation delivery of mRNA lipid nanoparticles immunogenicity in comparison with intramuscular injection (IM) in rats.

[0011] FIG. 4 is a graph illustrating ovalbumin (OVA) specific immunoglobin (Ig) G titers in rat serum at the 4-week blood sampling point after vaccine administration.

[0012] FIG. 5 is a graph illustrating OVA specific IgG titers in rat serum at the 4-week blood sampling point after vaccine administration.DETAILED DESCRIPTION OF THE DISCLOSURE

[0013] The present disclosure can be understood more readily by referencing the following detailed description, examples, drawings, and claims, and their previous and following descriptions. However, before the present devices, systems, and / or methods are disclosed and described, it is to be understood that this disclosure is not limited to the specific devices, systems, and / or methods disclosed unless otherwise specified. It is also to be understood that theterminology used herein is for the purpose of describing particular aspects only and is not necessarily intended to be limiting.

[0014] This description is provided as an enabling teaching of the disclosure. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various aspects of the disclosure described herein while still obtaining beneficial results. It will also be apparent that some of the desired benefits can be obtained by selecting some of the features described herein without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present description are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, this description is provided as illustrative of certain principles of the present disclosure and not in limitation thereof.Definitions

[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications referenced herein are incorporated by reference in their entirety' unless stated otherwise. In the event that there are a plurality' of definitions for a term herein, those in this section prevail unless stated otherwise.

[0016] As used throughout, the singular forms “a.” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to “a filament” can include tw o or more such filaments unless the context indicates otherwise.

[0017] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0018] As used herein, “animal” or “organism” refers to humans and other living organisms, including plants, to which the present disclosure may be applied.

[0019] As used herein, “analyte” means any chemical or biological material or compound suitable for passage through a biological membrane by the technology taught in this present disclosure or by technology previously known in the art of which an individual might want to know the concentration or activity inside the body. Glucose is a specific example of an analyte because it is a sugar suitable for passage through the biological membrane (e.g., skin). Individuals, for example, those with diabetes, might want to know their blood glucose levels. Other examplesof analytes include, but are not limited to, such compounds as sodium, potassium, bilirubin, urea, ammonia, calcium, lead, iron, lithium, salicylates, and the like.

[0020] As used herein, the term “biological fluid’' is defined as a fluid originating from a biological organism, including blood serum or whole blood as well as interstitial fluid.

[0021] As used herein, “enhancer.” “chemical enhancer,” “penetration enhancer,” “permeation enhancer,” and the like includes all enhancers that increase the flux of a permeant, analyte, or other molecules across the biological membrane and is limited only by functionality. In other words, all cell envelope-disordering compounds, solvents, and other chemical enhancement agents are intended to be included. Additionally, all active force enhancer technologies, such as the application of sonic energy, mechanical suction, pressure, local deformation of the tissues, iontophoresis, or electroporation, are included. One or more enhancer technologies may be combined sequentially or simultaneously . For example, a chemical enhancer may first be applied to permeabilize the capillary wall, and then an iontophoretic or sonic energy field may be applied to actively drive a permeant into those tissues surrounding and comprising the capillary bed.

[0022] As used herein, the term “interstitial fluid” is the clear fluid that occupies the space between the cells in the body.

[0023] As used herein, “lipid” refers to an organic compound that comprises an ester of fatty acid and is characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids, glycolipids, cationic lipids, non-cationic lipids, neutral lipids, and anionic lipids, all described in more detail herein; and (3) “derived lipids” such as steroids.

[0024] As used herein, “lipid particle” means a lipid formulation that can be used to deliver a therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, and the like). In some embodiments, the lipid particle is a nucleic acid-lipid particle, which is typically formed from a cationic lipid, a non-cationic lipid (e.g., a phospholipid), a conjugated lipid that prevents aggregation of the particle (e g., a PEG-lipid), and optionally cholesterol. Typically, the therapeutic nucleic acid (e.g., mRNA) may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0025] As used herein, “minimally invasive” refers to the use of mechanical, hydraulic, or electrical means that invade the stratum comeum to create a small hole or micropore without causing substantial damage to the underlying tissues.

[0026] As used herein, “non-invasive” means not requiring the entry of a needle, catheter, or other invasive medical instrument into a part of the body.

[0027] As used herein, “microporator” or “porato ’ refer to a component for a microporation device capable of microporation. Examples of a microporator or porator include, but are not limited to, a filament capable of conductively delivering thermal energy via direct contact to a biological membrane to cause the ablation of some portion of the membrane deep enough to form a micropore, an optically heated topical dye / absorber layer, an electromechanical actuator, a microlancet, an array of microneedles or lancets, a sonic energy ablator, a laser ablation system, a high-pressure fluid jet puncturer, and the like. As used herein, “microporator” and “porator” are used interchangeably.

[0028] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0029] As used herein, “penetration” means the controlled removal of cells caused by the thermal and kinetic energy released when the pyrotechnic element explodes which causes cells of the biological membrane and possibly some adjacent cells to be “blown away” from the site. As used herein, “fusible” and “fuse” refer to an element that could remove itself from and electrical circuit when a sufficient amount of energy7or heat has been applied to it. i.e., when a resistive, electrically activated poration element is designed to be a fusible element this means that upon activation, during or after the formation of the micropore in the biological membrane, the element breaks, stopping the current flow through it.

[0030] As used herein, “penetration enhancement” or “permeation enhancement” means an increase in the permeability of the biological membrane and / or tissue to a drug, bioactive composition, or other chemical molecule, compound, particle or substance (also called “permeant”), so as to increase the rate at which the drug, bio-active composition, or other chemical molecule, compound or particle permeates the biological membrane and / or tissue.

[0031] As used herein, “poration,” “microporation,” or any such similar term means the formation of a small hole or crevice (subsequently also referred to as a “micropore”) in or through the tissue or biological membrane, such as skin or mucous membrane, or the outer layer of an organism to lessen the barrier properties of this biological membrane for the passage of at least one permeant from one side of the biological membrane to the other for select purposes. In some embodiments, the hole or “micropore” so formed is approximately 1-1000 microns in diameter and extends into the biological membrane sufficiently to break the barrier properties of the stratum comeum without adversely affecting the underlying tissues. It is to be understood that the term “micropore” is used in the singular form for simplicity, but that the microporation devices described herein may form multiple artificial openings. Poration could reduce the barrier properties of a biological membrane into the body for selected purposes, or for certain medical orsurgical procedures. For the purposes of this application, “poration” and “microporation” are used interchangeably and mean the same thing.

[0032] As used herein, “stratum comeum” refers to the outermost layer of the skin, consisting of from about 15 to about 20 layers of cells in various stages of drying out. The stratum comeum provides a barrier to the loss of water from inside the body to the external environment and from attack from the external environment to the interior of the body.

[0033] As used herein, the term, “subcutaneous fluid” can include, without limitation, moisture, plasma, blood, one or more proteins, interstitial fluid, and any combination thereof. In one aspect, a subcutaneous fluid according to this description is a moisture source comprising water.

[0034] As used herein, “tissue” refers to an aggregate of cells of a particular kind, together with their intercellular substance, that forms a structural material. In the context of drug delivery to or through such tissue, at least one surface of the tissue is accessible to the transdermal delivery modality (e.g., poration device and / or patch). The tissue is the skin for various poration delivery modalities described herein. Other tissues suitable for use with this disclosure include mucosal tissue and soft organs.

[0035] As used herein, “immune cell” refers to help the body fight infections and other diseases. In skin, the epidermis comprises mostly epithelial cells, but also harbors Langerhans cells (LCs), dendritic cells, macrophages, mast cells, eosinophils, neutrophils, a. T lymphocytes, yo T lymphocytes (DETCs and dermal), non-y5 CD 1 -restricted lymphocytes, and B lymphocytes. Some immune cells migrate to lymph nodes to either induce peripheral tolerance to tissue selfantigens or initiate robust immune responses. In the event of a challenge, immune cells resident in the skin and those infiltrating from the periphery interact to create an intricate defense network.

[0036] As used herein, “transdermal” or “percutaneous” means passage of a permeant into and through the biological membrane to achieve effective therapeutic blood levels or local tissue levels of a permeant, or the passage of a molecule or fluid present in the body (“analyte”) out through the biological membrane so that the analyte molecule maybe collected on the outside of the body.

[0037] As used herein, the term “permeant,” “drug,” “permeant composition,” or “pharmacologically active agent” or any other similar term are used interchangeably to refer to any chemical or biological material or compound suitable for transdermal administration by the methods previously known in the art and / or by the methods taught in the present description, that induces a desired biological or pharmacological effect, which may include but is not limited to (1) having a prophylactic effect on the organism and preventing an undesired biological effect such as an infection, (2) alleviating a condition caused by a disease, for example, alleviating pain orinflammation, and / or (3) either alleviating, reducing, or completely eliminating the disease from the organism. The effect may be local, such as providing for a local anesthetic effect, or it may be systemic. Such substances include broad classes of compounds normally delivered into the body, including through body surfaces and membranes, including skin. In general, for example and not meant to be limiting, such substances can include any bioactive agents such as drug, chemical, or biological material that induces a desired biological or pharmacological effect. To this end, in one aspect, the permeant can be a small molecule agent. In another aspect, the permeant can be a macromolecular agent.

[0038] As used herein, an "effective’7amount of a pharmacologically active agent means a sufficient amount of a compound to provide the desired local or systemic effect and performance at a reasonable benefit / risk ratio attending any medical treatment. An "effective” amount of a permeation or chemical enhancer as used herein means an amount selected so as to provide the desired increase in biological membrane permeability, the desired depth of penetration, rate of administration, and amount of drug delivered.

[0039] As used herein, “transdermal flux rate” is the rate of passage of any analyte out through the skin of an individual, human or animal, or the rate of passage of any permeant, drug, pharmacologically active agent, dye, or pigment in and through the skin of an organism.

[0040] As used herein, "pharmaceutically acceptable carrier” refers to a carrier in which a substance such as a pharmaceutically acceptable drug could be provided for deliver. Pharmaceutically acceptable carriers are described in the art, for example, in “Remington: The Science and Practice of Pharmacy,” Mack Publishing Company, Pennsylvania, 1995, the disclosure of which is incorporated herein by reference. Carriers could include, for example, water and other aqueous solutions, saccharides, polysaccharides, buffers, excipients, and biodegradable polymers such as polyesters, polyanhydrides, polyamino acids, liposomes and mixtures thereof.

[0041] The term “pharmaceutically acceptable salt” as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid. Pharmaceutical salts can also be obtained by reacting a compound with an organic acid such as aliphatic or aromatic carboxylic or sulfonic acids, for example formic acid, acetic acid (AcOH), propionic acid, glycolic acid, pyruvic acid, malonic acid, maleic acid, fumaric acid, trifluoroacetic acid (TFA), benzoic acid, cinnamic acid,mandelic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluensulfonic acid, salicylic acid, stearic acid, muconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, 1,2- ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2- naphthalenesulfonic acid, or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a lithium, sodium or a potassium salt, an alkaline earth metal salt, such as a calcium, magnesium or aluminum salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D- glucamine. tris(hydroxymethyl)methylamine, (C1-C7 alkyl)amine, cyclohexylamine, dicyclohexylamine, triethanolamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, and salts with amino acids such as arginine and lysine; or a salt of an inorganic base, such as aluminum hydroxide, calcium hydroxide, potassium hy droxide, sodium carbonate, sodium hydroxide, or the like. In some embodiments, the compounds described herein may be in the form of a trifluoroacetate salt.

[0042] As used herein, ‘'reservoir” refers to a designated area or chamber within a device which is designed to contain a permeant for delivery' through an artificial opening in a biological membrane into an organism or may be designed to receive a biological fluid sample extracted from an organism through an artificial opening in a biological membrane. A reservoir may also contain excipient compounds which enhance the effect of a separately contained bioactive permeant. Additionally, a reservoir may contain or be treated with reactive enzymes or reagents designed to allow the measurement or detection of a selected analyte in an extracted biological fluid. A reservoir may be comprised of an open volume space, a gel, a flat planar space which has been coated or treated with a selected compound for subsequent release or reaction, or a matrix or permeable solid structure such as a pellet, tablet, powder, dried solid or porous polymer.

[0043] As used herein, a “tissue membrane” can be any one or more epidermal layers of a subject. For example, in one aspect, the tissue membrane is a skin layer that includes the outermost layer of the skin, i.e., the stratum comeum. In an alternative aspect, a skin layer can include one or more backing layers of the epidermis, commonly identified as stratum granulosum, stratum malpighii, and stratum germinativum layers. It will be appreciated by one of ordinary skill in the art that there is essentially little or no resistance to transport or to absorption of a permeant through the backing layers of the epidermis. Therefore, in one aspect, at least one formed pathway in a skin layer of a subject is a pathway in the stratum comeum layer of a subject. Further, as used herein, “stratum comeum” refers to the outermost layer of the skin, typically containing from about 15 to about 20 layers of cells in various stages of drying out. The stratum comeum providesa barrier to the loss of water from inside the body to the external environment and from attack from the external environment to the interior of the body. Still further, as used herein, “tissue membrane” can refer to an aggregate of cells of a particular kind, together with their intercellular substance, that forms a structural material. In various embodiments at least one surface of the tissue membrane is accessible to one or more of the poration devices and / or permeant compositions described herein. As noted above, the tissue membrane for various poration delivery modalities is the skin. Other tissues suitable for use with such devices and compositions include mucosal tissue and soft organs.

[0044] As used herein, “treat.” “treatment,” or “treating,” refers to administering a compound or pharmaceutical composition to a subject for prophylactic and / or therapeutic purposes. The term “prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a subject already suffering from a disease or condition.

[0045] In various embodiments, systems, devices, and methods that may be used and / or adapted for use with the compositions and methods described herein are described in one or more of U.S. Patent Nos. 6022316, 6142939, 6173202, 6183434. 6508785. 6527716, 6692456, 6730028, 7141034, 7392080, 7758561, 8016811, 8116860, and / or 9498609, all of which are hereby incorporated by reference in their entireties and particularly for the purpose of describing such systems and methods. In various embodiments, the systems and devices commercially available from PASSPORT® may be used or adapted for use in delivering the compositions described herein.Patch

[0046] In certain aspects, systems, devices and methods of the present disclosure may be used to transdermally deliver lipid nanoparticles and one or more RNA molecules across the skin. In some embodiments, the patch may comprise a top layer including an adhesive, a middle layer including a reservoir, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the middle layer further includes a drug delivery modifier. In some embodiments, the middle layer further includes an enhancer. In some embodiments, the patch includes a tissue interface layer. In some embodiments, the patch further includes a backing. In some embodiments, the release liner is configured to be removed before application to the subject’s skin. In some embodiments, the patch is configured as a film. In some embodiments, the patch is configured as a pellet.

[0047] In some embodiments, the lipid nanoparticles may include one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer conjugated lipids (e.g., pegylated lipid). In some embodiments, the lipid nanoparticles are not restricted to any particular morphology, and should be interpreted as to include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g.. in an aqueous environment and / or in the presence of a nucleic acid compound. For example, a liposome, a lipid complex, a lipoplex, and the like are within the scope of a lipid nanoparticle. In some embodiments, the lipid nanoparticles may include any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, such as mRNA, or in which the one or more nucleic acid molecules are encapsulated.

[0048] In some embodiments, the lipid nanoparticles have a mean diameter from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm. In some embodiments, the lipid nanoparticles include a lipid particle having a mean diameter from about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm. 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm. 100 nm, 105 nm, 110 nm, 115 nm, 120 nm. 125 nm, 130 nm, 135 nm. 140 nm, 145 nm, 150 nm, or ranges including and / or spanning the aforementioned values.

[0049] In some embodiments, the lipid nanoparticles are lipid encapsulated. “Lipid encapsulated” means a lipid particle that provides a therapeutic nucleic acid such as an RNA molecule (e.g.. an mRNA molecule) with full encapsulation, partial encapsulation, or both. In some embodiments, a nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticles.

[0050] Suitable methods of preparation of lipid nanoparticles are known in the art. For example, the methods disclosed in WO 2020 / 191103, WO 2022 / 036170, WO 2022 / 056413, and U.S. Patent No. 8,058,069 can be used to prepare a lipid nanoparticle of the present disclosure.Lipid Conjugates

[0051] In some embodiments, the lipid nanoparticles include a lipid conjugate. “Lipid conjugate” means a conjugated lipid that inhibits aggregation of lipid particles. The conjugated lipid may be useful in that it prevents the aggregation of particles. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic-polymer-lipid conjugates, and mixtures thereof. Furthermore, lipid delivery vehicles can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (Front Pharmacol. 2015 Dec 1; 6:286).

[0052] In some embodiments, the lipid conjugate is a PEG-lipid. The inclusion of polyethylene glycol (PEG) in a lipid formulation as a coating or surface ligand, a technique referred to as PEGylation, helps to protects nanoparticles from the immune system and their escape from RES uptake (Nanomedicine (Lond). 2011 Jun; 6(4):715-28). PEGylation has been widely used to stabilize lipid formulations and their payloads through physical, chemical, and biological mechanisms. Detergent-like PEG lipids (e.g., PEG-DSPE) can enter the lipid formulation to form a hydrated layer and steric barrier on the surface. Based on the degree of PEGylation, the surface layer can be generally divided into two types, brush-like and mushroomlike layers. For PEG-DSPE-stabilized formulations, PEG will take on the mushroom conformation at a low degree of PEGylation (usually less than 5 mol%) and will shift to brush conformation as the content of PEG-DSPE is increased past a certain level (Journal of Nanomaterials. 2011 ;2011 : 12). It has been shown that increased PEGylation leads to a significant increase in the circulation half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15; 13(): 507-30; J. Control Release. 2010 Aug 3; 145(3): 178-81). The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons. The average molecular weight may be any value or subvalue within the recited ranges, including endpoints. Such lipid conjugates may include, but are not limited to, PEG-lipid conjugates such as, e.g., PEG coupled to dialkydoxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers, and mixtures thereof. PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester-containing linker moieties and ester- containing linker moieties. In some embodiments, non-ester-containing linker moieties, such as amides or carbamates, are used. In one embodiment, the PEG-lipid comprises or is ALC-0159 (also known as 2-[(polyethylene glycol)-2000]-N.N-ditetradecylacetamide), which is N.N- dimyristy lamide of 2-hydroxy acetic acid, O-pegylated to a PEG chain mass of about 2 kilodaltons. In another embodiment, the PEG-lipid comprises or is DMG-PEG 2000, also known as 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol, which is a synthetic lipid formed by PEGylation of myristoyl diglyceride.

[0053] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from about 0. 1 mol% to about 2 mol%, from about 0.5 mol% to about 2 mol%, from about 1 mol% to about 2 mol%, from about 0.6 mol% to about 1.9 mol%, from about 0.7 mol% to about 1.8 mol%, from about 0.8 mol% to about 1.7 mol%, from about 0.9 mol% to about 1.6 mol%, from about 0.9 mol% to about 1.8 mol%, from about 1 mol% to about 1.8 mol%, from about 1 mol% to about 1.7 mol%, from about 1.2 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.7 mol%, from about 1.3 mol% to about 1.6 mol%, or from about 1.4 mol% to about 1.6 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid formulation. In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%. 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5%, (or any fraction thereof or range therein) of the total lipid present in the lipid formulation. The amount may be any value or subvalue within the recited ranges, including endpoints. The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid nanoparticles of the disclosure is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, for example, by ± 0.5 mol%. One of ordinary skill in the art will appreciate that the concentration of the lipid conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid formulation is to become fusogenic.

[0054] In some embodiments, the lipid nanoparticles include an amphipathic lipid. “Amphipathic lipid” means the material in which the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to. phospholipids, aminolipids, and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and [3- acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols.

[0055] In some embodiments, the lipid nanoparticles include a neutral lipid. ‘'Neutral lipid” means a lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0056] In some embodiments, the lipid nanoparticles include a non-cationic lipid. “Non-cationic lipid” means an amphipathic lipid or a neutral lipid or anionic lipid, and is described in more detail below.

[0057] In some embodiments, the lipid nanoparticles include an anionic lipid. “Anionic lipid” means a lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoylol eyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0058] In some embodiments, the lipid nanoparticles include a hydrophobic lipid. “Hydrophobic lipids'’ means compounds having apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N — N-dialkylamino. 1,2- diacryloxy -3 -aminopropane, and 1,2-dialky 1-3 -aminopropane.Cationic Lipids

[0059] In some embodiments, the lipid nanoparticles include a cationic lipid and amino lipid. Cationic lipids are widely studied for nucleic acid delivery due to their ability to bind to negatively charged membranes and induce uptake. Generally, cationic lipids are amphiphiles containing a positive hydrophilic head group and two (or more) lipophilic tails, or a steroid portion and a connector between these two domains. The terms “Cationic lipid” and “amino lipid” are used interchangeably to refer to lipids and salts thereof having one, two, three, or more fatty7acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group). The cationic lipid is typically protonated (positively charged) at a pH below the pKa of the cationic lipid and is substantially neutral at a pH above the pKa. The cationic lipids of the disclosure may also be referred to as titratable cationic lipids.

[0060] In some embodiments, the cationic lipid carries a net positive charge at physiological pH. In some embodiments, the cationic lipids may include a protonatable tertiaryamine (e.g., pH-titratable) head group; Cis alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains. In some embodiments, cationic lipids may include non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligonucleotides, and siRNA / small hairpin RNA (shRNA). Cationic lipids such as SM-102, ALC-0315. DOTAP (1,2- dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interaction, providing high in vitro transfection efficiency. Suitable cationic lipids include, but are not limited to: (15Z.18Z)-N,N-dimethyl-6-((9Z,12Z)- octadeca-9, 12-dien- 1 -y l)tetracosa- 15 , 18-dien- 1 -amine, ( 15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)- octadeca-9, 12-dien- 1 -y l)tetracosa-4, 15 , 18-trien- 1 -amine, (15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)- octadeca-9,12-dien- l-yl)tetracosa-5,l 5, 18-trien-l -amine, DSDMA, DODMA, DLinDMA, DLenDMA, y-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3), DLin-MP- DMA (also known as 1-B11). In some embodiments, the cationic lipid includes a commercially available cationic lipid. Examples include: LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1.2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.), LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(l-(2,3-dioleoyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N- dimethylammonium trifluoroacetate (DOSPA) and DOPE, from GIBCO / BRL), TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinoleny loxy-N,N- dimethylaminopropane (DLenDMA).

[0061] In some embodiments, the lipid nanoparticles include monocationic lipids. Monocationic lipids include, but are not limited to: l,2-dimyristoyl-sn-glycero-3- ethylphosphocholine (DMEPC), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), l,2-dioleoyl-3-trimethylammonium propane (DOTAP), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)- dimethylazanium bromide (DMRIE), Didodecyl(dimethyl)azanium bromide (DDAB), 1,2- dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 3(3-[N-(N,N- dimethylamino-ethane)carbamoyl] cholesterol (DC-Chol).

[0062] In the presently disclosed lipid nanoparticles, the cationic lipid may include, for example: N,N-dimethyl-N,N-di-9-cis-octadecenylammonium chloride (DODAC), N,N- distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP). N-(l-(2,3-dioleyloxy)propyl)-N,N.N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2.3-dioleyloxy)propylamine (DODMA). 1.2-Dilinoleyloxy-N.N- dimethylaminopropane (DLinDMA), 1 ,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y-DLenDMA), 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP), l,2-Dilinoleyloxy-3- (dimethylamino)acetoxypropane (Dlin-DAC), l,2-Dilinoleyloxy-3 -morpholinopropane (Dlin- MA), l,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3- dimethylaminopropane (Dlin-S-DMA), l-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (Dlin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (Dlin-TMA.Cl), 1,2- Dilinoleoyl-3-trimethylaminopropane chloride salt (Dlin-TAP.Cl), l,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (Dlin-MPZ), 3-(N,N-Dilinoleylamino)-l,2-propanediol (DlinAP), 3- (N,N-Dioleylamino)-l,2-propanediol (DOAP), l,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (Dlin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]- di oxolane (Dlin-K-DMA) or analogs thereof. (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z.28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), l,l’-(2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethylazanediyl)didodecan-2-ol (C 12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (Dlin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (Dlin-K- DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-l- amine (MC3 Ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N- dimethylbutan-1 -amine (MC4 Ether), N-(l-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), Dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxy ethyl ammonium bromide (DMRIE), 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (XTC), LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECT AMINE® (comprising DOSPA and DOPE, available from GIBCO / BRL). or combinations thereof.

[0063] Other suitable cationic lipids are disclosed in International Publication Nos.WO 2022 / 235972, WO 2022 / 235923, WO 2021 / 030701, WO 2015 / 074085, WO 2016 / 081029,WO 2017 / 117530, WO 2018 / 118102, WO 2018 / 119163, WO 2023 / 086514, WO 2022 / 235935,WO 2015 / 095340, WO 2009 / 086558. WO 2009 / 127060, WO 2010 / 048536, WO 2023 / 133946,WO 2010 / 054406, WO 2010 / 088537, WO 2010 / 129709, and WO 2011 / 153493; U.S. Patent Publication Nos. 2018 / 0170866, 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803; U.S. Patent Nos. 8,158,601,10,227,302; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are herein incorporated by reference.

[0064] Other suitable cationic lipids include those having alternative fatty acid groups and other dialkylamino groups, including those, in which the alkyl substituents are different (e.g., N-ethyl- N-methylamino-, and N-propyl-N-ethylamino-). These lipids are part of a subcategory of cationic lipids referred to as amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. In general, amino lipids having less saturated alkyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of filter sterilization. Amino lipids containing unsaturated fatty' acids with carbon chain lengths in the range of C 14 to C22 may be used. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.

[0065] In some embodiments, cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. Of course, it will be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipids be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the disclosure. In certain embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11. In some embodiments, the ionizable cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of an ionizable cationic lipid is about 6 to about 7.

[0066] In some embodiments, the lipid nanoparticles include a lipid vesicular core. The term "lipid vesicular core" refers to a lipid vesicle structure capable of encapsulating a therapeutically effective compound and capable of providing a binding surface on its outside for RNA. In other words, the outside of the lipid vesicular core is structured such that it can be covered by RNA and the inside of the lipid vesicular core is structured such that it faces a lumen, in which a therapeutically effective compound can be encapsulated. Lipid vesicle structures are substantially spherical structures usually made of materials having high amphiphilic lipid content. In some embodiments, the lipids of these spherical vesicles are organized in a lipid layer. In some embodiments, the lipids of these spherical vesicles are organized in lipid bilayers, which encapsulate a volume. In some embodiments, the volume is an aqueous volume. This volume provides a lumen, in which a therapeutically effective compound (e.g. water soluble compound)can be encapsulated. A therapeutically effective compound (e.g. water insoluble compound) can also be comprised in the lipid layer, particularly lipid bilayers, of said spherical vesicles, or a therapeutically effective compound (e.g. amphiphilic compound) can be comprised at the interface between the lipid layer, particularly lipid bilayers, and the encapsulated volume, aqueous volume, of said spherical vesicles. In some embodiments, the hpid vesicular core includes at least one cationic lipid.

[0067] In some embodiments, the at least one cationic lipid in the lipid vesicular core of the particles of the present disclosure amounts to at least about 10%, at least about 20%, at least about 30%. at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100%, or ranges including and / or spanning the aforementioned values. For example, the portion of ALC-0315, SM-102 or DOTMA in the lipid vesicular core of the particles of the present invention may amount to about 10%, about 20%, about 30%, about 40%, or about 50%.

[0068] In some embodiments, when the lipid vesicular core is a liposome, the liposome may be in the form of multilamellar vesicles (MLV), large unilamellar vesicles (LUV), small unilamellar vesicles (SUV) or multivesicular vesicles (MW) as well as in other bilayered forms known in the art. The size and lamellarity of the liposome will depend on the manner of preparation and the selection of the type of vesicles to be used will depend on the preferred mode of administration. In some embodiments, liposomes may have a diameter of between 50 and 150 nm (LUV or SUV) for systemic therapeutic purposes. In some embodiments, for local treatment, liposomes having larger diameters, such as MLV or MW, can be used. In some embodiments, the liposome may be further modified, for example, by an antibody, recognizing an antigen specifically expressed on the target cell structure and thereby improving the targeting of the liposome. In some embodiments, the liposome may be suitable fortransporting negatively charged molecules and for transfecting animal cells. In some embodiments, the animal cells are mammalian cells. In some embodiments, the animal cells are human cells.Helper Lipids and Sterols

[0069] In some embodiments, the lipid nanoparticles include a helper lipid. In some embodiments, the lipid vesicular core includes a helper lipid. The term "helper lipid" refers to a lipid capable of increasing the effectiveness of delivers’ of lipid-based particles such as cationic lipid-based particles to atarget. In some embodiments, the target is a cell. The helper lipid can be neutral, positively charged, or negatively charged. In some embodiments, the helper lipid is neutral or negatively charged. It has been found that lipid formulations, particularly cationic liposomes and hpid nanoparticles have increased cellular uptake if helper lipids are present in theformulation. (Curr. Drug Metab. 2014; 15(9): 882-92). Examples for helper lipids include 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE), cholesterol, l-palmitoyl-2-oleoyl-sn-glycero-3phosphocholin (POPC) and 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), but are not limited thereto. In some embodiments, the at least one helper lipid includes DSPC, DOPE, and / or cholesterol.

[0070] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the neutral lipid present in the lipid formulation comprises or consists of one or more phospholipids, e.g.. a cholesterol-free lipid formulation. In yet other embodiments, the neutral lipid present in the lipid formulation comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid formulation.

[0071] Other examples of helper lipids include nonphosphorous containing lipids such as, e g., stearylamine. dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.

[0072] In some embodiments, the lipid nanoparticle includes a cationic lipid of Formula (I) or (II):or a salt or isomer thereof, wherein, each of m and p is independently 1, 2. 3, 4. 5, 6 or 7, 1 is selected from 1, 2, 3, 4, and 5, Mi is M', Ri is — (CH2)nQ, in which Q is OH, and n is independently1, 2, 3, 4, or 5, M and M' are independently selected from — C(O)O — , and — OC(O) — , each of Ri, Rz and Ra is independently C1-17 alkyl, or C2-17 alkenyl, and R' is a C1-C12 linear or branched alkyl. In further embodiments, each of Ri, R2 and R3 is independently liner or branched C7-15 alkyl, liner or branched C9-13 alkyl, liner or branched C11 alkyl, liner or branched C7-15 alkenyl liner or branched C9-13 alkenyl, or liner or branched C11 alkenyl. In further embodiments, m is 3,4 or 5. Infurther embodiments, p is 5, 6 or 7. In further embodiments, 1 is 3,4 or 5. In further embodiments, n is 2, 3 or 4. In some embodiments, the lipid nanoparticles include an ionizable lipid of(also known as ALC-0315, [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)), or(also known as SM-102 or 9- heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate)), or a salt thereof. In some embodiments, the lipid nanoparticle comprising a compound of Formula (II) further includes a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn- glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphocholine (POPC), 1.2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1- hexadecyl-sn-glycero-3-phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1 ,2-diarachidonoyl-sn-gly cero-3-phosphocholine, 1 ,2-didocosahexaenoy 1-sn- glycero-3-phosphocholine. l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE). l,2-distearoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1 -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some embodiments, the lipid nanoparticles comprising a compound of Formula (II) further includes a cationic and / or ionizable lipid selected from the group consisting of 3-(didodecylamino)-Nl, Nl,4-tri dodecyl- 1- piperazineethanamine (KL 10), N 1 - [2-(didodecylamino)ethyl] -N 1 ,N4,N4-tridodecyl- 1 ,4- piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[l,3]- dioxolane (DLin-KC2-DMA), l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8- [(3 P)-cholest-5-en-3 -yloxy] octyl } oxy )-N,N -dimethy 1-3- [(9Z.12Z)-octadeca-9, 12-dien- 1 -y 1 oxy] propan- 1 -amine (Octyl-CLinDMA), (2R)-2-({8-[(3|3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Oct l-CLinDMA (2R)), and (2 S)-2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9, 12-dien-l -yloxy] propan- 1 -amine (Octyl-CLinDMA (2S)), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE- mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, and combinations thereof.

[0073] In any embodiments of the lipid nanoparticles described herein, the lipid nanoparticles may comprise one or more (optionally ionizable) cationic lipids (e.g., ALC-0315 or SM-102), one or more neutral lipids (e.g.. cholesterol), one or more PEG-lipids (e.g., ALC-0159 or DMG-PEG 2000), and one or more helper lipids (e.g., DSPC).

[0074] In some embodiments, the helper lipid comprises from about 2 mol% to about 20 mol%, from about 3 mol% to about 18 mol%, from about 4 mol% to about 16 mol%, about 5 mol% to about 14 mol%, from about 6 mol% to about 12 mol%, from about 5 mol% to about 10 mol%, from about 5 mol% to about 9 mol%, or about 2 mol%. about 3 mol%. about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation. The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ± 5 mol%.

[0075] The cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterolderivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 35 mol%, or about 28 mol% to about 35 mol%; or about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, or about 37 mol% or ranges including and / or spanning the aforementioned values of the total lipid present in the lipid formulation.

[0076] In some embodiments, the phospholipid component in the mixture may comprise from about 2 mol% to about 20 mol%, from about 3 mol% to about 18 mol%, from about 4 mol % to about 16 mol %, about 5 mol % to about 14 mol %, from about 6 mol % to about 12 mol%, from about 5 mol% to about 10 mol%, from about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.

[0077] In some embodiments, the lipid nanoparticles include about 30 mol % to about 60 mol % said active ingredient, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % cationic lipid, and about 0 mol % to about 10 mol % PEG lipid.

[0078] In some embodiments, the lipid nanoparticles include a lipid composition, comprising a nanoparticle or a bilayer of lipid molecules. In some embodiments, the lipid bilayer further includes a neutral lipid or a polymer. In some embodiments the lipid composition further encapsulates a nucleic acid. In some embodiments, the nucleic acid is capable of suppressing the expression of the target gene by utilizing RNA interference (RNAi), double-stranded RNA (siRNA) or anti-sense oligonucleotides. In some embodiments, the lipid composition further includes a nucleic acid and a neutral lipid or a polymer. In some embodiments, the lipid composition encapsulates the nucleic acid. In some embodiments, the nucleic acid is one or more RNA molecules. In some embodiments, the one or more RNA molecules includes a nucleotide sequence homologous to an mRNA in a target cell. In some embodiments, the nucleic acid is one or more therapeutic mRNA molecules encapsulated within the lipid particles.

[0079] In some embodiments, the one or more RNA molecules (e.g., mRNA molecules) are fully encapsulated w ithin the lipid portion of the lipid particles such that the RNA in the lipid particles is resistant in aqueous solution to nuclease degradation. In other embodiments, the lipid particles described herein are substantially non-toxic to mammals such as humans. In some embodiments, the lipid particles have a mean diameter of from 30 nm to 150 nm, from 40 nm to 150 nm, from 50 nm to 150 nm, from 60 nm to 130 nm, from 70 nm to 110 nm, or from 70 to 90 nm. In some embodiments, the lipid particles have a lipid:RNA ratio (mass / mass ratio) of from 1: 1 to 100: 1, from 1: 1 to 50: 1, from 2: 1 to 25: 1, from 3: 1 to 20: 1, from5: 1 to 15: 1, or from 5: 1 to 10: 1, or from 10: 1 to 14: 1, or from 9: 1 to 20: 1. In any embodiments of the RNA (e.g., mRNA) lipid nanoparticles described herein, the lipid particles have a lipid:RNA ratio (mass / mass ratio) of about or at least about 1: 1, 2: 1, 3:1, 4: 1, 5: 1, 6: 1, 7: 1, 8:1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 21 : 1, 22: 1, 23: 1, 24: 1, 25: 1, 26: 1, 27: 1, 28: 1. 29: 1 or 30: 1, or a range defined by any two of the preceding values. In one embodiment, the lipid particles have a lipid:RNA ratio (mass / mass ratio) of 12:1. In another embodiment, the lipid particles have a lipid:RNA ratio (mass / mass ratio) of 13: 1. In yet another embodiment, the lipid particles have a lipid:RNA ratio (mass / mass ratio) of about 4: 1. In yet another embodiment, the lipid particles have a lipid:RNA ratio (mass / mass ratio) of about 20: 1. In yet another embodiment, the lipid particles have a lipid:RNA ratio (mass / mass ratio) of about 26: 1.

[0080] In some embodiments, the lipid particles comprise one or more RNA molecules (e.g., mRNA molecules), a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The lipid particles can also include cholesterol. The lipid particles may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more RNA that express one or more polypeptides.

[0081] In some embodiments, the nucleic acid-lipid particles, the one or more RNA molecules (e.g., mRNA) may be fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In some embodiments, nucleic acid (e.g., mRNA) is fully encapsulated within the lipid portion of the lipid particles, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the mRNA in the lipid particles is not substantially degraded after exposure of the lipid particles to a nuclease at 37 °C. for at least 20, 30, 45, or 60 minutes. In certain other instances, the mRNA in the lipid particles is not substantially degraded after incubation of the particle in serum at 37° C. for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the mRNA is complexed with the lipid portion of the particles. One of the benefits of the formulations of the present disclosure is that the nucleic acid-lipid particle compositions are substantially non-toxic to mammals such as humans. “Fully encapsulated” means that the nucleic acid (e.g., mRNA molecule) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free RNA. In some embodiments, when fully encapsulated less than 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of free nucleic acid. In some embodiments, when fully encapsulated less than 10% of the nucleic acid in the particles is degraded in a treatment that would normally degrade 100% of free nucleic acid. In some embodiments, when fully encapsulated less than 5% of the nucleic acid in the particles isdegraded in a treatment that would normally degrade 100% of free nucleic acid. As used herein, “fully encapsulated” also means that the nucleic acid-lipid particles do not rapidly decompose into their component parts upon in vivo administration.

[0082] In some embodiments, full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid. Encapsulation is determined by adding the dye to a liposomal formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergent-mediated disruption of the liposomal bilayer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E=(Io-I) / Io, where / and Io refers to the fluorescence intensities before and after the addition of detergent.

[0083] In other embodiments, the present disclose provides a nucleic acid-lipid particle composition comprising a plurality of nucleic acid-lipid particles.

[0084] In some embodiments, the lipid nanoparticles comprise one or more RNA (e.g., mRNA) molecules that is fully encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%. In some embodiments, the lipid particle comprises RNA (e.g. mRNA) that is fully encapsulated within the lipid portion of the particles, from about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or ranges including and / or spanning the aforementioned values of the particles have the RNA encapsulated therein.

[0085] In some embodiments, the lipid nanoparticles include cationic lipid compounds. The compounds are particularly suitable for delivering polynucleotides to cells and tissues as demonstrated in subsequent sections. The lipomacrocycle compound described herein may be used for other purposes as well as, for example, recipients and additives.

[0086] The synthetic methods for the cationic lipid compounds can be synthesized with the skills in the art. Those skilled in the art will recognize other methods to produce these compounds, and also to produce the other compounds of the description.

[0087] The cationic lipid compounds may be combined with an agent to form microparticles, nanoparticles, liposomes, or micelles. The agent to be delivered by the particles, liposomes, or micelles may be in the form of a gas, liquid, or solid, and the agent may be a polynucleotide, protein, peptide, or small molecule. The lipomacrocycle compounds may be combined with other cationic lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, or lipids, to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

[0088] The present description provides novel cationic lipid compounds and drug delivery systems based on the use of such cationic lipid compounds. The system may be used in the pharmaceutical / drug delivery arts to deliver polynucleotides, proteins, small molecules, peptides, antigen, or drugs, to a patient, tissue, organ, or cell. These novel compounds may also be used as materials for coating, additives, excipients, materials, or bioengineering.

[0089] The cationic lipid compounds of the present description provide for several different uses in the drug delivery art. The amine-containing portion of the cationic lipid compounds may be used to complex polynucleotides, thereby enhancing the delivery of polynucleotide and preventing their degradation. The cationic lipid compounds may also be used in the formation of picoparticles, nanoparticles, microparticles, liposomes, and micelles containing the agent to be delivered. In some embodiments, the cationic lipid compounds are biocompatible and biodegradable, and the formed particles are also biodegradable and biocompatible and may be used to provide controlled, sustained release of the agent to be delivered. These and their corresponding particles may also be responsive to pH changes given that these are protonated at lower pH. They may also act as proton sponges in the delivery’ of an agent to a cell to cause endosome lysis.

[0090] In certain embodiments, the cationic lipid compounds are relatively non- cytotoxic. The cationic lipid compounds may be biocompatible and biodegradable. In some embodiments, the cationic lipid may have a measured pKa (in the formulation milieu) in the range of approximately 5.5 to approximately 7.5. In some embodiments, the cationic lipid may have a measured Ka (in the formulation milieu) in the range of approximately 6.0 to approximately 7.0. It may be designed to have a desired pKa between approximately 3.0 and approximately 9.0, or between approximately 5.0 and approximately 8.0. The cationic lipid compounds described herein are particularly attractive for drug delivery for several reasons: they contain amino groups for interacting with DNA, RNA, other polynucleotides, and other negatively charged agents, for buffering the pH, for causing endo-osmolysis, for protecting the agent to be delivered, they can be synthesized from commercially available starting materials; and / or they are pH responsive and can be engineered with a desired pKa. Non-limiting examples of non-cationic lipids includephospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidyl ethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG). dioleoylphosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanol amine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine. dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. In some embodiments, the acyl groups in these lipids include acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoy l. Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4'- hydroxy)-butyl ether.

[0091] In some embodiments, the non-cationic lipid present in lipid particles including a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the non-cationic lipid present in the lipid particles including one or more phospholipids, e.g., a cholesterol-free lipid particle formulation. In yet other embodiments, the non-cationic lipid present in the lipid particles includes a cholesterol or a derivative thereof, e.g., a phospholipid-free lipid particle formulation. Other examples of non-cationic lipids include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyoxylated fatty' acid amides, dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.

[0092] In some embodiments, the non-cationic lipid includes from 10 mol % to 60 mol %, from 20 mol % to 55 mol %, from 20 mol % to 45 mol %, 20 mol % to 40 mol %, from 25 mol% to 50 mol %, from 25 mol % to 45 mol %, from 30 mol % to 50 mol %, from 30 mol % to 45 mol %, from 30 mol % to 40 mol %, from 35 mol % to 45 mol %, from 37 mol % to 42 mol %, or 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, 45 mol % or ranges including and / or spanning the aforementioned values of the total lipid present in the particle.

[0093] In embodiments where the lipid particles contain a mixture of phospholipid and cholesterol or a cholesterol derivative, the mixture may include up to 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle. In some embodiments, the phospholipid component in the mixture may comprise from 2 mol % to 20 mol %, from 2 mol % to 15 mol %, from 2 mol % to 12 mol %, from 4 mol % to 15 mol %, or from 4 mol % to 10 mol %, or ranges including and / or spanning the aforementioned values of the total lipid present in the particle. In some embodiments, the phospholipid component in the mixture comprises from 5 mol % to 10 mol %, from 5 mol % to 9 mol %, from 5 mol % to 8 mol %, from 6 mol % to 9 mol %, from 6 mol % to 8 mol %, or 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, 10 mol %, or ranges including and / or spanning the aforementioned values of the total lipid present in the particle.

[0094] In other embodiments, the cholesterol component in the mixture may comprise from 25 mol % to 45 mol %, from 25 mol % to 40 mol %, from 30 mol % to 45 mol %, from 30 mol % to 40 mol %, from 27 mol % to 37 mol %, from 25 mol % to 30 mol %, or from 35 mol % to 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In some embodiments, the cholesterol component in the mixture comprises from 25 mol % to 35 mol %, from 27 mol % to 35 mol %, from 29 mol % to 35 mol %, from 30 mol % to 35 mol %, from 30 mol % to 34 mol %, from 31 mol % to 33 mol %, or 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.

[0095] In embodiments where the lipid particles are phospholipid-free, the cholesterol or derivative thereof may comprise up to 25 mol %, 30 mol %. 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.

[0096] In some embodiments, the cholesterol or derivative thereof in the phospholipid- free lipid particle formulation may comprise from 25 mol % to 45 mol %, from 25 mol % to 40 mol %, from 30 mol % to 45 mol %, from 30 mol % to 40 mol %, from 31 mol % to 39 mol %, from 32 mol % to 38 mol %, from 33 mol % to 37 mol %, from 35 mol % to 45 mol %, from 30 mol % to 35 mol %, from 35 mol % to 40 mol %, or 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, or ranges including and / or spanning the aforementioned values of the total lipid present in the particle.

[0097] In other embodiments, the non-cationic lipid comprises from 5 mol % to 90 mol %, from 10 mol % to 85 mol %, from 20 mol % to 80 mol %, 10 mol % (e.g., phospholipid only), or 60 mol % (e.g., phospholipid and cholesterol or derivative thereof) (or any fraction thereof or range therein) of the total lipid present in the particle.

[0098] The percentage of non-cationic lipid present in the lipid particles is a target amount, and that the actual amount of non-cationic lipid present in the formulation may vary, for example, by ±5 mol %.

[0099] In some embodiments, a composition containing a cationic lipid compound may be 30-70% cationic lipid compound, 0-60% cholesterol, 0-30% phospholipid and 1-10% polyethylene glycol (PEG). In some embodiments, the composition is 30-40% cationic lipid compound, 40-50% cholesterol, and 10-20% PEG. In some embodiments, the composition is 50- 75% cationic lipid compound, 20-40% cholesterol, and 5-10% phospholipid, and 1-10% PEG. In some embodiments, the composition may contain 60-70% cationic lipid compound, 25-35% cholesterol, and 5-10% PEG. In some embodiments, the composition may contain up to 90% cationic lipid compound and 2-15% helper lipid.

[0100] The formulation may be a lipid particle formulation, for example containing 8- 30% compound, 5-30% helper lipid, and 0-20% cholesterol; 4-25% cationic lipid, 4-25% helper lipid. 2-25% cholesterol. 10-35% cholesterol-PEG, and 5% cholesterol-amine; or 2-30% cationic lipid, 2-30% helper lipid, 1-15% cholesterol, 2-35% cholesterol-PEG, and 1-20% cholesterolamine; or up to 90% cationic lipid and 2-10% helper lipids, or even 100% cationic lipid.

[0101] In addition to cationic, the lipid particles described herein may further comprise a lipid conjugate. The conjugated lipid is useful in that it prevents the aggregation of particles. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic-polymer- lipid conjugates, and mixtures thereof.

[0102] In some embodiment, the lipid conjugate is a PEG-lipid. Examples of PEG- lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof. PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights: and include the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol- succinimidyl succinate (MePEG-S — NHS), monomethoxypolyethylene glycol-amine (MePEG- NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), monomethoxypoly ethylene glycol-imidazolyl-carbonyl (MePEG-IM), as well as such compounds containing a terminalhydroxyl group instead of a terminal methoxy group (e.g., HO-PEG-S, HO-PEG-S — NHS, HO- PEG-NH2). The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from 550 daltons to 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from 750 daltons to 5,000 daltons (e.g., from 1,000 daltons to 5.000 daltons, from 1.500 daltons to 3,000 daltons, from 750 daltons to 3,000 daltons, from 750 daltons to 2,000 daltons). In some embodiments, the PEG moiety has an average molecular weight of 2,000 daltons or 750 daltons.

[0103] In some embodiments, the PEG can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In some embodiment, the linker moiety is a non-ester-containing linker moiety. Suitable non-ester- containing linker moieties include, but are not limited to, amido ( — C(O)NH — ), amino ( — NR — ), carbonyl ( — C(O) — ), carbamate ( — NHC(O)O — ), urea ( — NHC(O)NH — ), disulphide ( — S — S — ), ether ( — O — ), succinyl ( — (OjCCEECEEC O) — ), succinimidyl ( —NHC(O)CH2CH2C(O)NH — ), ether, disulphide, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety). In some embodiments, a carbamate linker is used to couple the PEG to the lipid. In other embodiments, an ester-containing linker moiety is used to couple the PEG to the lipid. Suitable ester-containing linker moieties include, e.g., carbonate ( — OC(O)O — ), succinoyl, phosphate esters ( — O — (O)POH — O — ), sulfonate esters, and combinations thereof.

[0104] Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate. Such phosphatidylethanolamines are commercially available, or can be isolated or synthesized using conventional techniques known to those of skill in the art. In some embodiments, phosphatidylethanolamines may include saturated or unsaturated fatty acids with carbon chain lengths in the range of Cioto C20. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0105] The term "‘diacylglycerof’ or '‘DAG” includes a compound having 2 fatty acyl chains, R1and R2, both of which have independently between 2 and 30 carbons bonded to the 1- and 2-position of glycerol by ester linkages. The acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12),myristoyl (Cu), palmitoyl (Cie), stearoyl (Cis), and icosyl (C20). In some embodiments, R1and R2are the same, i.e., RJ and R2are both myristoyl (i.e., dimyristoyl), Rxand R2are both stearoyl (i.e., distearoyl).

[0106] The term “dialkyloxypropyT’ or “DAA” includes a compound having 2 alkyl chains. Ri and R2, both of which have independently between 2 and 30 carbons. The alkyl groups can be saturated or have varying degrees of unsaturation.

[0107] In some embodiments, the PEG-DAA conjugate is a PEG-dodecyloxy propyl (C10) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (Cie) conjugate, or a PEG-distearyloxypropyl (Cis) conjugate. In these embodiments, the PEG has an average molecular weight of 750 or 2,000 daltons. In particular embodiments, the terminal hydroxyl group of the PEG is substituted with a methyl group.

[0108] In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydirnethylacrylamide. polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0109] In some embodiments, the lipid conjugate (e.g., PEG-lipid) includes from 0.1 mol % to 2 mol %, from 0.5 mol % to 2 mol %, from 1 mol % to 2 mol %, from 0.6 mol % to 1.9 mol %, from 0.7 mol % to 1.8 mol %, from 0.8 mol % to 1.7 mol %, from 0.9 mol % to 1.6 mol %, from 0.9 mol % to 1.8 mol %, from 1 mol % to 1.8 mol %, from 1 mol % to 1.7 mol %, from 1.2 mol % to 1.8 mol %, from 1.2 mol % to 1.7 mol %, from 1.3 mol % to 1.6 mol %, or from 1.4 mol % to 1.5 mol %, or ranges including and / or spanning the aforementioned values of the total lipid present in the particle. In other embodiments, the lipid conjugate (e.g., PEG-lipid) includes from 0 mol % to 20 mol %, from 0.5 mol % to 20 mol %, from 2 mol % to 20 mol %, from 1.5 mol % to 18 mol %, from 2 mol % to 15 mol %, from 4 mol % to 15 mol %, from 2 mol % to 12 mol %, from 5 mol % to 12 mol %. or 2 mol % or ranges including and / or spanning the aforementioned values of the total lipid present in the particle.

[0110] In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from 4 mol % to 10 mol %, from 5 mol % to 10 mol %, from 5 mol % to 9 mol %, from 5 mol % to 8 mol %. from 6 mol % to 9 mol %, from 6 mol % to 8 mol %, or 5 mol %. 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % or ranges including and / or spanning the aforementioned values of the total lipid present in the particle.

[0111] The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid particles of the disclosure is a target amount, and the actual amount of lipid conjugate present in theformulation may vary, for example, by ±2 mol %. One of ordinary skill in the art will appreciate that the concentration of the lipid conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid particles are to become fusogenic.

[0112] By controlling the composition and concentration of the lipid conjugate, one can control the rate at which the lipid conjugate exchanges out of the lipid particles and, in turn, the rate at which the lipid particles become fusogenic. In addition, other variables including, e.g., pH, temperature, or ionic strength, can be used to vary and / or control the rate at which the lipid particles become fusogenic. Other methods which can be used to control the rate at which the lipid particles become fusogenic will become apparent to those of skill in the art upon reading this disclosure. Also, by controlling the composition and concentration of the lipid conjugate, one can control the lipid particle size.

[0113] In some embodiments, the mRNA sequence encodes an antigenic peptide or protein. In some embodiments, the mRNA encompasses mRNA generated in vitro, without chemical modifications or changes in the sequence. In some embodiments, the mRNA is natural and non-modified mRNA. In some embodiments, the mRNA is an artificial mRNA. In some embodiments, the artificial mRNA encompasses mRNA with chemical modifications, sequence modifications or non-natural sequences. In some embodiments, the mRNA does not comprise nucleoside modifications, in particular no base modifications. In a further embodiment, the mRNA compound does not comprise 1 -methylpseudouridine modifications. In one embodiment, the mRNA comprises only the naturally existing nucleosides. In one embodiment, the mRNA does not comprise any chemical modification and optionally comprises sequence modifications. In one embodiment, the mRNA only comprises the naturally existing nucleosides adenine, uracil, guanine and cytosine.

[0114] According to certain embodiments of the present disclosure, the mRNA sequence is mono-, bi-, or multicistronic. The coding sequences in a bi- or multicistronic mRNA may encode distinct peptides or proteins or a fragment or variant thereof. In some embodiments, the coding sequences encoding two or more peptides or proteins may be separated in the bi- or multicistronic mRNA by at least one IRES (internal ribosomal entry site) sequence. Thus, the term “encoding two or more peptides or proteins” may mean, without being limited thereto, that the bi- or even multicistronic mRNA, may encode e.g., at least two, three, four, five, six or more (in some embodiments different) peptides or proteins or their fragments or variants within the definitions provided herein. In some embodiments, without being limited thereto, the bi- or even multicistronic mRNA, may encode, for example, at least two, three, four, five, six or more (in some embodiments different) peptides or proteins as defined herein, or their fragments or variants as defined herein. In this context, a so-called IRES (internal ribosomal entry’site) sequence as defined above can function as a sole ribosome binding site, but it can also serve to provide a bi- or even multicistronic mRNA as defined above, which encodes several peptides or proteins which are to be translated by the ribosomes independently of one another. Examples of IRES sequences, which can be used according to this disclosure, are those from picomaviruses (e.g.. FMDV). pestiviruses (CFFV). polioviruses (PV). encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), mouse leukoma virus (MLV), simian immunodeficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0115] In some embodiments, the mRNA compound comprising an mRNA sequence of the present disclosure may thus be provided as a ‘'stabilized mRNA sequence”, that is to say as an mRNA that is essentially resistant to in vivo degradation (e.g., by an exo- or endo-nuclease). Such stabilization can be effected, for example, by a modified phosphate backbone of the mRNA of the present disclosure. A backbone modification in connection with the present disclosure is a modification in which phosphates of the backbone of the nucleotides contained in the mRNA are chemically modified. In some embodiments, nucleotides may include e.g., a phosphorothioate-modified phosphate backbone, for example at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom. Stabilized mRNAs may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form. Such backbone modifications ty pically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g., cytidine-5'-O-(l -thiophosphate)).

[0116] Examples of suitable tissue interface layers are described in U.S. Patent No. 7,392,080, which is hereby incorporated herein by reference in its entirety and particularly for the purpose of describing transdermal drug delivery patch systems.

[0117] In some embodiments, the top layer includes a backing. In some embodiments, the backing is a film, form, woven, or non-woven material. In some embodiments, the film includes a polyethylene (PE), polyethylene terephthalate (PET), polyurethane (PU), polyvinyl chloride (PVC), polychlorotrifluoroethylene (PCTFE) or cyclic olefin copolymers (COC) or polymers (COP). In some embodiments, the backing includes adhesive. In some embodiments, the adhesive is an acrylic, a silicone or a synthetic rubber such as Polyisobutylene (PIB) and Styrene-Isoprene-Styrene block copolymer (SIS). In some embodiments, the color of backing is transparent, semi- transparent, tan, white, or beige. In some embodiments, the backing is formedby thermoforming to make a cavity. In some embodiments, the backing is covered by adhesive tape.

[0118] In some embodiments, the bottom layer includes a release liner. In some embodiments, the release liner is a film. In some embodiments, the film is polyethylene terephthalate, polyethylene, paper, or aluminum foil. In some embodiments, the film includes a silicone or fluorosilicone coated layer. In some embodiments, the release liner is heat-sealed to the backing film.

[0119] In some embodiments, the drug delivery modifier is pH control agent. In some embodiments, the drug delivery modifier is an organic acid or base, a salt form of the organic acid or base, or a combination of thereof. In some embodiments, the organic acid is selected from ascorbic acid, citric acid, succinic acid, tartaric acid, maleic acid, lactic acid, benzoic acid, sorbic acid, amino acids, or a combination thereof. In some embodiments, the drug delivery' modifier is a non-organic acid. In some embodiments, the drug delivery modifier is a salt form of the non- organic acid. In some embodiments, the non-organic acid is hydrochloric acid, phosphoric acid, boric acid, acetic acid or a combination thereof. In some embodiments, the non-organic acid is evaporated during the manufacturing process. In some embodiments, the organic base is selected from sodium citrate, Tris, mono-sodium phosphate, di-sodium phosphate, tri-sodium phosphate, mono-potassium phosphate, di-potassium phosphate, tri-potassium phosphate, basic amino acids, or a combination thereof.

[0120] In some embodiments, the enhancer is a saccharide. In some embodiments, the saccharide comprises or is a sugar. In some embodiments, the enhancer is a non-reducing sugar. In some embodiments, the enhancer is a reducing sugar. In some embodiments, the saccharide is selected from mannitol, maltose, trehalose, xylitol, xylose, dextrose, lactose, sorbitol, sucrose, fructose, maltitol, erythritol, lactitol, isomalt, and cyclodextrin or a combination thereof. In some embodiments, the enhancer is sucrose. In some embodiments, the enhancer is lactose. In some embodiments, the enhancer is maltose. In some embodiments, the enhancer is a combination of sucrose and lactose. In some embodiments, the enhancer acts as stabilizer.

[0121] In some embodiments, the weight ratio of enhancer to lipid nanoparticles is greater than 0.005, greater than 0.02, greater than 0.05, greater than 0.1, greater than 0.2, or ranges including and / or spanning the aforementioned values. In some embodiments, the weight ratio of a sugar to lipid nanoparticles is greater than 0.02. In some embodiments, the weight ratio of a sugar to lipid nanoparticles is from about 0.02 to about 10. In some embodiments, the weight ratio of a sugar to lipid nanoparticle is from about 0.02 to about 0.4.

[0122] In some aspect, the middle layer of the patch includes a reservoir. In some embodiments, the reservoir includes about 1.0% to about 99.5% by weight lipid nanoparticles. Insome embodiments, the lipid nanoparticles include approximately 50 weight % to approximately 98 weight % of the middle layer, including amounts such as 50 weight %, 55 weight %, 70 weight %, 80 weight %, 90 weight %, 95 w eight %, 98 weight % of the middle layer, including any range of weight percentages derived from these values.

[0123] In some embodiments, the middle layer includes about 0.5% to about 99% by w eight of an enhancer. In some embodiments, the enhancer includes approximately 2 weight % to approximately 50 weight % of the middle layer, including amounts such as 2 weight %, 5 weight %, 10 weight %, 15 weight %, 30 weight %, 40 w eight %, 50 weight % of the middle layer, and including any range of weight percentages derived from these values.

[0124] In some embodiments, the middle layer includes about 5 to about 60% by weight of a drug delivery modifier. In some embodiments, the drug delivery modifier includes approximately 10 weight % to approximately 40 weight % of the middle layer, including additional amounts as 7.5 weight %, 15 weight %. 25 weight %, 40 weight % of the middle layer, and including any range of weight percentages derived from these values.

[0125] In some embodiments, the middle layer includes a drug delivery modifier in an area amount of at least 0.5 mg / cm2, at least 1 mg / cm2, at least 2 mg / cm2, at least 4 mg / cm2, at least 8 mg / cm2, or ranges including and / or spanning the aforementioned values, based on the surface area of the middle layer facing the bottom layer. In some embodiments, the middle layer includes at least 2 mg / cm2of disodium citrate. In some embodiments, the middle layer includes at least 4 mg / cm2of disodium citrate.

[0126] In some aspect, the middle layer includes a matrix support. In some embodiments, the matrix support includes at least one fiber. In some embodiments, the fiber is a nonwoven material. In some embodiments, the matrix support is a non-woven fabric. In some embodiments, the non-woven fabric is a polyethylene terephthalate. In some embodiments, the matrix support is a laminated material of film. In some embodiments, the film is a polyethylene terephthalate. In some embodiments, the matrix support is a laminated material of fiber. In some embodiments, the matrix is a laminated material of film and fiber.

[0127] In some embodiments, the thickness of the matrix support is less than 300 pm, less than 250 pm, less than 200 pm, less than 150 pm, less than 100 pm, less than 50 pm, or ranges including and / or spanning the aforementioned values.

[0128] In some embodiments, the areal weight of the fiber is less than 100 g / m2. less than 90 g / m2, less than 80 g / m2, less than 70 g / m2, less than 60 g / m2, less than 50 g / m2, less than 40 g / m2, less than 30 g / m2, less than 20 g / m2, less than 10 g / m2, or ranges including and / or spanning the aforementioned values.

[0129] In some embodiments, the reservoir or matrix support has a water holding capacity (WHC) of less than 20 mg / cm2, for example, from about 0.1 mg / cm2to about 20 mg / cm2, or from 1 mg / cm2to 10 mg / cm2based on the surface area of the reservoir or matrix support facing the bottom layer. The water holding capacity of the reservoir or matrix support means the amount of moisture the reservoir or matrix support can hold per 1 cm2of the transdermal surface. Specifically, a 1 cm2matrix is prepared, and this is immersed in a solution (phosphate buffered saline containing 0.1% surfactant (Tween® 80)) for a sufficiently long amount of time. Following this, the matrix support is slowly pulled out of the solution for around five seconds, the weight of the sample before immersion measured in advance is subtracted from the weight of the sample holding the liquid, and then it is possible to determine the water holding capacity of the matrix per unit area (1 cm2) of the transdermal surface. In some embodiments, the reservoir or matrix support water holding capacity is from about 10 mg / cm2or less. In some embodiments, the reservoir or matrix support water holding capacity is from about 1 mg / cm2to about 8 mg / cm2. In some embodiments, the reservoir or matrix water holding capacity is from about 2 mg / cm2to about 5 mg / cm2.

[0130] The water holding capacity of the reserv oir or matrix support may be controlled by adjusting the thickness and weight of the matrix support. In some embodiments, the matrix support has a thickness of 100 pm or less. In some embodiments, the matrix support has a thickness in the range of about 10 pm to about 100 pm. In some embodiments, the matrix support thickness is about 20 pm to about 90 pm. In some embodiments, the matrix support thickness is about 30 pm to about 80 pm. In some embodiments, the matrix support thickness is about 40 pm to about 60 pm.

[0131] In some embodiments, the matrix support areal weight is about 10 g / m2to about 100 g / m2. In some embodiments, the matrix support areal weight is about 15 g / m2to about 80 g / m2. In some embodiments, the matrix support areal weight is about 20 g / m2to about 60 g / m2. In some embodiments, the matrix support areal weight is about 25 g / m2to about 40 g / m2.

[0132] In some embodiments, the matrix support areal weight is about 0.1 mg / cm2to about 30 mg / cm2. In some embodiments, the matrix support areal weight is about.0.5 mg / cm2to about 30 mg / cm2. In some embodiments, the matrix a support real weight is about 0.5 mg / cm2to about 20 mg / cm2. In some embodiments, the matrix support areal weight is about 0.5 mg / cm2to about 10 mg / cm2.

[0133] In some embodiments, the size of the matrix is about 0.01 cm2to about 4 cm2In some embodiments, the size of the matrix is about 0.02 cm2to about 2 cm2. In some embodiments, the size of the matrix is about 0. 1 cm2to about 2 cm2. In some embodiments, the size of the matrix is about 0.25 cm2. In some embodiments, the size of the matrix is about 0.5 cm2.In some embodiments, the size of the matrix is about 1 cm2. In some embodiments, the size of the matrix is about 2 cm2.

[0134] In some embodiments, the total amount of lipid nanoparticles and enhancer per unit area of the matrix (surface area) is 0.01 mg / cm2to 200 mg / cm2In some embodiments, the total amount of lipid nanoparticles and enhancer per unit area of the matrix is 0.1 mg / cm2to 100 mg / cm2. In some embodiments, the total amount of lipid nanoparticles and enhancer per unit area of the matrix is 5 mg / cm2to 75 mg / cm2. In some embodiments, the total amount of lipid nanoparticles and enhancer per unit area of the matrix is 10 mg / cm2to 50 mg / cm2.

[0135] In some embodiments, the pH of the matrix ingredients is from about 3 to about 9. In some embodiments, the pH of the matrix is from about 4 to about 8. In some embodiments, the pH of the matrix is about 4. In some embodiments, the pH of the matrix is about 5. In some embodiments, the pH of the matrix is about 6. In some embodiments, the pH of the matrix is about 7.

[0136] In some aspect, the reservoir or the matrix includes from about 0.01 mg / cm2to about 200 mg / cm2lipid nanoparticles, based on the surface area of the reservoir / matrix facing the bottom layer. In some embodiments, the reservoir or the matrix includes from about 0. 1 mg / cm2to about 100 mg / cm2lipid nanoparticles. In some embodiments, the reservoir or the matrix includes from about 1 mg / cm2to about 50 mg / cm2. from about 0.1 mg / cm2to about 20 mg / cm2, from about 0.1 mg / cm2to about 10 mg / cm2, or from about 0.1 mg / cm2to about 5 mg / cm2lipid nanoparticles. In some embodiments, the reservoir or the matrix includes from about 5 mg / cm2to about 30 mg / cm2lipid nanoparticles. In some embodiments, the reservoir or the matrix includes from about 5 mg / cm2to about 25 mg / cm2lipid nanoparticles. In some embodiments, the reservoir or the matrix includes about 0.1 mg / cm2, 0.2 mg / cm2, 0.3 mg / cm2, 0.4 mg / cm2, 0.5 mg / cm2, 0.6 mg / cm2, 0.7 mg / cm2, 0.8 mg / cm2, 0.9 mg / cm2, 1.0 mg / cm2, 1.2 mg / cm2, 1.4 mg / cm2, 1.6 mg / cm2, 1.8 mg / cm2, 2.0 mg / cm2, 2.2 mg / cm2, 2.4 mg / cm2, 2.8 mg / cm2, 3.0 mg / cm2, 3.2 mg / cm2, 3.4 mg / cm2, 3.6 mg / cm2, 3.8 mg / cm2, 4.0 mg / cm2, 4.2 mg / cm2, 4.4 mg / cm2, 4.8 mg / cm2, or 5.0 mg / cm2, lipid nanoparticles. In any embodiments mentioned herein, the lipid nanoparticle may encapsulate one or more RNA molecules (e.g., mRNA molecules).

[0137] In some aspect, the reservoir or the matrix includes from about 0.05 mg / cm2to about 100 mg / cm2enhancer, based on the area of the matrix facing the bottom layer. In some embodiments, the reservoir or the matrix includes from about 0. 1 mg / cm2to about 50 mg / cm2enhancer. In some embodiments, the reservoir or the matrix includes from about 0.2 mg / cm2to about 5.0 mg / cm2enhancer. In some embodiments, the reservoir or the matrix includes from about 0.5 mg / cm2to about 10 mg / cm2enhancer. In some embodiments, the reservoir or the matrix includes from about 0.5 mg / cm2to about 4.0 mg / cm2enhancer. In some embodiments, thereservoir or the matrix includes from about 0.5 mg / cm2to about 2.0 mg / cm2enhancer. In some embodiments, the reservoir or the matrix includes about 0.5 mg / cm2enhancer. In some embodiments, the reservoir or the matrix includes about 1 mg / cm2enhancer. In some embodiments, the reservoir or the matrix includes about 2 mg / cm2enhancer. In some embodiments, the enhancer is at least selected from one or more oligosaccharides or polysaccharides, including but not limited to sucrose, lactose and maltose.

[0138] In some aspect, the patch further includes an anti-microbial agent. In some embodiments, the anti-microbial agent is selected from benzoic acid, methylparaben, propylparaben, benzalkonium chloride, chlorhexidine. cresol, salicylic acid, sorbic acid, benzethonium chloride, and salts and combinations thereof.

[0139] In some embodiments, the patch is configured as a dry patch formulation. In some embodiments, the dry7patch includes a backing, a matrix including lipid nanoparticles in a dry state, and a release liner. In some embodiments, the matrix further includes a drug delivery7modifier in a dry state. In some embodiments, the matrix further includes an enhancer in a dry state. In some embodiments, the matrix further includes an anti-microbial agent in a dry state. In some embodiments, the dry7patch is a heat dried film manufactured by dispensing or casting process. In some embodiments, the dry patch is a tablet or pellet manufactured by compressed process. In some embodiments, the dry patch formulation includes lipid nanoparticles with an encapsulated RNA (e g., mRNA molecule), sucrose, lactose, disodium citrate sesquihydrate, methylparaben, propylparaben, and benzalkonium chloride. In some embodiments, the dry7patch includes less than 10% of water in formulation. In some embodiments, the dry7patch includes less than 5% of water in formulation. In some embodiments, the dry patch includes less than 2% of water in formulation. In some embodiments, the dry patch includes less than 1% of water in formulation. In some embodiments, the dry patch includes less than 0.5% of water in formulation. In some embodiments, the dry patch includes less than 0. 1% of water in formulation.

[0140] In some embodiments, the patch is configured as a lyophilized patch formulation. In some embodiments, the lyophilized patch includes a backing, a matrix including lipid nanoparticles in a lyophilized state, and a release liner. In some embodiments, the lyophilized patch consists of a cavity7with spacer. In some embodiments, the lyophilized patch consists of a blister packaging. In some embodiments, the matrix further includes a drug delivery7modifier in a lyophilized state. In some embodiments, the matrix includes an enhancer in a lyophilized state. In some embodiments, the matrix further includes an anti-microbial agent in a lyophilized state. In some embodiments, the lyophilized patch is lyophilized by processes known to those skilled in the art. In some embodiments, the lyophilized patch is a tablet or pellet manufactured by compressed process. In some embodiments, the lyophilized patch formulationincludes lipid nano particle with an encapsulated mRNA, sucrose, lactose, disodium citrate sesquihydrate, methylparaben, propylparaben, and benzalkonium chloride. In some embodiments, the lyophilized patch includes less than 10% of water in formulation. In some embodiments, the lyophilized patch includes less than 5% of water in formulation. In some embodiments, the lyophilized patch includes less than 2% of water in formulation. In some embodiments, the lyophilized patch includes less than 1% of water in formulation. In some embodiments, the lyophilized patch includes less than 0.5% of water in formulation. In some embodiments, the lyophilized patch includes less than 0. 1% of water in formulation.

[0141] In some embodiments, the patch is configured as a tablet patch. In some embodiments, the tablet patch is a thin solid tablet. Thin solid tablets can be in various wafer- or plate-like shapes such as elliptical, circular, triangular, rectangular, square, pentagonal, hexagonal, irregular, etc. In various embodiments thin solid tablets are substantially flat. In an embodiment, a substantially flat thin solid tablet is slightly curved or bowed to a degree that facilitates handling, e.g., as compared to a flat thin solid tablet that is more difficult to pick up from a flat surface. In various embodiments, a thin solid tablet as described herein has an area density of more than 30 mg / cm2, more than 40 mg / cm2, more than 50 mg / cm2, more than 60 mg / cm2, more than 70 mg / cm2, more than 80 mg / cm2, more than 90 mg / cm2, or more than 100 mg / cm2; less than 400 mg / cm2, less than 350 mg / cm2. less than 300 mg / cm2. less than 250 mg / cm2, or less than 200 mg / cm2; or in any range having endpoints defined by any two of the aforementioned values. For example, in various embodiments, the thin solid tablet has an area density of more than 30 mg / cm2and less than 400 mg / cm2; more than 40 mg / cm2and less than 400 mg / cm2; or more than 30 mg / cm2and less than 400 mg / cm2.

[0142] In various embodiments, a thin solid tablet as described herein has a thickness (depending on the area density and the area of a face) of about 0.01 mm or greater, about 0.02 mm or greater, about 0.03 mm or greater, about 0.04 mm or greater, about 0.05 mm or greater, about 0.05 mm or greater, about 0.1 mm or greater, about 0.2 mm or greater, about 0.5 mm or greater, or about 1 mm or greater; about 10 mm or less, about 5 mm or less; about 2 mm or less; or about 1 mm or less; or in any range having endpoints defined by any two of the aforementioned values. For example, in various embodiments, the thin solid tablet has a thickness in the range of about 0.01 mm to about 10 mm or in the range of about 0. 1 mm to about 5 mm.

[0143] In various embodiments, the thin solid tablet has a face in a manner analogous to the front or back face of a coin. In various embodiments, a face of the thin solid tablet has an area of about 0.01 cm2or greater, about 0.05 cm2or greater, about 0. 1 cm2or greater, about 0.25 cm2or greater, about 0.5 cm2or greater, about 0.75 cm2or greater, or about 1 cm2or greater; or about 50 cm2or less, about 25 cm2or less, about 15 cm2or less, about 10 cm2or less, about 5cm2or less, or about 2 cm2or less, or in any range having endpoints defined by any two of the aforementioned values. For example, in various embodiments, a face of a thin solid tablet has an area in the range of about 0.01 cm2to about 25 cm2, about 0.1 cm2to about 10 cm2, or about 0.15 cm2to about 5 cm2.

[0144] In some embodiments, the patch is configured as a reservoir patch. In some embodiments, the reservoir patch includes a backing, a spacer to make a cavity, a matrix including lipid nanoparticles in a cavity, and a release liner. The matrix can be in various shapes such as elliptical, circular, triangular, rectangular, square, pentagonal, hexagonal, irregular, etc. In an embodiment, the depth of cavity is between about 0.5 mm and 10 mm. In some embodiments, the depth of cavity is between about 0.5 mm and 5 mm. In some embodiments, the depth of cavity is between 1 mm and 3 mm.

[0145] In some embodiments, the patch is configured as a lyophilized dry patch. In some embodiments, the lyophilized dry patch includes a backing, a middle layer comprising a matrix including lyophilized nanoparticles and mRNA, and a release liner. The matrix can be in various shapes such as elliptical, circular, triangular, rectangular, square, pentagonal, hexagonal, irregular, etc. In an embodiment, the matrix is between about 0.5 mm and 10 mm. In some embodiments, the matrix is between about 0.5 mm and 5 mm. In some embodiments, the matrix is between 1 mm and 3 mm.

[0146] In some embodiments, the patch is configured as a tablet patch. In some embodiments, the tablet patch includes a backing, a middle layer comprising a tablet including lipid nanoparticles and mRNA, and a release liner. The matrix can be in various shapes such as elliptical, circular, triangular, rectangular, square, pentagonal, hexagonal, irregular, etc. In an embodiment, the matrix is between about 0.5 mm and 10 mm. In some embodiments, the tablet is between about 0.5 mm and 5 mm. In some embodiments, the tablet is between 1 mm and 3 mm.

[0147] In some embodiments, the patch is configured as a solid dispersed dry patch. In some embodiments, the solid dispersed dry patch includes a backing, a middle layer comprising a matrix including dry state lipid nanoparticles and mRNA, and a release liner. The matrix can be in various shapes such as elliptical, circular, triangular, rectangular, square, pentagonal, hexagonal, irregular, etc. In an embodiment, the matrix is between about 0.5 mm and 10 mm. In some embodiments, the tablet is between about 0.5 mm and 5 mm. In some embodiments, the matrix is between 1 mm and 3 mm.

[0148] In some embodiments, the matrix includes lipid nanoparticles which comprise RNA, wherein the number of positive charges in the lipid nanoparticles does not exceed the number of negative charges in the nanoparticles and / or the nanoparticles have a neutral or netnegative charge and / or the charge ratio of positive charges to negative charges in the nanoparticles is 1.4: 1 of less and / or the zeta potential of the nanoparticles is 0 or less.Method / Uses

[0149] Additional aspect disclosed herein relates to administering to a subject in need an effective amount of lipid nanoparticles as disclosed elsewhere herein. Some embodiments pertain to treating a disease or condition through administration of a patch as disclosed herein. In some embodiments, the patch of the present disclosure may deliver lipid nanoparticles into a subject transdermally through one or more micropores formed by a transdermal delivery system as described herein. In some embodiments, the method may include perforating the outermost stratum comeum and epidermis, and then applying a patch thereon, such that the lipid nanoparticles in the patch pass through the epidermis, deliver to immune cells in skin. In some embodiments, the method may include perforating the outermost stratum comeum and epidermis, and then applying a patch thereon, such that the lipid nanoparticles in the patch pass through the epidermis, diffuse into the capillary dermis, and enter systemic circulation. Thus, embodiments of a patch of the present disclosure may provide drug administration means to replace injections, which can be suitably used for immediate release applications or sustained release of lipid nanoparticles, with a higher immune reactions or bioavailability compared to injections or passive transdermal administration.

[0150] In some embodiments, the patch of the present disclosure can also be used in a method for delivering an RNA (e.g., an mRNA) to a subject through a target biological membrane, wherein the method includes a step for forming one or more micropores on a biological membrane, and a step for placing a patch as described herein so as to be in physical contact with the one or more micropores, such that at least a portion of the lipid nanoparticles is soluble in biological moisture received from the target through the one or more micropores. In some embodiments, the biological membrane is a dermal membrane. In some embodiments, the dermal membrane is a skin that includes the dermal layers and any layers or membranes deeper than the dermis. The dermal layers refer to the connective tissue layer beneath the epidermis, which includes the papillary7and reticular layers. Any additional layers may include those that lie beneath the dermis, such as the subcutaneous tissue and other underlying structures. In some embodiments, a dermal layer includes the epidermis and the dermis. In some embodiments, a dermal layer includes the epidermis, the dermis, and the hypodermis. In some embodiments, a dermal layer is the epidermis.

[0151] In some embodiments, a method of treating, reducing, or ameliorating a subject with an infection includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject’s skin over the micropores for a period of time effective to result intransdermal delivery of the lipid nanoparticles as disclosed elsewhere herein. In some embodiments, the patch includes a top layer including an adhesive, a middle layer including lipid nanoparticles and one or more RNA molecules, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles and one or more RNA molecules through the plurality of micropores. In some embodiments, the RNA is encapsulated in lipid nanoparticles molecule capable of treating the infection or immunotherapy.

[0152] In some embodiments, a method for providing a subject with an immune response in a subject in need thereof includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal delivery of the lipid nanoparticles as disclosed elsewhere herein. In some embodiments, the patch includes a top layer including an adhesive, a middle layer including lipid nanoparticles and one or more RNA molecules, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles and one or more RNA molecules through the plurality7of micropores. In some embodiments, the lipid nanoparticles include one or more RNA molecules (e.g., mRNA) capable of providing an immunotherapeutic response.

[0153] In some embodiments, a method of providing a subj ect with an immunotherapy includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal delivery7of the lipid nanoparticles as disclosed elsewhere herein. In some embodiments, the patch includes a top layer including an adhesive, a middle layer including lipid nanoparticles and one or more RNA molecules, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles and one or more RNA molecules through the plurality of micropores. In some embodiments, the lipid nanoparticles include one or more RNA molecules (e.g., mRNA) capable of providing an immunotherapeutic response.

[0154] In some embodiments, a method of treating, reducing, or ameliorating a subject with a coronavirus infection includes opening a plurality7of micropores in the skin of the subject; and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal delivery7of the lipid nanoparticles. In some embodiments, the patch includes atop layer including an adhesive, a middle layer including lipid nanoparticles, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles throughthe plurality of micropores. In some embodiments, the lipid nanoparticles include one or more mRNA molecules capable of treating the coronavirus infection.

[0155] In some embodiments, a method of treating, reducing, or ameliorating subject with an influenza infection includes opening a plurality of micropores in the skin of the subject; and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal delivery of the lipid nanoparticles. In some embodiments, the patch includes atop layer including an adhesive, a middle layer including lipid nanoparticles, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles through the plurality of micropores. In some embodiments, the lipid nanoparticles include one or more mRNA molecules capable of treating the influenza infection.

[0156] In some embodiments, a method of treating, reducing, preventing, or ameliorating a disease, condition or disorder in a subject includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal delivery of the lipid nanoparticles as disclosed elsewhere herein. In some embodiments, the patch includes a top layer including an adhesive, a middle layer including lipid nanoparticles and one or more RNA molecules, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles and one or more RNA molecules through the plurality of micropores. In some embodiments, the lipid nanoparticles include one or more RNA molecules (e.g., mRNA) capable of treating, reducing, preventing, or ameliorating a disease, condition or disorder in the subject.

[0157] In some embodiments, a method of treating, reducing, or ameliorating a subject with a pathogenic antigen includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal delivery of the lipid nanoparticles as disclosed elsewhere herein. In some embodiments, the lipid nanoparticles include an mRNA, the mRNA including a sequence encoding an antigenic peptide or protein, or a fragment, variant or derivative thereof. In some embodiments, these antigenic peptides or proteins may be derived from pathogenic antigens, tumor antigens, allergenic antigens or autoimmune self-antigens. Such pathogenic antigens are derived from pathogenic organisms, in particular bacterial, viral or protozoological (multicellular) pathogenic organisms, which evoke an immunological reaction by subject, in particular a mammalian subject, more particularly a human. More specifically, pathogenic antigens are preferably surface antigens, e.g.. proteins (or fragments of proteins, e.g. the exterior portion of a surface antigen) located at the surface of the virus or the bacterial or protozoological organism.

[0158] Pathogenic antigens are peptide or protein antigens derived from a pathogen associated with infectious disease which may be selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus. Astroviridae. Babesia genus. Bacillus anthracis. Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp. Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus. Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus. Enterovirus genus. Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus (EBV). Escherichia coh O157:H7, 0111 and O104:H4, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus. Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulation, HIV (Human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumo virus (hMPV), Human papillomavirus (HPV). Human parainfluenza viruses (HP1V). Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis , Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum. Molluscum contagiosum vims (MCV), Mumps vims, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family(Influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus wester mam. Parvovirus Bl 9, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii. Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus. Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus. Sin Nombre virus. Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes. Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borne encephalitis virus (TBEV). Toxocara canis or Toxocara cati. Toxoplasma gondii, Treponema pallidum, Trichinella spiralis. Trichomonas vaginalis. Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (N7N), Varicella zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae. West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus. Yersinia enter ocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0159] In some embodiments, the antigens for the pathogens are selected from Influenza virus, respiratory syncytial virus (RSV), Herpes simplex virus (HSV). human Papilloma virus (HPV), Human immunodeficiency virus (HIV). Plasmodium, Staphylococcus aureus, Dengue virus. Chlamydia trachomatis, Cytomegalovirus (CMV), Hepatitis B virus (HBV), Mycobacterium tuberculosis, Rabies virus, and Yellow Fever Virus.

[0160] In some embodiments, the pathogenic antigen (antigen derived from a pathogen associated with infect ous disease) may be selected from the following antigens: Outer membrane protein A OmpA, biofilm associated protein Bap, transport protein MucK (Acinetobacter baumannii, Acinetobacter infections)); variable surface glycoprotein VSG, microtubule-associated protein MAPP 15, trans-sialidase TSA (Trypanosoma brucei, African sleeping sickness (African trypanosomiasis)); HIV p24 antigen, HIV envelope proteins (Gpl20, Gp41, Gpl60). polyprotein GAG, negative factor protein Nef, trans-activator of transcription Tat (HIV (Human immunodeficiency virus), AIDS (Acquired immunodeficiency syndrome)); galactose-inhibitable adherence protein GIAP, 29 kDa antigen Eh29, Gal / GalNAc lectin, protein CRT. 125 kDa immunodominant antigen, protein M17, adhesin ADH112, protein STIRP (Entamoeba histolytica, Amoebiasis); Major surface proteins 1-5 (MSP la, MSP lb, MSP2, MSP3, MSP4, MSP5), type IV secreotion system proteins (VirB2, VirB7, VirBl l, VirD4) (Anaplasma genus, Anaplasmosis); protective Antigen PA, edema factor EF, lethal facotor LF, the S-lay er homology proteins SLH (Bacillus anthracis. Anthrax); acranolysin, phospholipase D, collagen-binding protein CbpA (Arcanobacterium haemolyticum, Arcanobacteriumhaemolyticum infection); nucleocapsid protein NP, glycoprotein precursor GPC, glycoprotein GP1, glycoprotein GP2 (Junin virus, Argentine hemorrhagic fever); chitin-protein layer proteins, 14 kDa suarface antigen A14, major sperm protein MSP, MSP polymerization-organizing protein MPOP. MSP fiber protein 2 MFP2, MSP polymerization-activating kinase MPAK, ABA- 1 -like protein ALB, protein ABA-1, cuticulin CUT-1 (Ascaris lumbricoides . Ascariasis); 41 kDa allergen Asp v!3, allergen Asp 13, major conidial surface protein rodlet A, protease Peplp, GPI- anchored protein Gel Ip, GPI-anchored protein Crap (Aspergillus genus, Aspergillosis); family VP26 protein, VP29 protein (Astroviridae, Astrovirus infection); Rhoptr -associated protein 1 RAP-1, merozoite surface antigens MSA-1. MSA-2 (al, a2, c). 12D3, 1105, 21134, P29, variant erythrocyte surface antigen VESA1, Apical Membrane Antigen 1 AMA-1 (Babesia genus. Babesiosis); hemolysin, enterotoxin C, PX01-51, glycolate oxidase, ABC-transporter, penicillin- bingdn protein, zinc transporter family protein, pseudouridine synthase Rsu, plasmid replication protein RepX, oligoendopeptidase F, prophage membrane protein, protein HemK, flagellar antigen H, 28.5-kDa cell surface antigen (Bacillus cereus, Bacillus cereus infection); large T antigen LT, small T antigen, capsid protein VP1, capsid protein VP2 (BK virus, BK virus infection); 29 kDa-protein, caspase-3 -like antigens, glycoproteins (Blastocystis hominis, Blastocystis hominis infection); yeast surface adhesin WI-1 (Blastomyces dermatitidis, Blastomycosis); nucleoprotein N. polymerase L. matrix protein Z, glycoprotein GP (Machupo virus, Bolivian hemorrhagic fever); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin binding protein A DbpA, decorin binding protein B DbpB, flagellar filament 41 kDa core protein Fla, basic membrane protein A precursor BmpA (Immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE (Borrelia genus, Borrelia infection); Botulinum neurotoxins BoNT / Al, BoNT / A2, BoNT / A3, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, recombinant botulinum toxin F He domain FHc (Clostridium botulinum, Botulism (and Infant botulism)); nucleocapsid, glycoprotein precursor (Sabia virus, Brazilian hemorrhagic fever); copper / Zinc superoxide dismutase SodC, bacterioferritin Bfr, 50S ribosomal protein RplL, OmpA-like transmembrane domain-containing protein 0mp31, immunogenic 39-kDa protein M5 P39, zinc ABC transporter periplasmic zinc-bnding protein znuA, periplasmic immunogenic protein Bp26, 30S ribosomal protein S12 RpsL, glyceraldehyde-3-phosphate dehydrogenase Gap, 25 kDa outer-membrane immunogenic protein precursor Omp25. invasion protein B lalB, trigger factor Tig, molecular chaperone DnaK, putative peptidyl-prolyl cis-trans isomerase SurA, lipoprotein 0mpl9, outer membrane protein MotY 0mpl6, conserved outer membrane protein DI 5, malate dehydrogenase Mdh, component of the Type-IV secretion system (TOSS) VirJ, lipoprotein of unknown function BAB 1 0187 (Brucella genus, Brucellosis); members of the ABCtransporter family (LoIC, OppA, and PotF), putative lipoprotein releasing system transmembrane protein LoIC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial Elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA coding protein, boaB coding protein (Burkholderia cepacia and other Burkholderia species, Burkholderia infection); mycolyl- transferase Ag85A, heat-shock protein Hsp65, protein TB10.4, 19 kDa antigen, protein PstS3, heat-shock protein Hsp70 (Mycobacterium ulcerans, Buruli ulcer); norovirus major and minor viral capsid proteins VP1 and VP2, genome polyprotein, Sapoviurus capsid protein VP1, protein Vp3, geome polyprotein (Caliciviridae family, Calicivirus infection (Norovirus and Sapovirus)); major outer membrane protein PorA, flagellin FlaA. surface antigen CjaA, fibronectin binding protein CadF, aspartate / glutamate-binding ABC transporter protein PeblA, protein FspAL protein FspA2 (Campylobacter genus, Campylobacteriosis); glycolytic enzyme enolase, secreted aspartyl proteinases SAP1-10, glycophosphatidylinositol (GPI)-linked cell wall protein, protein Hyrl, complement receptor 3-related protein CR3-RP, adhesin Als3p, heat shock protein 90 kDa hsp90, cell surface hydrophobicity protein CSH (usually Candida albicans and other Candida species, Candidiasis); 17-kDa antigen, protein P26, trimeric autotransporter adhesins TAAs, Bartonella adhesin A BadA, variably expressed outer-membrane proteins Vomps, protein Pap3, protein HbpA, envelope-associated protease HtrA, protein OMP89, protein GroEL, protein LalB, protein OMP43, dihydrolipoamide succinyltransferase SucB (Bartonella henselae, Catscratch disease); amastigote surface protein-2, amastigote-specific surface protein SSP4, cruzipain, trans-sialidase TS, try pomas tigote surface glycoprotein TSA-1, complement regulatory protein CRP-10, protein G4, protein G2, paraxonemal rod protein PAR2, paraflagellar rod component Part, mucin- Associated Surface Proteins MPSP (Trypanosoma cruzi. Chagas Disease (American trypanosomiasis)); envelope glycoproteins (gB, gC, gE, gH, gl, gK, gL) (Varicella zoster virus (VZV), Chickenpox); major outer membrane protein MOMP, probable outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock proteins Hsp60 HSP10, protein IncA, proteins from the type III secretion system, ribonucleotide reductase small chain protein NrdB. plasmid protein Pgp3. chlamydial outer protein N CopN, antigen CT521, antigen CT425, antigen CT043, antigen TC0052, antigen TC0189, antigen TC0582, antigen TC0660, antigen TC0726, antigen TC0816, antigen TC0828 (Chlamydia trachomatis. Chlamydia),' low calcium response protein E LCrE, chlamydial outer protein N CopN, serine / threonine-protein kinase PknD, acyl-carrier-protein S-malonyltransferase FabD, singlestranded DNA-binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 0mp2, polymorphic membrane protein family (Pmpl, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8. Pmp9, PmplO, Pmpl l, Pmpl2, Pmpl3, Pmpl4, Pmpl5, Pmpl6, Pmpl7, Pmpl8, Pmpl9. Pmp20, Pmp21) (Chlamydophila pneumoniae, Chlamydophilapneumoniae infection); cholera toxin B CTB, toxin coregulated pilin A TcpA, toxin coregulated pilin TcpF, toxin co-regulated pilus biosynthesis ptrotein F TcpF, cholera enterotoxin subunit A, cholera enterotoxin subunit B, Heat-stable enterotoxin ST, mannose-sensitive hemagglutinin MSHA, outer membrane protein U Porin ompU, Poring B protein, polymorphic membrane protein-D (Vibrio cholerae, Cholera); propionyl-CoA carboxylase PCC. 14-3-3 protein, prohibitin, cysteine proteases, glutathione transferases, gelsolin, cathepsin L proteinase CatL, Tegumental Protein 20.8 kDaTP20.8, tegumental protein 31.8 kDa TP31.8, lysophosphatidic acid phosphatase LPAP, (Clonorchis sinensis, Clonorchiasis); surface layer proteins SLPs, glutamate dehydrogenase antigen GDH. toxin A, toxin B, cysteine protease Cwp84, cysteine protease Cwpl3, cysteine protease Cwpl9, Cell Wall Protein CwpV, flagellar protein FliC, flagellar protein FliD (Clostridium difficile, Clostridium difficile infection); rhinoviruses: capsid proteins VP1, VP2, VP3, VP4; coronaviruses: sprike proteins S, envelope proteins E, membrane proteins M, nucleocapsid proteins N (usually rhinoviruses and coronaviruses. Common cold (Acute viral rhinopharyngitis; Acute coryza)); prion protein Prp (CJD prion, Creutzfeldt-Jakob disease (CJD)); envelope protein Gc, envelope protein Gn, nucleocapsid proteins (Crimean-Congo hemorrhagic fever virus, Crimean-Congo hemorrhagic fever (CCHF)); virulence-associated DEAD-box RNA helicase VAD1, galactoxylomannan-protein GalXM, glucuronoxylomannan GXM, mannoprotein MP (Cryptococcus neoformans, Cryptococcosis); acidic ribosomal protein P2 CpP2, mucin antigens Mucl, Muc2, Muc3 Muc4, Muc5, Muc6, Muc7, surface adherence protein CP20, surface adherence protein CP23, surface protein CP 12, surface protein CP21, surface protein CP40, surface protein CP60, surface protein CP 15, surface-associated glycopeptides gp40, surface- associated glycopeptides gpl5, oocyst wall protein AB, profilin PRF. apyrase (Cryptosporidium genus. Cryptosporidiosis); fatty acid and retinol binding protein-1 FAR-1, tissue inhibitor of metalloproteinase TIMP (TMP), cysteine proteinase ACEY-1, cysteine proteinase ACCP-1, surface antigen Ac-16, secreted protein 2 ASP-2, metalloprotease 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, adult-specific secreted factor Xa serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1 (usually Ancylostoma braziliense,' multiple other parasites. Cutaneous larva migrans (CLM)); cathepsin L-like proteases, 53 / 25-kDa antigen, 8 kDa family members, cysticercus protein with a marginal trypsin-like activity TsAg5, oncosphere protein TSOL18, oncosphere protein TSOL45- 1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia solium, Cysticercosis); pp65 antigen, membrane protein ppi 5, capsid-proximal tegument protein ppi 50, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glcoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99 (Cytomegalovirus (CMV), Cytomegalovirus infection); capsid protein C, premembrane protein prM, membraneprotein M, envelope protein E (domain I, domain II, domain II), protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A, protein 2K, protein NS4B, protein NS5 (Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4)-Flaviviruses, Dengue fever); 39 kDa protein (Dientamoeba fragilis, Dientamoebiasis); diphtheria toxin precursor Tox, diphteria toxin DT, pilin-specific sortase SrtA, shaft pilin protein SpaA, tip pilin protein SpaC. minor pilin protein SpaB, surface-associated protein DIP 1281 (Corynebacterium diphtheriae, Diphtheria); glycoprotein GP, nucleoprotein NP, minor matrix protein VP24, major matrix protein VP40, transcription activator VP30. polymerase cofactor VP35, RNA polymerase L (Ebolavirus (EBOV). Ebola hemorrhagic fever); prion protein (vCJD prion, Variant Creutzfeldt-Jakob disease (vCJD, nvCJD)); UvrABC system protein B, protein Flpl, protein Flp2, protein Flp3, protein TadA, hemoglobin receptor HgbA, outer membrane protein TdhA, protein CpsRA, regulator CpxR, protein SapA, 18 kDa antigen, outer membrane protein NcaA, protein LspA, protein LspAl. protein LspA2, protein LspB, outer membrane component DsrA, lectin DItA, lipoprotein Hip, major outer membrane protein OMP, outer membrane protein 0mpA2 (Haemophilus ducreyi, Chancroid); aspartyl protease 1 Pepl, phospholipase B PLB, alpha-mannosidase 1 AMN1, glucanosyltransferase GEL1, urease URE, peroxisomal matrix protein Pmpl, proline-rich antigen Pra, humal T-cell reative protein TcrP (Coccidioides immitis and Coccidioides posadasii, Coccidioidomycosis); allergen Tri r 2. heat shock protein 60 Hsp60, fungal actin Act, antigen Tri r2, antigen Tri r4, antigen Tri tl, protein IV, glycerol-3 -phosphate dehydrogenase Gpdl, osmosensor HwSholA, osmosensor HwSholB, histidine kinase HwHhk7B, allergen Mala s 1, allergen Mala s 11, thioredoxin Trx Mala s 13, allergen Mala f, allergen Mala s (usually Trichophyton spp, Epidermophyton spp., Maias sezia spp., Hortaea werneckii,Dermatophytosis); protein EG95, protein EG10, protein EG18, protein EgA31, protein EM18, antigen EPCI, antigen B, antigen 5, protein P29, protein 14-3-3, 8-kDa protein, myophilin, heat shock protein 20 HSP20, glycoprotein GP-89, fatty acid binding protein FAPB (Echinococcus genus, Echinococcosis); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2. outer membrane protein OMP. outer membrande protein 19 OMP- 19, major antigenic protein MAPI, major antigenic protein MAP 1-2, major antigenic protein MAP1B, major antigenic protein MAPI-3, Erum2510 coding protein, protein GroEL, protein GroES, 30-kDA major outer membrane proteins. GE 100-kDa protein, GE 130- kDa protein, GE 160-kDa protein (Ehrlichia genus, Ehrlichiosis); secreted antigen SagA, sagA- like proteins Sal A and SalB, collagen adhesin Scm, surface proteins Fmsl (EbpA(fm), Fms5 (EbpB(fm), Fms9 (EpbC(fm) and FmslO, protein EbpC(fm), 96 kDa immunoprotective glycoprotein G1 (Enterococcus genus, Enterococcus infection); genome polyprotein, polymerase 3D, viral capsid protein VP1. viral capsid protein VP2, viral capsid protein VP3, viral capsidprotein VP4, protease 2A. protease 3C (Enterovirus genus. Enterovirus infection); outer membrane proteins OM, 60 kDa outer membrane protein, cell surface antigen OmpA, cell surface antigen OmpB (sca5), 134 kDa outer membrane protein, 31 kDa outer membrane protein, 29.5 kDa outer membrane protein, cell surface protein SCA4, cell surface protein Adri (RP827), cell surface protein Adr2 (RP828), cell surface protein SCA1. Invasion protein invA, cell division protein fts, secretion proteins sec Ofamily, virulence proteins virB, tlyA, tlyC, parvulm-like protein Pip, preprotein translocase SecA, 120-kDa surface protein antigen SPA, 138 kD complex antigen, major 100-kD protein (protein I), intracytoplasmic protein D, protective surface protein antigen SPA (Rickettsia prowazekii. Epidemic typhus); Epstein-Barr nuclear antigens (EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP)), latent membrane proteins (LMP-1, LMP-2A, LMP-2B), early antigen EBV-EA, membrane antigen EBV-MA, viral capsid antigen EBV-VCA, alkaline nuclease EBV-AN, glycoprotein glycoprotein gp350, glycoprotein gpl lO, glycoprotein gp42, glycoprotein gHgL. glycoprotein gB (Epstein- Barr Virus (EBV), Epstein-Barr Virus Infectious Mononucleosis); cpasid protein VP2, capsid protein VP1, major protein NS1 (Parvovirus B19, Erythema infectiosum (Fifth disease)); pp65 antigen, glycoprotein 105, major capsid protein, envelope glycoprotein H, protein U51 (Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Exanthem subitum); thioredoxin- glutathione reductase TGR, cathepsins LI and L2. Kunitz-type protein KTM, leucine aminopeptidase LAP, cysteine proteinase Fast, saposin-like protein-2 SAP-2, thioredoxin peroxidases TPx, Prx-1, Prx-2, cathepsin I cysteine proteinase CL3, protease cathepsin L CL1, phosphoglycerate kinase PGK, 27-kDa secretory protein, 60 kDa protein HSP35alpha, glutathione transferase GST, 28.5 kDa tegumental antigen 28.5 kDa TA, cathepsin B3 protease CatB3, Type I cystatin stefin-1, cathepsin L5, cathepsin Llg and cathepsin B, fatty acid binding protein FABP, leucine aminopeptidases LAP (Fasciola hepatica and Fasciola giganiica. Fasciolosis); prion protein (FFI prion, Fatal familial insomnia (FFI)); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, vespid allergen homologue VAH, thiordoxin peroxidase 2 TPX-2. antigenic protein SXP (peptides N, Nl, N2, and N3), activation associated protein-1 ASP-L Thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferases GST, myosin, vespid allergen homologue VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen Col-4, secreted larval acidic proteins SLAPs. chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose-l,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode specific gene product OvB20, onchocystatin CPI-2, Cox-2 (Filarioidea superfamily, Filariasis); phospholipase C PLC, heat-labile enterotoxin B, Iota toxin component lb, protein CPE1281 pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, perfringolysin 0 Pfo. glyceraldehyde-3 -phosphatedehydrogenase GapC, Fructose-bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha toxoid ATd, epsilon-toxoid ETd, protein HP, large cytotoxin TpeL, endo-beta-N-acetylglucosaminidase Naglu, phosphoglyceromutase Pgm (Clostridium perfringens, Food poisoning by Clostridium perfringens) leukotoxin IktA, adhesion FadA, outer membrane protein RadD, high-molecular weight arginine-binding protein (Fusobacterium genus, Fusobacterium infection); phospholipase C PLC, heat-labile enterotoxin B, Iota toxin component lb, protein CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, perfringolysin 0 Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, fructose- bisphosphate aldolase Alf2. Clostridium perfringens enterotoxin CPE, alpha toxin AT. alpha toxoid ATd, epsilon-toxoid ETd, protein HP, large cytotoxin TpeL, endo-beta-N- acetylglucosaminidase Naglu, phosphoglyceromutase Pgm (usually Clostridium perfringens,' other Clostridium species, Gas gangrene (Clostridial myonecrosis)); lipase A, lipase B, peroxidase Decl (Geotrichum candidum, Geotrichosis); prion protein (GSS prion, Gerstmann- Straussler-Scheinker syndrome (GSS)); cyst wall proteins CWP1, CWP2, CWP3, variant surface protein VSP, VSP1, VSP2, VSP3, VSP4, VSP5, VSP6, 56 kDa antigen, pyruvate ferredoxin oxidoreductase PFOR, alcohol dehydrogenase E ADHE, alpha-giardin, alpha8-giardin, alphal- guiardin, beta-giardin, cystein proteases. glutathione-S-transferase GST, arginine deiminase ADI, fructose- 1,6-bisphosphat aldolase FBA, Giardia trophozoite antigens GTA (GTA1, GTA2), ornithine carboxyl transferase OCT, striated fiber-asseblin-like protein SALP, uridine phosphoryl- like protein UPL, alpha-tubulin, beta-tubulin (Giardia intestinalis , Giardiasis); members of the ABC transporter family (LoIC, OppA. and PotF), putative lipoprotein releasing system transmembrane protein LoIC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial Elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA coding protein (Burkholderia mallei, Glanders); cyclophilin CyP, 24 kDa third-stage larvae protien GS24, excretion-secretion products ESPs (40, 80, 120 and 208 kDa) (Gnathostoma spinigerum and Gnathostoma hispidum, Gnathostomiasis); pilin proteins, minor pilin-associated subunit pilC. major pilin subunit and variants pilE, pilS. phase variation protein porA. Porin B PorB, protein TraD, Neisserial outer membrane antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin binding protein TbpA, transferrin binding protein TbpB PBP2, mtrR coding protein, ponA coding protein, membrane permease FbpBC, FbpABC protein system. LbpAB proteins, outer membrane protein Opa, outer membrane transporter FetA, iron-repressed regulator MpeR (Neisseria gonorrhoeae, Gonorrhea); outer membrane protein A OmpA, outer membrane protein C OmpC, outer membrane protein KI 7 0mpK17 (Klebsiella granulomatis. Granuloma inguinale (Donovanosis)); fibronectin-binding protein Sfb, fibronectin / fibrinogen-binding protein FBP54,fibronectin-binding protein FbaA, M protein type 1 Emml, M protein type 6 Emm6, immunoglobulin-binding protein 35 Sib35, Surface protein R28 Spr28, superoxide dismutase SOD, C5a peptidase ScpA, antigen I / II Agl / II, adhesin AspA, G-related alpha2-macroglobulin- binding protein GRAB, surface fibrillar protein M5 (Streptococcus pyogenes, Group A streptococcal infection); C protein P antigen, arginine deiminase proteins, adhesin BibA, 105 kDA protein BPS, surface antigens c, surface antigens R, surface antigens X, trypsin-resistant protein Rl, trypsin-resistant protein R3, trypsin-resistant protein R4, surface immunogenic protein Sip, surface protein Rib, Leucine-rich repeats protein LrrG, serine-rich repeat protein Srr-2, C protein alpha-antigen Bea, Beta antigen Bag, surface antigen Epsilon, alpha-like protein ALP1. alpha-like protein ALP5 surface antigen delta, alpha-like protein ALP2, alpha-like protein ALP3, alpha-like protein ALP4, Cbeta protein Bac (Streptococcus agalactiae, Group B streptococcal infection); transferrin-binding protein 2 Tbp2, phosphatase P4, outer membrane protein P6, peptidoglycan- associated lipoprotein Pal, protein D. protein E, adherence and penetration protein Hap, outer membrane protein 26 Omp26, outer membrane protein P5 (Fimbrin), outer membrane protein D15, outer membrane protein OmpP2, 5'-nucleotidase NucA, outer membrane protein Pl, outer membrane protein P2, outer membrane lipoprotein Pep, Lipoprotein E, outer membrane protein P4, fuculokinase FucK, [Cu,Zn] -superoxide dismutase SodC, protease HtrA, protein 0145, alphagalactosylceramide (Haemophilus influenzae, Haemophilus influenzae infection); polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Hand, foot and mouth disease (HFMD)); RNA polymerase L. protein L, glycoprotein Gn, glycoprotein Gc. nucleocapsid protein 5. envelope glycoprotein Gl, nucleoprotein NP, protein N, polyprotein M (Sin Nombre virus, Hantavirus, Hantavirus Pulmonary Syndrome (HPS)); heat shock protein HspA, heat shock protein HspB, citrate synthase GItA, protein UreB, heat shock protein Hsp60, neutrophil-activating protein NAP, catalase KatA, vacuolating cytotoxin Vac A, urease alpha UreA, urease beta Ureb, protein CpnlO, protein groES, heat shock protein HsplO, protein MopB. cytotoxicity -associated 10 kDa protein CAG. 36 kDa antigen, beta-lactamase HcpA, Beta-lactamase HcpB (Helicobacter pylori, Helicobacter pylori infection); integral membrane proteins, aggregation-prone proteins, 0-antigen, toxin-antigens Stx2B, toxin-antigen StxlB, adhesion-antigen fragment Int28, protein EspA, protein EspB, Intimin, protein Tir, protein IntC300, protein Eae (Escherichia co / / O157:H7. 0111 and 0104:H4. Hemolytic-uremic syndrome (HUS)); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein 5, envelope glycoprotein Gl, nucleoprotein NP, protein N, polyprotein M (Bunyaviridae family, Hemorrhagic fever with renal syndrome (HFRS)); glycoprotein G, matrix protein M, nucleoprotein N. fusion protein F, polymerase L, protein W, proteinC, phosphoprotein p, non-structural protein V (Henipavirus (Hendra virus Nipah virus), Henipavirus infections); polyprotein, glycoproten Gp2, hepatitis A surface antigen HBAg, protein 2 A, virus protein VP1, virus protein VP2, virus protein VP3, virus protein VP4, protein P1B, protein P2A, protein P3AB, protein P3D (Hepatitis A Virus, Hepatitis A); hepatitis B surface antigen HBsAg, Hepatitis B core antigen HbcAg, polymerase, protein Hbx. preS2 middle surface protein, surface protein L, large S protein, virus protein VP1, virus protein VP2, virus protein VP3, virus protein VP4 (Hepatitis B Vims (HBV), Hepatitis B); envelope glycoprotein El gp32 gp35 envelope glycoprotein E2 NS1 gp68 gp70, capsid protein C core protein Core, polyprotein, virus protein VP1, virus protein VP2, virus protein VP3, vims protein VP4, antigen G, protein NS3, protein NSSA, (Hepatitis C Vims, Hepatitis C); virus protein VP1, virus protein VP2, virus protein VP3, vims protein VP4, large hepaptitis delta antigen, small hepaptitis delta antigen (Hepatitis D Virus, Hepatitis D); virus protein VP1, vims protein VP2, vims protein VP3, virus protein VP4, capsid protein E2 (Hepatitis E Vims, Hepatitis E); glycoprotein L ULI, uracil-DNA glycosylase UL2. protein UL3, protein UL4, DNA replication protein UL5, portal protein UL6, virion maturation protein UL7, DNA helicase UL8, replication origin-binding protein UL9, glycoprotein M UL10, protein UL11, alkaline exonuclease UL12, serine-threonine protein kinase UL13, tegument protein UL14, terminase ULI 5, tegument protein ULI 6, protein ULI 7. capsid protein VP23 ULI 8, major capsid protein VP5 ULI 9, membrane protein UL20. tegument protein UL21. Glycoprotein H (UL22), Thymidine Kinase UL23, protein UL24, protein UL25, capsid protein P40 (UL26, VP24, VP22A), glycoprotein B (UL27), ICP18.5 protein (UL28), maj or DNA-binding protein ICP8 (UL29), DNA polymerase UL30, nuclear matrix protein UL31, envelope glycoprotein UL32, protein UL33, inner nuclear membrane protein UL34, capsid protein VP26 (UL35), large tegument protein UL36, capsid assembly protein UL37, VP19C protein (UL38), ribonucleotide reductase (Large subunit) UL39, ribonucleotide reductase (Small subunit) UL40, tegument protein / virion host shutoff VHS protein (UL41), DNA polymerase processivity factor UL42, membrane protein UL43, glycoprotein C (UL44), membrane protein UL45, tegument proteins VP11 / 12 (UL46), tegument protein VP 13 / 14 (UL47), virion maturation protein VP 16 (UL48. Alpha-TIF), envelope protein UL49, dUTP diphosphatase UL50, tegument protein UL51, DNA helicase / primase complex protein UL52, glycoprotein K (UL53), transcriptional regulation protein 1E63 (ICP27, UL54), protein UL55, protein UL56, viral replication protein ICP22 (1E68, US1), protein U52, serine / threonine-protein kinase U53. glycoprotein G (U54). glycoprotein J (U55), glycoprotein D (U56), glycoprotein I (U57), glycoprotein E (U58), tegument protein U59, capsid / tegument protein US10, Vmw21 protein (US11), ICP47 protein (IE12, US12), major transcriptional activator ICP4 (1E175, RSI), E3 ubiquitin ligase ICPO (IE110), latency-related protein 1 LRP1, latency -related protein 2 LRP2. neurovirulence factor RL1 (ICP34.5). latency-associatedtranscript LAT (Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Herpes simplex); heat shock protein Hsp60, cell surface protein H1C, dipeptidyl peptidase type IV DppIV, M antigen, 70 kDa protein, 17 kDa histone-like protein (Histoplasma capsulation, Histoplasmosis); fatty' acid and retinol binding protein- 1 FAR-1, tissue inhibitor of metalloproteinase TIMP (TMP), cysteine proteinase ACEY-1, cysteine proteinase ACCP-1. surface antigen Ac- 16. secreted protein 2 ASP- 2, metalloprotease 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, surface-associated antigen SAA-2, adult-specific secreted factor Xa, serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1, 5-transferase GST, aspartic protease APR-1, acetylcholinesterase AChE (Ancylostoma duodena le wiA Necator americanus, Hookyvorm infection); protein NS1, protein NP1, protein VP1, protein VP2, protein VP3 (Human bocavirus (HBoV), Human bocavirus infection); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrande protein 19 OMP- 19, major antigenic protein MAPI, major antigenic protein MAP1-2, major antigenic protein MAP1B, major antigenic protein MAPI -3, Erum2510 coding protein, protein GroEL, protein GroES, 30-kDA major outer membrane proteins, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa protein (Ehrlichia ewingii, Human ewingii ehrlichiosis); major surface proteins 1-5 (MSPla, MSPlb, MSP2. MSP3, MSP4, MSP5), type IV secreotion system proteins VirB2, VirB7. VirBl l, VirD4 (Anaplasma phagocy tophilum, Human granulocytic anaplasmosis (HGA)); protein NS1, small hydrophobic protein N52, SH protein, fusion protein F, glycoprotein G, matrix protein M, matrix protein M2-1, matrix protein M2-2, phosphoprotein P, nucleoprotein N, polymerase L (Human metapneumovirus (hMPV), Human metapneumovirus infection); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrande protein 19 OMP-19, major antigenic protein MAP 1 , maj or antigenic protein MAP 1-2, maj or antigenic protein MAP 1 B, maj or antigenic protein MAPI-3, Erum2510 coding protein, protein GroEL, protein GroES, 30-kDA major outer membrane proteins, GE 100-kDa protein. GE 130-kDa protein, GE 160-kDa protein (Ehrlichia chaffeensis. Human monocytic ehrlichiosis); replication protein EE. regulatory protein E2, protein E3, protein E4, protein ES, protein E6, protein E7, protein E8, major capsid protein LI, minor capsid protein L2 (Human papillomavirus (HPV), Human papillomavirus (HPV) infection); fusion protein F, hemagglutinin-neuramidase HN. glycoprotein G, matrix protein M, phosphoprotein P, nucleoprotein N, polymerase L (Human parainfluenza viruses (HP IV), Human parainfluenza virus infection); Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), Ml protein, M2 protein, NS1 protein, NS2 protein (NEP protein: nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein and PB2 protein (Orthomyxoviridae family, Influenza virus (flu)); genome polyprotein, protein E. protein M,capsid protein C (Japanese encephalitis virus, Japanese encephalitis); RTX toxin, type IV pili, major pilus subunit PilA, regulatory transcription factors PilS and PilR, protein sigma54, outer membrane proteins (Kingella kingae, Kingella kingae infection); prion protein (Kuru prion, Kuru); nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP (Lassa virus, Lassa fever); peptidoglycan-associated lipoprotein PAL. 60 kDa chaperonin Cpn60 (groEL. HspB). type IV pilin PilE, outer membrane protein MIP, major outer membrane protein MompS, zinc metalloproteinase MSP (Legionella pneumophila, Legionellosis (Legionnaires' disease, Pontiac fever)); P4 nuclease, protein WD, ribonucleotide reductase M2, surface membrane glycoprotein Pg46, cysteine proteinase CP, glucose-regulated protein 78 GRP-78, stage-specific S antigen-like protein A2, ATPase Fl, beta-tubulin, heat shock protein 70 Hsp70, KMP-11, glycoprotein GP63, protein BT1, nucleoside hydrolase NH, cell surface protein Bl, ribosomal protein Pl-like protein Pl, sterol 24-c-methyltransferase SMT, LACK protein, histone Hl, SPB1 protein, thiol specific antioxidant TSA, protein antigen STH, signal peptidase SP, histone H2B, suface antigen PSA-2, cy stein proteinase b Cpb (Leishmania genus, Leishmaniasis); major membrane protein I, serine- rich antigen-45 kDa, 10 kDa caperonin GroES, HSP kDa antigen, amino-oxononanoate synthase AONS, protein recombinase A RecA, AcetyL / propionyl-coenzyme A carboxylase alpha, alanine racemase, 60 kDa chaperonin 2, ESAT-6-like protein EcxB (L-ESAT-6), protein Lsr2, protein ML0276. Heparin-binding hemagglutinin HBHA, heat-shock protein 65 Hsp65, mycPl or ML0041 coding protein htrA2 or ML0176 coding protein htrA4 or ML2659 coding protein, gcp or ML0379 coding protein, clpC or ML0235 coding protein (Mycobacterium leprae and Mycobacterium lepromatosis, Leprosy); outer membrane protein LipL32, membrane protein LIC10258. membrane protein LP30, membrane protein LIC12238. Ompa-like protein Lsa66, surface protein LigA, surface protein LigB, major outer membrane protein OmpLl, outer membrane protein LipL41, protein LigAni, surface protein LcpA, adhesion protein LipL53, outer membrane protein UpL32, surface protein Lsa63, flagellin FlaBl, membran lipoprotein LipL21, membrane protein pL40, leptospiral surface adhesin Lsa27, outer membrane protein OmpL36, outer membrane protein OmpL37, outer membrane protein OmpL47. outer membrane protein OmpL54, acyltransferase LpxA (Leptospira genus, Leptospirosis); listeriolysin 0 precursor Hly (LLO), invasion-associated protein lap (P60), Listeriolysin regulatory protein PrfA, Zinc metalloproteinase Mpl, Phosphatidylinositol-specific phospholipase C PLC (PIcA, PlcB), 0- acetyltransferase Oat, ABC-transporter permease Im.G 1771. adhesion protein LAP, LAP receptor Hsp60, adhesin LapB, haemolysin listeriolysin 0 LLO, protein ActA, Intemalin A InIA, protein InIB (Listeria monocytogenes. Listeriosis); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin binding protein A DbpA, decorin binding protein B DbpB. flagellar filament 41 kDa core protein Fla. basic membrane protein A BmpA(Immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE (usually Borrelia burgdorferi and other Borrelict species, Lyme disease (Lyme borreliosis)); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, vespid allergen homologue VAH, thiordoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N, NL N2, and N3), activation associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferases GST, myosin, vespid allergen homologue VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen Col-4, Secreted Larval Acidic Proteins SLAPs, chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose- 1,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode specific gene product OvB20, onchocystatin CPL2, protein Cox-2 (Wuchereria bcmcrofti and Brugia malayi, Lymphatic filariasis (Elephantiasis)); glycoprotein GP, matrix protein polymerase L, nucleoprotein N (Lymphocytic choriomeningitis virus (LCMV), Lymphocytic choriomeningitis); thrombospondin-related anonymous protein TRAP, SSP2 Sporozoite surface protein 2, apical membrane antigen 1 AMA1, rhoplry membrane antigen RMA1, acidic basic repeat antigen ABRA, cell-traversal protein PF, protein Pvs25, merozoite surface protein 1 MSP-1, merozoite surface protein 2 MSP-2, ring-infected erythrocyte surface antigen RESALiver stage antigen 3 LSA-3. protein Eba-175. serine repeat antigen 5 SERA-5, circumsporozoite protein CS. merozoite surface protein 3 MSP3, merozoite surface protein 8 MSP5, enolase PF10, hepatocyte erythrocyte protein 17 kDa HEP 17, erythrocyte membrane protein 1 EMP1, protein Kbetamerozoite surface protein 4 / 5 MSP 4 / 5, heat shock protein Hsp90, glutamate-rich protein GLURP, merozoite surface protein 4 MSP-4, protein STARP, circumsporozoite protein-related antigen precursor CRA (Plasmodium genus. Malaria); nucleoprotein N, membrane-associated protein VP24, minor nucleoprotein VP30, polymerase cofactor VP35, polymerase L, matrix protein VP40, envelope glycoprotein GP (Marburg virus, Marburg hemorrhagic fever (MHF)); protein C, matrix protein M, phosphoprotein P, non-structural protein V, hemagglutinin glycoprotein H, polymerase L, nucleoprotein N. fusion protein F (Measles virus. Measles); members of the ABC transporter family (LoIC, OppA, and PotF), putative lipoprotein releasing system transmembrane protein LoIC / E, flagellin FliC, Burkholderia intracellular motility' A BimA, bacterial Elongation factor- Tu EF-Tu, 17 kDa OmpA-like protein, boaA coding protein, boaB coding protein (Burkholderia pseudomallei, Melioidosis (Whitmore's disease)); pilin proteins, minor pilin-associated subunit pilC, major pilin subunit and variants pilE, pilS, phase variation protein porA, Porin B PorB, protein TraD, Neisserial outer membrane antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin binding protein TbpA, transferrin binding protein TbpB PBP2, mtrR coding protein, ponA codingprotein, membrane permease FbpBC, FbpABC protein system, LbpAB proteins, outer membrane protein Opa, outer membrane transporter FetA, iron-repressed regulator MpeR, factor H-binding protein fHbp, adhesin NadA, protein NhbA, repressor FarR (Neisseria meningitidis, Meningococcal disease); 66 kDa protein, 22 kDa protein (usually Metagonimus yokagawai, Metagonimiasis); polar tube proteins (34, 75, and 170 kDa in Glugea, 35, 55 and 150 kDa in Encephalitozoon), kinesin-related protein, RNA polymerase II largest subunit, similar of integral membrane protein YIP A, anti-silencing protein 1, heat shock transcription factor HSF, protein kinase, thymidine kinase, NOP-2 like nucleolar protein (Microsporidia phylum, Microsporidiosis); CASP8 and FADD-like apoptosis regulator, Glutathione peroxidase GPX1, RNA helicase NPH-II NPH2, Poly(A) polymerase catalytic subunit PAPL, Major envelope protein P43K, early transcription factor 70 kDa subunit VETFS, early transcription factor 82 kDa subunit VETFL, metalloendopeptidase Gl-type, nucleoside triphosphatase I NPH1, replication protein A28-like MC134L, RNA poly mease 7 kDa subunit RPO7 (Molluscum contagiosum virus (MCV), Molluscum contagiosum (MC)); matrix protein M, phosphoprotein P / V, small hydrophobic protein SH, nucleoprotein N, protein V, fusion glycoprotein hemagglutininneuraminidase HN, RNA polymerase L (Mumps virus, Mumps); Outer membrane proteins OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, intracytoplasmic protein D, crystalline surface layer protein SLP. protective surface protein antigen SPA (Rickettsia typhi, Murine typhus (Endemic typhus)); adhesin Pl, adhesion P30, protein pl 16, protein P40, cytoskeletal protein HMW1, cytoskeletal protein HMW2, cytoskeletal protein HMW3, MPN152 coding protein, MPN426 coding protein, MPN456 coding protein, MPN-500coding protein (Mycoplasma pneumoniae, Mycoplasma pneumonia); NocA, Iron dependent regulatory protein, VapA, VapD, VapF, VapG, caseinolytic protease, filament tip-associated 43-kDa protein, protein P24, protein P61, 15-kDa protein, 56-kDa protein (usually Nocardia asteroides and other Nocardia species, Nocardiosis); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT- 2, thioredoxin peroxidase TPX, vespid allergen homologue VAH, thiordoxin peroxidase 2 TPX- 2, antigenic protein SXP (peptides N, Nl, N2, and N3), activation associated protein-1 ASP-1, Thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferases GST, myosin, vespid allergen homologue VAH, 175 kDa collagenase, glyceraldehyde-3 -phosphate dehydrogenase GAPDH. cuticular collagen Col-4, Secreted Larval Acidic Proteins SLAPs. chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose-l,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode specific gene product OvB20, onchocy statin CPI-2, Cox-2 (Onchocerca volvulus, Onchocerciasis (River blindness)); 43 kDa secreted glycoprotein, glycoprotein gpO, glycoprotein gp75, antigen Pb27, antigen Pb40, heat shock protein Hsp65, heatshock protein Hsp70, heat shock protein Hsp90, protein PIO, triosephosphate isomerase TPI, N- acetyl-glucosamine-binding lectin Paracoccin, 28 kDa protein Pb28 (Paracoccidioides brasiliensis , Paracoccidioidomycosis (South American blastomycosis)); 28-kDa cruzipain-like cystein protease Pw28CCP (usually Paragonimus westermani and other Paragonimus species, Paragonimiasis); outer membrane protein OmpH, outer membrane protein Omp28. protein PM1539, protein PM0355, protein PM1417, repair protein MutL, protein BcbC, prtein PM0305, formate dehydrogenase-N, protein PM0698, protein PM1422, DNA gyrase. lipoprotein PIpE, adhesive protein Cp39, heme aquisition system receptor HasR, 39 kDa capsular protein, iron- regulated OMP IROMP. outer membrane protein OmpA87. fimbrial protein Ptf, fimbrial subunit protein PtfA, transferrin binding protein Tbpl, esterase enzyme MesA, Pasteurella multocida toxin PMT, adhesive protein Cp39 (Pasteurella genus, Pasteurellosis); ‘’filamentous hemagglutinin FhaB, adenylate cy clase CyaA, pertussis toxin subunit 4 precursor PtxD, pertactin precursor Pm, toxin subunit 1 PtxA, protein Cpn60, protein brkA, pertussis toxin subunit 2 precursor PtxB, pertussis toxin subunit 3 precursor PtxC, pertussis toxin subunit 5 precursor PtxE, pertactin Pm, protein Fim2, protein Fim3;” (Bordetella pertussis. Pertussis (Whooping cough)); “Fl capsule antigen, virulence-associated V antigen, secreted effector protein LcrV, V antigen, outer membrane protease Pla, secreted effector protein YopD, putative secreted protein- ty rosine phosphatase YopH, needle complex major subunit YscF, protein kinase YopO, putative autotransporter protein YapF, inner membrane ABC-transporter YbtQ (Irp7), putative sugar binding protein YP00612, heat shock protein 90 HtpG, putative sulfatase protein YdeN, outermembrane lipoprotein carrier protein LoIA, secretion chaperone YerA, putative lipoprotein YP00420, hemolysin activator protein HpmB, pesticin / yersiniabactin outer membrane receptor Psn, secreted effector protein YopE, secreted effector protein YopF, secreted effector protein YopK, outer membrane protein YopN outer membrane protein YopM, Coagulase / fibrinolysin precursor Pla;’' (Yersinia pestis, Plague); protein PhpA, surface adhesin PsaA, pneumolysin Ply, ATP-dependent protease CIp, lipoate-protein ligase LpIA, cell wall surface anchored protein psrP, sortase SrtA. glutamyl-tRNA synthetase GItX, choline binding protein A CbpA, pneumococcal surface protein A PspA, pneumococcal surface protein C PspC, 6-phosphogluconate dehydrogenase Gnd, iron-binding protein PiaA, Murein hydrolase LytB, proteon LytC, protease Al (Streptococcus pneumoniae. Pneumococcal infection); major surface protein B, kexin-like protease KEX1, protein A12, 55 kDa antigen P55, major surface glycoprotein Msg (Pneumocystis jirovecii, Pneumocystis pneumonia (PCP)); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (Poliovirus, Poliomyelitis); protein Nfal, exendin-3, secretory' lipase, cathepsin B-like protease, cysteine protease, cathepsin, peroxiredoxin, protein CrylAc (usually Naegleriafowleri, Primary amoebic meningoencephalitis (PAM)); agnoprotein, large T antigen, small T antigen, major capsid protein VP1, minor capsid protein Vp2 (JC virus, Progressive multifocal leukoencephalopathy); low calcium response protein E LCrE, chlamydial outer protein N CopN, serine / threonine-protein kinase PknD, acyl-carrier-protein S-ma lonyltransferase FabD, singlestranded DNA-binding protein Ssb, major outer membrane protein MOMP. outer membrane protein 2 0mp2, polymorphic membrane protein family (Pmpl, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, PmplO, Pmpl l, Pmpl2, Pmpl3, Pmpl4, Pmpl5, Pmpl6, Pmpl7, Pmpl8, Pmpl9, Pmp20, Pmp21) (Chlamydophila psittaci, Psittacosis); outer membrane protein Pl. heat shock protein B HspB. peptide ABC transporter, GTP-binding protein, protein IcmB, ribonuclease R, phosphatas SixA, protein DsbD, outer membrane protein ToIC, DNA-binding protein PhoB, ATPase DotB, heat shock protein B HspB, membrane protein Coml, 28 kDa protein, DNA-3-methyladenine glycosidase I, pouter membrane protein OmpH, outer membrane protein AdaA, glycine cleavage system T-protein (Coxiella burnetii. Q fever); nucleoprotein N, large structural protein L, phophoprotein P, matrix protein M, glycoprotein G (Rabies virus, Rabies); fusionprotein F, nucleoprotein N, matrix protein M, matrix protein M2-1, matrix protein M2-2, phophoprotein P, small hydrophobic protein SH, major surface glycoprotein G, polymerase L, non-structural protein 1 NS1, non-structural protein 2 NS2 (Respiratory syncytial virus (RSV), Respiratory’ syncytial virus infection); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (Rhinovirus, Rhinovirus infection); outer membrane proteins OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5). cell surface protein SCA4. cell surface protein SCAl, protein PS 120, intracytoplasmic protein D. protective surface protein antigen SPA (Rickettsia genus, Rickettsial infection); outer membrane proteins OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCAl, intracytoplasmic protein D (Rickettsia akari. Rickettsialpox); envelope glycoprotein GP, polymerase L, nucleoprotein N, non-structural protein NSS (Rift Valley fever virus. Rift Valley fever (RVF)); outer membrane proteins OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCAl, intracytoplasmic protein D (Rickettsia rickelisii. Rocky mountain spotted fever (RMSF)); non-structural protein 6 N56, non- structural protein 2 N52, intermediate capsid protein VP6, inner capsid protein VP2, non- structural protein 3 NS3, RNA-directed RNA polymerase L, protein VP3, non-structural protein 1 NS1, non-structural protein 5 N55, outer capsid glycoprotein VP7, non-structural glycoprotein 4N54, outer capsid protein VP4; (Rotavirus, Rotavirus infection); polyprotein P200, glycoprotein El, glycoprotein E2, protein N52, capsid protein C (Rubella virus, Rubella); chaperonin GroEL (MopA), inositol phosphate phosphatase SopB, heat shock protein HslU, chaperone protein DnaJ,protein TviB, protein IroN, flagellin FliC, invasion protein SipC, glycoprotein gp43, outer membrane protein LarnB, outer membrane protein PagC, outer membrane protein ToIC, outer membrane protein NmpC, outer membrane protein FadL, transport protein SadA, transferase WgaP, effector proteins SifA, SteC, SseL, SseJ and SseF (Salmonella genus. Salmonellosis'),- "‘protein 14, non-structural protein NS7b, non-structural protein NS8a. protein 9b. protein 3a, nucleoprotein N, non-structural protein NS3b, non-structural protein N56, protein 7a, non- structural protein NS8b, membrane protein M, envelope small membrane protein EsM, replicase polyprotein la, spike glycoprotein S. replicase polyprotein lab; SARS coronavirus, SARS (Severe Acute Respiratory Syndrome)); serin protease, Atypical Sarcoptes Antigen 1 ASAI. glutathione 5-transferases GST, cystein protease, serine protease, apolipoprotein Sarcoptes scabiei. Scabies); glutathione 5-transferases GST, paramyosin, hemoglbinase SM32, major egg antigen, 14 kDa fatty7acid-binding protein Sml4. major larval surface antigen P37, 22.6 kDa tegumental antigen, calpain CANP. triphospate isomerase Tim, surface protein 9B, outer capsid protein VP2, 23 kDa integral membrane protein Sm23, Cu / Zn-superoxide dismutase, glycoprotein Gp, myosin (Schistosoma genus, Schistosomiasis (Bilharziosis)); 60 kDa chaperonin, 56 kDa type-specific antigen, pyruvate phosphate dikinase, 4-hydroxybenzoate octaprenyltransferase (Orientia tsutsugamushi, Scrub typhus); dehydrogenase GuaB, invasion protein Spa32, invasin IpaA, invasin IpaB, invasin IpaC, invasin IpaD, invasin IpaH. invasin IpaJ (Shigella genus, Shigellosis (Bacillary dysentery)); protein P53, virion protein US 10 homolog, transcriptional regulator 1E63, transcriptional transactivator 1E62, protease P33, alpha trans-inducing factor 74 kDa protein, deoxyuridine 5'-triphosphate nucleotidohydrolase, transcriptional transactivator 1E4, membrane protein UL43 homolog, nuclear phosphoprotein UL3 homolog, nuclear protein UL4 homolog, replication origin-binding protein, membrane protein 2, phosphoprotein 32, protein 57, DNA polymerase processivity factor, portal protein 54, DNA primase, tegument protein ULI 4 homolog, tegument protein UL21 homolog, tegument protein UL55 homolog, tripartite terminase subunit UL33 homolog, tripartite terminase subunit UL15 homolog, capsid-binding protein 44, virionpackaging protein 43 (Varicella zoster virus (VZV), Shingles (Herpes zoster)); truncated 3-beta hydroxy-5-ene steroid dehydrogenase homolog, virion membrane protein A13, protein A19, protein A31, truncated protein A35 homolog, protein A37.5 homolog, protein A47, protein A49, protein A51 , semaphorin-like protein A43, serine proteinase inhibitor 1 , serine proteinase inhibitor 2, serine proteinase inhibitor 3. protein A6, protein B15, protein Cl, protein C5, protein C6, protein F7, protein F8, protein F9, protein Fl 1, protein F14, protein F15, protein F16 (Variola major or Variola minor, Smallpox (Variola)); adhesin / gly coprotein gp70, proteases (Sporothrix schenckii, Sporotrichosis); heme-iron binding protein IsdB, collagen adhesin Cna, clumping factor A ClfA, protein MecA, fibronectin-binding protein A FnbA. enterotoxin type A EntA, enterotoxintype B EntB, enterotoxin type C EntCl, enterotoxin type C EntC2, enterotoxin t pe D EntD, enterotoxin type E EntE, Toxic shock syndrome toxin-1 TSST-1, Staphylokinase, Penicillin binding protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus genus, Staphylococcal food poisoning); heme-iron binding protein IsdB, collagen adhesin Cna, clumping factor A ClfA, protein MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntCl, enterotoxin type C EntC2, enterotoxin t pe D EntD, enterotoxin type E EntE, Toxic shock syndrome toxin-1 TSST-1, Staphylokinase, Penicillin binding protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus genus e.g. aureus, Staphylococcal infection); antigen Ss-IR. antigen NIE, strongylastacin, Na+-K+ ATPase Sseat-6, tropomysin SsTmy-1, protein LEC-5, 41 kDa aantigen P5, 41-kDa larval protein, 31-kDa larval protein, 28-kDa larval protein (Strongyloides stercoralis, Strongy loidiasis); glycerophosphodiester phosphodiesterase GlpQ (Gpd), outer membrane protein TmpB, protein Tp92, antigen TpFl, repeat protein Tpr, repeat protein F TprF, repeat protein G TprG, repeat protein I TprL repeat protein J TprJ, repeat protein KTprK, treponemal membrane protein A TmpA, lipoprotein, 15 kDaTppl5, 47 kDa membrane antigen, miniferritin TpFl, adhesin Tp0751, lipoprotein TP0136, protein TpN17, protein TpN47, outer membrane protein TP0136, outer membrane protein TP0155, outer membrane protein TP0326, outer membrane protein TP0483, outer membrane protein TP0956 (Treponema pallidum. Syphilis); Cathepsin L-like proteases, 53 / 25-kDa antigen, 8 kDa family members, cysticercus protein with a marginal trypsin-like activity' TsAg5, oncosphere protein TSOL18, oncosphere protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia genus, Taeniasis): tetanus toxin TetX, tetanus toxin C TTC, 140 kDa S layer protein, flavoprotein beta-subunit CT3, phospholipase (lecithinase), phosphocarrier protein HPr (Clostridium tetani, Tetanus (Lockjaw)); genome polyprotein, protein E, protein M, capsid protein C (Tick-bome encephalitis virus (TBEV), Tick-bome encephalitis); 58-kDa antigen, 68-kDa antigens, Toxocara larvae excretory- secretory antigen TES, 32-kDa glycoprotein, glycoprotein TES-70, glycoprotein GP31, excretory- secretory antigen TcES-57, perienteric fluid antigen Pe. soluble extract antigens Ex, excretory / secretory larval antigens ES, antigen TES-120, polyprotein allergen TBA-1, cathepsin L-like cysteine protease c-cpl-1, 26-kDa protein (Toxocara canis or Toxocara cati, Toxocariasis (Ocular Larva Migrans (OLM) and Visceral Lan a Migrans (VLM))); microneme proteins (MIC 1, MIC2, MIC3, MIC4. MIC5, MICE, MIC 7, MICE), rhoptry protein Rop2. rhoptry proteins (Ropl, Rop2, Rop3, Rop4, Rop5, Rop6, Rop7, Ropl6, Rjopl7), protein SRI, surface antigen P22, major antigen p24, major surface antigen p30, dense granule proteins (GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRAB, GRA9, GRA10), 28 kDa antigen, surface antigen SAG1, SAG2 related antigen, nucleoside-triphosphatase 1, nucleoside-triphosphatase 2, protein Stt3, HesB-likedomain-containing protein, rhomboid-like protease 5, toxomepsin 1 {Toxoplasma gondii, Toxoplasmosis); 43 kDa secreted glycoprotein, 53 kDa secreted glycoprotein, paramyosin, antigen Ts21, antigen Ts87, antigen p46000, TSL-1 antigens, caveolin-1 CAV-1, 49 kDa newborn larv a antigen, prosaposin homologue, serine protease, serine proteinase inhibitor, 45 -kDa glycoprotein Gp45 {Trichinella spiralis, Trichinellosis); Myb-like transcriptional factors (Mybl, Myb2, Myb3), adhesion protein AP23, adhesion protein AP33, adhesin protein AP33-3, adhesins AP51, adhesin AP65, adhesion protein AP65-1, alpha-actinin, kinesin-associated protein, teneurin, 62 kDa proteinase, subtilisin-like serine protease SUB1, cysteine proteinase gene 3 CP3, alpha-enolase Enol, cysteine proteinase CP30. heat shock proteins (Hsp70, Hsp60) immunogenic protein P270, {Trichomonas vaginalis. Trichomoniasis); beta-tubulin, 47-kDa protein, secretory leucocyte-like proteinase-1 SLP-1, 50-kDa protein TT50, 17 kDa antigen, 43 / 47 kDa protein {Trichuris trichiura, Trichuriasis (Whipworm infection)); protein ESAT-6 (EsxA), 10 kDa filtrate antigen EsxB, secreted antigen 85-B FBPB, fibronectin-binding protein A FbpA (Ag85A), serine protease PepA, PPE family protein PPE18, fibronectin-binding protein D FbpD, immunogenic protein MPT64, secreted protein MPT51, catalase-peroxidase-peroxynitritase T KATG, periplasmic phosphate-binding lipoprotein PSTS3 (PBP-3, Phos-1), iron-regulated heparin binding hemagglutinin Hbha, PPE family protein PPE14, PPE family protein PPE68, protein Mtb72F, protein Apa, immunogenic protein MPT63. periplasmic phosphate-binding lipoprotein PSTS1 (PBP-1), molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permease protein pstA, 14 kDa antigen, fibronectin-binding protein C FbpCl, Alanine dehydrogenase TB43, Glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTBE. Rpf-like proteins, protein MTB32, protein MTB39, crystallin, heat-shock protein HSP65, protein PST- S(usually Mycobacterium tuberculosis, Tuberculosis); outer membrane protein FobA, outer membrane protein FobB, intracellular growth locus IgICl, intracellular growth locus IgIC2, aminotransferase Wbtl, chaperonin GroEL, 17 kDa major membrane protein TUL4, lipoprotein LpnA, chitinase family 18 protein, isocitrate dehydrogenase, Nif3 family protein, type IV pili glycosylation protein, outer membrane protein toIC, FAD binding family protein, type IV pilin multimeric outer membrane protein, two component sensor protein KdpD, chaperone protein DnaK, protein TolQ {Francisella tularensis, Tularemia); “MB antigen, urease, protein GyrA, protein GyrB, protein ParC, protein ParE, lipid associated membrane proteins LAMP, thymidine kinase TK, phospholipase PL-A1, phospholipase PL-A2, phospholipase PL-C, surface-expressed 96-kDa antigen;” {Ureaplasma urealyticum, Ureaplasma urealyticum infection); non-structural polyprotein, structural polyprotein, capsid protein CP, protein El, protein E2, protein E3, protease Pb, protease P2, protease P3 (Venezuelan equine encephalitis virus, Venezuelan equineencephalitis); glycoprotein GP, matrix protein Z, polymerase L, nucleoprotein N (Guanarito virus, Venezuelan hemorrhagic fever); polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, brotein NS4A, protein NS4B, protein NS5 (West Nile virus, West Nile Fever); cpasid protein CP, protein El, protein E2, protein E3, protease P2 (Western equine encephalitis virus, Western equine encephalitis); genome polyprotein. protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (Yellow fever virus, Yellow fever); putative Yop targeting protein Y obB, effector protein Y opD, effector protein Y opE, protein Y opH, effector protein Y opJ, protein translocation protein YopK, effector protein YopT, protein YpkA, flagellar biosyntheses protein FlhA, peptidase M48, potassium efflux system KefA, transcriptional regulatoer RovA, adhesin Ifp, translocator portein LcrV, protein PcrV, invasin Inv, outer membrane protein OmpF- like porin, adhesin YadA, protein kinase C, phospholipase Cl, protein PsaA, mannosy Itransferase- like protein WbyK, protein YscU, antigen YPMa {Yersinia pseudotuberculosis. Yersinia pseudotuberculosis infection); effector protein YopB, 60 kDa chaperonin, protein WbcP, tyrosin- protein phosphatase YopH, protein YopQ, enterotoxin, Galactoside permease, reductaase NrdE, protein YasN, Invasin Inv, adhesin YadA, outer membrane porin F OmpF, protein UspAl, protein EibA, protein Hia, cell surface protein Ail, chaperone SycD, protein LcrD, protein LcrG, protein LcrV, protein SycE, protein YopE, regulator protein TyeA, protein YopM, protein YopN, protein YopO, protein YopT, protein YopD, protease CIpP, protein MyfA, protein FilA, and protein PsaA {Yersinia enierocoliiica. Y ersiniosis). The brackets in the preceding section indicate the particular pathogen or the family of pathogens of which the antigen(s) is / are derived and the infectious disease with which the pathogen is associated.

[0161] In some embodiments, a method of treating, reducing, or ameliorating subject with an influenza infection includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal delivery of the lipid nanoparticles as described herein. In some embodiments, the patch includes a top layer including an adhesive, a middle layer including lipid nanoparticles and one or more RNA molecules, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles and one or more RNA molecules through the plurality of micropores. In some embodiments, the lipid nanoparticles include one or more mRNA molecules capable of treating the influenza infection. In some embodiments, the mRNA comprises a coding region, encoding at least one antigenic peptide or protein derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (Ml), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2(NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2) of an influenza virus or a fragment or variant thereof. In some embodiments, the amino acid sequence of the at least one antigenic peptide or protein may be selected from any peptide or protein derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (Ml), matrix protein 2 (M2), non- structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2) of an influenza virus or a fragment or variant or from any synthetically engineered influenza virus peptide or protein.

[0162] In some embodiments, the coding sequence of the mRNA of the present disclosure encodes at least one antigenic peptide or protein which is derived from a hemagglutinin (HA) protein of an influenza A virus; or a hemagglutinin (HA) protein of an influenza B virus; or a neuraminidase (NA) protein of an influenza A virus; or a neuraminidase (NA) protein of an influenza B virus; or a fragment or variant thereof, wherein the hemagglutinin (HA) protein of an influenza A virus or the hemagglutinin (HA) protein of an influenza B virus or the neuraminidase (NA) protein of an influenza A virus or the neuraminidase (NA) protein of an influenza B virus is selected from the hemagglutinin (HA) proteins or the neuraminidase (NA) proteins as listed in the sequence listing of the present disclosure.

[0163] In some embodiments, a method of treating, reducing, or ameliorating subject with an Ebola infection includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal delivery’ of the lipid nanoparticles as described herein. In some embodiments, the patch includes a top layer including an adhesive, a middle layer including lipid nanoparticles and one or more RNA molecules, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles and one or more RNA molecules through the plurality’ of micropores. In some embodiments, the lipid nanoparticle includes an RNA molecule capable of treating the Ebola infection. In some embodiments, the mRNA includes a coding region, encoding at least one antigenic peptide or protein derived from the glycoprotein (GP) and / or the matrix protein 40 (VP40) and / or the nucleoprotein (NP) of a virus of the genus Ebolavirus or Marburgvirus or a fragment, variant or derivative thereof. In some embodiments, the amino acid sequence of the at least one antigenic peptide or protein may be selected from any peptide or protein derived from glycoprotein (GP) and / or the matrix protein 40 (VP40) and / or the nucleoprotein (NP) a glycoprotein of an Ebola virus or a fragment or variant or from any synthetically engineered Ebola virus peptide or protein. In some embodiments, the coding regionencodes at least one antigenic peptide or protein derived from a glycoprotein of an Ebola virus or a fragment or variant thereof. In this context, the at least one coding region encodes at least one full-length protein of a glycoprotein of an Ebola virus or a variant thereof.

[0164] The patch of the present disclosure may be applied to a subject with a disease or condition where an immediate effect or a sustained effect is expected. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles may have a PK profile comparable to subcutaneous injection. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles may have a PK profile superior to intravenous and subcutaneous injection. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles may have a PK profile with a bioavailability greater than 100%, greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 750%, greater than 1000%, greater than 1500%, greater than 2000%, greater than 2500%, greater than 3000%, greater than 3500%, greater than 4000%, greater than 4500%, greater than 5000% compared to intravenous (IV) administration, or ranges including and / or spanning the aforementioned values. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles may have a PK profile with a bioavailability from about 350% to about 4800% compared to IV.

[0165] In some embodiments, a method of treating, reducing, or ameliorating a subject with a coronavirus infection includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal delivery of the lipid nanoparticles as described herein. In some embodiments, the patch includes a top layer including an adhesive, a middle layer including lipid nanoparticles, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles through the plurality of micropores. In some embodiments, the lipid nanoparticles include an mRNA molecule capable of treating the coronavirus infection.

[0166] In some embodiments, a method of treating, reducing, or ameliorating subject with a tumor includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject’s skin over the micropores for a period of time effective to result in transdermal deliver}' of the lipid nanoparticles as disclosed elsewhere herein. In some embodiments, the patch includes a top layer including an adhesive, a middle layer including lipid nanoparticles, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles through the plurality of micropores. In some embodiments, the lipid nanoparticles include an mRNA capable of treating the tumor. In some embodiments, the mRNA includes a coding region, encoding a tumor antigen. In some embodiments, the tumor antigenmay be selected from, but is not limited to, the group consisting of 1 A01_HLA-A / m (UniProtKB: P30443); 1A02 (UniProtKB: P01892); 5T4 (UniProtKB: Q13641); ACRBP (UniProtKB: Q8NEB7); AFP (UniProtKB: P02771); AKAP4 (UniProtKB: Q5JQC9); alpha-actinin-_4 / m (UniProtKB: B4DSX0); alpha-actinin-_4 / m (UniProtKB: B4E337); alpha-actinin-_4 / m (UniProtKB: 043707); alpha-methylacyl-coenzyme_A_racemase (UniProtKB: A0A024RE16); alpha-methylacyl-coenzyme_A_racemase (UniProtKB: A8KAC3); ANDR (UniProtKB: P10275); ART-4 (UniProtKB: Q9ULX3); ARTCl / m (UniProtKB: P52961); AURKB (UniProtKB: Q96GD4); B2MG (UniProtKB: P61769); B3GN5 (UniProtKB: Q9BYGO); B4GN1 (UniProtKB: Q00973); B7H4 (UniProtKB: Q7Z7D3); BAGE-1 (UniProtKB: Q13072); BASI (UniProtKB: P35613); BCL-2 (UniProtKB: A9QXG9); bcr / abl (UniProtKB: A9UEZ4); bcr / abl (UniProtKB: A9UEZ7); bcr / abl (UniProtKB: A9UEZ8); bcr / abl (UniProtKB: A9UEZ9); bcr / abl (UniProtKB: A9UF00); bcr / abl (UniProtKB: A9UF01); bcr / abl (UniProtKB: A9UF03); bcr / abl (UniProtKB: A9UF04); bcr / abl (UniProtKB: A9UF05); bcr / abl (UniProtKB: A9UF06); bcr / abl (UniProtKB: A9UF08); beta-catenin / m (UniProtKB: P35222); beta-catenin / m (UniProtKB: Q8WYA6); BING-4 (UniProtKB: 015213); BIRC7 (UniProtKB: Q96CA5); BRCAl / m (UniProtKB: A0A024R1V0); BRCAl / m (UniProtKB: A0A024R1V7); BRCAl / m (UniProtKB: A0A024R1Z8); BRCAl / m (UniProtKB: A0A068BFX7); BRCAl / m (UniProtKB: C6YB45); BRCAl / m (UniProtKB: C6YB47); BRCAl / m (UniProtKB: G3XAC3); BY55 (UniProtKB: 095971); calreticulin (UniProtKB: B4DHR1); calreticulin (UniProtKB: B4E2Y9); calreticulin (UniProtKB: P27797); calreticulin (UniProtKB: Q96L12); CAMEL (UniProtKB: 095987); CASP-8 / m (UniProtKB: Q14790); CASPA (UniProtKB: Q92851-4); cathepsin B (UniProtKB: A0A024R374); cathepsin_B (UniProtKB: P07858); cathepsin_L (UniProtKB: A0A024R276); cathepsin L (UniProtKB: P07711); cathepsin L (UniProtKB: Q9HBQ7); CD1A (UniProtKB: P06126); CD1B (UniProtKB: P29016); CD1C (UniProtKB: P29017); CD1D (UniProtKB: P15813); CD1E (UniProtKB: P15812); CD20 (UniProtKB: Pl 1836); CD22 (UniProtKB: 060926); CD22 (UniProtKB: P20273); CD22 (UniProtKB: QOEAF5); CD276 (UniProtKB: Q5ZPR3); CD33 (UniProtKB: B4DF51); CD33 (UniProtKB: P20138); CD33 (UniProtKB: Q546G0); CD3E (UniProtKB: P07766); CD3Z (UniProtKB: P20963); CD44_Isoform_l (UniProtKB: P16070); CD44_Isoform_6 (UniProtKB: P16070-6); CD4 (UniProtKB: P01730); CD52 (UniProtKB: P31358); CD52 (UniProtKB: Q6IBDO); CD52 (UniProtKB: V9HWN9); CD55 (UniProtKB: B1AP15); CD55 (UniProtKB: D3DT85); CD55 (UniProtKB: D3DT86); CD55 (UniProtKB: P08174); CD56 (UniProtKB: P 13591); CD80 (UniProtKB: AONOP2); CD80 (UniProtKB: P33681); CD86 (UniProtKB: P42081); CD8A (UniProtKB: P01732); CDC127 / m (UniProtKB: G5EA36); CDC127 / m (UniProtKB: P30260); CDE30 (UniProtKB: P28908); CDK4 / m (UniProtKB: A0A024RBB6); CDK4 / m (UniProtKB: Pl 1802); CDK4 / m (UniProtKB:Q6LC83); CDK4 / m (UniProtKB: Q96BE9); CDKN2A / m (UniProtKB: D1LYX3); CDKN2A / m (UniProtKB: G3XAG3); CDKN2A / m (UniProtKB: K7PML8); CDKN2A / m (UniProtKB: E8E941); CDKN2A / tn (UniProtKB: Q8N726); CEA (ReESeq: NP_004354); CEAM6 (UniProtKB: P40199); CH3L2 (UniProtKB: Q15782); CLCA2 (UniProtKB: Q9UQC9); CME28 (UniProtKB: Q9NQT4); CML66 (UniProtKB: Q96RS6); COA-l / m (UniProtKB: Q5T124); coactosin-like_protein (UniProtKB: Q14019); collagen XXIII (UniProtKB: L8EAS4); collagen XXIII (UniProtKB: Q86Y22); COX-2 (UniProtKB: Q6ZYK7); CP1B1 (UniProtKB: QI 6678); CSAG2 (UniProtKB: Q9Y5P2-2); CSAG2 (UniProtKB: Q9Y5P2); CT45A1 (UniProtKB: Q5HYN5); CT55 (UniProtKB: Q8WUE5); CT- 9 / BRD6 (UniProtKB: Q58F21); CTAG2_Isoform_LAGE-l A (UniProtKB: 075638-2); CTAG2_Isoform_LAGE-lB (UniProtKB: 075638); CTCFL (UniProtKB: Q8NI51); Cten (UniProtKB: Q8IZW8); cyclin Bl (UniProtKB: P14635); cyclin Dl (UniProtKB: P24385); cyp-B (UniProtKB: P23284); DAM-10 (UniProtKB: P43366); DEP1A (UniProtKB: Q5TB30); E7 (UniProtKB: P03129); E7 (UniProtKB: P06788); E7 (UniProtKB: P17387); E7 (UniProtKB: P06429); E7 (UniProtKB: P27230); E7 (UniProtKB: P24837); E7 (UniProtKB: P21736); E7 (UniProtKB: P26558); E7 (UniProtKB: P36831); E7 (UniProtKB: P36833); E7 (UniProtKB: Q9QCZ1); E7 (UniProtKB: Q81965); E7 (UniProtKB: Q80956); EFl A2 (UniProtKB: Q05639); EFTUD2 / m (UniProtKB: Q15029); EGFR (UniProtKB: A0A0B4J1Y5); EGFR (UniProtKB: E7BSVO); EGFR (UniProtKB: LOR6G1); EGFR (UniProtKB: P00533-2); EGFR (UniProtKB: P00533); EGFR (UniProtKB: Q147T7); EGFR (UniProtKB: Q504U8); EGFR (UniProtKB: Q8NDU8); EGLN3 (UniProtKB: Q9H6Z9); ELF2 / m (UniProtKB: B7Z720); EMMPRIN (UniProtKB: Q54A51); EpCam (UniProtKB: P16422); EphA2 (UniProtKB: P29317); EphA3 (UniProtKB: P29320): EphA3 (UniProtKB: Q6P4R6); ErbB3 (UniProtKB: B3KWG5); ErbB3 (UniProtKB: B4DGQ7); ERBB4 (UniProtKB: Q15303); ERG (UniProtKB: Pl 1308); ETV6 (UniProtKB: P41212); EWS (UniProtKB: Q01844); EZH2 (UniProtKB: F2YMM1); EZH2 (UniProtKB: G3XAE2); EZH2 (UniProtKB: LOR855); EZH2 (UniProtKB: Q15910); EZH2 (UniProtKB: S4S3R8); FABP7 (UniProtKB: 015540); FCGR3A_Version_l (UniProtKB: P08637); FCGR3A_Version_2 (CCDS: CCDS1232.1); FGFS (UniProtKB: P12034); FGFS (UniProtKB: Q60518); FGFR2 (UniProtKB: P21802); fibronectin (UniProtKB: A0A024R5I6); fibronectin (UniProtKB: A0A024RB01); fibronectin (UniProtKB: A0A024RDT9); fibronectin (UniProtKB: A0A024RDV5); fibronectin (UniProtKB: A6NH44); fibronectin (UniProtKB: A8K6A5); fibronectin (UniProtKB: B2R627); fibronectin (UniProtKB: B3KXM5); fibronectin (UniProtKB: B4DIC5); fibronectin (UniProtKB: B4DN21); fibronectin (UniProtKB: B4DS98); fibronectin (UniProtKB: B4DTH2); fibronectin (UniProtKB: B4DTK1); fibronectin (UniProtKB: B4DU16); fibronectin (UniProtKB: B7Z3W5); fibronectin (UniProtKB: B7Z939); fibronectin (UniProtKB: G5E9X3); fibronectin (UniProtKB: Q9H382); FOS(UmProtKB: P01100); FOXP3 (UniProtKB: Q9BZS1); FUT1 (UniProtKB: P19526); G250 (UniProtKB: Q16790); GAGE-1 (Genbank: AAA82744); GAGE-2 (UniProtKB: Q6NT46); GAGE-3 (UniProtKB: Q13067); GAGE-4 (UniProtKB: Q13068); GAGE-5 (UniProtKB: Q13069); GAGE-6 (UniProtKB: Q13070); GAGE7b (UniProtKB: 076087); GAGE-8_(GAGE- 2D) (UniProtKB: Q9UEU5); GASR (UniProtKB: P32239); GnT-V (UniProtKB: Q09328); GPC3 (UniProtKB: I6QTG3); GPC3 (UniProtKB: P51654); GPC3 (UniProtKB: Q8IYG2); GPNMB / m (UniProtKB: A0A024RA55); GPNMB / m (UniProtKB: Q14956); GPNMB / m (UniProtKB: Q8IXJ5); GPNMB / m (UniProtKB: Q96F58); GRM3 (UniProtKB: Q14832); HAGE (UniProtKB: Q9NXZ2); hepsin (UniProtKB: B2ZDQ2); hepsin (UniProtKB: P05981); Her2 / neu (UniProtKB: B4DTR1); Her2 / neu (UniProtKB: L8E8G2); Her2 / neu (UniProtKB: P04626); Her2 / neu (UniProtKB: Q9UK79); HLA-A2 / m (UniProtKB: Q95387); HLA-A2 / m (UniProtKB: Q9MYF8); homeobox_NKX3.1 (UniProtKB: Q99801); HOM-TES-85 (UniProtKB: B2RBQ6); HOM-TES- 85 (UniProtKB: Q9P127); HPG1 (Pubmed: 12543784); HS71 A (UniProtKB: PODMV8); HS71B (UniProtKB: PODMV9); HST-2 (UniProtKB: P10767); hTERT (UniProtKB: 094807); 1CE (UniProtKB: 000748); IF2B3 (UniProtKB: 000425); IL10 (UniProtKB: P22301); IL-13Ra2 (UniProtKB: Q14627); IL2-RA (UniProtKB: P01589); IL2-RB (UniProtKB: P14784); IL2-RG (UniProtKB: P31785); IL-5 (UniProtKB: P05113); IMP3 (UniProtKB: Q9NV31); ITA5 (UniProtKB: P08648); ITB1 (UniProtKB: P05556); ITB6 (UniProtKB: P18564); kallikrein-2 (UniProtKB: A0A024R4J4); kallikrein-2 (UniProtKB: A0A024R4N3); kallikrein-2 (UniProtKB: BOAZU9); kallikrein-2 (UniProtKB: B4DU77); kallikrein-2 (UniProtKB: P20151); kallikrein-2 (UniProtKB: Q6T774); kallikrein-2 (UniProtKB: Q6T775); kallikrein-4 (UniProtKB: A0A0C4DFQ5); kallikrein-4 (UniProtKB: Q5BQA0); kallikrein-4 (UniProtKB: Q96PTO); kallikrein-4 (UniProtKB: Q96PT1); kallikrein-4 (UniProtKB: Q9Y5K2); KI20A (UniProtKB: 095235); KIAA0205 (UniProtKB: Q92604); KIF2C (UniProtKB: Q99661); KK-LC-1 (UniProtKB: Q5H943); LDLR (UniProtKB: P01130); LGMN (UniProtKB: Q99538); LIRB2 (UniProtKB: Q8N423); LY6K (UniProtKB: Q17RY6); MAGAS (UniProtKB: P43359); MAGA8 (UniProtKB: P43361); MAGAB (UniProtKB: P43364); MAGE-A10 (UniProtKB: A0A024RC14); MAGE-A12 (UniProtKB: P43365); MAGE-A1 (UniProtKB: P43355); MAGE- A2 (UniProtKB: P43356); MAGE-A3 (UniProtKB: P43357); MAGE-A4 (UniProtKB: A0A024RC12); MAGE-A4 (UniProtKB: P43358); MAGE-A4 (UniProtKB: Q1RN33); MAGE- A6 (UniProtKB: A8K072); MAGE-A6 (UniProtKB: P43360); MAGE-A6 (UniProtKB: Q6FHI5); MAGE-A9 (UniProtKB: P43362); MAGE-B10 (UniProtKB: Q96LZ2); MAGE-B16 (UniProtKB: A2A368); MAGE-B17 (UniProtKB: A8MXT2); MAGE- Bl (UniProtKB: Q96TG1); MAGE-B2 (UniProtKB: 015479); MAGE-B3 (UniProtKB: 015480); MAGE-B4 (UniProtKB: 015481); MAGE-B5 (UniProtKB: Q9BZ81); MAGE-B6 (UniProtKB: Q8N7X4);MAGE-C1 (UniProtKB: 060732); MAGE-C2 (UniProtKB: Q9UBF1); MAGE-C3 (UniProtKB: Q8TD91); MAGE-D1 (UniProtKB: Q9Y5V3); MAGE-D2 (UniProtKB: Q9UNF1); MAGE-D4 (UniProtKB: Q96JG8); MAGE- El (UniProtKB: Q6IAI7); MAGE-El (MAGEl) (UniProtKB: Q9HCI5); MAGE-E2 (UniProtKB: Q8TD90); MAGE-F1 (UniProtKB: Q9HAY2); MAGE-H1 (UniProtKB: Q9H213); MAGEL2 (UniProtKB: Q9U355); mammaglobin A (UniProtKB: Q13296); mammaglobin_A (UniProtKB: Q6NX70); MART-l / melan-A (UniProtKB: Q16655); MART-2 (UniProtKB: Q5VTY9); MC1 R (UniProtKB: Q01726); MC1 R (UniProtKB: Q1JUE4); MC1 R (UniProtKB: Q1JUL6); MC1 R (UniProtKB: Q1JUL8); MCI R (UniProtKB: Q1JUL9); MCI R (UniProtKB: Q1JUM0); MCI R (UniProtKB: Q1JUM2); MC1 R (UniProtKB: Q1JUM3); MC1_R (UniProtKB: Q1JUM4); MC1_R (UniProtKB: Q1JUM5); MCI R (UniProtKB: Q6UR92); MC1 R (UniProtKB: Q6UR94); MCI R (UniProtKB: Q6UR95): MC1 R (UniProtKB: Q6UR96); MC1 R (UniProtKB: Q6UR97); MCI R (UniProtKB: Q6UR98); MCI R (UniProtKB: Q6UR99); MC1 R (UniProtKB: Q6URA0); MC1_R (UniProtKB: Q86YW1); MC1_R (UniProtKB: V9Q5S2); MC1_R (UniProtKB: V9Q671); MCI R (UniProtKB: V9Q783); MC1 R (UniProtKB: V9Q7F1); MC1 R (UniProtKB: V9Q8N1); MC1_R (UniProtKB: V9Q977); MC1_R (UniProtKB: V9Q9P5); MC1 R (UniProtKB: V9Q9R8); MC1 R (UniProtKB: V9QAE0); MCI R (UniProtKB: V9QAR2); MCI R (UniProtKB: V9QAW3); MCI R (UniProtKB: V9QB02); MC1 R (UniProtKB: V9QB58); MCI R (UniProtKB: V9QBY6); MC1 R (UniProtKB: V9QC17); MC1 R (UniProtKB: V9QC66); MCI R (UniProtKB: V9QCQ4); MCI R (UniProtKB: V9QDF4); MCI R (UniProtKB: V9QDN7); MC1 R (UniProtKB: V9QDQ6); M- CSF (UniProtKB: P09603); mesothelin (UniProtKB: Q13421); MITF (UniProtKB: 075030-8); MITF (UniProtKB: 075030-9); MITF (UniProtKB: 075030); MMPl l (UniProtKB: B3KQS8); MMP7 (UniProtKB: P09237); MUC-1 (Genbank: AAA60019); MUM-l / m (RefSeq: NP_116242); MUM-2 / m (UniProtKB: Q9Y5R8); MYCN (UniProtKB: P04198); MY01A (UniProtKB: Q9UBC5); MY01B (UniProtKB: 043795); MY01C (UniProtKB: 000159); MYOID (UniProtKB: 094832); MY01E (UniProtKB: Q12965); MY01F (UniProtKB: 000160); MY01G (UniProtKB: B0I1T2); MY01H (RefSeq: NP_001094891); NA17 (UniProtKB: Q3V5E5); NA88- A (Pubmed: 10790436); Neo-PAP (UniProtKB: Q9BWT3); NFYC / m (UniProtKB: Q13952); NGEP (UniProtKB: Q6IWH7); NPM (UniProtKB: P06748); NRCAM (UniProtKB: Q92823); NSE (UniProtKB: P09104); NUF2 (UniProtKB: Q9BZD4); NY-ESO-1 (UniProtKB: P78358); 0A1 (UniProtKB: P51810); OGT (UniProtKB: 015294); OS-9 (UniProtKB: B4DH11); OS-9 (UniProtKB: B4E321); OS-9 (UniProtKB: B7Z8E7); OS-9 (UniProtKB: Q13438); osteocalcin (UniProtKB: P02818); osteopontin (UniProtKB: A0A024RDE2); osteopontin (UniProtKB: A0A024RDE6); osteopontin (UniProtKB: A0A024RDJ0); osteopontin (UniProtKB: B7Z351);osteopontin (UniProtKB: F2YQ21); osteopontin (UniProtKB: P10451); p53 (UniProtKB: P04637); PAGE-4 (UniProtKB: 060829); PAI-1 (UniProtKB: P05121); PAI-2 (UniProtKB: P05120); PAP (UniProtKB: Q06141); PAP (UniProtKB: Q53S56); PATE (UniProtKB: Q8WXA2); PAX3 (UniProtKB: P23760); PAXS (UniProtKB: Q02548); PD1L1 (UniProtKB: Q9NZQ7); PDCD1 (UniProtKB: Q15116); PDEF (UniProtKB: 095238); PECA1 (UniProtKB: P16284); PGCB (UniProtKB: Q96GW7); PGFRB (UniProtKB: P09619); Pim-1_-Kinase (UniProtKB: A0A024RD25); Pin-1 (UniProtKB: 015428); Pin-1 (UniProtKB: Q13526); Pin-1 (UniProtKB: Q49AR7); PLAC1 (UniProtKB: Q9HBJ0); PMEL (UniProtKB: P40967); PML (UniProtKB: P29590); POTEF (UniProtKB: A5A3E0); POTE (UniProtKB: Q86YR6); PRAME (UniProtKB: A0A024R1E6); PRAME (UniProtKB: P78395); PRDX5 / m (UniProtKB: P30044); PRM2 (UniProtKB: P04554); prostein (UniProtKB: Q96JT2); proteinase-3 (UniProtKB: D6CHE9); proteinase-3 (UniProtKB: P24158); PSA (UniProtKB: P55786); PSB9 (UniProtKB: P28065); PSCA (UniProtKB: D3DWI6); PSCA (UniProtKB: 043653); PSGR (UniProtKB: Q9H255); PSM (UniProtKB: Q04609); PTPRC (RefSeq: NP 002829); RAB8A (UniProtKB: P61006); RAGE-1 (UniProtKB: Q9UQ07); RARA (UniProtKB: P10276); RASH (UniProtKB: P01112); RASK (UniProtKB: P01116); RASN (UniProtKB: P01111); RGSS (UniProtKB: 015539); RHAMM / CD168 (UniProtKB: 075330); RHOC (UniProtKB: P08134); RSSA (UniProtKB: P08865); RU1 (UniProtKB: Q9UHJ3); RU2 (UniProtKB: Q9UHG0); RUNX1 (UniProtKB: Q01196); S-100 (UniProtKB: V9HW39); SAGE (UniProtKB: Q9NXZ1); SART-_1 (UniProtKB: 043290); SART-2 (UniProtKB: Q9UL01); SART-3 (UniProtKB: Q15020); SEPR (UniProtKB: Q12884): SERPINBS (UniProtKB: P36952); SIA7F (UniProtKB: Q969X2); SIA8A (UniProtKB: Q92185); SIAT9 (UniProtKB: Q9UNP4): SIRT2 / m (UniProtKB: A0A024ROG8); SIRT2 / m (UniProtKB: Q8IXJ6); SOX10 (UniProtKB: P56693); SP17 (UniProtKB: Q15506); SPNXA (UniProtKB: Q9NS26); SPXN3 (UniProtKB: Q5MJ09); SSX-1 (UniProtKB: Q16384); SSX-2 (UniProtKB: Q16385); SSX3 (UniProtKB: Q99909); SSX-4 (UniProtKB: 060224); ST1A1 (UniProtKB: P50225); STAG2 (UniProtKB: Q8N3U4-2); STAMP-1 (UniProtKB: Q8NFT2); STEAP-1 (UniProtKB: A0A024RA63); STEAP-1 (UniProtKB: Q9UHE8): Survivin- 2B (UniProtKB: 015392-2); survivin (UniProtKB: 015392); SYCP1 (UniProtKB: A0A024R0I2); SYCP1 (UniProtKB: B7ZLS9); SYCP1 (UniProtKB: Q15431); SYCP1 (UniProtKB: Q3MHC4); SYT-SSX-1 (UniProtKB: A4PIV7); SYT-SSX-1 (UniProtKB: A4PIV8); SYT-SSX-2 (UniProtKB: A4PIV9); SYT-SSX-2 (UniProtKB: A4PIWO); TARP (UniProtKB: QOVGM3); TCRg (UniProtKB: A2JGV3); TF2AA (UniProtKB: P52655); TGFB1 (UniProtKB: P01137); TGFR2 (UniProtKB: P37173); TGM-4 (UniProtKB: B2R7D1); TIE2 (UniProtKB: Q02763); TKIL1 (UniProtKB: P51854); TPI / m (UniProtKB: P60174); TRGV11 (UniProtKB: Q99601); TRGV9 (UniProtKB: A4D1X2): TRGV9 (UniProtKB: Q99603); TRGV9 (UniProtKB: Q99604);TRPC1 (UniProtKB: P48995); TRP-p8 (UniProtKB: Q7Z2W7); TSG10 (UniProtKB: Q9BZW7); TSPY1 (UniProtKB: Q01534); TVC_(TRGV3) (Genbank: M13231.1); TX101 (UniProtKB: Q9BY14-2); tyrosinase (UniProtKB: A0A024DBG7); tyrosinase (UniProtKB: L8B082); tyrosinase (UniProtKB: L8B086); tyrosinase (UniProtKB: L8B0B9); tyrosinase (UniProtKB: 075767); tyrosinase (UniProtKB: P 14679); tyrosinase (UniProtKB: U3M8N0); tyrosinase (UniProtKB: U3M9D5); tyrosinase (UniProtKB: U3M9J2); TYRP1 (UniProtKB: P17643); TYRP2 (UniProtKB: P40126); UPA (UniProtKB: Q96NZ9); VEGFR1 (UniProtKB: B5A924); WT1 (UniProtKB: A0A0H5AUY0); WT1 (UniProtKB: P19544); WT1 (UniProtKB: Q06250); XAGE1 (UniProtKB: Q9HD64).

[0167] In some embodiments, a method of providing an immune checkpoint inhibitor to a subject in need thereof, the method includes opening a plurality of micropores in the skin of the subject, and applying the patch to the subject's skin over the micropores for a period of time effective to result in transdermal delivery of the lipid nanoparticles as disclosed elsewhere herein. In some embodiments, the patch includes a top layer including an adhesive, a middle layer including lipid nanoparticles, and a bottom layer. In some embodiments, the bottom layer includes a release liner. In some embodiments, the period of time is selected to deliver a therapeutically effective amount of the lipid nanoparticles through the plurality of micropores. In some embodiments, the lipid nanoparticles include an RNA molecule capable of checkpoint modulation. In some embodiments, the mRNA includes a coding region, encoding at least a portion of an inhibitory checkpoint molecule. In some embodiments, the mRNA includes a coding region, encoding an entire inhibitory checkpoint molecule. In some embodiments, the inhibitory' checkpoint molecule includes PD-1, PD-L1. CTLA-4, PD-L2, LAG3, TIM3 / HAVCR2, 2B4, A2aR, B7H3, B7H4, BTLA, CD30, CD160, CD155, GAL9, HVEM, IDOL ID02, KIR, LAIR1 and VISTA. In some embodiments, lipid nanoparticles and mRNA as described elsewhere herein is used in combination with a checkpoint modulator. In some embodiments, the checkpoint modulator selected from the group consisting of the checkpoint modulator is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a TIGIT-inhibitor, an 0X40 stimulator, a 4-lBB stimulator, a CD40L stimulator, a CD28 stimulator and a GITR stimulator.

[0168] In some embodiments, the opening of the plurality of micropores in the skin of the subject includes applying a transdermal mi croporation apparatus to the subject's skin. In some embodiments, the transdermal microporation apparatus includes a conductive member including an array of conductive fdaments. In some embodiments, the transdermal microporation apparatus includes a conductive member including an array of conductive filaments. In some embodiments, the transdermal microporation opens the micropores by thermal tissue ablation. In someembodiments, the transdermal microporation creates micropores through the stratum comeum to the epidermis.

[0169] The patch of the present disclosure may be applied to a subject with a disease or condition where an immediate effect or a sustained effect is expected. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles may have a PK / PD profile comparable to injections. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles may have a PK / PD profile superior to injections. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles may have a PK / PD profile with a bioavailability / pharmacological activities greater than 100%, greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 750%, greater than 1000%, greater than 1500%, greater than 2000%, greater than 2500%, greater than 3000%, greater than 3500%, greater than 4000%, greater than 4500%, greater than 5000% compared to injections, or ranges including and / or spanning the aforementioned values. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles may have a PK profile with a bioavailability from about 100% to about 5000% compared to injections.

[0170] In some embodiments, the patch and transdermal delivery' of the lipid nanoparticles may provide an enhanced delivery of the lipid nanoparticles through the skin of the subject. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles may provide a longer lasting delivery of the lipid nanoparticles to the subject as compared to injections. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles area under the curve (AUC) in plasma for the lipid nanoparticles is about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 times higher than intravenous injections, or ranges including and / or spanning the aforementioned values. In some embodiments, the patch and transdermal delivery of the lipid nanoparticles area under the curve (AUC) in plasma for the lipid nanoparticles is 5.4 times higher than intravenous injections. In some embodiments, the patch and transdermal delivery7of the lipid nanoparticles area under the curve (AUC) in plasma for the lipid nanoparticles is 8.9 times higher than intravenous injections.

[0171] In some embodiments, the patch and transdermal delivery of an effective amount of the lipid nanoparticles into the subject is achieved at least 0.5 hour after administration, about 1 hour after administration, about 2 hours after administration, about 3 hours after administration, about 4 hours after administration, about 5 hours after administration, about 6 hours after administration, about 7 hours after administration, about 8 hours after administration, or ranges including and / or spanning the aforementioned values.

[0172] In some embodiments, the patch and transdermal delivery of an effective amount of the lipid nanoparticles into the subject was maintained more than 3 hours afteradministration, more than 4 hours after administration, more than 5 hours after administration, more than 6 hours after administration, more than 7 hours after administration, more than 8 hours after administration, more than 9 hours administration, more than 10 hours after administration, or ranges including and / or spanning the aforementioned values. In some embodiments, immune responses last over several months.

[0173] In some embodiments, the patch and transdermal delivery of the lipid nanoparticles into the skin achieves a Tmax about 1 hour after administration, about 2 hours after administration, about 3 hours after administration, about 4 hours after administration, about 5 hours after administration, about 5 hours after administration, or ranges including and / or spanning the aforementioned values.

[0174] In some embodiments, a subject receives sufficient lipid nanoparticles from multiple dosages before high levels of lipid nanoparticles are achieved. One can readily and immediately envision a regimen wherein a subject is administered a first patch, and the subject receives one or more subsequent patches. Such a regimen may continue such that the subject receives a third patch after the subject receives the second patch. In some embodiments, a subject may receive: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more patches during treatment. One or more additional patches as described herein may be administered before the first patch dose, or before one or more subsequent patch dosages.

[0175] In some instances, a period of time passes between administering one or more patches to a subject. In some embodiments, the time period between one or more patches administered is equal to or at least about: twice daily, 1 day, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months. 6 months, 7 months. 8 months, 9 months, 10 months, 11 months, 1 year, or ranges including and / or spanning the aforementioned values. In some embodiments, one or more additional therapeutic agents are administered to the subject during the period between the subject’s administrations of the composition.

[0176] A subject in need of receiving a patch as disclosed herein to improve the subject’s health need not always be identified prior to receiving a first treatment with the patch described herein. For example, a subject may be predetermined to develop a disease or condition in the future, prior to showing any present signs or symptoms of a disease or condition (such as contact with another person known to have tested positive for coronavirus). Alternatively, the subject may receive treatment prophylactically if he or she is at risk or not at risk of a disease or condition (e.g., once a patient reaches an age of equal to or greater than 50, 60, 70, etc.). Accordingly, in some embodiments, the patch is administered to the subject after the subject receives an early diagnosis. In some embodiments, not every subject is a candidate for such administration and identification of treatment subjects may be desirable. It is understood thatpatient selection depends upon a number of factors within the skill of the ordinarily skilled physician. Thus, some embodiments disclosed herein further comprise identifying a subject as one that will benefit from administering an effective amount of lipid nanoparticles or composition including the same to increase longevity, increase survival time, increase life span, or improve upon immunization. Subjects may be identified on the basis of physiological factors specific to the subject according to the subject’s age, present medical condition, present medical treatment, prescribed medical treatment, or in some embodiments, the subject being diagnosed with a disease or condition. In some embodiments, treatment of a disease or condition includes preventing, reducing, and / or slowing the infection of a virus.

[0177] In some embodiments, a method of stimulating an immune response in a subject includes administering a patch as described herein. In some embodiments, a method of stimulating a neutralizing antibody response in a subject includes administering a patch as described herein. In some embodiments, a method of inducing a protective immune response in a subject includes administering a patch as described herein. In some embodiments, a method of stimulating a directed immune response in a subject includes administering a patch as described herein to a subject.

[0178] In some embodiments, as discussed elsewhere herein, the subject is a human. However, the methods are not limited to the treatment of humans and are equally applicable to the treatment of mammals. In such instances of treating non-human mammals, patient selection depends upon a number of factors within the skill of the ordinarily skilled veterinarian or research scientist.Svstem / Apparatus

[0179] The basic configurations of microporation drug delivery systems are known to those skilled in the art and thus do not require further elaboration herein. For example, transdermal permeant delivery systems are described in U.S. Patent No. 8.116,860, which is hereby incorporated herein by reference and particularly for the purpose of describing various features of such microporation drug delivery systems. As described therein, the microporation drug delivery system of U. S. Patent No. 8, 11 ,860 (referred to therein using the reference number "TO”) includes basic features that include a filament array (referred to therein using the reference number "70") configured to create the one or more pathways or micropores in a patient’s skin and one or more transdermal patches (referred to therein using the reference number ’TOO”) containing at least one drug formulation. Other similar microporation drug delivery7systems including such basic features are known to those skilled in the art. Various microporation drug delivery systems having suchbasic features are known to those skilled in the art and may be used or adapted for use by those skilled in the art guided by the teachings provided herein.

[0180] In some embodiments, the mi croporation device may be defined by a total area of pathways (for example, micropores) created in the skin by the one or more filaments and a total energy delivered to the one or more filaments to create the pathways. In some embodiments, the microporation device creates pathways such that the total area of pathways in the skin is between approximately 0.01 and 4.0 of a square centimeter (cm) of the skin. In some embodiments, the filament density is about 100, 125, 150. 175, 200, 225. 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 filament / cm2In some embodiments, the filament density is about 200 filament / cm2. In some embodiments, the filament density is from about 300 filament / cm2. In some embodiments, the filament density is 400 filament / cm2or ranges including and / or spanning the aforementioned values. In some embodiments, the pathways in the skin may include comprise approximately 0.5 to 12.5% of the skin area for each square centimeter of the skin exposed to the microporation device. In some embodiments, the pathways in the skin may include comprise approximately 1.25 to 10% of the skin area for each square centimeter of the skin exposed to the microporation device.

[0181] In some embodiments, the energy delivered to the one or more filaments to create the pathways may be in the range of 0.0067 pj / pm3- 0.0400 pj / pm3. The energy delivered to the one or more filaments may be delivered in pulses of between 2 and 12 milliseconds (ms) for sufficient energy to create consistent pathways to be delivered. Characteristics of pathways that efficiently and safely deliver a drug through the skin, for example from a patch as described herein, may vary between in vitro and in vivo embodiments. For example, in some embodiments, the one or more filaments creates the pathway when energy of between 2 mJ / filament and 12 mJ / filament is applied to the one or more filaments for a pulse of between 2 and 16 ms. In some embodiments, the energy' applied to the one or more filaments to create the pathway is between 2 mJ / filament and 8 mJ / filament, 2 mJ / filament and 6 mJ / filament or between 2 mJ / filament and 4 mJ / filament. In some embodiments, the duration of the pulse is between 2 and 12 ms. In such embodiments, the one or more filaments may comprise or substantially be formed from stainless steel and having a volume (V) of 300,000 pm3(0.0067 pJ / pm3-0.0400 mJ / pm3) for 2 mJ / filament- 12mJ / filament.

[0182] In some embodiments, the one or more filaments, arranged in a filament array, can create between 25 and 500 pathways / cm2of the biological membrane (e.g., skin) to which the one or more filaments are exposed. In some embodiments, the one or more filaments arranged in a filament array, can create between 50 and 400 pathways / cm2of the skin to which the one or more filaments are exposed. In some embodiments, the one or more filament array is 100pathways / cm2In some embodiments, the one or more filament array is 200 pathways / cm2. In some embodiments, the one or more filament array is 300 pathways / cm2. In some embodiments, the one or more filament array is 400 pathways / cm2. In some embodiments, the one or more filament array is 500 pathways / cm2.

[0183] In some embodiments, an accumulated (or summed) depth of all the pathways formed by the one or more filaments is between approximately 2500 and 30000 pm per square centimeter of skin exposed to the one or more filaments. In some embodiments, an accumulated or summed volume of all the pathways formed by the one or more filaments is between approximately 0.05 and 0.35 mm3per square centimeter of skin.

[0184] In some embodiments, the patch described herein may have one or more characteristics that enhance the optimal drug release and diffusion of the lipid nanoparticles into the body in conjunction with the pathways created by the microporation device.

[0185] In some embodiments, the improvements to the microporation device and the patch as described herein enable the microporation drug delivery system to effectively and safely provide delivery of the lipid nanoparticles in a manner that provides improved bioavailability and / or transferability' of the lipid nanoparticles directly immune cells in skin or from the blood to the desired location of the lipid nanoparticles in the subject. In some embodiments, the microporation drug delivery system for delivery of lipid nanoparticles can include a substrate having an upper substrate surface and defining the poration area, the substrate comprising a filament array having a plurality of filaments that are disposed in the poration area, wherein teach filament is capable of conductively delivering thermal energy via direct contact to the tissue membrane to form a plurality of micropores in a micropore area. In some embodiments, the substrate is the skin of the subject. Embodiments of the microporation drug delivery system described herein may provide for improved patient compliance and enhanced drug delivery capabilities. Embodiments of the microporation drug delivery system may also provide for reduced risks of adverse effects caused by uncontrolled delivery and reduce development terms and costs for drugs for patients. Embodiments of the microporation drug delivery system also enables painless and needle-free self-administration of corresponding drugs by the patient a location of patient’s choice, which leads to improved compliance and reduced costs (less visits to health care professionals). Embodiments of the microporation drug delivery system as described herein may be used for patients with a wide range of skin types, conditions, and so forth with a lower variation on individual drug delivery results.EXAMPLES

[0186] Various embodiments and alternatives are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.Example 1

[0187] This example provides a general manufacture of patches for the transdermal microporation delivery of mRNA lipid nanoparticles.

[0188] Patch Manufacturing. A patch was manufactured based on the procedure known by those skilled in the art. Individual formulations were utilized based on different ingredients as described herein. The patch includes a top layer (i.e. a backing with adhesive), a middle layer (i.e. a reservoir or matrix including lipid nanoparticles, an mRNA, and ingredients disposed within the reservoir or matrix, or a tablet containing lipid nanoparticles and an mRNA and ingredients), and a bottom layer (i.e. a release liner), wherein the release liner is configured to be removed before application to the subject's skin. The four types of patches (reservoir patch, lyophilized dry patch, tablet patch and solid dispersed dry patch) are described in detail in FIG. 1.

[0189] mRNA lipid nanoparticles were prepared by a microfluidic mixing device.

[0190] As illustrated in FIG. 1, patches produced according to certain embodiments of the methods described herein may comprises a top layer 101, a middle layer 103. and a bottom layer 105. In some embodiments, the top layer 101 may comprise a backing optionally with an adhesive. In some embodiments, the middle layer 103 comprises a matrix with a spacer. In some embodiments, the middle layer 103 does not comprise a matrix. In some embodiments, the bottom layer 105 comprises a release liner.

[0191] In the reservoir method, the reservoir patches were prepared using a spacer 115 and 117 to make a cavity in the middle layer 103 of the patch. In some embodiments, a matrix 107 comprising a liquid containing mRNA encapsulated lipid nanoparticles is disposed in the cavity. The reservoir patch may also include a water insoluble material (liquid absorbing material). These materials, chosen to hold a liquid containing the mRNA solution, can include woven and nonwoven gauzes, fiber membranes, films, or combinations of these. All ingredients, including mRNA lipid nanoparticles, are filled in a cavity in the middle layer 103 of the patch.

[0192] In the lyophilized method, all ingredients, including mRNA lipid nanoparticles, were filled in a cavity' formed by a spacer 119 and 121 in the middle layer of the patch. Then, the patch was lyophilized without a release liner to form a lyophilized dry patch according to FIG. 1. In some embodiments, a matrix 109 comprising lyophilized mRNA and lipid nanoparticles is disposed in the cavity.

[0193] In the tableting method, the dry ingredients, such as sugars, are mixed and weighted as a designated amount. The weighed powder was transferred into the designated sizeof the die punch for the compression machine. The die was set to the machine and compressed. The thin square tablet 111 was taken and placed on the non-woven pad located in a cavity formed by the spacer 123 and 125 in the middle layer 103 of the patch. The patch was covered by a release liner. The release liner was removed and the remaining ingredients, including mRNA lipid nanoparticles, are filled onto the tablet 11 1 in the patch to form a tablet patch according to FIG. 1.

[0194] In the dry patch dry ing method, all ingredients, including the mRNA lipid nanoparticles preparation, were dispersed in a solvent. The predetermined amount of ingredients was dispensed onto a non-woven pad 111 of the middle layer 103 of the solid dispersed dry patch. The dispensed patch was moved to the drying desiccator or drying oven until dried. The dried patch was removed from the drying desiccator or drying oven, and the patch was covered by a release liner to form a solid dispersed dry' patch according to FIG. 1. The individual patch was packed with a desiccant in the aluminum laminated pouch.

[0195] As illustrated in FIG. 2, a lyophilized dry patch may also be prepared by forming, the top layer 201 (a backing) similar to a blister pouch to hold all ingredients, including mRNA lipid nanoparticles in a middle layer 203. After lyophilization, the patch was covered by the bottom layer 205 comprising a release liner. In some embodiments, the lyophilized dry patch may also comprise a spacer 207 adjacent to the middle layer 203. The individual patch was packed with a desiccant in an aluminum laminated pouch.Example 2

[0196] The transdermal microporation delivery (PassPort: PP) of mRNA lipid nanoparticles was tested and evaluated immunogenicity in comparison with intramuscular injection (IM) in rats.

[0197] An ovalbumin (OVA) mRNA sequence w as used as a model mRNA. The lipid nanoparticle was purchased from Cayman (Lipid Nanoparticle (LNP-0315) Exploration Kit.

[0198] mRNA lipid nanoparticles were prepared by a microfluidic mixing device (Lilac Pharma, LiNAS-M System). The lipid nanoparticle manufacturing conditions are shown in Table 1. 0.9 mL OVA mRNA aqueous solution and 0.3 mL ethanol lipid solution were used.Table 1.

[0199] The ethanol lipid solution contains ALC-0315, L2-DSPC, cholesterol and ALC-0159 was prepared according to the following procedure: prepare individual lipid stock solutions of the three lipids supplied as crystalline solids in absolute ethanol. ALC-0315 is ready to use as supplied; bring all stock solution to room temperature prior to use and ensure they are well-dissolved: transfer the appropriate volume of each lipid mixture component to a single tube as listed in Table 1 A below to prepare the ethanol lipid mixture; mixing by pipetting several times.Table 1A. Lipid Solution Preparation

[0200] The mRNA aqueous solution was prepared by mixing 0.4797 mL of OVA mRNA stock solution (1 mg / mL) with 0.4203 mL of water to obtain 0.9 mL final aqueous solution having a concentration of OVA mRNA of 533 pg / mL.

[0201] The formulated mRNA (OVA) lipid nanoparticle was analyzed via Dynamic Light Scattering (DLS) for Hydrodynamic diameter (Z-Avg) and Poly dispersity Index (Pdl). The results showed a Z-Avg of 87.68 d.nm and a Pdl of 0.170.

[0202] The PP patches were prepared according to Example 1. The solution containing OVA mRNA lipid nanoparticle was filled in a reservoir or tablet patch just before in vivo study. The dry patch was stored in the refrigerator after preparation until starting in vivo study.

[0203] A prime vaccination was administered by IM and PP on Day-0. The groups consisted of saline IM (Group 1 as a base line control), IM (Group 2 containing OVA mRNA) and mRNA PP-1-4 (Groups 3 to 6 containing OVA mRNA).Table 2, In vivo Immunization Study in Rats

[0204] Blood samples were taken at 2 and 4 weeks after a prime vaccination. After four weeks, the rats were sacrificed, and blood samples were collected for anti-OVA IgG ELISA analysis using the Rat Ovalbumin Specific IgG ELISA Kit (MyBioSource). FIG. 3 depicts immunogenicity results from the study. All PP groups showed a similar IgG increase levels compared to IM (Group 2). PP-1 (Group 3). PP-3 (Group 5), and PP-4 (Group 6) showed similar percentage change of antibody production compared to IM. PP-2 (Group 4) showed a higher increase compared to the IM.Example 3

[0205] The PP delivery of OVA mRNA lipid nanoparticles was tested under different PP conditions to evaluate immunogenicity in rats. The OVA mRNA Lipid Nanoparticle (NanOZ LNP-mRNA(OVA), Cat #: LNP11000MRNA41) used was purchased from OZ Biosciences USA Inc.

[0206] The PP patches were prepared according to Example 1. The solution containing OVA mRNA lipid nanoparticles was filled into a reservoir and non-woven reservoir patches before the in vivo study. The dry' patch was prepared in a desiccator before the in vivo study.

[0207] A prime vaccination was administered on Day 0, and a booster vaccination was administered on Day 14. Blood samples were taken at 4 weeks after the prime vaccination. The titer amount of serum OVA-specific IgG was measured by ELISA. FIG. 4 and FIG. 5 depict the immunogenicity results from the study.

[0208] The materials used in the ELISA is summarized in Table 2A. OVA slgG Titer ELISA assay procedure is described as the following1) Add 100 uL of the 10 ug / mL OVA solution onto the 96 well plate (ThermoFisher MaxiSorp Plate) and incubate overnight at 4 °C;2) After overnight incubation, wash plate three times with 280 uL wash buffer 3 times;3) Add 200 uL / well of 1% BSA in PBS and incubate for 2h at room temperature;4) Wash blocked plate with 280uL wash buffer 3 times, add 50uL of diluted sample in each well and incubate for 2h at room temperature;5) Wash plate with 280uL wash buffer 3 times;6) Dilute Anti-IgG, Rat Goat-Poly, HRP by using 1 / 10 Block ace (1:4000), take lOuL of stock antibody and dilute with 39.990 uL of 1 / 10 block ace. and add lOOuL / well of Dilute Anti-IgG, Mouse Goat-Poly, HRP; then incubate for Ih at room temperature under dark condition;7) Wash plate with 280uL wash buffer 3 times and add lOOuL / well of TMB solution, and incubate for 25 minutes at room temperature under dark condition;8) Add IM sulfuric acid lOOuL / well;9) Measure the absorbance at 450 nm;10) The maximum dilution ratio where the absorbance gives a higher AU value then the threshold (2x the average of the blank AU values) is taken as the sample's antibody titer.Table 2A. EUISA Materials

[0209] The study consisted of Groups 1 to 15. Group 1 was the PBS IM group (negative control), Groups 2 to 10 containing 1 pg OVA mRNA, and Groups 11 to 15 containing 5 pg OVA mRNA as seen in Tables 3 and 4. According to Tables 3 and 4 below, groups 3-10 and 12-15 are vaccine (for example, OVA mRNA in UNP) administered using the mi croporation drug delivery system with different administration parameters (i.e.. patch type, patch area, formulation, and dose). Table 3 provides parameters for patches of groups 1-10. Table 4 provides parameters for patches of groups 11-15.Table 3. In vivo Immunization Study in Rats (1 pg mRNA)Table 4. In vivo Immunization Study in Rats (5 pg mRNA)

[0210] FIG. 4 shows a bar graph indicating a total amount of antibody titers in rats for groups 2 to 10 of vaccines administered l ug OVA mRNA groups according to the parameters in Table 3 at the 4-week blood sampling point. All groups (G2-10) showed higher levels compared to the negative control group (Gl). For the PassPort groups. Group 5 (Reservoir Patch (1 .0 cm2)), Group 6 (Reservoir Patch (0.2 cm2) with a liquid absorbing material), Group 9 (Dry Patch (0.2 cm2) with Img lactose), and Group 10 (Dry Patch (1.0 cm2)) overall showed the highest antibody response at the Ipg dose. Comparing the reservoir patch groups (Groups 3, 4, and 5) the larger reservoir area yielded a slightly higher or similar antibody response. Comparing the dry patch groups (Groups 7, 8, 9, and 10), the dry patch with added lactose (1 mg) and the 1.0cm2dry patch yielded higher antibody response compared to all other PP groups.

[0211] FIG. 5 shows a bar graph indicating a total amount of antibody titers in rats for groups 11 to 15 of vaccines administered 5 pg OVA mRNA according to the parameters in Table 4 at the 4-week blood sampling point. All groups (Gl 1-1 ) showed higher levels compared to the negative control group (Gl). Group 12 (Dry Patch (0.5 cm2)) showed the higher antibody response out of the PP reservoir patch groups at the 5 g dose.

[0212] Furthermore, although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, it will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but rather to also cover all modification and alternatives coming with the true scope and spirit of the disclosure.

Claims

WHAT IS CLAIMED IS:

1. A patch for delivering RNA into a skin of a subject, the patch comprising: a backing; a reservoir comprising lipid nanoparticles and one or more RNA molecules; and a release liner, wherein, the release liner is configured to be removed before application of the patch to the subject’s skin.

2. The patch of claim 1, wherein the lipid nanoparticles comprise one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, an anionic lipid, a neutral lipid, an amino lipid, a sphingolipid, a helper lipid, a lipid vesicular core, a phospholipid, and a conjugated lipid, and combinations thereof.

3. The patch of claim 2, wherein the cationic lipid is selected from the group consisting of ALC-0315, SM-102, (15Z, 18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-l-yl) tetracosa-15.18-dien-l-amine, (15Z, 18Z)-N,N-dimethyl-6- ((9Z,12Z)-octadeca-9,12-dien-l-yl) tetracosa-4.15.18-trien-l -amine, (15Z,18Z)-N,N-dimethyl-6- ((9Z,12Z)-octadeca-9,12-dien-l-yl) tetracosa-5,15,18-trien- 1 -amine, DSDMA, DLin-K-DMA, , DLin-K-C3-DMA, DLin-K-C4- DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA, DLin-M-C3-DMA, DLin- MP-DMA. l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC). l,2-dioleoyl-3- trimethylammonium propane (DOTAP), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 3p-[N-(N,N- dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol), N.N-dimethyl-N,N-di-9-cis- octadecenylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N- dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-Dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N.N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y-DLenDMA). 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP), l,2-Dilinoleyloxy-3- (dimethylamino)acetoxypropane (Dlin-DAC), l,2-Dilinoleyloxy-3 -morpholinopropane (Dlin- MA). l,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3- dimethylaminopropane (Dlin-S-DMA), l-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (Dlin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (Dlin-TMA.Cl), 1,2- Dilinoleoyl-3-trimethylaminopropane chloride salt (Dlin-TAP.Cl), l,2-Dilinoleyloxy-3-(N- methylpiperazinojpropane (Dlin-MPZ), 3-(N,N-Dilinoleylamino)-l,2-propanediol (DlinAP), 3- (N,N -Dioley lamino)- 1 ,2-propanediol (DO AP), 1 ,2-Dilinoleyloxo-3 -(2-N,N -dimethylamino)ethoxy propane (Dlin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]- di oxolane (Dlin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), l,l’-(2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethylazanediyl)didodecan-2-ol (Cl 2-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (Dlin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (Dlin-K- DMA), 3-((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yloxy)-N,N-dimethylpropan-l- amine (MC3 Ether). 4-((6Z,9Z.28Z,31Z)-heptatriaconta-6.9.28,31-tetraen-19-yloxy)-N.N- dimethylbutan- 1 -amine (MC4 Ether), N-(l-(2,3-dioleyloxy)propyl)-N-2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), Dioctadecylamidoglycyl carboxy spermine (DOGS), l,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxy ethyl ammonium bromide (DMRIE), 2,2-Dilinoleyl-4-dimethylaminoethyl-tl,3J-dioxolane (XTC), and combinations thereof.

4. The patch of claim 2, wherein the anionic lipid is selected from the group consisting of phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N- dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines. N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and combinations thereof.

5. The patch of claim 2, wherein the helper lipid is selected from the group consisting of 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE), cholesterol, l-palmitoyl-2-oleoyl-sn-glycero-3phosphocholin (POPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and combinations thereof.

6. The patch of claim 2, wherein the phospholipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidyl serine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoylol eyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE). monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine.

7. The patch of any one of claims 1 to 6, wherein the one or more RNA molecules are encapsulated in the lipid nanoparticles.

8. The patch of any one of claims 1 to 7, wherein the one or more RNA molecules comprise an mRNA encoding a biologically active protein, or a nucleotide sequence homologous to an mRNA in a target cell.

9. The patch of any one of claims 1 to 8, wherein the lipid nanoparticles are in an amount in the range from about 0.01 mg / cm2to about 200 mg / cm2, from about 0. 1 mg / cm2to about 20 mg / cm2, or from about 0. 1 mg / cm2to about 5 mg / cm2in the reservoir.

10. The patch of any one of claims 1 to 9, wherein the reservoir further comprises at least one sugar, a drug delivery modifier, and a preservative.

11. The patch of claim 10, wherein the at least one sugar is sucrose, trehalose, mannitol, sorbitol, lactose, maltose, or a combination thereof.

12. The patch of claim 10 or 11, wherein the weight ratio of the at least one sugar to the lipid nanoparticles is greater than 0.02.

13. The patch of claim 12, wherein the weight ratio of the at least one sugar to the lipid nanoparticles is from about 0.02 to about 0.4.

14. The patch of any one of claims 10 to 13, wherein the drug delivery modifier is an organic acid, a salt thereof, or a combination thereof.

15. The patch of claim 14, wherein the drug delivery’ modifier is citric acid or its salt form, or a combination thereof.

16. The patch of any one of claims 10 to 15, wherein the preservative is an anti-microbial agent.

17. The patch of claim 16, wherein the anti-microbial agent is selected from the group consisting of methyl paraben, propylparaben, benzalkonium chloride, and sodium benzoate, and combinations thereof.

18. The patch of any one of claims 1 to 17, wherein the reservoir comprises at least one of sucrose, lactose, disodium citrate sesquihydrate, methylparaben, propylparaben, and benzalkonium chloride.

19. The patch of any one of claims 1 to 18, wherein the reservoir comprises at least one fiber or a laminated material of film, or a combination thereof.

20. The patch of claim 19, wherein the at least one fiber is a non-woven fiber having a thickness of less than 300 pm.

21. The patch of claim 19 or 20, wherein the at least one fiber has a weight of less than 100 g / m222. The patch of any one of claims 1 to 21, wherein the reservoir has a water-holding capacity of less than 20 mg / cm2.

23. A device for delivering RNA into a skin of a subject through a plurality of micropores, the device comprising: a porator comprising an array of conductive filaments; an applicator electrically connected to the conductive filaments and configured to supply a predetermined electrical energy to the array of conductive filaments to create the plurality of micropores in an micropore area of the skin by heating the filaments; and a patch according to any one of claims 1 to 22.

24. The device of claim 23, wherein the device is configured to generate thermal energy based on a current flowing through the array of conductive filaments and provide the thermal energy to a dermal membrane positioned adjacent to the device.

25. The device of any one of claims 23 or 24, wherein the applicator is configured to supply a predetermined electrical energy' to the array of conductive filaments for creating the plurality of micropores.

26. The device of any one of claims 23 to 25, wherein the device creates between about 25 to about 500 micropath way s / cm2.

27. The device of any one of claims 23 to 26, wherein the device has a filament density from about 300 to about 500 filament / cm2or about 400 filament / cm228. The device of any one of claims 23 to 27, wherein the device has a poration energy’ from about 2 mJ / filament to about 10 mJ / filament or about 4 mJ / filament to about 8 mJ / filament.

29. The device of any one of claims 23 to 28 wherein the porator is configured to open at least one channel in the subject’s skin, and has an area from about 0.01 cm2to about 4 cm2.

30. The device of claim 29, wherein the porator is configured to open at least one channel in the subject’s skin and has an area of about or less than about 0.1 cm2, about 0.2 cm2, about 0.25 cm2, about 0.65 cm2, or about 1.0 cm2.

31. The device of claim 29, wherein the at least one channel is one or more micropores, wherein one micropore is about 0.5% to about 12.5% of the total microporation area.

32. A system for transdermal RNA delivery into a skin of a subject through a plurality of micropores, the system comprising: a substrate having an upper substrate surface and defining a poration area, the substrate comprises a filament array having a plurality of filaments that are disposed in the poration area, wherein each filament is capable of conductively delivering thermal energy’via direct contact to the skin to form a plurality of micropores in a micropore area of the skin; an applicator electrically connected to the filament array and configured to supply a predetermined electrical energy to the filaments to create the plurality of micropores in the micropore area of the skin by heating the filaments; a power supply circuit configured to provide electric current to the applicator; and a patch according to any one of claims 1 to 22 configured for application on the micropore area.

33. The system of claim 32, wherein the applicator is configured to generate thermal energy based on the electric current flowing through the array of conductive filaments.

34. The system of any one of claims 32 or 33, wherein the applicator creates between about 25 to about 500 micropathways / cm2or from about 400 micropathways / cm2.

35. The system of any one of claims 32 to 34, wherein the applicator has a filament density’ from about 300 to about 500 filament / cm2or from about 400 filament / cm236. The system of any one of claims 32 to 35, wherein the applicator has a poration energy from about 2 to about 10 mJ / filament or about 4 mJ / filament to about 8 mJ / filament.

37. The system of any one of claims 32 to 36, wherein the substrate is configured to open at least one channel in the subject’s skin, wherein the substrate has an area from about 0.01 cm2to about 4 cm2.

38. The system of claim 37, wherein the substrate is configured to open at least one channel in the subject’s skin and the substrate has an area of about or less than about 0.1 cm2, about 0.2 cm2, about 0.25 cm2, about 0.65 cm2, or about 1.0 cm2.

39. The system of any one of claims 32 to 38 wherein the one or more micropores is about 0.5 to about 12.5% of the total poration area.

40. The system of any one of claims 32 or 39, wherein the applicator produces at least 50 pores in a subject's skin.

41. A method of providing RNA to a subject, the method comprising: opening at least one channel in the subject’s skin; and applying the patch of any one of claims 1 to 22 to the subject’s skin.

42. A method for triggering an immune response in a subject in need thereof, the method comprising: opening at least one channel in the subject’s skin; and applying the patch of any one of claims 1 to 22 to the subject’s skin.

43. The method of claim 41 or 42, wherein opening at least one channel in the subject’s skin comprises applying a transdermal microporation device according to any one of claims to 23 to 31 to the subject’s skin.

44. The method of claim 41, wherein the RNA is mRNA comprising a sequence encoding an antigenic peptide or protein, or a fragment, variant or derivative thereof.

45. The method of claim 44, wherein the sequence encoding the antigenic peptide or protein, or the fragment, variant or derivative thereof is selected from the group consisting of pathogenic antigens, tumor antigens, allergenic antigens and autoimmune self-antigens.

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